key: cord- -p abodr authors: backofen, rolf; fricke, markus; marz, manja; qin, jing; stadler, peter f. title: distribution of graph-distances in boltzmann ensembles of rna secondary structures date: journal: algorithms in bioinformatics doi: . / - - - - _ sha: doc_id: cord_uid: p abodr large rna molecules often carry multiple functional domains whose spatial arrangement is an important determinant of their function. pre-mrna splicing, furthermore, relies on the spatial proximity of the splice junctions that can be separated by very long introns. similar effects appear in the processing of rna virus genomes. albeit a crude measure, the distribution of spatial distances in thermodynamic equilibrium therefore provides useful information on the overall shape of the molecule can provide insights into the interplay of its functional domains. spatial distance can be approximated by the graph-distance in rna secondary structure. we show here that the equilibrium distribution of graph-distances between arbitrary nucleotides can be computed in polynomial time by means of dynamic programming. a naive implementation would yield recursions with a very high time complexity of o(n ( )). although we were able to reduce this to o(n ( )) for many practical applications a further reduction seems difficult. we conclude, therefore, that sampling approaches, which are much easier to implement, are also theoretically favorable for most real-life applications, in particular since these primarily concern long-range interactions in very large rna molecules. the distances distribution within an rna molecule is of interest in various contexts. most directly, the question arises whether panhandle-like structures (in which ' and ' ends of long rna molecules are placed in close proximity) are the rule or an exception. panhandles have been reported in particular for many rna virus genomes. several studies [ , , , ] agree based on different models that the two ends of single-stranded rna molecules are typically not far apart. on a more technical level, the problem to compute the partition function over rna secondary structures with given end-to-end distance d, usually measured as the number of external bases (plus possibly the number of structural domains) arises for instance when predicting nucleic acid secondary structure in the presence of single-stranded binding proteins [ ] or in models of rna subjected to pulling forces (e.g. in atom force microscopy or export through a small pore) [ , , ] . it also plays a role for the effect of loop energy parameters [ ] . in contrast to the end-to-end distance, the graph-distance between two arbitrarily prescribed nucleotides in a larger rna structure does not seem to have been studied in any detail. however, this is of particular interest in the analysis of singlemolecule fluorescence resonance energy transfer (smfret) experiments [ ] . this technique allows to monitor the distance between two dye-labeled nucleotides and can reveal details of the kinetics of rna folding in real time. it measures the nonradiative energy transfer between the dye-labeled donor and acceptor positions. the efficiency of this energy transfer, e fret , strongly depends on the spatial distance r according to e fret = ( + (r/r ) ) − . the förster radius r sets the length scale, e.g. r ≈ Å for the cy -cy dye pair. a major obstacle is that, at present, there is no general and efficient way to link smfret measurements to interpretations in terms of explicit molecular structures. to solve this problem, a natural first step to compute the distribution of spatial distances for an equilibrium ensemble of d structures. since this is not feasible in practice despite major progress in the field of rna d structure prediction [ ] , we can only resort to considering the graph-distances on the ensemble of rna secondary structures instead. although a crude approximation of reality, our initial results indicate that the graph distance can be related to the smfret data such as those reported by [ ] . from a computer science point of view, furthermore, we show here that the distance distribution can be computed exactly using a dynamic programming approach. an rna secondary structure is a vertex labeled outerplanar graph g(v, ξ, e), where v = { , , . . . , n} is a finite ordered set (of nucleotide positions) and ξ : { , , . . . , n} → {a, u, g, c}, i → ξ i assigns to each vertex at position i (along the rna sequence from ' to ') the corresponding nucleotide ξ i . we write ξ = ξ . . . ξ n for the sequence underlying secondary structure and use ξ[i . . . j] = ξ i . . . ξ j to denote the subsequence from i to j. the edge set e is subdivided into backbone edges of the form {i, i + } for ≤ i < n and a set b of base pairs satisfying the following conditions: . the first condition allows base pairs only for watson-crick and gu base pairs. the second condition implements the minimal steric requirement for an rna to bend back on itself. the third condition enforces that b forms a matching in the secondary structure. the last condition (nesting condition) forbids crossing base pairs, i.e. pseudoknots. the nesting condition results in a natural partial order in the set of base pairs b defined as {i, j} ≺ {k, l} if k < i < j < l. in particular, given an arbitrary vertex k, the set b k = {{i, j} ∈ b|i ≤ k ≤ j} of base pairs enclosing k is totally ordered. note that k is explicitly allowed to be incident to its enclosing base pairs. consider a fixed secondary structure g, for a given base pair {i, j} ∈ b, we say a vertex k is accessible from {i, j} if i < k < j and there is no other pair {i , j } ∈ b such that i < i < k < j < j. the unique subgraph l i,j induced by i, j, and all the vertices accessible from {i, j} is known as the loop of {i, j}. the type of a loop l i,j is unique determined depending on whether {i, j} is external or not, and the numbers of unpaired vertices and base pairs. for details, see [ ] . each secondary structure g has a unique set of loops {l i,j |{i, j} ∈ b}, which is called the loop decomposition of g. the free energy f (g) of a given secondary structure, according to the standard energy model [ ] , is defined as the sum of the energies of all loops in its unique loop decomposition. the relative location of two vertices v and w in g is determined by the base pairs b v and b w that enclose them. if b v ∩ b w = ∅, there is a unique ≺-minimal base pair {i v,w , j v,w } that encloses both vertices and thus a uniquely defined loop l {iv,w ,jv,w } in the loop associated with v and w. that enclose v and w, respectively. this simple partition holds the key to computing distance distinguished partition functions below. it will be convenient in the following to introduce edge weights ω i,j = a if j = i + , i.e., for backbone edges, and ω i,j = b for {i, j} ∈ b. given a path p, we define the weight of the path d(p) as the sum of the weights of edges in the path. the (weighted) graph-distance d g v,w in g is defined as the weight of the path p connecting v and w with d(p) being minimal. for the weights, we require the following condition: (w) if i and j are connected by an edge, then {i, j} ∈ e is the unique shortest path between i and j. this condition ensures that single edges cannot be replaced by detours of shorter weight. condition (w) and property (ii) of the secondary structure graphs implies b < a because the closing base pair must be shorter than a hairpin loop. furthermore, considering a stacked pair we need b < b + a, i.e. a > . we allow the degenerate case b = that neglects the traversals of base pairs. for a fixed structure g, d g v,w is easy to compute. here, we are interested in the distribution p r[d g v,w |ξ] and its expected value as first shown in [ ] , q and related quantities can be computed in cubic time. a crucial quantity for our task is the restricted partition function for a given pair v, w of positions in a given rna sequence ξ. a simple but tedious [ ] . for the ease of presentation we describe in the following only the recursion for the simplified energy model for the "circular maximum matching" matching, in which energy contributions are associated with individual base pairs rather than loops. our approach easily extends to the full model by using separating the partition functions into distinct cases for the loop types. we use the letter z to denote partition functions with distance constraints, while q is used for quantities that appear in mccaskill's algorithm and are considered as pre-computed here. before we continue with the calculation of the partition function, let's first look into problem formulation in more detail. for the fret application, it is well-known that the rate which with fret occurs is correlated with the distance. therefore, only a limited range of distance changes (e.g. Å − Å for cy -cy ) can be reported by the fret experiments. thus the more useful formulation of our problem is not to use the full expected quantity for all positions. instead, we are interested in the average for all distances within some threshold θ d . as the space and time complexity will depend on the number of distances we consider, we will parametrise our complexity by the number of nucleotides n and the number of overall distances considered d = θ d + , as well. an important special case assumes that both v and w are external. this is case e.g. when v and w are bound by proteins. in particular, the problem of computing end-to-end distances, i.e., v = and w = n, is of this type. assuming (w), the shortest path between two external vertices v, w consists of the external vertices and their backbone connections together with the external base pairs. we call this path the inside path of i, j since it does not involve any vertices "outside" the subsequence ξ[i..j]. for efficiently calculating the internal distance between any two vertices v, w, we denote by z i i,j [d] the partition function over all secondary structures on ξ[i..j] with end-to-end distance exactly d. furthermore, let q b i,j denote the partition function over all secondary structures on ξ[i..j] that are enclosed by the base pair {i, j}. we will later also need the partition function q i,j over the sub-sequence ξ[i..j], regardless of whether {i, j} is paired or not. now note that any structure on ξ[i..j] starts either with an unpaired base or with a base pair connecting i to some position k satisfying i < k ≤ j. in the first can be split as follows, this gives the recursion [ ] , the investigate of loop entropy dependence [ ] , the analysis of fret signals in the presence of single-stranded binding proteins [ ] , as well as in mathematical studies of rna panhandle-like structures [ , ] . in the following it will be convenient to define also a special terms for the empty structure. setting allows us to formally write an individual backbone edge as two edges flanking the empty structure and hence to avoid the explicit treatment of special cases. this definition of z i also includes the case that i and j are base paired in the recursion ( ). this is covered by the case k = j, where we evaluate is the only admissible value here, this refers to z i j+ ,j [−a], which has the correct value of due to our definition. later on, we will also need z i under the additional condition that the path starts and end with a backbone edge. we therefore introduce z i defined as from v (green) to w (blue) is not an inside path: inside emphasizes that, in contrast to the shortest path (cyan arrows) between the red region and w, it is not contained in the interval determined by its end points. by our initialization of z i , we can simply define z i by this recursion requires o(dn ) time and space. it is possible to reduce the complexity in this special case by a linear factor. the trick is to use conditional probabilities for arcs starting at i or the conditional probability for i to be singlestranded, which can be determined from the partition function for rna folding [ ] , see appendix b. the minimal distance between two positions that are covered by an arc can be realized by inside paths and outside paths. this complicates the algorithmic approach, since both types of paths must be controlled simultaneously. consider fig. . the shortest path between the green and blue regions includes some vertices outside the interval between these two regions. the basic idea is to generalize equation ( ) to computing the partition function z v,w [d] . the main question now becomes how to recurse over decompositions of both the inside and the outside paths. fig. shows that the outside paths are important for the green region, i.e., the region that is covered by an arc. hence, we have to consider the different cases that the two positions v and w are covered by arcs. the set Ω of all secondary structures on ξ can be divided into two disjoint subclasses that have to be treated differently: note that this bipartition explicitly depends on v and w. in the following, we will first introduce the recursions that are required in Ω structures to compute contribution of Ω structures to z v,w [d] one example of this case is given in fig. with the red and blue region, where v (vertex in green region) is covered by an arc, and w (vertex in blue region) is external. denote the ≺-maximal base pair enclosing v by {i, j}. since at most one of v and w is covered by an arc, we know that j < w. hence, every path p from v to w, and hence also the shortest paths (not necessarily unique) must run through the right end j of the arc {i, j}. more precisely, there must sub-paths p and overall, the contribution to z v,w [d] for structures in Ω is given by note that for splitting the distance, we reuse the same indices (e.g., the j in where as for the remaining partition function, we use successive indices (e.g.,the i in . this difference comes from the fact that splitting a sequence into subsequences is done naturally between two successive indices, whereas splitting a distance is naturally done by splitting at an individual position. we have only to guarantee that the substructures which participate in the split do agree on the structural context of the split position. this is guaranteed by requiring that z i starts and ends with a backbone edge. we note that the incorporation of the full dangling end parameters makes is more tedious to handle the splitting positions. this results in a complexity of o(n d ) time and o(n d) space. however, we do not need to split in i, j, k, j simultaneously. instead, we could split case (c) at position j and introduce for all v ≤ j and k ≤ w the auxiliary variables finally, we can replace recursion ( ) by ( ) can be derived, with the additional complication since we have to take care of the additional outside distance due to the arc (p, q). thus, we obtain the following splitting: again we can avoid the complexity of simultaneously splitting at {i, j} and {k, l} by doing a major split after j. thus, we get the equivalent recursions as in eqns. ( ) ( ) ( ) : overall, we get the following recursion: there are only few base pairs that are likely to form and that cover v, w, especially for v, w where the internal distance of v, w is large enough such that an outside path has to be considered at all. if we assume a constant number of such long-range base-pairs, then the complexity is reduced by an n -factor. for the complexity in terms of distance, recall that d is typically small. as a black box. in order to compute these terms, we distinguish the limiting cases a.) v = i, b.) v = j, c.) is external from the generic case d.): starting from the limiting cases, we initialize z b,v v,j [ , d r ] as follows: o t h e r w i s e and analogously for finally, we have the following recursion for i = v = j, d > and d r > : where q b i,j is the external partition function over all structures on the union of the intervals ξ [ . .i] ∪ ξ[j..n] so that {i, j} is a base pair. this is equivalent the theoretical analysis of the distance distribution problem shows that, while polynomial-time algorithms exist, they probably cannot the improved to space and time complexities that make them widely applicable to large rna molecules. due to the unfavorable time complexity of the current algorithm and the associated exact implementation in c, a rather simple and efficient sampling algorithm has been implemented. we resort to sampling boltzmann-weighted secondary structures with rnasubopt -p [ ] , which uses the same stochastic backtracing approach as sfold [ ] . as the graph-distance for a pair of nucleotides in a given secondary structure can be computed in o(n log n) time, even large samples can be evaluated efficiently . in which, the minimum free energy structure is showed in (a), (c) is the real secondary structure which is ranked as the rd best sub-optimal structure with rnasubopt -e. he graphic representations of these structures are produced with varna [ ] . (b) the corresponding smfret efficiency (e f ret ) histograms are reported in [ ] . from these data, three separate states of the dase ribozyme can be distinguished, the unfolded (u), intermediate (i) and folded (f) states. (c) the graph distance distribution in the ensemble which is approximated with rnasubopt -p at temperature • c. as we pointed out in the introduction, the graph distance measure introduced in this paper can serve as a first step towards a structural interpretation of smfret data. as an example, we consider the graph distance distribution of a diels-alderase (dase) ribozyme (fig. (a) ). histograms of smfret efficiency (e fret ) for this nt long catalytic rna are reported in [ ] for a large number of surface-immobilized ribozyme molecules as a function of the mg + concentration in the buffer solution. a sketch of their histograms is displayed in fig. (b) . the dyes are attached to sequence positions (cy ) and (cy ) and hence do not simply reflect the end-to-end distance, fig. (a)(c). in this example, we observe the the expected correspondence small graph distances with a strong smfret signal. this is a particular interesting example, since the minimal free energy (mfe) structure (fig. (a)(a) ) predicted with rnafold is not identified with the real secondary structure (fig. (a)(c) ). in fact, the ground state secondary structure is ranked as the rd best sub-optimal structure derived via rnasubopt -e. the free energy difference between these two structures is only . kcal/mol. however, their graph distances show a relatively larger difference. the nd best sub-optimal structure (fig. (a)(b) ) looks rather similar with the rd structure, in particular, they share the same graph distance value. the smfret data of [ ] indicate the presence of three sub-populations, corresponding to three different structural states: folded molecules (state f), intermediate conformation (state i) and unfolded molecules (state u). in the absence of mg + , the i state dominates, and only small fractions are found in states u and f. unfortunately, the salt dependence of rna folding is complex [ , ] and currently is not properly modeled in the available folding programs. we can, however, make use of the qualitative correspondence of low salt concentrations with high temperature. in fig. (c) we therefore re-compute the graph distance distribution in the ensemble at an elevated temperature of • c. here, the real structure becomes the second best structure with free energy − . kcal/mol and we observe a much larger fraction of (nearly) unfolded structures with longer distances between the two beacon positions. qualitatively, this matches the smfret data showed in fig. (b) . long-range interactions play an important role in pre-mrna splicing and in the regulation of alternative splicing [ , ] , bringing splice donor, acceptor, branching site into close spatial proximity. fig. (a) shows for d. melanogaster pre-mrnas that the distribution of graph-distances between donor and acceptor sites shifted towards smaller values compared to randomly selected pairs of positions with the same distance. although the effect is small, it shows a clear difference between the real rna sequences and artificial sequences that were randomized by di-nucleotide shuffling. the spatial organization of the genomic and sub-genomic rnas is important for the processing and functioning of many rna viruses. this goes far beyond the well-known panhandle structures. in coronavirus the interactions of the ' trs-l cis-acting element with body trs elements has been proposed as an important determinant for the correct assembly of the coronavirus genes in the host [ ] . the matrix of expected graph-distances in fig. (b) shows that trs-l and trs-b are indeed placed near each other. more detailed information is provided in appendix (d). our first results show that the systematic analysis of the graph-distance distribution both for individual rnas and their aggregation over ensembles of structures can provide useful insights into structural influences on rna function. these may not be obvious directly from the structures due to the inherent difficulties of predicting long-range base pairs with sufficient accuracy and the many issues inherent in comparing rna structures of very disparate lengths. due the complexity of algorithm we have refrained from attempting a direct implementation in an imperative programming language. instead, we are aiming at an implementation in haskell that allows us to make use of the framework of algebraic dynamic programming [ ] . the graph distance measure and the associated algorithm can be extended in principle to of rna secondary structures with additional tertiary structural elements such as pseudoknots [ ] and g-quadruples [ ] . rna-rna interaction structures [ ] also form a promising area for future extensions. we note finally, that the fourier transition method introduced in [ ] could be employed to achieve a further speedup. a stem structure in fibroblast growth factor receptor transcripts mediates cell-type-specific splicing by approximating intronic control elements expected distance between terminal nucleotides of rna secondary structures varna: interactive drawing and editing of the rna secondary structure automated de novo prediction of native-like rna tertiary structures a statistical sampling algorithm for rna secondary structure prediction structure and functional relevance of a transcription-regulating sequence involved in coronavirus discontinuous rna synthesis impact of loop statistics on the thermodynamics of rna folding the end-to-end distance of rna as a randomly self-paired polymer modeling the interplay of single-stranded binding proteins and nucleic acid secondary structure force-induced denaturation of rna. biophys translocation of structured polynucleotides through nanopores algebraic dynamic programming the '- ' distance of rna secondary structures mg +-dependent folding of a diels-alderase ribozyme probed by single-molecule fret analysis ion-rna interactions thermodynamic analysis of the effects of mono-and divalent ions on rna conformational equilibria rna-rna interaction prediction based on multiple sequence alignments viennarna package . . alg d meets g: g-quadruplexes in rna secondary structure prediction expanded sequence dependence of thermodynamic parameters improves prediction of rna secondary structure incorporating chemical modification constraints into a dynamic programming algorithm for prediction of rna secondary structure the equilibrium partition function and base pair binding probabilities for rna secondary structure rna structure and the mechanisms of alternative splicing the secondary structure of rna under tension topology and prediction of rna pseudoknots a practical guide to single-molecule fret from sequences to shapes and back: a case study in rna secondary structures using the fast fourier transform to accelerate the computational search for rna conformational switches the ends of a large rna molecule are necessarily close this work was supported in part by the deutsche forschungsgemeinschaft proj. nos. ba / - , sta / - , spp and ma / - . key: cord- -livz zuu authors: gayathri, p.; satheshkumar, p.s.; prasad, k.; nair, smita; savithri, h.s.; murthy, m.r.n. title: crystal structure of the serine protease domain of sesbania mosaic virus polyprotein and mutational analysis of residues forming the s -binding pocket date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: livz zuu sesbania mosaic virus (semv) polyprotein is processed by its n-terminal serine protease domain. the crystal structure of the protease domain was determined to a resolution of . Å using multiple isomorphous replacement and anomalous scattering. the semv protease domain exhibited the characteristic trypsin fold and was found to be closer to cellular serine proteases than to other viral proteases. the residues of the s -binding pocket, h , t and n were mutated to alanine in the Δn -protease–vpg polyprotein, and the cis-cleavage activity was examined. the h a and t a mutants were inactive, while the n a mutant was partially active, suggesting that the interactions of h and t with p -glutamate are crucial for the e–t/s cleavage. a region of exposed aromatic amino acids, probably essential for interaction with vpg, was identified on the protease domain, and this interaction could play a major role in modulating the function of the protease. sesbania mosaic virus (semv) is an ss-rna plant sobemovirus found infecting sesbania grandiflora in india. its genome of nucleotides codes for four open reading frames (orfs) (lokesh et al., ) . orf codes for an -kda protein predicted to be a movement protein (sivakumaran et al., ) . the orf codes for a polyprotein, which encompasses more than one functional domain, while orf is present as an internal orf in orf and is expressed via ribosomal frame shifting mechanism (lokesh et al., ) . the orf is expressed from a sub-genomic rna and codes for the coat protein of the virus. polyprotein processing is one of the major strategies employed by both animal and plant viruses to generate more than one functional protein from the same polypeptide chain (wellink and van kammen, ) . to accomplish cleavage at specific sites, viruses employ one or more proteases with unique cleavage specificities. in an earlier study, we have shown that in semv, the processing is mediated by the nterminal protease domain coded by orf (satheshkumar et al., ) . it is a serine protease, similar to cellular proteases like trypsin and chymotrypsin (gorbalenya et al., ) . the catalytic residues are h , d and s . the protease cleaves the orf polyprotein at three different positions, at e -t , e -t and e -s to release four different domains-protease, vpg (viral protein genome linked), p and rdrp (rna-dependent rna polymerase) (satheshkumar et al., ) . in most other viruses that have vpg at the v end of their genome, the domain arrangement is vpg -protease, whereas, in sobemoviruses, it is protease -vpg. recent biochemical studies on the protease -vpg domains of semv have shown that the interaction of vpg with the protease domain modulates the protease activity (satheshkumar et al., ) . the serine protease domain lacking the amino acids (dn -pro) was found to be inactive in trans. however, the presence of vpg at the c-terminus of dn -pro rendered the polyprotein active in cis and trans. by mutational analysis, it was demonstrated that interaction of w in the vpg domain with the protease was responsible for both cis and trans proteolytic activities and the associated conformational changes. it was suggested that the natively unfolded vpg is an activator of the protease and could regulate the polyprotein processing. in the present study, the crystal structure of semv protease domain was determined to a resolution of . Å by multiple isomorphous replacement coupled with anomalous scattering, with a view to identify the residues involved in substrate binding as well as protease -vpg interactions. based on structural alignment of the s -binding pocket with proteases of glu/gln specificity, h , t and n were identified as glutamate-binding residues, and their role has been established by mutational analysis. a region of exposed aromatic residues, probably essential for interaction with the vpg domain, has been identified on the protease domain. this is the first report of the structure of a non-structural protein from the genus sobemovirus and provides the framework for understanding polyprotein processing in the genus. cloning and expression of the full-length protease domain (residues - of the polyprotein) in escherichia coli resulted in insoluble aggregates. examination of the amino acid sequence of the protease domain suggested that its amino terminal residues might correspond to a transmembrane region. it was found that the solubility and the activity of the protease were enhanced by the deletion of (dn -pro) and (dn -pro) residues. the dn -pro and dn -pro domains of the polyprotein were cloned, expressed and purified as described earlier (satheshkumar et al., ) . both dn -pro and dn -pro domains were crystallized using microbatch method in the presence of . m tris, ph . , . m ammonium sulphate, . m , -hexane diol, mm hmercaptoethanol and % glycerol. the protein crystals were shown to belong to the space group p . the cell parameters were determined to be a = b = . Å , c = . Å , a = b = -, c = -. the asymmetric unit of the crystal was compatible with a monomer of the protease with a matthew's coefficient of . Å /da and a solvent content of % (matthews, ) . a very mosaic diffraction pattern of limited resolution was observed with many crystals. the quality of diffraction of such crystals could be substantially improved by repeated soaking of the crystals in the cryoprotectant solution (crystallization buffer with % ethylene glycol) followed by flash freezing in liquid nitrogen. interestingly, the data from the annealed and non-annealed crystals scaled well. hence, both types of datasets were combined and used for structure solution. the use of a similar annealing procedure to improve diffraction has been reported earlier (kriminski et al., ) . surprisingly, both the dn -pro and dn -pro crystals had identical cell parameters even though dn -pro was amino acids longer than dn -pro. on mass spectroscopic analysis, it was observed that both had the same mass of¨ , da suggesting an internal cleavage. n-terminal sequencing of the proteins suggested a possible cleavage between the residues a and v (data not shown) in both the mutants. the ntermini of both dn -pro and dn -pro therefore correspond to the residue v . henceforth, the dn -pro and dn -pro were considered indistinguishable, and data from both constructs were combined for phasing. semv protease does not have any significant sequence similarity to any of the cellular or viral serine proteases. hence, molecular replacement method could not be used for phase determination, and data from heavy atom derivatives and anomalous scattering were used to determine the structure. the data collection statistics for the native and derivative datasets and phasing statistics are shown in tables a and b, respectively. most of the residues in the final map are in good electron density, except for a few residues at the protein surface. the side chains of a few lysines, arginines and aspartates with poor density have been truncated according to the extent of density observed for them. a section of the electron density map is shown in fig. . electron density is absent for the first three residues at the n-terminus and two residues at the c-terminus. hence, the final model includes residues to . the electron density for the stretch of residues from to is poor, and these residues are omitted in the model. the loop region comprising of residues to is partially disordered, and there is a break in the electron density at s . a total of water molecules have been added. the structure has been refined to a resolution of . Å though the data extended to . Å , due to the presence of very few test reflections in the last resolution bin. table c lists the refinement statistics. semv protease belongs to the trypsin-like family of serine proteases. the overall fold exhibits the characteristic features of the trypsin fold. fig. shows the overall fold of the protease and a topology diagram with the secondary structure elements labeled according to the convention followed for trypsin. it consists of two h barrels (domains i and ii) connected by a long inter-domain loop. both the domains belong to the all h class of proteins. the active site and the substrate-binding cleft occur in between the two domains and are fairly exposed to the solvent. there are only three helices in semv protease. the barrel formed by the h-strands ai, bi, ci, di, ei and fi constitutes the domain i. the first h-strand ai of domain i begins at l . the active site residue h occurs in the small helix in the segment connecting ci and di strands (fig. ) . strand ei extends from residues to and consists of two strands eia and eib connected by the stretch of residues from to in an extended but irregular conformation. d of the catalytic triad occurs at the end of the loop ei -fi. the loop connecting the two domains is a long stretch of residues extending from to . this region contains a small helix, formed by the residues to . the domain ii consists of the strands aii, bii, cii, dii, eii, fii and the c-terminal helix. the strand cii forms part of the wall of the s -binding pocket. there is an intramolecular disulfide bond connecting strands aii and cii. the loop connecting cii and dii forms the oxyanion hole and contains the active site s . the polypeptide chain ends in the c-terminal helix formed by residues ranging from to . the asymmetric unit consists of a monomer of semv protease. however, it was observed that there is an intermolecular disulfide bond between c of -fold symmetry related molecules. this bond was formed despite the presence of mm h-mercaptoethanol in the crystallization buffer. fig. shows the two molecules connected by the disulfide. this disulfide is likely to be an artifact of crystal packing as gel filtration studies have shown that the protease is a monomer in solution. this is further reflected in the area of the interface formed between the molecules ( . Å ), which is not very extensive. hence, the protease can be considered as a monomer in the crystal also, consistent with its behavior in solution. similar intermolecular disulfide bonds have been observed in the tobacco etch virus (tev) nia protease structure (phan et al., ) and in a few other cases. a comparison of the three-dimensional structure of semv protease domain with all the available entries in the protein data bank was carried out using the dali server (holm and sander, ) . the dali server identifies significant similarities in the three-dimensional structures of polypeptides irrespective of the sequence similarities between them. the highest z score ( . ) was observed for the heparin-binding protein ( a s), a serine protease homolog. the list of the top structures from the dali server output is shown in table a . the z scores and rmsd values suggest that the semv protease is closer to the non-viral proteases than to the viral proteases. the structures listed in table a and other viral proteases of known three-dimensional structure (table b ) were compared to the semv protease by pair wise alignment. the salient features of such a comparison are described below. a helix is present at the n-terminus in all the viral proteases except the capsid-forming proteases of sindbis, semilki forest and venezeulan equine encephalitis viruses ( svp, vcp and ep respectively). this n-terminal helix is not part of the canonical trypsin fold and is absent in the case of semv protease. the n-terminal residues of the semv protease pack against the bii strand of domain ii and have characteristics of h-strand-forming residues. main chain o and n of s form hydrogen bonds with the backbone atoms of the bii strand residue s . this conformation of the n-terminus agrees with those of the cellular and bacterial proteases namely heparinbinding protein ( a s), bacillus intermedius glutamyl endopeptidase ( p c), bovine beta trypsin ( ptp) and staphylococcus aureus epidermolytic toxin a ( agj). the rhino and polio virus proteases cqq and l n harbor the conserved rna-binding motif kfrdir (mosimann et al., ) in the helical segment occurring in the inter-domain loop. although this motif is absent in semv, the residues h , k and k constitute a positively charged patch in this region. however, the rna-binding properties of semv protease have not been investigated. another region, which shows considerable variability among the different families of serine proteases, is the loop connecting bii and cii. in many of the viral serine-like cysteine proteases of picornaviruses ( cqq, l n, hav and hrv), this loop has a long insertion that forms a h-hairpin, which extends to the substratebinding site. it contributes to the peptide binding and shields the active site from the solvent. the peptide substrate binds as a bridging strand between eii and the h-hairpin. semv protease does not have this insertion, and this leads to an exposed active site. this h-hairpin is not present in most of the non-viral proteases. the loop is also not found in the viral serine proteases equine arteritis virus nsp protease, hepatitis c virus protease ( mbm and jxp, respectively) and the capsid-forming proteases ( ep , vcp and svp), suggesting that an exposed active site is a characteristic of the viral serine proteases. the active site residue h forms hydrogen bonds with both s and d , the other two residues of the catalytic triad. the active site and the hydrogen bonds involved are shown in fig. a . the side chain of d is stabilized by two hydrogen bonds-between its oy and the main chain n of h , and its oy and ny of h . the n( of h is at a hydrogen bonding distance from og of s , although the geometry for the hydrogen bond is not optimal. the electron density for the imidazole ring of the h is not very clear, implying conformational flexibility. this is a feature found in the active site histidines of other serine proteases also. in the hrv protease, the active site h exists in alternate conformations (petersen et al., ) , while in the polio viral c protease, the electron density for the side chain is weak, and the mean temperature factor of the atoms is above the average (mosimann et al., ) . the oxyanion hole, a highly conserved region of the active site in terms of sequence (gxsg) and structure, is important for the stabilization of the oxyanion intermediate formed during the reaction. in semv protease, the main chain amide nitrogens of g and s are involved in the oxyanion hole formation. the structure is in conformity with the earlier mutational studies on the active site residues of semv protease (satheshkumar et al., ) . viral proteases involved in polyprotein processing are known to have very stringent substrate specificities. mutational analyses have demonstrated that the serine protease domain of semv cleaves at e -t occurring between protease -vpg, and vpg-p domains, and also at e -s between p and rdrp (satheshkumar et al., ) . in order to understand the structural basis of the stringent cleavage specificity of the enzyme, a comparison was carried out between semv protease and the structures of other proteases that display glutamate specificity (table c ). it was observed that the histidine that binds to the carbonyl oxygen of glu/gln is conserved in all glu/gln-specific proteases except the s. griseus proteases ( sga and sgp) where a positively charged arginine at the base of the substrate-binding pocket stabilizes the glutamate (read et al., ; read and james, ) . the corresponding residue in semv protease is h , and it is directed towards the active site. ny of h forms a hydrogen bond with n( of h (fig. a) . the conformation of h side chain is, in turn, stabilized by a hydrogen bond from ny to the main chain n of the disulfidebonded c . in the glutamate-specific s. griseus protease ( hpg), the role of the histidine triad, h , h and h ( hpg numbering), in stabilizing the charge on glutamic acid has been emphasized (nienaber et al., ) . in semv protease, a corresponding histidine triad does not exist, and h hydrogen bonded to the conserved h is not in the same position as that of h in the s. griseus protease. however, the chain of hydrogen bonds is maintained. this can possibly help in the stabilization of the charge of the glutamic acid side chain. the residues present in the proposed glutamate-binding site or the s pocket are t , a , h , f and n (fig. a) . out of these residues, only h and t are conserved across most of the glu/gln-binding proteins. in a few cases, thr is replaced by ser, whose og substitutes for the thr og. his and thr have been implicated to have a major role in glu/ gln binding, and the mutations of these have resulted in inactive enzymes in poliovirus c protease (ivanoff et al., ) . in the crystal structure of tev complexed with substrate, it has been observed that the carboxyamide group of glutamine forms hydrogen bonds with both his and thr (phan et al., ) . in the present structure, electron density observed near the oxyanion hole has been interpreted as a glycerol molecule. fig. b shows the glycerol bound near the s -binding pocket with the f o Àf c electron density around it contoured at . r level. it is possible that the orientation of the glycerol is related to the mode of glutamate binding. the main chain carboxyl group of glu should be directed towards the amide nitrogens of gly and ser at the oxyanion hole (perona and craik, ) . accordingly, the o of glycerol forms hydrogen bond with the main chain n of g (fig. ) . at the other end, o forms hydrogen bonds with both n( of h and main chain o of a . a water molecule, wat , is present between h and the glycerol. this water molecule is close to the aromatic ring of f and comes within hydrogen bonding distance from h and t side chain atoms. however, f aromatic ring and the water molecule are not in well-defined density. f is in a position equivalent to s of tev protease and s of equine arteritis virus nsp protease. these serine residues are involved in substrate binding at the p site (phan et al., ; barrette-ng et al., ) . further, all glu/glnbinding proteases, except semv protease, have either a ser or gly at this position. the absence of well-defined density for f in semv protease suggests that its side chain might undergo substantial displacement on binding of the substrate or on conformational changes induced by the interaction of the protease domain with vpg. tev and equine arteritis virus proteases, in which a serine residue occurs at the position corresponding to f , are active in trans. mutation of this f to serine, the corresponding residue in other glu/glnbinding proteases, might therefore bestow activity in trans to semv protease, even in the absence of vpg domain. an extended c-terminus results in a buried cleft in the case of tev protease. the absence of this extension makes semv substrate-binding site significantly different from that of tev protease. the residues in this region constitute the specificity pocket in tev protease. in semv protease, the cleft is solvent exposed, and the loop - appears to be closest to the substrate-binding region. the other side of the substratebinding cleft is similar in tev and semv proteases and is lined by the h strand eii. this is a common feature in most of the proteases and mainly involves main chain interactions. as mentioned earlier, the dn -pro or dn -pro was not active in trans. hence, in order to confirm the role of the proposed glutamate-binding residues (fig. a ) in protease activity, the residues h , t and n were mutated to ala in dn -protease -vpg (dn pv) fusion protein, and the cis-cleavage of the expressed protein into protease and vpg was monitored as described in the materials and methods section. the results obtained were confirmed by a western blot analysis using anti-protease antibodies (fig. d) . expression of dn pv showed a band of size kda corresponding to the protease domain (fig. a, lane ; fig. d , lane ) confirming that nearly complete cleavage of dn pv ( kda) had occurred. the active site mutant dn pv-s a (satheshkumar et al., ) showed a prominent band corresponding to kda on expression (fig. a, lane ) . the h a mutant gave a band of size kda corresponding to the dn pv (fig. a, lane ; fig. d, lane ) , confirming that the mutation had affected the cleavage activity of dn pv. similarly t a mutant also did not show any cis-cleavage activity (fig. b, lane ; fig. d, lane ) . however, n to a mutation in the dn pv resulted only in a partial loss of the protease activity (fig. b, lane ; fig. d, lane ) . the mutant dn pv-n a partially cleaved the dn pv giving a -kda dn pv band and a -kda dn -protease band, implying that n is not absolutely essential for the substrate binding, but contributes to the proper binding of the glutamate residue and hence to the optimal activity. disulfides are generally implicated in the stability of the three-dimensional structures of serine proteases (wang et al., ) . most of the prokaryotic and eukaryotic trypsins have highly conserved disulfide bonds. on the contrary, disulfide bonds have not been observed in any of the viral protease structures reported so far. in the poliovirus and hepatitis c virus proteases ( l n and jxp), binding of a metal ion contributes to the structural integrity (de francesco et al., ) . in semv protease, c forms a disulfide bond with c . this disulfide bond connects the h strands aii and cii in domain ii (fig. a) . it holds the walls of the s specificity pocket and could have a role in the maintenance of its rigid conformation. a disulfide bond is responsible for the rigidity of s -binding pocket in many of the mammalian trypsins. though the disulfide bond in semv protease does not correspond to any of the conserved disulfide bonds in trypsin, it involves the cii strand of domain ii. the same strand in trypsin has the conserved disulfide between the residues and (wang et al., ) . in order to assess the role of the unique disulfide bond in semv protease in the maintenance of the s -binding pocket, c was mutated to alanine in dn pv, and the effect of the mutation on cis-cleavage was examined. as shown in fig. c , lane , and fig. d , lane , the -kda dn pv-c a mutant was completely cleaved in cis to -kda band like the wild-type dn pv (fig. a, lane ; fig. d , lane ). hence, it can be concluded that this disulfide bond is not essential for the maintenance of the rigidity of the s -binding pocket. an interesting observation in the crystal structure of semv protease is the occurrence of a stretch of aromatic residues exposed to the surface (fig. ) . these aromatic residues f , w , y and y are not consecutive in sequence but form a stack near the c-terminus of the protein. two of these residues form part of the c-terminal helix. these residues may be of functional significance to the polyprotein. semv residues f and w are conserved across genomes of sobemoviruses. the presence of exposed aromatic residues is believed to indicate a protein -protein interaction interface. it has been demonstrated that there are extensive interactions between protease and vpg, and the conformational changes that accompany these interactions enhance and regulate protease activity (satheshkumar et al., ) . w of vpg was shown to mediate aromatic interactions with the protease, which results in a positive peak at nm in the cd spectrum of protease -vpg fusion protein, but the interacting partner in protease was not identified. the presence of exposed aromatic residues in the present structure strongly suggests that this might be the site of protease -vpg interaction. it is probable that w of protease interacts with w of vpg domain. the positive peak observed at nm in the cd spectrum of protease -vpg fusion protein might be the result of this interaction. further mutational studies are required to delineate the significance of the aromatic stretch of residues in protease activity. the crystal structure of semv protease provides insights on the possibilities of cis (intramolecular)-or trans (intermolecular)-cleavages. the c-terminal helix ends at residue , and this is followed by a stretch of five residues. the helix is a stable structure and is closely packed against the rest of the protein. the presence of a c-terminal helix is a characteristic of many serine proteases. the disordered short segment at the cterminus is not long enough to reach the active site suggesting the possibility of intermolecular cleavage between protease and vpg (trans-cleavage). however, it is possible that there could be a major conformational change because of the presence of the natively unfolded vpg at the c-terminus, and this may position the residues for cis-cleavage at the active site. biochemical evidences (data not shown) as well as the present structure suggest that a cleavage occurs between a and v at the n-terminus. this is an unexpected finding, as this site does not correspond to the canonical site for substrate cleavage. the relative positions of the n-terminus and the active site suggest that an intramolecular cleavage is indeed possible. the n-terminus in the crystal structure is disordered, and clear electron density is observed only from s . a further extension of approximately four residues is long enough to approach the active site through the cleft below the bii-cii loop of domain ii. this gains additional support by the observation that the n-terminus of semv protease superposes very well with the glutamyl endopeptidase from bacillus intermedius ( p c), as shown in fig. . in the case of p c, the n-terminus extends to the active site, and this extension is essential for zymogen activation and charge compensation for glutamate specificity (meijers et al., ) . therefore, based on the relative positions of the n-and c-termini and the active site in the crystal structure, it can be concluded that the cleavage between protease and vpg could occur in cis or trans, while the n-terminal cleavage could be cis. the non-specific cleavage at ala-val could be due to the conformation of the polypeptide, which positions the a -v peptide bond optimally for the cleavage. the residues ala and val are small and could be accommodated in the active site without steric hindrance. these observations also suggest that the specificity of the protease depends not only on sequence but also on the conformation of the polypeptide. a non-specific cleavage on the polypeptide chain has been observed at the cterminus of tev protease also (nunn et al., ) . there have also been reports in the literature on other viral proteases such as rhinovirus hrv - a protease, where the specificity requirements for cis-cleavage are less stringent than for trans-cleavage. the entropy term for substrate binding is favorable in ciscleavage, and hence, the specificity requirements are less stringent (petersen et al., ) . in spite of this, most viral proteases are extremely specific, and they function mainly in the processing of the respective polyproteins. their activities are modulated by the presence of vpg domains either at the n-or cterminus. determination of the structure of the protease with the vpg domain could shed light on the molecular mechanism of such regulations. the dn -protease (dn -pro) and dn -protease (dn -pro) domains of the polyprotein were cloned in prset c vector fig. . superposition of semv protease (blue) and bacillus intermedius glutamyl endopeptidase (pink). the superposition was generated using the output from dali server. at nhei and bamhi sites. the cloning strategy resulted in the addition of amino acids from the vector sequence at the nterminus including the hexa-histidine residues inserted for affinity purification. the proteins were expressed in e. coli bl (de ) plyss cells by the addition of . mm iptg at a culture density . od at nm and purified using ni-nta affinity chromatography (novagen) as previously described (satheshkumar et al., ) . the final protein preparation was in mm tris, ph . , buffer containing mm nacl. both dn -pro and dn -pro domains formed small crystals in the presence of . m tris, ph . , . m ammonium sulphate, . m , -hexane diol, mm hmercaptoethanol and % glycerol (crystallization buffer). larger crystals could be obtained using the microbatch method when a mixture of silicone and mineral oils in the ratio : was layered over the crystallization droplets ( al of mg/ml protein and al of crystallization buffer). the crystals appeared from a layer of precipitate within weeks and grew to a size of approximately .  .  . mm during a period of month. the crystals were transferred to the crystallization buffer containing % ethylene glycol as the cryoprotectant for a few seconds and then mounted in a cryo-loop. the crystals were exposed to cu-k a radiation at liquid nitrogen temperature ( k). x-ray diffraction data were collected using a rotating anode x-ray generator and a mar research image plate detector system. the datasets were processed using denzo, and the resulting intensities were scaled using scalepack (otwinowsky, ) . the frames were processed in the space group p . systematic absences indicated the presence of a or screw along the c axis. the structure solution was attempted by multiple isomorphous replacement (mir). four isomorphous mercury derivatives were obtained by soaking the crystals in p-chloro mercury benzene sulfonate (pcmbs), mercuric iodide, mercuric chloride and thiomersal (ethyl mercury thio salicylic acid or emts). the native and derivative datasets were scaled using the scaleit program in the ccp program suite version . . (ccp , ). both isomorphous and anomalous difference patterson maps were calculated for these derivatives using ccp program suite and harker sections for the space group p were plotted. initially, one site in the pcmbs derivative was obtained using the isomorphous differences in the program suite solve version . (terwilliger, ) . the rest of the sites in all the derivatives were identified from the difference fourier maps using mlphare in ccp program suite ( ) . the fractional coordinates of all the sites, their occupancies and anomalous occupancies were refined in mlphare for all the derivatives to a resolution of . Å . the b-factors of the sites were not refined and were fixed at the values obtained from the wilson plots. the space group was confirmed to be p , as this choice resulted in an interpretable map with the correct handedness. the experimental phases obtained from isomorphous and anomalous signals were used in the program resolve (terwilliger, ) to perform phase extension to . Å by density modification followed by automated model building. this resulted in the placement of residues with side chains and without side chains into the electron density map. the coordinates of the partial model and the phases from resolve were used as input for arp/warp version . (morris et al., ) for building the rest of the model. % of the residues were built by the program, which included residues - , - , - , - and - . the rest of the residues were built manually in subsequent cycles of refinement using coot (emsley and cowtan, ) and refmac . (murshudov et al., ) . the progress of the refinement was monitored by using % ( ) of the total independent reflection measurements for the calculation of the free r-factor. the hendrickson -lattman coefficients were used as restraints during the refinement (skubak et al., ) . individual b-factors of non-hydrogen atoms were refined. all structural alignments were done using the output from dali server (holm and sander, ) . the figures were prepared using molscript (kraulis, ) and bobscript (esnouf, ) and rendered using raster d (merritt and murphy, ) . the topology diagram for the protease was prepared using topdraw (bond, ) . site directed mutagenesis was performed by pcr-based approach (weiner et al., ) . the sense and antisense primers were designed with desired changes in the nucleotides (table ) , and restriction sites were incorporated in the primers to enable easy screening. the pcr was carried out using deep vent polymerase (new england biolabs) according to the manufacturer's instructions. the pcr product was treated with . al dpni enzyme at -c for min to remove the template dna and transformed into aliquots of dh a competent cells. the cells were plated after transformation in antibiotic containing plates. the colonies obtained were inoculated separately. plasmid isolation was carried out and digested with the appropriate restriction enzyme. the mutants screened by restriction enzyme digestion were further confirmed for the presence of mutation by dna sequencing. e. coli bl (de ) plyss cells were transformed with the recombinant clones, and the proteins were expressed by induction with . mm iptg for - h at -c. the cell pellet was resuspended in buffer a ( mm tris -hcl, ph . , mm nacl, . % triton x , % glycerol), sonicated and the expression was checked by sds polyacrylamide gel electrophoresis. the cleavage activity was monitored by the appearance of bands of expected size on the sds polyacrylamide gel. the results were confirmed by western blot analysis as described in satheshkumar et al. ( ) . the coordinates and structure factors for semv protease have been submitted to the protein data bank, and the structure has been assigned the accession code zyo. structure of arterivirus nsp . the smallest chymotrypsin-like proteinase with an alpha/beta c-terminal extension and alternate conformations of the oxyanion hole topdraw: a sketchpad for protein structure topology cartoons the ccp suite: programs for protein crystallography a zinc binding site in viral serine proteinases coot: model-building tools for molecular graphics further additions to molscript version . , including reading and contouring of electron-density maps sobemovirus genome appears to encode a serine protease related to cysteine proteases of picornaviruses protein structure comparison by alignment of distance matrices expression and site-specific mutagenesis of the poliovirus c protease in escherichia coli molscript: a program to produce both detailed and schematic plots of protein structures flash-cooling and annealing of protein crystals complete nucleotide sequence of sesbania mosaic virus: a new virus species of the genus sobemovirus solvent content of protein crystals the crystal structure of glutamyl endopeptidase from bacillus intermedius reveals a structural link between zymogen activation and charge compensation raster d version . . a program for photorealistic molecular graphics arp/warp and automatic interpretation of protein electron density maps refined x-ray crystallographic structure of the poliovirus c gene product refinement of macromolecular structures by the maximum-likelihood method a glutamic acid specific serine protease utilizes a novel histidine triad in substrate binding crystal structure of tobacco etch virus protease shows the protein c terminus bound within the active site processing of x-ray diffraction data collected in oscillation mode structural basis of substrate specificity in the serine proteases the structure of the a proteinase from a common cold virus: a proteinase responsible for the shut-off of host-cell protein synthesis structural basis for the substrate specificity of tobacco etch virus protease structure of the complex of streptomyces griseus protease b and the third domain of the turkey ovomucoid inhibitor at . -a resolution refined crystal structure of streptomyces griseus trypsin at . a resolution polyprotein processing: cis and trans proteolytic activities of sesbania mosaic virus serine protease natively unfolded'' vpg is essential for sesbania mosaic virus serine protease activity identification of viral genes required for cell-to-cell movement of southern bean mosaic virus direct incorporation of experimental phase information in model refinement solve and resolve: automated structure solution, density modification and model building the role of the cys -cys disulfide bond in trypsin: new targets for engineering substrate specificity site-directed mutagenesis of double-stranded dna by the polymerase chain reaction proteases involved in the processing of viral polyproteins. brief review mrnm and hss thank the department of science and technology (dst) and the department of biotechnology (dbt) of the government of india for financial support. diffraction data were collected at the x-ray facility for structural biology at molecular biophysics unit, indian institute of science (iisc), supported by dst and dbt. we thank the staff in the x-ray laboratory and supercomputer education and research centre of iisc for their co-operation during the course of these investigations. pg and pss acknowledge the council for scientific and industrial research, government of india, for the fellowships. key: cord- - otxft authors: altman, russ b.; mooney, sean d. title: bioinformatics date: journal: biomedical informatics doi: . / - - - _ sha: doc_id: cord_uid: otxft why is sequence, structure, and biological pathway information relevant to medicine? where on the internet should you look for a dna sequence, a protein sequence, or a protein structure? what are two problems encountered in analyzing biological sequence, structure, and function? how has the age of genomics changed the landscape of bioinformatics? what two changes should we anticipate in the medical record as a result of these new information sources? what are two computational challenges in bioinformatics for the future? ular biology and genomics-have increased dramatically in the past decade. history has shown that scientific developments within the basic sciences tend to lag about a decade before their influence on clinical medicine is fully appreciated. the types of information being gathered by biologists today will drastically alter the types of information and technologies available to the health care workers of tomorrow. there are three sources of information that are revolutionizing our understanding of human biology and that are creating significant challenges for computational processing. the most dominant new type of information is the sequence information produced by the human genome project, an international undertaking intended to determine the complete sequence of human dna as it is encoded in each of the chromosomes. the first draft of the sequence was published in (lander et al., ) and a final version was announced in coincident with the th anniversary of the solving of the watson and crick structure of the dna double helix. now efforts are under way to finish the sequence and to determine the variations that occur between the genomes of different individuals. essentially, the entire set of events from conception through embryonic development, childhood, adulthood, and aging are encoded by the dna blueprints within most human cells. given a complete knowledge of these dna sequences, we are in a position to understand these processes at a fundamental level and to consider the possible use of dna sequences for diagnosing and treating disease. while we are studying the human genome, a second set of concurrent projects is studying the genomes of numerous other biological organisms, including important experimental animal systems (such as mouse, rat, and yeast) as well as important human pathogens (such as mycobacterium tuberculosis or haemophilus influenzae). many of these genomes have recently been completely determined by sequencing experiments. these allow two important types of analysis: the analysis of mechanisms of pathogenicity and the analysis of animal models for human disease. in both cases, the functions encoded by genomes can be studied, classified, and categorized, allowing us to decipher how genomes affect human health and disease. these ambitious scientific projects not only are proceeding at a furious pace, but also are accompanied in many cases by a new approach to biology, which produces a third new source of biomedical information: proteomics. in addition to small, relatively focused experimental studies aimed at particular molecules thought to be important for disease, large-scale experimental methodologies are used to collect data on thousands or millions of molecules simultaneously. scientists apply these methodologies longitudinally over time and across a wide variety of organisms or (within an organism) organs to watch the evolution of various physiological phenomena. new technologies give us the abilities to follow the production and degradation of molecules on dna arrays (lashkari et al., ) , to study the expression of large numbers of proteins with one another (bai and elledge, ) , and to create multiple variations on a genetic theme to explore the implications of various mutations on biological function (spee et al., ) . all these technologies, along with the genome-sequencing projects, are conspiring to produce a volume of biological information that at once contains secrets to age-old questions about health and disease and threatens to overwhelm our current capabilities of data analysis. thus, bioinformatics is becoming critical for medicine in the twentyfirst century. the effects of this new biological information on clinical medicine and clinical informatics are difficult to predict precisely. it is already clear, however, that some major changes to medicine will have to be accommodated. with the first set of human genomes now available, it will soon become cost-effective to consider sequencing or genotyping at least sections of many other genomes. the sequence of a gene involved in disease may provide the critical information that we need to select appropriate treatments. for example, the set of genes that produces essential hypertension may be understood at a level sufficient to allow us to target antihypertensive medications based on the precise configuration of these genes. it is possible that clinical trials may use information about genetic sequence to define precisely the population of patients who would benefit from a new therapeutic agent. finally, clinicians may learn the sequences of infectious agents (such as of the escherichia coli strain that causes recurrent urinary tract infections) and store them in a patient's record to record the precise pathogenicity and drug susceptibility observed during an episode of illness. in any case, it is likely that genetic information will need to be included in the medical record and will introduce special problems. raw sequence information, whether from the patient or the pathogen, is meaningless without context and thus is not well suited to a printed medical record. like images, it can come in high information density and must be presented to the clinician in novel ways. as there are for laboratory tests, there may be a set of nondisease (or normal) values to use as comparisons, and there may be difficulties in interpreting abnormal values. fortunately, most of the human genome is shared and identical among individuals; less than percent of the genome seems to be unique to individuals. nonetheless, the effects of sequence information on clinical databases will be significant. . new diagnostic and prognostic information sources. one of the main contributions of the genome-sequencing projects (and of the associated biological innovations) is that we are likely to have unprecedented access to new diagnostic and prognostic tools. single nucleotide polymorphisms (snps) and other genetic markers are used to identify how a patient's genome differs from the draft genome. diagnostically, the genetic markers from a patient with an autoimmune disease, or of an infectious pathogen within a patient, will be highly specific and sensitive indicators of the subtype of disease and of that subtype's probable responsiveness to different therapeutic agents. for example, the severe acute respiratory syndrome (sars) virus was determined to be a corona virus using a gene expression array containing the genetic information from several common pathogenic viruses. in general, diagnostic tools based on the gene sequences within a patient are likely to increase greatly the number and variety of tests available to the physician. physicians will not be able to manage these tests without significant computational assistance. moreover, genetic information will be available to provide more accurate prognostic information to patients. what is the standard course for this disease? how does it respond to these medications? over time, we will be able to answer these questions with increasing precision, and will develop computational systems to manage this information. several genotype-based databases have been developed to identify markers that are associated with specific phenotypes and identify how genotype affects a patient's response to therapeutics. the human gene mutations database (hgmd) annotates mutations with disease phenotype. this resource has become invaluable for genetic counselors, basic researchers, and clinicians. additionally, the pharmacogenomics knowledge base (pharmgkb) collects genetic information that is known to affect a patient's response to a drug. as these data sets, and others like them, continue to improve, the first clinical benefits from the genome projects will be realized. . ethical considerations. one of the critical questions facing the genome-sequencing projects is "can genetic information be misused?" the answer is certainly yes. with knowledge of a complete genome for an individual, it may be possible in the future to predict the types of disease for which that individual is at risk years before the disease actually develops. if this information fell into the hands of unscrupulous employers or insurance companies, the individual might be denied employment or coverage due to the likelihood of future disease, however distant. there is even debate about whether such information should be released to a patient even if it could be kept confidential. should a patient be informed that he or she is likely to get a disease for which there is no treatment? this is a matter of intense debate, and such questions have significant implications for what information is collected and for how and to whom that information is disclosed (durfy, ; see chapter ). a brief review of the biological basis of medicine will bring into focus the magnitude of the revolution in molecular biology and the tasks that are created for the discipline of bioinformatics. the genetic material that we inherit from our parents, that we use for the structures and processes of life, and that we pass to our children is contained in a sequence of chemicals known as deoxyribonucleic acid (dna). the total collec- r. b. altman and s. d. mooney tion of dna for a single person or organism is referred to as the genome. dna is a long polymer chemical made of four basic subunits. the sequence in which these subunits occur in the polymer distinguishes one dna molecule from another, and the sequence of dna subunits in turn directs a cell's production of proteins and all other basic cellular processes. genes are discreet units encoded in dna and they are transcribed into ribonucleic acid (rna), which has a composition very similar to dna. genes are transcribed into messenger rna (mrna) and a majority of mrna sequences are translated by ribosomes into protein. not all rnas are messengers for the translation of proteins. ribosomal rna, for example, is used in the construction of the ribosome, the huge molecular engine that translates mrna sequences into protein sequences. understanding the basic building blocks of life requires understanding the function of genomic sequences, genes, and proteins. when are genes turned on? once genes are transcribed and translated into proteins, into what cellular compartment are the proteins directed? how do the proteins function once there? equally important, how are the proteins turned off ? experimentation and bioinformatics have divided the research into several areas, and the largest are: ( ) genome and protein sequence analysis, ( ) macromolecular structure-function analysis, ( ) gene expression analysis, and ( ) proteomics. practitioners of bioinformatics have come from many backgrounds, including medicine, molecular biology, chemistry, physics, mathematics, engineering, and computer science. it is difficult to define precisely the ways in which this discipline emerged. there are, however, two main developments that have created opportunities for the use of information technologies in biology. the first is the progress in our understanding of how biological molecules are constructed and how they perform their functions. this dates back as far as the s with the invention of electrophoresis, and then in the s with the elucidation of the structure of dna and the subsequent sequence of discoveries in the relationships among dna, rna, and protein structure. the second development has been the parallel increase in the availability of computing power. starting with mainframe computer applications in the s and moving to modern workstations, there have been hosts of biological problems addressed with computational methods. the human genome project was completed and a nearly finished sequence was published in . the benefit of the human genome sequence to medicine is both in the short and in the long term. the short-term benefits lie principally in diagnosis: the availability of sequences of normal and variant human genes will allow for the rapid identification of these genes in any patient (e.g., babior and matzner, ) . the long-term benefits will include a greater understanding of the proteins produced from the genome: how the proteins interact with drugs; how they malfunction in disease states; and how they participate in the control of development, aging, and responses to disease. the effects of genomics on biology and medicine cannot be understated. we now have the ability to measure the activity and function of genes within living cells. genomics data and experiments have changed the way biologists think about questions fundamental to life. where in the past, reductionist experiments probed the detailed workings of specific genes, we can now assemble those data together to build an accurate understanding of how cells work. this has led to a change in thinking about the role of computers in biology. before, they were optional tools that could help provide insight to experienced and dedicated enthusiasts. today, they are required by most investigators, and experimental approaches rely on them as critical elements. twenty years ago, the use of computers was proving to be useful to the laboratory researcher. today, computers are an essential component of modern research. this is because advances in research methods such as microarray chips, drug screening robots, x-ray crystallography, nuclear magnetic resonance spectroscopy, and dna sequencing experiments have resulted in massive amounts of data. these data need to be properly stored, analyzed, and disseminated. the volume of data being produced by genomics projects is staggering. there are now more than . million sequences in genbank comprising more than billion digits. but these data do not stop with sequence data: pubmed contains over million literature citations, the pdb contains three-dimensional structural data for over , protein sequences, and the stanford microarray database (smd) contains over , experiments ( million data points). these data are of incredible importance to biology, and in the following sections we introduce and summarize the importance of sequences, structures, gene expression experiments, systems biology, and their computational components to medicine. sequence information (including dna sequences, rna sequences, and protein sequences) is critical in biology: dna, rna, and protein can be represented as a set of sequences of basic building blocks (bases for dna and rna, amino acids for proteins). computer systems within bioinformatics thus must be able to handle biological sequence information effectively and efficiently. one major difficulty within bioinformatics is that standard database models, such as relational database systems, are not well suited to sequence information. the basic problem is that sequences are important both as a set of elements grouped together and treated in a uniform manner and as individual elements, with their relative locations and functions. any given position in a sequence can be important because of its own identity, because it is part of a larger subsequence, or perhaps because it is part of a large set of overlapping subsequences, all of which have different significance. it is necessary to support queries such as, "what sequence motifs are present in this sequence?" it is often difficult to represent these multiple, nested relationships within standard relational database schema. in addition, the neighbors of a sequence element are also critical, and it is important to be able to perform queries such as, "what sequence elements are seen elements to the left of this element?" for these reasons, researchers in bioinformatics are developing object-oriented databases (see chapter ) in which a sequence can be queried in different ways, depending on the needs of the user (altman, ) . the sequence information mentioned in section . . is rapidly becoming inexpensive to obtain and easy to store. on the other hand, the three-dimensional structure information about the proteins that are produced from the dna sequences is much more difficult and expensive to obtain, and presents a separate set of analysis challenges. currently, only about , three-dimensional structures of biological macromolecules are known. these models are incredibly valuable resources, however, because an understanding of structure often yields detailed insights about biological function. as an example, the structure of the ribosome has been determined for several species and contains more atoms than any other to date. this structure, because of its size, took two decades to solve, and presents a formidable challenge for functional annotation (cech, ) . yet, the functional information for a single structure is vastly outsized by the potential for comparative genomics analysis between the structures from several organisms and from varied forms of the functional complex, since the ribosome is ubiquitously required for all forms of life. thus a wealth of information comes from relatively few structures. to address the problem of limited structure information, the publicly funded structural genomics initiative aims to identify all of the common structural scaffolds found in nature and grow the number of known structures considerably. in the end, it is the physical forces between molecules that determine what happens within a cell; thus the more complete the picture, the better the functional understanding. in particular, understanding the physical properties of therapeutic agents is the key to understanding how agents interact with their targets within the cell (or within an invading organism). these are the key questions for structural biology within bioinformatics: . how can we analyze the structures of molecules to learn their associated function? approaches range from detailed molecular simulations (levitt, ) to statistical analyses of the structural features that may be important for function (wei and altman, ). bioinformatics for more information see http://www.rcsb.org/pdb/. . how can we extend the limited structural data by using information in the sequence databases about closely related proteins from different organisms (or within the same organism, but performing a slightly different function)? there are significant unanswered questions about how to extract maximal value from a relatively small set of examples. . how should structures be grouped for the purposes of classification? the choices range from purely functional criteria ("these proteins all digest proteins") to purely structural criteria ("these proteins all have a toroidal shape"), with mixed criteria in between. one interesting resource available today is the structural classification of proteins (scop), which classifies proteins based on shape and function. the development of dna microarrays has led to a wealth of data and unprecedented insight into the fundamental biological machine. the premise is relatively simple; up to , gene sequences derived from genomic data are fixed onto a glass slide or filter. an experiment is performed where two groups of cells are grown in different conditions, one group is a control group and the other is the experimental group. the control group is grown normally, while the experimental group is grown under experimental conditions. for example, a researcher may be trying to understand how a cell compensates for a lack of sugar. the experimental cells will be grown with limited amounts of sugar. as the sugar depletes, some of the cells are removed at specific intervals of time. when the cells are removed, all of the mrna from the cells is separated and converted back to dna, using special enzymes. this leaves a pool of dna molecules that are only from the genes that were turned on (expressed) in that group of cells. using a chemical reaction, the experimental dna sample is attached to a red fluorescent molecule and the control group is attached to a green fluorescent molecule. these two samples are mixed and then washed over the glass slide. the two samples contain only genes that were turned on in the cells, and they are labeled either red or green depending on whether they came from the experimental group or the control group. the labeled dna in the pool sticks or hybridizes to the same gene on the glass slide. this leaves the glass slide with up to , spots and genes that were turned on in the cells are now bound with a label to the appropriate spot on the slide. using a scanning confocal microscope and a laser to fluoresce the linkers, the amount of red and green fluorescence in each spot can be measured. the ratio of red to green determines whether that gene is being turned off (downregulated) in the experimental group or whether the gene is being turned on (upregulated). the experiment has now measured the activity of genes in an entire cell due to some experimental change. figure . illustrates a typical gene expression experiment from smd. computers are critical for analyzing these data, because it is impossible for a researcher to comprehend the significance of those red and green spots. currently scientists are using gene expression experiments to study how cells from different organ- isms compensate for environmental changes, how pathogens fight antibiotics, and how cells grow uncontrollably (as is found in cancer). a new challenge for biological computing is to develop methods to analyze these data, tools to store these data, and computer systems to collect the data automatically. with the completion of the human genome and the abundance of sequence, structural, and gene expression data, a new field of systems biology that tries to understand how proteins and genes interact at a cellular level is emerging. the basic algorithms for analyzing sequence and structure are now leading to opportunities for more integrated analysis of the pathways in which these molecules participate and ways in which molecules can be manipulated for the purpose of combating disease. a detailed understanding of the role of a particular molecule in the cell requires knowledge of the context-of the other molecules with which it interacts-and of the sequence of chemical transformations that take place in the cell. thus, major research areas in bioinformatics are elucidating the key pathways by which chemicals are transformed, defining the molecules that catalyze these transformations, identifying the input compounds and the output compounds, and linking these pathways into bioinformatics networks that we can then represent computationally and analyze to understand the significance of a particular molecule. the alliance for cell signaling is generating large amounts of data related to how signal molecules interact and affect the concentration of small molecules within the cell. there are a number of common computations that are performed in many contexts within bioinformatics. in general, these computations can be classified as sequence alignment, structure alignment, pattern analysis of sequence/structure, gene expression analysis, and pattern analysis of biochemical function. as it became clear that the information from dna and protein sequences would be voluminous and difficult to analyze manually, algorithms began to appear for automating the analysis of sequence information. the first requirement was to have a reliable way to align sequences so that their detailed similarities and distances could be examined directly. needleman and wunsch ( ) published an elegant method for using dynamic programming techniques to align sequences in time related to the cube of the number of elements in the sequences. smith and waterman ( ) published refinements of these algorithms that allowed for searching both the best global alignment of two sequences (aligning all the elements of the two sequences) and the best local alignment (searching for areas in which there are segments of high similarity surrounded by regions of low similarity). a key input for these algorithms is a matrix that encodes the similarity or substitutability of sequence elements: when there is an inexact match between two elements in an alignment of sequences, it specifies how much "partial credit" we should give the overall alignment based on the similarity of the elements, even though they may not be identical. looking at a set of evolutionarily related proteins, dayhoff et al. ( ) published one of the first matrices derived from a detailed analysis of which amino acids (elements) tend to substitute for others. within structural biology, the vast computational requirements of the experimental methods (such as x-ray crystallography and nuclear magnetic resonance) for determining the structure of biological molecules drove the development of powerful structural analysis tools. in addition to software for analyzing experimental data, graphical display algorithms allowed biologists to visualize these molecules in great detail and facilitated the manual analysis of structural principles (langridge, ; richardson, ) . at the same time, methods were developed for simulating the forces within these molecules as they rotate and vibrate (gibson and scheraga, ; karplus and weaver, ; levitt, ) . the most important development to support the emergence of bioinformatics, however, has been the creation of databases with biological information. in the s, structural biologists, using the techniques of x-ray crystallography, set up the protein data bank (pdb) of the cartesian coordinates of the structures that they elucidated (as well as associated experimental details) and made pdb publicly available. the first release, in , contained structures. the growth of the database is chronicled on the web: the pdb now has over , detailed atomic structures and is the primary source of information about the relationship between protein sequence and protein structure. similarly, as the ability to obtain the sequence of dna molecules became widespread, the need for a database of these sequences arose. in the mid- s, the genbank database was formed as a repository of sequence information. starting with sequences and , bases in , the genbank has grown by much more than million sequences and billion bases. the genbank database of dna sequence information supports the experimental reconstruction of genomes and acts as a focal point for experimental groups. numerous other databases store the sequences of protein molecules and information about human genetic diseases. included among the databases that have accelerated the development of bioinformatics is the medline database of the biomedical literature and its paper-based companion index medicus (see chapter ). including articles as far back as and brought online free on the web in , medline provides the glue that relates many high-level biomedical concepts to the low-level molecule, disease, and experimental methods. in fact, this "glue" role was the basis for creating the entrez and pubmed systems for integrating access to literature references and the associated databases. perhaps the most basic activity in computational biology is comparing two biological sequences to determine ( ) whether they are similar and ( ) how to align them. the problem of alignment is not trivial but is based on a simple idea. sequences that perform a similar function should, in general, be descendants of a common ancestral sequence, with mutations over time. these mutations can be replacements of one amino acid with another, deletions of amino acids, or insertions of amino acids. the goal of sequence alignment is to align two sequences so that the evolutionary relationship between the sequences becomes clear. if two sequences are descended from the same ancestor and have not mutated too much, then it is often possible to find corresponding locations in each sequence that play the same role in the evolved proteins. the problem of solving correct biological alignments is difficult because it requires knowledge about the evolution of the molecules that we typically do not have. there are now, however, well-established algorithms for finding the mathematically optimal alignment of two sequences. these algorithms require the two sequences and a scoring system based on ( ) exact matches between amino acids that have not mutated in the two sequences and can be aligned perfectly; ( ) partial matches between amino acids that have mutated in ways that have preserved their overall biophysical properties; and ( ) gaps in the alignment signifying places where one sequence or the other has undergone a deletion or insertion of amino acids. the algorithms for determining optimal sequence alignments are based on a technique in computer science known as dynamic programming and are at the heart of many computational biology applications (gusfield, ) . figure . shows an example of a smith-waterman matrix. unfortunately, the dynamic programming algorithms are computationally expensive to apply, so a number of faster, more heuristic methods have been developed. the most popular algorithm is the basic local alignment search tool (blast) (altschul et al., ) . blast is based on the observations that sections of proteins are often conserved without gaps (so the gaps can be ignored-a critical simplification for speed) and that there are statistical analyses of the occurrence of small subsequences within larger sequences that can be used to prune the search for matching sequences in a large database. another tool that has found wide use in mining genome sequences is blat (kent, ) . blat is often used to search long genomic sequences with significant performance increases over blast. it achieves its -fold increase in speed over other tools by storing and indexing long sequences as nonoverlapping k-mers, allowing efficient storage, searching, and alignment on modest hardware. one of the primary challenges in bioinformatics is taking a newly determined dna sequence (as well as its translation into a protein sequence) and predicting the structure of the associated molecules, as well as their function. both problems are difficult, being fraught with all the dangers associated with making predictions without hard experimental data. nonetheless, the available sequence data are starting to be sufficient to allow good predictions in a few cases. for example, there is a web site devoted to the assessment of biological macromolecular structure prediction methods. recent results suggest that when two protein molecules have a high degree (more than percent) of sequence similarity and one of the structures is known, a reliable model of the other can be built by analogy. in the case that sequence similarity is less than percent, however, performance of these methods is much less reliable. when scientists investigate biological structure, they commonly perform a task analogous to sequence alignment, called structural alignment. given two sets of threedimensional coordinates for a set of atoms, what is the best way to superimpose them so that the similarities and differences between the two structures are clear? such computations are useful for determining whether two structures share a common ancestry and for understanding how the structures' functions have subsequently been refined during evolution. there are numerous published algorithms for finding good structural alignments. we can apply these algorithms in an automated fashion whenever a new structure is determined, thereby classifying the new structure into one of the protein families (such as those that scop maintains). one of these algorithms is minrms (jewett et al., ) . minrms works by finding the minimal root-mean-squared-distance (rmsd) alignments of two protein structures as a function of matching residue pairs. minrms generates a family of alignments, each with different number of residue position matches. this is useful for identifying local regions of similarity in a protein with multiple domains. minrms solves two problems. first, it determines which structural superpositions, or alignment, to evaluate. then, given this superposition, it determines which residues should be bioinformatics considered "aligned" or matched. computationally, this is a very difficult problem. minrms reduces the search space by limiting superpositions to be the best superposition between four atoms. it then exhaustively determines all potential four-atommatched superpositions and evaluates the alignment. given this superposition, the number of aligned residues is determined, as any two residues with c-alpha carbons (the central atom in all amino acids) less than a certain threshold apart. the minimum average rmsd for all matched atoms is the overall score for the alignment. in figure . , an example of such a comparison is shown. a related problem is that of using the structure of a large biomolecule and the structure of a small organic molecule (such as a drug or cofactor) to try to predict the ways in which the molecules will interact. an understanding of the structural interaction between a drug and its target molecule often provides critical insight into the drug's mechanism of action. the most reliable way to assess this interaction is to use experimental methods to solve the structure of a drug-target complex. once again, these experimental approaches are expensive, so computational methods play an important role. typically, we can assess the physical and chemical features of the drug molecule and can use them to find complementary regions of the target. for example, a highly electronegative drug molecule will be most likely to bind in a pocket of the target that has electropositive features. prediction of function often relies on use of sequential or structural similarity metrics and subsequent assignment of function based on similarities to molecules of known r. b. altman and s. d. mooney function. these methods can guess at general function for roughly to percent of all genes, but leave considerable uncertainty about the precise functional details even for those genes for which there are predictions, and have little to say about the remaining genes. analysis of gene expression data often begins by clustering the expression data. a typical experiment is represented as a large table, where the rows are the genes on each chip and the columns represent the different experiments, whether they be time points or different experimental conditions. within each cell is the red to green ratio of that gene's experimental results. each row is then a vector of values that represent the results of the experiment with respect to a specific gene. clustering can then be performed to determine which genes are being expressed similarly. genes that are associated with similar expression profiles are often functionally associated. for example, when a cell is subjected to starvation (fasting), ribosomal genes are often downregulated in anticipation of lower protein production by the cell. it has similarly been shown that genes associated with neoplastic progression could be identified relatively easily with this method, making gene expression experiments a powerful assay in cancer research (see guo, , for review) . in order to cluster expression data, a distance metric must be determined to compare a gene's profile with another gene's profile. if the vector data are a list of values, euclidian distance or correlation distances can be used. if the data are more complicated, more sophisticated distance metrics may be employed. clustering methods fall into two categories: supervised and unsupervised. hand. usually, the method begins by selecting profiles that represent the different groups of data, and then the clustering method associates each of the genes with the be performed automatically. two such unsupervised learning methods are the hierarchical and k-means clustering methods. hierarchical methods build a dendrogram, or a tree, of the genes based on ing close neighbors into a cluster. the first step often involves connecting the closest profiles, building an average profile of the joined profiles, and repeating until the entire tree is built. k-means clustering builds k clusters or groups automatically. the algorithm begins by picking k representative profiles randomly. then each gene is associated with the representative to which it is closest, as defined by the distance metric being employed. then the center of mass of each cluster is determined using all of the member gene's profiles. depending on the implementation, either the center of mass or the nearest member to it becomes the new representative for that cluster. the algorithm then iterates until the new center of mass and the previous center of mass are within some threshold. the result is k groups of genes that are regulated similarly. one drawback of k-means is that one must chose the value for k. if k is too large, logical "true" clusters may be split into pieces and if k is too small, there will be clusters that are bioinformatics commonly applied because these methods require no knowledge of the data, and can supervised learning methods require some preconceived knowledge of the data at representative profile to which they are most similar. unsupervised methods are more their expression profiles. these methods are agglomerative and work by iteratively join-merged. one way to determine whether the chosen k is correct is to estimate the average distance from any member profile to the center of mass. by varying k, it is best to choose the lowest k where this average is minimized for each cluster. another drawback of k-means is that different initial conditions can give different results, therefore it is often prudent to test the robustness of the results by running multiple runs with different starting configurations (figure . ) . the future clinical usefulness of these algorithms cannot be understated. in , van't veer et al. ( found that a gene expression profile could predict the clinical outcome of breast cancer. the global analysis of gene expression showed that some can- r. b. altman and s. d. mooney cers were associated with different prognosis, not detectable using traditional means. another exciting advancement in this field is the potential use of microarray expression data to profile the molecular effects of known and potential therapeutic agents. this molecular understanding of a disease and its treatment will soon help clinicians make more informed and accurate treatment choices. biologists have embraced the web in a remarkable way and have made internet access to data a normal and expected mode for doing business. hundreds of databases curated by individual biologists create a valuable resource for the developers of computational methods who can use these data to test and refine their analysis algorithms. with standard internet search engines, most biological databases can be found and accessed within moments. the large number of databases has led to the development of meta-databases that combine information from individual databases to shield the user from the complex array that exists. there are various approaches to this task. the entrez system from the national center for biological information (ncbi) gives integrated access to the biomedical literature, protein, and nucleic acid sequences, macromolecular and small molecular structures, and genome project links (including both the human genome project and sequencing projects that are attempting to determine the genome sequences for organisms that are either human pathogens or important experimental model organisms) in a manner that takes advantages of either explicit or computed links between these data resources. the sequence retrieval system (srs) from the european molecular biology laboratory allows queries from one database to another to be linked and sequenced, thus allowing relatively complicated queries to be evaluated. newer technologies are being developed that will allow multiple heterogeneous databases to be accessed by search engines that can combine information automatically, thereby processing even more intricate queries requiring knowledge from numerous data sources. the main types of sequence information that must be stored are dna and protein. one of the largest dna sequence databases is genbank, which is managed by ncbi. genbank is growing rapidly as genome-sequencing projects feed their data (often in an automated procedure) directly into the database. figure . shows the logarithmic growth of data in genbank since . entrez gene curates some of the many genes within genbank and presents the data in a way that is easy for the researcher to use (figure . ) . year the exponential growth of genbank total number of bases figure . . the exponential growth of genbank. this plot shows that since the number of bases in genbank has grown by five full orders of magnitude and continues to grow by a factor of every years. in addition to genbank, there are numerous special-purpose dna databases for which the curators have taken special care to clean, validate, and annotate the data. the work required of such curators indicates the degree to which raw sequence data must be interpreted cautiously. genbank can be searched efficiently with a number of algorithms and is usually the first stop for a scientist with a new sequence who wonders "has a sequence like this ever been observed before? if one has, what is known about it?" there are increasing numbers of stories about scientists using genbank to discover unanticipated relationships between dna sequences, allowing their research programs to leap ahead while taking advantage of information collected on similar sequences. a database that has become very useful recently is the university of california santa cruz genome assembly browser (figure . ) . this data set allows users to search for specific sequences in the ucsc version of the human genome. powered by the similarity search tool blat, users can quickly find annotations on the human genome that contain their sequence of interest. these annotations include known variations (mutations and snps), genes, comparative maps with other organisms, and many other important data. although sequence information is obtained relatively easily, structural information remains expensive on a per-entry basis. the experimental protocols used to determine precise molecular structural coordinates are expensive in time, materials, and human power. therefore, we have only a small number of structures for all the molecules characterized in the sequence databases. the two main sources of structural information are the cambridge structural database for small molecules (usually less than atoms) and the pdb for macromolecules (see section . . ), including proteins and nucleic acids, and combinations of these macromolecules with small molecules (such as drugs, cofactors, and vitamins). the pdb has approximately , high-resolution structures, but this number is misleading because many of them are small variants on the same structural architecture (figure . ) . if an algorithm is applied to the database to filter out redundant structures, less than , structures remain. there are approximately , proteins in humans; therefore many structures remain unsolved (e.g., burley and bonanno, ; gerstein et al., ) . in the pdb, figure . . a stylized diagram of the structure of chymotrypsin, here shown with two identical subunits interacting. the red portion of the protein backbone shows α-helical regions, while the blue portion shows β-strands, and the white denotes connecting coils, while the molecular surface is overlaid in gray. the detailed rendering of all the atoms in chymotrypsin would make this view difficult to visualize because of the complexity of the spatial relationships between thousands of atoms. each structure is reported with its biological source, reference information, manual annotations of interesting features, and the cartesian coordinates of each atom within the molecule. given knowledge of the three-dimensional structure of molecules, the function sometimes becomes clear. for example, the ways in which the medication methotrexate interacts with its biological target have been studied in detail for two decades. methotrexate is used to treat cancer and rheumatologic diseases, and it is an inhibitor of the protein dihydrofolate reductase, an important molecule for cellular reproduction. the three-dimensional structure of dihydrofolate reductase has been known for many years and has thus allowed detailed studies of the ways in which small molecules, such as methotrexate, interact at an atomic level. as the pdb increases in size, it becomes important to have organizing principles for thinking about biological structure. scop provides a classification based on the overall structural features of proteins. it is a useful method for accessing the entries of the pdb. the ecocyc project is an example of a computational resource that has comprehensive information about biochemical pathways. ecocyc is a knowledge base of the metabolic capabilities of e. coli; it has a representation of all the enzymes in the e. coli genome and of the compounds on which they work. it also links these enzymes to their position on the genome to provide a useful interface into this information. the network of pathways within ecocyc provides an excellent substrate on which useful applications can be built. for example, they could provide: ( ) the ability to guess the function of a new protein by assessing its similarity to e. coli genes with a similar sequence, ( ) the ability to ask what the effect on an organism would be if a critical component of a pathway were removed (would other pathways be used to create the desired function, or would the organism lose a vital function and die?), and ( ) the ability to provide a rich user interface to the literature on e. coli metabolism. similarly, the kyoto encyclopedia of genes and genomes (kegg) provides pathway datacets for organism genomes. a postgenomic database bridges the gap between molecular biological databases with those of clinical importance. one excellent example of a postgenomic database is the online mendelian inheritance in man (omim) database, which is a compilation of known human genes and genetic diseases, along with manual annotations describing the state of our understanding of individual genetic disorders. each entry contains links to special-purpose databases and thus provides links between clinical syndromes and basic molecular mechanisms (figure . ). the smd is another example of a postgenomic database that has proven extremely useful, but has also addressed some formidable challenges. as discussed previously in several sections, expression data are often represented as vectors of data values. in addition to the ratio values, the smd stores images of individual chips, complete with annotated gene spots (see figure . ). further, the smd must store experimental conditions, the type and protocol of the experiment, and other data associated with the experiment. arbitrary analysis can be performed on different experiments stored in this unique resource. a critical technical challenge within bioinformatics is the interconnection of databases. as biological databases have proliferated, researchers have been increasingly interested in linking them to support more complicated requests for information. some of these links are natural because of the close connection of dna sequence to protein structure (a straightforward translation). other links are much more difficult because the semantics of the data items within the databases are fuzzy or because good methods for linking certain types of data simply do not exist. for example, in an ideal world, a protein sequence would be linked to a database containing information about that sequence's function. unfortunately, although there are databases about protein function, it is not always easy to assign a function to a protein based on sequence information alone, and so the databases are limited by gaps in our understanding of biology. some excellent recent work in the integration of diverse biological databases has been done in connection with the ncbi entrez/pubmed systems, the srs resource, discoverylink, and the biokleisli project. the human genome sequencing projects will be complete within a decade, and if the only raison d'etre for bioinformatics is to support these projects, then the discipline is not well founded. if, on the other hand, we can identify a set of challenges for the next generations of investigators, then we can more comfortably claim disciplinary status for the field. fortunately, there is a series of challenges for which the completion of the first human genome sequence is only the beginning. with the first human genome in hand, the possibilities for studying the role of genetics in human disease multiply. a new challenge immediately emerges, however: collecting individual sequence data from patients who have disease. researchers estimate that more than percent of the dna sequences within humans are identical, but the remaining sequences are different and account for our variability in susceptibility to and development of disease states. it is not unreasonable to expect that for particular disease syndromes, the detailed genetic information for individual patients will provide valuable information that will allow us to tailor treatment protocols and perhaps let us make more accurate prognoses. there are significant problems associated with obtaining, organizing, analyzing, and using this information. there is currently a gap in our understanding of disease processes. although we have a good understanding of the principles by which small groups of molecules interact, we are not able to fully explain how thousands of molecules interact within a cell to create both normal and abnormal physiological states. as the databases continue to accumulate information ranging from patient-specific data to fundamental genetic information, a major challenge is creating the conceptual links between these databases to create an audit trail from molecular-level information to macroscopic phenomena, as manifested in disease. the availability of these links will facilitate the identification of important targets for future research and will provide a scaffold for biomedical knowledge, ensuring that important literature is not lost within the increasing volume of published data. an important opportunity within bioinformatics is the linkage of biological experimental data with the published papers that report them. electronic publication of the biological literature provides exciting opportunities for making data easily available to scientists. already, certain types of simple data that are produced in large volumes are expected to be included in manuscripts submitted for publication, including new sequences that are required to be deposited in genbank and new structure coordinates that are deposited in the pdb. however, there are many other experimental data sources that are currently difficult to provide in a standardized way, because the data either are more intricate than those stored in genbank or pdb or are not produced in a volume sufficient to fill a database devoted entirely to the relevant area. knowledge base technology can be used, however, to represent multiple types of highly interrelated data. knowledge bases can be defined in many ways (see chapter ); for our purposes, we can think of them as databases in which ( ) the ratio of the number of tables to the number of entries per table is high compared with usual databases, ( ) the individual entries (or records) have unique names, and ( ) the values of many fields for one record in the database are the names of other records, thus creating a highly interlinked network of concepts. the structure of knowledge bases often leads to unique strategies for storage and retrieval of their content. to build a knowledge base for storing information from biological experiments, there are some requirements. first, the set of experiments to be modeled must be defined. second, the key attributes of each experiment that should be recorded in the knowledge base must be specified. third, the set of legal values for each attribute must be specified, usually by creating a controlled terminology for basic data or by specifying the types of knowledge-based entries that can serve as values within the knowledge base. the development of such schemes necessitates the creation of terminology standards, just as in clinical informatics. the riboweb project is undertaking this task in the domain of rna biology (chen et al., ) . riboweb is a collaborative tool for ribosomal modeling that has at its center a knowledge base of the ribosomal structural literature. riboweb links standard bibliographic references to knowledge-based entries that summarize the key experimental findings reported in each paper. for each type of experiment that can be performed, the key attributes must be specified. thus, for example, a cross-linking experiment is one in which a small molecule with two highly reactive chemical groups is added to an ensemble of other molecules. the reactive groups attach themselves to two vulnerable parts of the ensemble. because the molecule is small, the two vulnerable areas cannot be any further from each other than the maximum stretched-out length of the small molecule. thus, an analysis of the resulting reaction gives information that one part of the ensemble is "close" to another part. this experiment can be summarized formally with a few features-for example, target of experiment, cross-linked parts, and cross-linking agent. the task of creating connections between published literature and basic data is a difficult one because of the need to create formal structures and then to create the necessary content for each published article. the most likely scenario is that biologists will write and submit their papers along with the entries that they propose to add to the knowledge base. thus, the knowledge base will become an ever-growing communal store of scientific knowledge. reviewers of the work will examine the knowledge-based elements, perhaps will run a set of automated consistency checks, and will allow the knowledge base to be modified if they deem the paper to be of sufficient scientific merit. riboweb in prototype form can be accessed on the web. one of the most exciting goals for computational biology and bioinformatics is the creation of a unified computational model of physiology. imagine a computer program that provides a comprehensive simulation of a human body. the simulation would be a complex mathematical model in which all the molecular details of each organ system would be represented in sufficient detail to allow complex "what if ?" questions to be asked. for example, a new therapeutic agent could be introduced into the system, and its effects on each of the organ subsystems and on their cellular apparatus could be assessed. the side-effect profile, possible toxicities, and perhaps even the efficacy of the agent could be assessed computationally before trials are begun on laboratory animals or human subjects. the model could be linked to visualizations to allow the teaching of medicine at all grade levels to benefit from our detailed understanding of physiological processes-visualizations would be both anatomic (where things are) and functional (what things do). finally, the model would provide an interface to human genetic and biological knowledge. what more natural user interface could there be for exploring physiology, anatomy, genetics, and biochemistry than the universally recognizable structure of a human that could be browsed at both macroscopic and microscopic levels of detail? as components of interest were found, they could be selected, and the available literature could be made available to the user. the complete computational model of a human is not close to completion. first, all the participants in the system (the molecules and the ways in which they associate to form higher-level aggregates) must be identified. second, the quantitative equations and symbolic relationships that summarize how the systems interact have not been elucidated fully. third, the computational representations and computer power to run such a simulation are not in place. researchers are, however, working in each of these areas. the genome projects will soon define all the molecules that constitute each organism. research in simulation and the new experimental technologies being developed will give us an understanding of how these molecules associate and perform their functions. finally, research in both clinical informatics and bioinformatics will provide the computational infrastructure required to deliver such technologies. bioinformatics is closely allied to clinical informatics. it differs in its emphasis on a reductionist view of biological systems, starting with sequence information and moving to structural and functional information. the emergence of the genome sequencing projects and the new technologies for measuring metabolic processes within cells is beginning to allow bioinformaticians to construct a more synthetic view of biological processes, which will complement the whole-organism, top-down approach of clinical informatics. more importantly, there are technologies that can be shared between bioinformatics and clinical informatics because they both focus on representing, storing, and analyzing biological data. these technologies include the creation and management of standard terminologies and data representations, the integration of heterogeneous databases, the organization and searching of the biomedical literature, the use of machine learning techniques to extract new knowledge, the simulation of biological processes, and the creation of knowledge-based systems to support advanced practitioners in the two fields. the proceedings of one of the principal meetings in bioinformatics, this is an excellent source for up-to-date research reports. other important meetings include those sponsored by the this introduction to the field of bioinformatics focuses on the use of statistical and artificial intelligence techniques in machine learning introduces the different microarray technologies and how they are analyzed dna and protein sequence analysis-a practical approach this book provides an introduction to sequence analysis for the interested biologist with limited computing experience this edited volume provides an excellent introduction to the use of probabilistic representations of sequences for the purposes of alignment, multiple alignment this primer provides a good introduction to the basic algorithms used in sequence analysis, including dynamic programming for sequence alignment algorithms on strings, trees and sequences: computer science and computational biology gusfield's text provides an excellent introduction to the algorithmics of sequence and string analysis, with special attention paid to biological sequence analysis problems artificial intelligence and molecular biology this volume shows a variety of ways in which artificial intelligence techniques have been used to solve problems in biology genotype to phenotype this volume offers a useful collection of recent work in bioinformatics another introduction to bioinformatics, this text was written for computer scientists the textbook by stryer is well written, and is illustrated and updated on a regular basis. it provides an excellent introduction to basic molecular biology and biochemistry what ways will bioinformatics and medical informatics interact in the future? will the research agendas of the two fields merge will the introduction of dna and protein sequence information change the way that medical records are managed in the future? which types of systems will be most affected (laboratory, radiology, admission and discharge, financial it has been postulated that clinical informatics and bioinformatics are working on the same problems, but in some areas one field has made more progress than the other why should an awareness of bioinformatics be expected of clinical informatics professionals? should a chapter on bioinformatics appear in a clinical informatics textbook? explain your answers one major problem with introducing computers into clinical medicine is the extreme time and resource pressure placed on physicians and other health care workers. will the same problems arise in basic biomedical research? why have biologists and bioinformaticians embraced the web as a vehicle for disseminating data so quickly, whereas clinicians and clinical informaticians have been more hesitant to put their primary data online? key: cord- -y t pux authors: brzezinski, dariusz; kowiel, marcin; cooper, david r.; cymborowski, marcin; grabowski, marek; wlodawer, alexander; dauter, zbigniew; shabalin, ivan g.; gilski, miroslaw; rupp, bernhard; jaskolski, mariusz; minor, wladek title: covid‐ .bioreproducibility.org: a web resource for sars‐cov‐ ‐related structural models date: - - journal: protein sci doi: . /pro. sha: doc_id: cord_uid: y t pux the covid‐ pandemic has triggered numerous scientific activities aimed at understanding the sars‐cov‐ virus and ultimately developing treatments. structural biologists have already determined hundreds of experimental x‐ray, cryo‐em, and nmr structures of proteins and nucleic acids related to this coronavirus, and this number is still growing. to help biomedical researchers, who may not necessarily be experts in structural biology, navigate through the flood of structural models, we have created an online resource, covid .bioreproducibility.org, that aggregates expert‐verified information about sars‐cov‐ ‐related macromolecular models. in this paper, we describe this web resource along with the suite of tools and methodologies used for assessing the structures presented therein. this article is protected by copyright. all rights reserved. the spread of the novel coronavirus around the world has triggered an unprecedented response from the scientific community. six months into the pandemic, pubmed already listed over , scientific papers with the terms covid- or sars-cov- in the title, and tens of analyses are reported daily in mass media around the globe. understandably, firstline research findings, including molecular structure determinations, depositions in the protein data bank (pdb), and related results, are often made public on biorxiv or medrxiv before formal peer review. this approach delivers the latest results to scientists that develop treatments and vaccines without any delay but at the cost of elevated risk of mistakes and errors, which can mislead scientists performing follow-up research and misinform the general public. the world health organization has even coined the portmanteau 'infodemic` to describe the phenomenon of potentially misleading information overload. as of july , , the pdb has amassed structural models of sars-cov- -related macromolecules, including proteins and rna fragments. structure-based drug design depends on such molecular models, especially of complexes with candidate drugs slated for further development. however, the rapidly growing number of structures without corresponding publications and the potential mistakes associated with pandemic-driven research can create confusion among biomedical researchers and could impede, rather than accelerate, drug development. indeed, an analysis of the "entry history" of structures deposited to the pdb between january and july , showed that as many as out of the ( . %) sars-cov- structures (excluding pandda fragment screening deposits) required a major revision of the initial model, whereas only of the other , ( . %) structures deposited during that time period had any major revisions. for of the sars-cov- structures, the revisions were significant and involved replacement of the atomic coordinates. some of these revisions were triggered by our resource. part be due to the celerity of the research, and in part to this and similar projects that requested original diffraction data, which prompted the authors of these structures to revisit their models. an additional factor which may impede the use of molecular structures in biomedical research is that they are sometimes presented in a way geared toward modeling and theoretical chemistry, but not for biomedical scientists that are not necessarily experts in protein crystallography. in this paper, we present covid- .bioreproduciblity.org, a web resource that organizes sars-cov- related structural information in a way that should be understandable and useful for a wider scientific community, and not only for structural biologists. the website also serves as a repository for examined and, if found to be suboptimal, corrected versions of pdb structures of sars-cov- proteins and rna fragments, with a focus on assessing the smallmolecule ligands modeled in those structures. moreover, we strive to re-deposit the optimized structure models in the pdb, always in collaboration with the original authors. the validation tools and re-refinement protocols used in this project can serve as a template for future molecular structure assessment efforts. due to the rapid response in time of the pandemic, the covid- .bioreproducibility.org web resource was created in an agile, fast-prototyping manner, focusing on speedy delivery and flexibility to accommodate changes. as a result, several new features are still being bound. ligands that may affect the protein function are called "functional ligands" (as opposed, e.g. to the ligands which are artifacts of the protein purification or crystallization process, and there are no indications that their binding may affect the way the protein functions). users can also quickly filter for structures with or without functional ligands, protein-protein complexes, pathogen-host interactions, and fragment screening results. the website also allows text searches [ fig. crystallography or, if significant improvement might be expected, contact the authors with the request to submit the diffraction images. moreover, we extract quality metrics related to structures by querying a locally installed copy of the pdbj's vrpt database schema. after the data have been automatically gathered from the above-mentioned sources, they are processed by geometry checking, statistical, and validation tools, most of which were developed in-house by the laboratories collaborating on this project. finally, the structures are evaluated by a team of expert structural biologists who use a combination of the mined data, validation reports, and manual inspection of the protein models and associated electron density to examine potential problems. careful attention is paid to all functional ligands and inhibitors contained in the structures. if potential problems are spotted, the diffraction data are re-processed (whenever the raw data are available) and the models are re-refined. the corrected models are made publicly available on our webserver. in addition, we always this article is protected by copyright. all rights reserved. attempt to contact the original authors and encourage them to jointly re-deposit the optimized models to the pdb. the details of the application of our structure evaluation tools and our structure correction protocol, are discussed in the following sections. to make an informed decision whether a structure should be re-refined or not, we use several criteria and tools to assess its quality. we check the overall geometry (ramachandran outliers and rotamer outliers), the correlation between model and electron density map (especially for ligands), the presence of large peaks in difference electron density map, the placement of the macromolecular model in the unit cell, and whether r and rfree are reasonable for the reported resolution. additionally, to provide a simple quantitative overview of the quality of x-ray structures, we calculate and show on our website the pq metric. pq is the structure's quality percentile (from to , the higher the better) based on rfree, rsrz score, clashscore, ramachandran outliers and rotamer outliers. being a hybrid reciprocal and space-real space global metric, pq can be easily used to sort structures and compare their overall quality. the pq metric is recalculated weekly for each structure. equipped with the above-mentioned validation tools, expert structural biologists may decide to manually inspect each structure in coot. if diffraction data are available, the potential gains of their manual re-processing are analyzed. using the calculated electron density maps, the main chain and side chains can be easily reviewed with coot or molstack. special emphasis is put on unmodeled electron density blobs. based on such a review, a decision whether to re-refine the structure is made. full re-refinement is a laborious process and sometimes requires contact with the deposition authors. in case of deposits that do not have primary citations, the identification of the principal investigator (pi) is not always an easy task. for that reason, the commission on biological macromolecules of the international union of crystallography (iucr), together with the iucr committee on data, have asked the pdb to publicly disclose the e-mail address of the pi (or depositing author) of each deposit. this article is protected by copyright. all rights reserved. once the decision to re-refine has been made, we use the following protocol to improve the model. some aspects of this protocol are general in nature, and the exact values may be changed for a particular structure. the protocol and decisions made for each structure are based on our extensive experience in protein structure determination, - crystallographic software development, , published guidelines on structure refinement and structure quality, , , and previous campaigns of pdb structure re-refinement: , if raw diffraction data are available, the results of automatic processing of images by hkl- auto are examined to verify that the structure was determined in the correct space group and at optimal resolution. in case of inconsistent results, we use the hkl- program suite with the implementation of corrections for x-ray absorption, radiation decay, and anisotropic diffraction. each structure under inspection is placed in a standardized way in the reference unit cell using achesym. even though crystal structures can be presented with the molecular models located in various crystallographically equivalent locations, we seek to facilitate the process of comparisons of analogous structures for noncrystallographers by placing the models as close to the origin of the unit cell as possible. the achesym server takes into account the equivalence of the space group symmetry positions and adjusts the location of the model in the unit cell. as a result, the atomic coordinates and electron density maps of the re-refined versions of isomorphous structures, i.e., structures in the same space group and with differences for cell parameters a/b/c within . % and cell angles within %, are standardized to the same location. this means that the macromolecules occupy similar positions in their corresponding unit cells and both the coordinates and electron density maps of isomorphous structures can be easily viewed already superposed using any current computer. the models subjected to restrained maximum posterior refinement in refmac with hydrogen atoms added in riding positions. for all standard protein residues, the refmac dictionary is used as a source of ideal stereochemical targets, after each round of refmac refinement, the atomic model is manually inspected and corrected according to the following checklist: a. review unmodeled electron density blobs, which might represent ligands or residues missing from the polymer model. b. inspect all difference electron density peaks above . rmsd ( . rmsd if there are too many peaks). inspect the strongest negative density peaks. c. inspect rotamer outliers, which may indicate incorrect placement of side chains, as well as residues with missing atoms. this article is protected by copyright. all rights reserved. d. review density fit graphs and inspect poorly fitting residues; verify terminal residue placement; and inspect any gaps in the sequence. e. inspect ramachandran outliers. f. once large electron density blobs have been modeled and major issues with the protein backbone have been addressed, look for potential water molecules to add, with peaks above . rmsd in the mfo-dfc map and distances to protein h-bonded atoms ranging from . - . Å. if unmodeled electron density blobs are found during manual corrections, they are considered as potential ligands. in such cases, we try to identify the ligand with the help of checkmyblob, fit the ligand in the density, and run no fewer than refmac cycles. if the ligand does not have proper stereochemical description in the standard refmac dictionary, new geometrical restraints are generated using the grade web server and carefully checked before use. since ligands originally modeled in the deposition may be incorrect, they are inspected visually and, if questionable, validated using checkmyblob. similarly, it has been shown that a significant fraction of metal-containing structures in the pdb have incorrect metal assignment or modeling. therefore, special attention is given to metal identification by the checkmymetal validation server and, when possible, using anomalous maps calculated with data collected above and below the x-ray absorption edge. challenging cases are discussed by at least two team members. many structures are inspected by at least one other expert after the refinement has been completed. the revised structures are stored in the web resource described here, along with a description of the identified issues and changes made. however, if the changes are significant, the goal is to redeposit the re-refined structure in the pdb, preferably together with the original authors, using the mechanism of re-versioning. an example report for a structure re-processed from original data and re-refined according to the above protocol is presented in figure and in figure s . a report showing a case when the original diffraction data were not available is presented in figures s and s . the goal of the covid- .bioreproducibility.org web resource is to gather macromolecular structures related to the sars-cov- virus and assess them using state-of-the-art tools. additionally, we aim to provide information that can be easily used by non-structural biologists. that is why the structures are categorized according to the experimental method, virus type, protein type, and ligand category. we also attempt to facilitate quick overall structure assessment for general users by calculating aggregated quality metrics, such as the pq percentile. finally, we make sure that isomorphous structures solved in the same space group can be easily compared, by moving them into standardized location of the reference unit cell. although non-uniform model placement in the unit cell may not seem to be a serious issue for trained crystallographers, for many biomedical researchers it makes comparison harder and structures may appear to be completely different, leading to confusion and misinterpretations. during the work on the server, we made several disturbing observations. first, in several cases, the deposited images were clearly not compatible with the diffraction data used for structure refinement. second, some of the contacted scientists claimed that the diffraction data were deleted immediately after processing in order to save disk space. third, several scientists did not respond to our request to provide their data, despite the iucr recommendation and an appeal from the community to make diffraction data related to sars-cov- public (http://phenix-online.org/pipermail/phenixbb/ -march/ ). however, in one case our request resulted in the original authors re-depositing an optimized structure instead of depositing the diffraction data. all of the above facts show that the struggle for reproducibility of scientific results is an uphill battle, and suggest that leading scientific journals should do more , than run editorials about the need to improve the reproducibility of scientific results. it is worth noting that the described web resource is not the only project established with the aim of validating, correcting, or providing additional information on covid- with vaccines in late-stage development , and the first reports of drugs increasing survival chances, the covid- pandemic will hopefully end soon. however, this may not necessarily be the end of the sars-cov- coronavirus, as it may evolve in yet unforeseen ways to evade vaccines and treatments. therefore, we will keep improving the web resource presented herein, with the hope that it will remain useful to biologists for years to come and that it will set standards for any future health crises. this structure will be re-deposited to the pdb under a new pdb id, due to significant changes rcsb protein data bank: sustaining a living digital data resource that enables breakthroughs in scientific research and biomedical education biorxiv: the preprint server for biology new preprint server for medical research coronavirus sars-cov- : filtering fact from fiction in the infodemic a multi-crystal method for extracting obscured crystallographic states from conventionally uninterpretable electron density ligand-centered assessment of sars-cov- drug target models in the protein data bank on the evolution of the quality of macromolecular models in the pdb drugbank . : a major update to the drugbank database data structures for statistical computing in python pdbj mine: design and implementation of relational database interface for protein data bank japan a public database of macromolecular diffraction experiments current developments in coot for macromolecular model building of electron cryo-microscopy and crystallographic data molstack: a platform for interactive presentations of electron density and cryo-em maps and their interpretations structural, biochemical, and evolutionary characterizations of glyoxylate/hydroxypyruvate reductases show their division into two distinct subfamilies albumin-based transport of nonsteroidal anti-inflammatory drugs in mammalian blood plasma biomolecular crystallography: principles, practice, and application to structural biology conformation-dependent restraints for polynucleotides: the sugar moiety hkl- : the integration of data reduction and structure solution -from diffraction images to an initial model in minutes refining the macromolecular model -achieving the best agreement with the data from x-ray diffraction experiment assessment of crystallographic structure quality and protein-ligand complex structure validation a close look onto structural models and primary ligands of metallo-β-lactamases detect, correct, retract: how to manage incorrect structural models processing of x-ray diffraction data collected in oscillation mode diffraction data analysis in the presence of radiation damage weak data do not make a free lunch, only a cheap meal how good are my data and what is the resolution? linking crystallographic model and data quality achesym : an algorithm and server for standardized placement of macromolecular models in the unit cell on optimal placement of molecules in the unit cell overview of refinement procedures within refmac : utilizing data from different sources accurate bond and angle parameters for x-ray protein structure refinement conformation-dependent restraints for polynucleotides: i. clustering of the geometry of the phosphodiester group accurate geometrical restraints for watson-crick base pairs tlsmd web server for the generation of multi-group tls models significance tests on the crystallographic r factor features and development of coot molprobity: more and better reference data for improved all-atom structure validation automatic recognition of ligands in electron density by machine learning magnesium-binding architectures in rna crystal structures: validation, binding preferences, classification and motif detection checkmymetal : a macromolecular metal-binding validation tool characterizing metal-binding sites in proteins with x-ray crystallography findable accessible interoperable re-usable (fair) diffraction data are coming to protein crystallography no raw data, no science: another possible source of the reproducibility crisis faculty opinions recommendation of [miyakawa t safety and immunogenicity of the chadox ncov- vaccine against sars-cov- : a preliminary report of a phase / , single-blind immunogenicity and safety of a recombinant adenovirus type- -vectored covid- vaccine in healthy adults aged years or older: a randomised, double-blind effect of dexamethasone in hospitalized patients with covid- : preliminary report this article is protected by copyright. all rights reserved one of the authors (wm) notes that he has also been involved in the development of software and data management and mining tools; some of them were commercialized by hkl research and are mentioned in the paper. wm is the co-founder of hkl research and a member of the board. the authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. key: cord- -x q dwc authors: worrall, jonathan ar; luisi, ben f title: information available at cut rates: structure and mechanism of ribonucleases date: - - journal: curr opin struct biol doi: . /j.sbi. . . sha: doc_id: cord_uid: x q dwc ribonucleases are counterweights in the balance of gene expression and are also involved in the maturation of functional rna. recent structural data reveal how ribonucleases recognize and cleave targets, in most cases with the catalytic assistance of metal cofactors. many of these enzymes are ‘processive’, in that they make multiple scissions following the binding of substrates; crystallographic data can account for this solution behaviour. these data not only explain how ribonucleases turn over transcripts, but also provide hints about how they often play dual roles in quality control checks on structured rna. only a few decades ago, when the genetic code was still in its infancy of abstraction, crick, brenner, jacob and monod reasoned that the information encoded by dna must be converted into protein through an intermediary that is short-lived and consequently does not accumulate in the cell. this message was subsequently shown to be composed of rna and its short life is a well-known curse of experimentalists who try to extract it from cells. the instability is caused principally by the activity of ribonucleases, which are abundant in cells and seem to lie eagerly in wait of vulnerable substrates. seemingly nuisances, these enzymes in fact play important roles in the regulation of gene expression, for example, in the course of development or in response to environmental change [ ] . in prokaryotes, nucleases affect the differential processing and rate of degradation of polycistronic transcripts, and thus may contribute to the coordinated stoichiometric synthesis of the subunits of multicomponent complexes [ ] . the apparent wanton destruction of rna by ribonucleases poses several questions: how do cells maintain folded rna structures, such as trna and ribosomes, in the presence of all these keen ribonucleases? conversely, how are unstructured or damaged rnas identified and turned over? lastly, many different types of structured rnas are synthesized as precursors that must be trimmed to form the mature species. are there dedicated, highly specific nucleases that do this controlled trimming? perhaps the answer to all these questions stems from the observation that the turnover of mrna and the processing of structural rna share common steps; for instance, in escherichia coli, the processing of rrna and the degradation of mrna both start with endonucleolytic cleavage followed by exonucleolytic attack on the fragments [ ] . many of the ribonucleases that turn over transcripts play dual roles in the degradation of stable rna and the processing or fidelity checking and stresstriggered decay of structured rna. as proposed by deutscher [ ] , the structure of the rna itself may be the signal that steers it to a course of maturation or to a fate of destruction; we shall explore how these implicit signals are recognized. as is often the case, eukaryotes are more complicated and might have dedicated complex machinery for the different tasks of degradation and quality control [ ] . here, the choice of processing versus turnover may be affected by the time lag of processing, in analogy to kinetic proofreading of translational fidelity [ ] . we summarize some recent structural and functional data that provide insight into these processes, as represented by key bacterial ribonucleases and a few from archaea and eukaryotes. for convenience and perspective, we group these ribonucleases into the context of enzymes from e. coli, an organism for which we are rapidly approaching comprehensive coverage (table ) . not all of our questions about ribonucleases are answered by these structures, but some good hints are provided. the role of rna structural determinants is a unifying theme, but the related catalytic chemistries also provide another connection between these new structures. in e. coli, the endoribonuclease rnase e plays a key role in initiating transcript turnover [ ] . rnase e is an essential enzyme that affects the balance and composition of the mrna population [ , ] . rnase e targets specific transcripts for destruction, in conjunction with the destruction of small regulatory rnas [ ] . in addition to its role in destroying transcripts, rnase e also plays a creative role in generating certain rna species by maturing larger precursors. these include rrna ( s rna), trna, tmrna, which is required to rescue stalled ribosomes, and rnase p, a conserved ribozyme involved in trna processing [ ] [ ] [ ] [ ] [ ] . the crystal structure of the rnase e catalytic domain has been solved in complex with rna substrate at . Å (figure a ) [ ] . the ribonuclease is a composite of structure and mechanism of ribonucleases worrall and luisi to ssrna exoribonuclease using a phosphorolytic cleavage mechanism. gene duplication of rnase ph fold found in pnpase, a processive ssrna exoribonuclease. compact a/b: the rnase ph fold. homologues of rnase ph found in the archaeal and eukaryotic exosome, with a similar protomer arrangement to that seen in pnpase, which itself has a modular organisation of s and kh domains and an internal duplication of the rnase ph fold. (figure a) , and an s subdomain, which occurs in many different rna-binding proteins. in rnase e, the s subdomain clamps down on the rna substrate and appears to be flexibly tethered to the body of the nuclease (see the summary in figure ). the catalytic site is situated in the dnase i subdomain, where a single metal-binding site is found ( figure b ); however, it is possible that there is a second metal-binding site in the transition state. (the use of two-metal clusters as catalytic centres is a recurrent theme in other ribonucleases; see table .) cleavage is by nucleophilic attack on the phosphate backbone within a single-stranded a/u-rich region, generating fragments with a free -oh (on the side of cleavage) and a -monophosphate (on the side). the rnase e structure suggests that recognition of the end of the substrate induces a conformational switch that results in the phosphate backbone being oriented for attack by an oh group activated by a coordinated magnesium ion. in e. coli and probably many other related gram-negative bacteria, rnase e is part of a multienzyme assembly known as the rna degradosome [ , ] . the other components of the degradosome are the glycolytic enzyme enolase, an atp-dependent rna helicase (rhlb) and the phosphorolytic exoribonuclease polynucleotide phosphorylase (pnpase). the physical association of these proteins within the degradosome coordinates their enzymatic activities. pnpase is a pro-cessive phosphorolytic exonuclease that may work together with rnase e to ensure the cooperative destruction of substrates once the first cleavage is made; it is also involved in checking the fidelity of rrna precursors as a quality control mechanism [ ] . rhlb is required to unwind structured rna substrates so that they become suitable substrates for the nucleases of the degradosome. the role of enolase is unclear, but the available evidence indicates that its recruitment into the degradosome assembly is required to control levels of transcripts for the glucose transporter [ , ] . an enolase recognition motif occurs in rnase e homologues from other gramnegative bacteria, including pathogens such as salmonella, and it seems likely that, in these organisms, the enolase interacts with rnase e [ ] . the exoribonuclease rnase ii is representative of an extensive enzyme family found in all three domains of life, whose members play roles in the maturation, turn-structure and mechanism of ribonucleases worrall and luisi schematic summary of the modes of rna processing of some of the endoribonucleases (rnase e) and exoribonucleases discussed in the text and in table over and quality control of certain species of structured rna [ ] . rnase ii cuts single-stranded (ss)rna processively in a to direction, using a hydrolytic mechanism, and releases nucleotide monophosphates as it gallops along (summarized in figure ). crystal structures of the apo form of e. coli rnase ii and of an inactive mutant in complex with ssrna have been reported [ , ] (figure a ). this revealed another multidomain molecular montage, composed of two cold-shock domains (csds) at the n-terminal end, followed by a catalytic domain and, finally, an s domain. the x-ray data reveal that the single-stranded substrate lies deep in a channel at the bottom of which is the catalytic site [ ] ( figure a) . one magnesium ion is present in the active site, coordinated by aspartates. the inactivating mutation in rnase ii lies at a putative metal-coordinating residue, d n, and frazão et al. [ ] propose that two metals may be involved in the activation of a water molecule for hydrolysis of the terminal phosphodiester, analogous to the active sites of polymerases and perhaps some ribozymes [ ] . this hypothesis is corroborated by the mutagenesis studies of zuo et al [ ] . the organization of the active site of rnase ii has striking parallels with the active site of the endoribonuclease rnase h; thus, even though the folds differ, the enzymes are likely to share similar catalytic chemistries. rnase r is a homologue of rnase ii, but with an intriguing and distinctive property: it appears to have a built-in ability to unwind the secondary structure of rna substrates, even if they contain many strong gc base pairs. in a striking parallel with certain types of rna helicases, rnase r can sense the polarity of the phosphodiester backbone of the substrate. thus, the ribonuclease greatly prefers substrates with single-stranded regions at the end. the ribonuclease can processively degrade structured substrates, provided they have a single-stranded overhang that is seven or more nucleotides in length [ ] . cleavage occurs at the singlestranded overhang and proceeds processively in the direction, ploughing straight through the secondary structure ( figure ). whereas dead-box helicases use the free energy of atp binding and hydrolysis to disrupt secondary structure, rnase r transduces the favourable free energy of rna backbone hydrolysis into mechanical work that translates the single-stranded substrate further into the catalytic pocket, much like a ratchet. the linkage between rna cleavage and unwinding of secondary structure is most likely indirect, and may involve changes in the energy of rna binding to the catalytic pocket and to the s subdomain. one hypothesis is that the energydependent ratcheting mechanism pulls the duplex rna against the apex of the tunnel, into which it cannot fit, thus causing it to unwind. it is not presently clear why rnase r manages to perform this operation, whereas homologous rnase ii does not. nevertheless, rnase r seems to have evolved a very clever mechanism to con-vert the energy of hydrolysis into work of unwinding, and thus seems to be a model of energy conservation. in eukaryotes, the rnase r homologue is a component of the exosome assembly, which we will discuss below. both rnase h and rnase iii are representative of components of the rna interference (rnai) machinery, which is described elsewhere in this issue [ ] . we mention them here briefly to emphasize the role of metals in catalysis, one common theme of the ribonucleases presented here (table ) . rnase h enzymes cleave duplex rna or rna-dna hybrids, and are representative of a large family whose members include transposases and the argonaute ribonuclease, which is involved in rna silencing. rnase iii cleaves double-stranded (ds)rna and the e. coli enzyme is a model system for the entire family, which includes eukaryotic enzymes such as dicer (involved in the rnai mechanism). the protein uses induced fit to recognize rna substrates [ ] ( figure ). as seen in the other ribonucleases, divalent metals are again the key components of the catalytic site. the structure of rnase h from mouse leukaemia virus has been solved at high resolution [ ] and corroborates the presence of a magnesium-binding site in the catalytic site, consistent with the two-magnesium mechanism proposed earlier by yang et al. [ ] for e. coli rnase h. a recent crystallographic analysis reveals the stepwise participation of the two magnesium metals in the rnase h mechanism, with the first participating in nucleophilic activation of water and the second metal stabilizing the transition state [ , ] . reflecting the distinct roles of the two metals, they are coordinated in non-equivalent ways. by contrast, metal coordination is symmetrical in the rnase-h-like transposases; this is required because the metals have equivalent roles in the successive steps of nucleophilic activation of water during strand cleavage and -oh activation during strand transfer [ ] . rnase z is a conserved endonuclease that cleaves trna precursors at the end in preparation for the addition of a cca aminoacylation motif. the fold belongs to the blactamase structural family and the active site contains two coordinated zinc ions that participate in the hydrolysis of the rna backbone. structures are available for the apo and trna-bound forms from bacillus subtilis (figure b ), revealing that substrate binding causes conformational moulding of both macromolecules to organize the catalytic site [ , ] . this induced fit appears to result in recognition of the shape and contour of the trna, as well as direct recognition of two conserved guanine bases. b. subtilis has two other endoribonucleases that are proposed to have a similar metallo-b-lactmase fold: rnase j and rnase j [ ]. these two enzymes appear to be rnase e homologues in function, but emphatically not in sequence or fold. in eukaryotes and archaea, the multienzyme exosome plays many key roles in rna processing and turnover, rnai and quality control surveillance. the central component of the exosome is a phosphorolytic ribonuclease that is structurally homologous to the bacterial pnpase of the degradosome. the subunits of the exosome and the subdomains of pnpase closely resemble the ancient fold found in the phosphorolytic exoribonuclease rnase ph [ ] . crystal structures of the core of the exosomes from the archaea sulfolobus solfataricus and archaeoglobus fulgidus have recently become available, revealing a hexameric ring comprising two types of rnase-ph-like subunits, known as rrp and rrp (rrna-processing proteins and ; see figure a ) [ , , ] . three of the exosome subunits are likely to have catalytic activity for phosphorolysis, whereas the other three might not. instead, these non-catalytic subunits might provide a surface for the recruitment of the other exosome components. in the phosphorolytic reaction, the last phosphodiester bond of the substrate is attacked by phosphate, to leave a -oh and nucleotide diphosphates. in addition to the hexameric core, the archaeal exosome contains the subunits known as rrp , which have the s fold (also present in rnase e and rnase ii) and the kh fold, another ancient and ubiquitous rna-binding motif. the rnase ph, s and kh domains are organized into a quaternary structure that is remarkably similar to bacterial pnpase (figure b ). although not yet elucidated, it is likely that this structure is also conserved in the eukaryotic exosome. the active site lies within the central channel of the hexameric ring and is - Å from the exterior surface that engages the rrp subunit, suggesting that a product of - nucleotides is the limit of digestion; thus, auxiliary components might be needed to complete the processing of undigested residual fragments ( figure ). the auxiliary components of the eukaryotic exosome include atp-dependent helicases and hydrolytic ribonucleases, such as homologues of rnase r (described earlier) and the metal-dependent exoribonuclease rnase d (described below) ( figure ). in eukaryotes, exosomes are found in the cytoplasm and nucleus, where they have different auxiliary components and specialized function. the cytoplasmic exosome is involved in the turnover of both normal and defective transcripts [ ] . the nuclear exosome is involved in processing precursors of structured rna, such as . s rrna, and in quality control surveillance of many different types of rna [ ] . the structure of the nuclear auxiliary factor rrp p has been solved [ ], confirming that this enzyme is related to the rnase d family of exonucleases; this family includes the proofreading subdomain of dna polymerase i [ ] and the poly(a)-specific ribonuclease [ ] . in these enzymes, characterised by conserved acidic residues, two divalent metal ions activate a water molecule for hydrolysis of the terminal phosphodiester. the interaction of a c-terminal domain of rrp p with the exosome core might modulate its function [ ] . in eukaryotes, additional hydrolytic exoribonucleases function to hydrolyze substrates in ribbon or surface representation of (a) the archaeal core exosome from a. fulgidus (pdb code ba ) and (b) pnpase from streptomyces antibioticus (pdb code e p) viewed along the threefold rotation axis. in the exosome, a ring of alternating rrp and rrp subunits (green and blue) forms a hexameric core structure, with three rrp subunits (orange) binding to the top face. the core domains of the exosome share the same rnase ph fold as the bacterial pnpase. the s and kh domains, common to both the exosome and pnpase structures, are indicated. the s domains of the rrp subunits are ideally situated to guide the end of ssrna into the pore ( Å diameter) for processing. in the pnpase structure, the s domains are not well defined, suggesting high mobility in the absence of bound rna. in vertebrates, small nucleolar rnas (snornas) function in ribosome processing in the nucleolus; some have a role in the modification of bases or cutting of rrna precursors [ ] . some snornas are processed by endonucleolytic cleavage, through the action of enzymes such as the manganese-dependent endoribonuclease xendou from the amphibian xenopus laevis [ ] . sequence alignment reveals similar proteins in other eukaryotes (including human, drosophila melanogaster, caenorhabditis elegans and arabidopsis thaliana), nidoviruses and, remarkably, the cyanobacterium nostoc punctiforme [ ] . this might indicate the versatile use of this fold in rna processing. a homologous enzyme, known as nendou, is a component of the genomic replication apparatus of the sars virus and other members of the coronavirus family [ ] . these enzymes, which we refer to as belonging to the endou family, are specific for uracil and the product has a - cyclic phosphate; this is usually an indication that the ribose sugar -oh is oriented and activated to act as a nucleophile. crystal structures have become available for the nendou enzymes from sars virus [ ] and mouse hepatitis virus [ ] , and for xendou from x. laevis [ ] . these data reveal a common, unique fold that distinguishes the endou family from any other ribonuclease family identified thus far ( figure ) . remarkably, the position of the key catalytic residues in the active sites of xendou and nendou mimics the position of corresponding residues in the ribonuclease rnase a (two histidines, which serve as general acid/general base, and a lysine) ( figure ). this congruence is achieved with different folds and serves as a striking example of convergent evolution. the re-invention of a catalytic site usually indicates an important function, but the precise role of the viral protein is currently not well understood. the use of metals to activate a nucleophile is a common theme for both endonucleases and exonucleases of the hydrolytic class. these hydrolytic reactions are highly exergonic; by contrast, phosphorolysis, in which inorganic phosphate attacks the backbone to drive bond cleavage, corresponds to a much smaller free energy change. it seems reasonable to expect that selective pressure would affect the balance of one reaction over the other. in the course of the evolution of eukaryotes, the associated genomic expansion and the tremendous amount of non-coding rna necessitates significant investment of resources and energy. perhaps the exosome represents an energy-efficient means of coping with the recycling of structure and mechanism of ribonucleases worrall and luisi overall fold of two representatives of a new architectural class of endoribonuclease -the endou family. (a) nendou, the nsp protein from the sars coronavirus (pdb code h ). (b) xendou from x. laevis (pdb code c w). a close-up view of a region of the active site of xendou is shown (inset). the active site is situated in a groove formed by b-strand and a-helix , where a phosphate ion, shown in stick form, was found in the crystal. active site residues his and his are required for substrate cleavage; arg (found in one of two conformations) is proposed to form a stabilizing interaction with the negatively charged substrate. in nendou, a similar groove-like feature housing the catalytic residues is found at the c-terminal end of the protein, between a-helix and b-strand . oligonucleotides, compared with the costs associated with hydrolytic decay. some hydrolytic enzymes, such as rnase r, can use the free energy of hydrolysis to 'drive through' secondary structure, so that this does not require the assistance of an energy-consuming rna helicase. most rnas are spared this expensive mode of destruction unless they are specified by a single-stranded tail of minimal length or an exposed monophosphate together with a single-stranded region. as the structure rather than the sequence itself is recognized, the fold must be under considerable evolutionary scrutiny and might be optimised to fine-tune the levels of populations of transcripts, so that the enzymes they encode have an extra layer of regulatory connections. therefore, one speculation is that the targeting of selected transcript classes for turnover by ribonucleases might also permit the coordinated regulation of different genes and thus might deepen connections between metabolic processes [ ] . this hypothesis awaits testing. the enzymes and control of eukaryotic mrna turnover transcriptional organization and posttranscriptional regulation of the bacillus subtilis branched-chain amino acid biosynthesis genes this well-written review presents a clear and cogent summary of ribonuclease function, and proposes a dual role for ribonucleases in processing rna and in turnover rna-quality control by the exosome a comprehensive review of the role of the exosome and its auxiliary components in rna turnover and quality control checks in eukaryotes the phylogenetic distribution of bacterial ribonucleases global analysis of escherichia coli rna degradosome function using dna microarrays rnase g complementation of rne null mutation identifies functional interrelationships with rnase e in escherichia coli coupled degradation of a small regulatory rna and its mrna targets in escherichia coli processing of the precursor to the catalytic rna subunit of rnase p from escherichia coli initiation of trna maturation by rnase e is essential for cell viability in escherichia coli rnase g (cafa protein) and rnase e are both required for the maturation of s ribosomal rna rnase e is required for the maturation of ssra rna and normal ssra rna peptide-tagging activity regulation of s rna biogenesis by switching utilization of both sigma factors and endoribonucleases structure of escherichia coli rnase e catalytic domain and its implications for rna turnover and processing the first crystal structure of a member of the rnase e/rnase g family is described the escherichia coli rna degradosome: structure, function and relationship in other ribonucleolytic multienzyme complexes the rna degradosome: life in the fast lane of adaptive molecular evolution quality control of ribosomal rna mediated by polynucleotide phosphorylase and rnase r enolase in the rna degradosome plays a crucial role in the rapid decay of glucose transporter mrna in response to phosphosugar stress in escherichia coli rnase e-based ribonucleoprotein complexes: mechanical basis of mrna destabilization mediated by bacterial noncoding rnas recognition of enolase in the escherichia coli rna degradosome unravelling the dynamics of rna degradation by ribonuclease ii and its rna-bound complex the first reported crystal structure of a member of the rnase ii/rnase r family structural basis for processivity and single-strand specificity of rnase ii the crystal structure of e. coli rnase ii is presented, together with a proposal for the processing and catalytic mechanism a general two-metal-ion mechanism for catalytic rna substrate recognition and catalysis by the exoribonuclease rnase r this analysis of enzyme cutting rates and affinity for rna substrates established the key determinants of recognition ribonuclease revisited: structural insights into ribonuclease iii family enzymes structural insight into the mechanism of double-stranded rna processing by ribonuclease iii the structure of a eukaryotic viral rnase h, which cleaves rna-dna hybrids structure of ribonuclease h phased at Å resolution by mad analysis of the selenomethionyl protein stepwise analyses of metal ions in rnase h catalysis from substrate destabilization to product release crystal structures of rnase h bound to an rna/dna hybrid: substrate specificity and metal-dependent catalysis making and breaking nucleic acids: two-mg + -ion catalysis and substrate specificity this continuation of their earlier structural studies [ ] reveals structural moulding of rna substrate and enzyme upon engagement structural basis for substrate binding, cleavage and allostery in the trna maturase rnase z putzer h: ribonucleases j and j : two novel endoribonucleases in b. subtilis with functional homology to e. coli rnase e a proposal for the fold of the family of b. subtilis rnase j and j , which are functional homologues of rnase e running rings around rna: a superfamily of phosphatedependent rnases structural basis of end rna recognition and exoribonucleolytic cleavage by an exosome rnase ph core the archaeal exosome core is a hexameric ring structure with three catalytic subunits the first structure of an archaebacterial exosome demonstrates the arrangement of rnase-ph-like subunits in a ring that resembles the pnpase architecture structural framework for the mechanism of archaeal exosomes in rna processing structure of the nuclear exosome component rrp p reveals an interplay between the active site and the hrdc domain the crystal structure of a component of the nuclear exosome from yeast (saccharomyces cerevisiae) confirms its structural relationship with the bacterial rnase d exonuclease and corroborates its mode of metal binding crystal structure of escherichia coli rnase d, an exoribonuclease involved in structured rna processing the structure of e. coli rnase d reveals a structural relationship with the exonuclease domain of dna polymerase i structural insight into poly(a) binding and catalytic mechanism of human parn biogenesis of small nucleolar ribonucleoproteins purification, cloning, and characterization of xendou, a novel endoribonuclease involved in processing of intron-encoded small nuclear rnas in xenopus laevis the structure of the endoribonuclease xendou: from small nucleolar rna processing to severe acute respiratory syndrome coronavirus replication this paper presents the structure and postulated mechanism of xendou from the amphibian x. laevis, representing the vertebrate homologue of a new structural class of manganese-dependent endoribonuclease major genetic marker of nidoviruses encodes a replicative endoribonuclease crystal structure and mechanistic determinants of sars coronavirus nonstructural protein define an endoribonuclease family new antiviral target revealed by the hexameric structure of mouse hepatitis virus non-structural protein nsp structural basis for double-stranded rna processing by dicer crystal structure of argonaute and its implications for risc slicer activity crystal structure of the trna processing endoribonuclease trnase z from thermotoga maritima a duplicated fold is the structural basis for polynucleotide phosphorylase catalytic activity, processivity and regulation crystal structure of the phosphorolytic exoribonuclease rnase ph from bacillus subtilis and implications for its quaternary structure and trna binding crystal structure of the trna processing enzyme rnase ph from aquifex aeolicus crystal structure of a bacterial ribonuclease p rna bacterial rnase p: a new view of an ancient enzyme crystal structure of the rna component of bacterial ribonuclease p crystal structure of the specificity domain of ribonuclease p the authors are supported by the wellcome trust. we thank gadi schuster, kiyoshi nagai, chris smith, murray deutscher, beatrice vallone, maria arménia carrondo, richard j jackson and aj carpousis for helpful discussions. we also thank maria armé nia carrondo and colin mcvey for kindly providing the figure of rnase ii. key: cord- -ng zr authors: klebe, gerhard title: virtual ligand screening: strategies, perspectives and limitations date: - - journal: drug discov today doi: . /j.drudis. . . sha: doc_id: cord_uid: ng zr in contrast to high-throughput screening, in virtual ligand screening (vs), compounds are selected using computer programs to predict their binding to a target receptor. a key prerequisite is knowledge about the spatial and energetic criteria responsible for protein–ligand binding. the concepts and prerequisites to perform vs are summarized here, and explanations are sought for the enduring limitations of the technology. target selection, analysis and preparation are discussed, as well as considerations about the compilation of candidate ligand libraries. the tools and strategies of a vs campaign, and the accuracy of scoring and ranking of the results, are also considered. in the late s and early s, experimental high-throughput screening (hts) and combinatorial chemistry were aggressively developed to overcome the lead discovery bottleneck in drug development. using sophisticated large-scale automation, it was anticipated that this would generate an unprecedented number of novel leads, resulting in a substantial increase in novel drug entities launched to the market per year. however, in reality, the opposite was the case [ , ] . frequently, the discovered hits could not be validated and further optimized into actual leads and preclinical candidates. thus, the initial euphoria surrounding these approaches has subsided owing to the disappointingly low hit rates and significant costs involved [ , ] . such situations fuel the consideration and development of alternative techniques. the expression 'virtual screening' (vs) was coined in the late s; however, the techniques involved are much older. in an effort to show that searching for lead candidates using a computer is a serious alternative to hts, the term 'vs' was adopted by the community. in contrast to hts, which is largely phenomenological and technology driven, in vs, compounds are selected by predicting their binding to a macromolecular target using computer programs (in drug discovery, the term 'target' or 'receptor' is used frequently to describe the macromolecule to which a drug binds, which is usually a protein but can also be dna or rna). the compounds studied do not necessarily exist, and their 'testing' does not consume valuable substance material. experimental deficiencies, such as limited solubility, aggregate formation or any sort of influence that could possibly interfere with experimentally applied assay conditions do not need to be considered in the initial computational screen. in contrast to hts, vs requires as a key prerequisite knowledge about the spatial and energetic criteria responsible for the binding of a particular candidate ligand to the receptor under investigation. in consequence, either the three-dimensional ( d) structure of the macromolecular target -as given by crystal structure analyses, nmr or sophisticated homology modelling -or, at the very least, a rigid reference ligand with a known bioactive conformation mapping out the putative receptor binding site must be available [ ] . this defines vs as a knowledgedriven approach. even though it is likely that multiple screening campaigns have been performed in parallel in industry [ ] [ ] [ ] [ ] , it was only recently that mcmaster university launched an open and unbiased competitive screening against escherichia coli dihydrofolate reductase to detect inhibitors [ ] , to assess how well vs can enrich candidate ligands compared with hts random screening [ , ] . the original sample set of compounds used for screening was split into two, and one fraction was tested by hts. obtained hits were reported and served as a training set to validate and tailor applied vs tools in different research groups, who entered into a competition to retrieve hits also detected by the experimental screen in the second portion of the screening sample [ ] [ ] [ ] [ ] [ ] . subsequently, in a totally unbiased fashion, the second part of the data sample was evaluated by hts and vs in parallel. unexpectedly, the second portion did not show any hits as competitive inhibitors, as would be expected for ligands docked into the substrate binding site of a target protein. interestingly, brenk et al. [ ] reported on some promising hits found by docking that were not detected as inhibitors in the initial hts. retesting at higher concentration indicated weak inhibition. this observation could be seen as supporting the view that vs and hts are complementary approaches and that they might find candidates missed by the other [ , ] . as mentioned above, the methods involved in vs are much older than the approach itself. initial attempts to find ligands by docking or by mapping them onto ligand-based pharmacophore models were the generic prototypes of a vs approach. however, this term was not used at that time, probably because computers and algorithms were not fast enough to enable large scale applications. focusing on approaches that actually make use of an available protein structure, one of the first systems to be studied by docking was hiv protease. initial versions of the program dock, developed over many years by kuntz's group [ , ] , tried to dock rigid entries from the cambridge crystallographic database into the protein receptor, focusing primarily on shape complementarity and later considering chemical complementarity. in , the kuntz group retrieved the neuroleptic drug haloperidol as a potential 'lead' from a docking screen using a database of known drug molecules as input. however, this compound would have had to be administered at a very high dose -far beyond a toxicologically tolerable concentration -to be an effective inhibitor of the protease [ ] . nevertheless, haloperidol as a 'lead' gave rise to some new ideas for developing a derivative possessing mm inhibition of hiv protease [ ] . later, at dupont-merck, a d database search retrieved a substituted terphenyl derivative as a putative lead for inhibiting this protease. further optimization via six-and sevenmembered rings resulted in the class of cyclic ureas that are able to replace the crucial structural water molecule in the protease, at the same time targeting the carboxy groups of the two catalytic aspartates via two appropriately placed hydroxy functionalities [ ] . since these early vs attempts, a plethora of case studies has been performed and the list of success stories is steadily growing. despite the fact that vs is still a young discipline, it has been reviewed frequently [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , most recently in a comprehensive overview by kubinyi [ ] . if one excludes purely retrospective studies, in which the potential of a method is demonstrated by its ability to enrich putatively active molecules from a sample of anticipated nonactive ones, targets have been studied to date, and reports on the discovery of mostly micromolar binding ligands in a truly predictive fashion are available (table ) . still under development, and far from mature, the number of strategies followed in vs is nearly as large as the number of reported screen- ing campaigns. owing to space constraints, this review is unable to give a comprehensive overview of success stories or investigated targets, or to review all details of currently applied vs protocols; accordingly, the reader is recommended to consult the abovementioned surveys [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . instead, this review attempts to summarize and comment on the agreed concepts and prerequisites for performing successful vs runs. furthermore, it attempts to analyse the limitations of the approach in a frank and plain manner and seeks to explain why such limitations still exist. accordingly, in the following sections, first the target selection, analysis and preparation are discussed, followed by considerations about the compilation of a candidate ligand screening sample. this is followed by a discussion of the actual requirements, tools and strategies of a vs campaign, and, finally, remarks concerning the accuracy of the scoring and ranking of the screening results. being a knowledge-driven approach, the scope of vs strongly depends on the amount and quality of information available about the system under investigation. clearly, the availability of the target receptor is of great benefit compared with situations where only a rigid reference ligand is known. the target receptor could be any macromolecular biomolecule, either a protein, rna or dna. in terms of the rigid reference ligand, either a substrate, inhibitor molecule, agonist or antagonist (e.g. a steroid hormone) could serve. because the structures of target receptors are becoming increasingly available, this review concentrates solely on such situations. however, it does not mean that vs cannot be applied to situations in which no structural information about the target receptor is available [ ] . with respect to dna and rna as target receptors, the development of appropriate docking and scoring tools has been initiated only recently, and, accordingly, vs applications on such targets are still rare [ , ] . by contrast, the scope of applications addressing proteins ranges from enzymes to gprotein-coupled receptors and ion channels (table ) . assessing the druggability of the target receptor the first issue to be considered is the druggability of the selected target. does the selected protein exhibit a binding pocket that can be successfully targeted by small molecule ligands? clearly, characteristics such as pocket size and geometry, surface complexity and roughness, exposure of recognition properties and their complementarity in shape and polarity with respect to a putative druglike ligand, are of importance. because such criteria are not immediately available, a pragmatic, but hardly generally applicable, approach would be to correlate gene families. if one member of such a family is able to bind a drug, other members might also be able to bind druglike ligands with related physicochemical properties [ ] . this assumption is based simply on the fact that members of a gene family usually operate on related substrates or recognize similar endogenous ligands. however, the setup for a vs study requires more conclusive information about the actual bindingsite architecture. recently, hajduk et al. [ ] suggested several very decisive indices that help to discriminate druggable from nondruggable binding pockets. for druggability, the total surface area and a portion of a polar contact area below Å appear to be beneficial, along with an appropriate pocket compactness, surface roughness and complexity. this suggests that there is an optimal size and composition of a protein-binding pocket that is best suited to recognize and accommodate small organic ligands. however, the analysis by hajduk et al. also showed that no single index consistently dominates the correlation. this fact points to the complexity of the interrelationships among the various discriminatory indices and explains why, at present, our concepts for predicting druggability are still rudimentary. selection of the most relevant geometry of the target receptor the selection of an appropriate d geometry for the target is another important issue when setting up a vs run. the most powerful method for learning about the spatial structure of proteins is crystal structure analysis. the accuracy and reliability of this method strongly depends on the resolution of the diffraction data. an alternative experimental technique to determine protein structures is nmr; there the accuracy of the structural determination depends strongly on the local distribution of the nuclear overhauser effect distance information along the protein chain (this effect involves transfer of magnetization through space and gives information about distances between nuclei in a d structure). furthermore, in the past, significant methodological enhancements in homology modelling have improved the quality of protein models [ ] [ ] [ ] ; accordingly, several successful vs screening campaigns have been reported based on model-built protein structures [ ] [ ] [ ] . homology modelling strongly depends on the availability of related proteins for which a crystal structure has been determined. the model will be particularly precise in those areas where the homology with the experimentally determined references is high, which is usually the case in the conserved core regions. however, functional binding sites, to be addressed by vs, are generally located in loop regions where, even among homologues of a gene family, significant differences are experienced (apart from catalytic residues in enzymes that are spatially highly conserved). to enhance the accuracy of model-built structures in regions where the binding sites are found, new algorithms have been suggested that consider the putative binding orientation of known ligands during the homology modelling process. these algorithms feed the information about the binding properties of possibly bound ligands back into the homology building process. improved binding site geometries can be expected from such approaches [ , ] . another obstacle that complicates vs attempts is molecular flexibility. ligands and proteins possess internal degrees of freedom and can adopt various conformational states. with respect to the target protein, several methods have been described to simulate flexibility [ ] . for vs, it is important to obtain an estimate of the protein conformers competent at accommodating a ligand. once these conformers have been determined, a vs run can either be performed by considering the flexibility of the protein instantly during the calculation, or by addressing an ensemble of several rigid receptor conformations [ ] [ ] [ ] [ ] . ligand-binding competent conformers can either be sampled by exhaustive conformational searches (e.g. using molecular dynamics simulations [ , ] ) or by examining multiple conformational states, observed in crystal structures with different ligands bound, to obtain insight into the relevant conformations [ , ] . the latter approach appears tempting because it can be assumed that a sample of experimentally observed protein conformers corresponds to stable, low freeenergy states. even though the crystal structure is usually considered as the 'gold standard' for learning about the geometry of a protein, it is highly questionable how representative a single structure determination really is. mcgovern and shoichet [ ] reported on increasing information decay, irrespective of whether the geometry of a ligand-bound or ligand-free crystal structure was used or a model-built structure was considered. principally, crystalline protein-ligand complexes can be prepared by exposing preformed crystals of a protein to the solution of a ligand, which can then diffuse into the crystals and find its way to the binding site. this process is called 'soaking'. alternatively, in a cocrystallization experiment, the protein-ligand complex is formed by equilibrating them in solution, and then the assembled complex is crystallized. we recently observed, depending on the soaking protocol applied, different conformational states of the protein aldose reductase complexed by the inhibitor zopolrestat [ ] . in one structure, the protein forms a hydrogen bond with the ligand via one of its binding site-exposed amide bonds ( figure ). in a second structure, the amide bond is rotated off from the binding site, and the same ligand can no longer form this hydrogen bond. such differences have a significant impact on docking and scoring results in vs. depending on the crystallization conditions, ligands have been observed to accommodate a binding pocket with reversed orientation [ ] ; in other cases, ligand-induced conformational adaptations of the protein are observed [ ] ( figure ). increasing the concentration of a ligand in the soaking buffer will enhance the opportunity to accommodate it in the binding pocket. however, it is likely that soaking depicts some kind of kinetic trap for ligands; consequently, surprising differences in cocrystallization have been observed (several ligands bound at one time, multiple binding modes [ ] ). before selecting a particular crystal structure as a reference for a vs run, detailed analysis of parameters such as population of the bound ligand, b-factors next to the binding site (b-factors indicate thermal motion in a crystal structure; however, they are highly correlated with the population of a ligand in the crystal) or consistency of the hydrogen bond network is advisable. in addition, most programs used for the actual computer screens require properly defined protonation states of the active-site residues. this is by no means easy to perform because local dielectric drug discovery today volume , numbers / july reviews induced-fit adaptations in two different crystal forms. two crystal structures of benzamidine (in both cases, accommodated deeply buried in the s pocket, which is indicated as a deep depression in the left-and right-hand images) bound to a trypsin mutant exhibiting a binding pocket related to factor xa. both crystal structures differ in terms of the conformational state of phe (in the left-and right-hand images at the left rim of the binding pocket with the red surface), which is exposed ('up') in one structure (orange and on the left) and buried ('down') in the other (green and on the right). this adaptation involves partial unwinding of a short three-turn helix (in the centre, helix at the left-hand side of the binding pocket) and rearrangement of a disulfide bond (in the centre, yellow bond to the back) [ , ] . binding mode differs with soaking and co-crystallization conditions. crystal structure of aldose reductase with the inhibitor zopolrestat is shown. the crystal structure determined after one day of soaking (yellow) differs from the structure obtained after six days of soaking (magenta) but is identical to that obtained by cocrystallization (light blue). whereas in the one day-soaked structure (yellow) the amide bond between ala and leu orientates its nh group towards the inhibitor to form a hydrogen bond (dotted yellow line) with the bound ligand, in the latter two structures (magenta and light blue) this amide bond rotates away from the inhibitor and no such hydrogen bond is observed [ ] . www.drugdiscoverytoday.com conditions can modulate pk a values of functional groups by several orders of magnitude. even more complex and, at present, difficult to calculate are pk a shifts of residues during the course of ligand binding. isothermal titration calorimetry [ ] measurements can make such changes apparent; these changes probably occur more frequently than we presently admit [ ] , and can easily turn an acceptor functional group into a donor or a charge-assisted hydrogen bond into a neutral one. these changes are of significance during various validation steps of a vs run. we have recently observed that even the protonation states of the residues in the binding pocket of inhibitor-bound aldose reductase can change depending on whether the cofactor nadph/nadp + is present in the oxidized or reduced state ( figure ) (o. krämer and g. klebe, unpublished results). with respect to protein flexibility, the decision has to be taken as to whether a vs run should consider one single protein conformer or multiple binding-competent states of the receptor. different strategies with respect to docking and scoring will be the consequence (see below). experience shows that conformational adaptations observed in a protein-binding pocket are usually related to the structural modulations potentially needed by the protein to accomplish its functional role [ ] . such functional adaptations must correspond to low-energy transformations, otherwise dramatic shortcomings in the functional performance of the protein would be the consequence. accordingly, enzymes that operate on a large palette of structurally diverse substrates (cf. aldose reductase and short chain dehydrogenases) or perform substantial conformational adaptations during catalysis (cf. kinases) will experience multiple bindingcompetent states that have to be considered in vs. thus, a profound understanding of the functional properties of a protein might be the best option for predicting the conformational adaptability of a protein. such information is not usually available at the beginning of a drug development project, when vs is used. because proteins occur in gene families of closely related members, the analysis across different entries of the family might provide some insight into the flexibility properties inherently shared by the members of the family. however, it might also be that some family members display rigid solutions, as required by their function (e.g. trypsin and factor xa among the serine proteases), whereas others possess pronounced flexible behaviour, as appropriate to their function (e.g. the serine protease factor viia) [ ] . protonation states can change upon ligand binding. depending on the oxidation state of the bound cofactor nadph (left) and nadp + (right), aldose reductase binds to a carboxylate-type inhibitor (e.g. idd ) by placing its acid functional group (red) into the catalytic centre; the complex thereby formed remains either in an unchanged protonation state (left) or picks up protons upon binding (right). in the centre, the crystal structure of the complex is shown. a short contact distance between the carboxylate function of the inhibitor (shown with a yellow surface) and the nicotinamide portion of the cofactor (shown with an atom-type coloured surface, where oxygen is red, nitrogen is blue, carbon is white, sulfur is yellow and phosphorous is orange) is formed. high resolution x-ray crystal structure analysis and neutron diffraction has provided evidence that the inhibitor binds with its carboxylate function in the deprotonated state, and the active site his is present in the neutral state. it remains unresolved where the proton goes in the case of the nadp + -bound complex, or whether the residues of the catalytic centre exhibit deviating protonation states in the uncomplexed and inhibited situation. with respect to vs, a unique and precise assignment of the protonation states of the ligand and protein functional groups in a pk a range between and is essential because, for example, for docking it is important whether such a group is considered as donor or acceptor of a hydrogen bond (krämer and klebe, unpublished). after the reliability and relevance of the protein conformer(s) of the selected target protein have been assessed, its binding-site properties should be mapped before blindly starting a vs run (see below). this strategy helps the user to evaluate the properties of the target protein better and subsequently to examine the relevance of docking solutions suggested by vs. several tools have been described to elucidate the 'hot spots' of binding in a particular binding pocket [ ] . these methods are either based on thoroughly parameterized force fields [ ] or on well-selected empirical information (superstar [ ] , drugscore [ ] ; figure ). most importantly, this analysis has to be performed on all multiple conformational states of a binding pocket because this will provide a composite picture of how the molecular recognition properties of a binding site might change upon protein adaptation. water, the nasty third binding partner in proteinligand complexes finally, a very crucial decision with respect to the setup of the protein reference for a vs run concerns the consideration of water molecules in a binding site. the analysis of several thousand crystal structures of ligand-protein complexes using the waterbase module in relibase [ , ] revealed that, in about two-thirds of all cases, a water molecule is involved in ligand binding, frequently mediating contacts between protein and ligand. thus, any approach based on the prediction of binding modes of putative candidate ligands, as required in vs, must take water into consideration. one possible approach for learning about water molecules tightly bound to the protein is the analysis of crystallographic data with respect to the repeated occurrence of water molecules in structurally related binding sites (e.g. in a gene family) or multiple structural determinations of the same protein with many distinct ligands [ ] . if water is present in all structures analysed at more or less the same location, this is a strong indica-tion that this water molecule is tightly bound, and in a vs run could be considered as an integral part of the target structure. as with hts, vs needs a thoughtfully designed and thoroughly compiled sample of small molecule candidate ligands for screening. pharmaceutical companies will primarily screen their own proprietary compound collections, with the advantage that detected hits will be exclusive and will cover molecules for which the synthesis is well-established at their site. however, are such sample pools biased? how well is the available chemical space covered? these considerations have led large pharma companies to complement their inhouse collections with compounds offered through commercial suppliers. starting with the available chemical directory as the initial prototype (see mdl, http://www.mdli.com), there are currently more than million unique purchasable compounds on offer [ ] . how well do they cover chemical space, and which portion of this space represents druglike molecules? some dramatic figures have been proposed concerning the number of organic molecules that can be considered as druglike. usually, molecules meet these criteria if they are composed only of the elements h, c, n, o, p, s, cl and br, and possess a molecular weight < da [ ] . how diverse should the entries of a compound database used for screening be? what physicochemical properties have to be met by the candidates to guarantee sufficient bioavailability? the concept of a well-balanced and homogeneously populated space of diverse druglike molecules appears very tempting; however, there is no proper definition of what descriptors to use as coordinates on the axes of such a compound space. what is 'diverse' in this context? as with the criterion 'similarity', the expression 'diversity' is a relative measure that relates to a reference point. in vs, the reference point is the target receptor and the difference in binding mapping the hot spots of binding. hotspot analysis using drugscore for the binding pocket of t-rna guanine transglycosylase (surface of the binding pocket indicated in white). regions energetically favourable for the binding of a hydrogen bond donor group (represented by an nh group) or an acceptor group (represented by a c=o group), or a hydrophobic molecular portion (represented by aromatic carbon atoms, c.ar) have been analysed and contoured on three subsequent levels (indicated using three different colours) above the deepest energy minimum found for each atom type in the maps [ ] . the crystallographically determined binding mode of an inhibitor is shown superimposed. www.drugdiscoverytoday.com affinity of two ligands with respect to this structure. this defines whether two molecules should be classified as 'similar' or 'diverse'. it can be imagined that, for one target, a correctly placed methyl group on a ligand might have a dramatic effect on binding affinity, whereas for another it will be tolerated with no change in binding affinity. in the first case, the ligands with and without a methyl group would have to be termed 'diverse', whereas in the second, they will be called 'similar'. however, consulting a 'diversity' scoring that concentrates solely on the topology of a ligand, the methylated and unsubstituted derivatives will probably be ranked as 'highly similar'. is there an optimal size for candidate ligands to be submitted to screening? in the past, combinatorial chemistry in particular has enabled pharmaceutical companies to develop screening libraries of several million candidate molecules; thus, in principle, the sheer number of test compounds is no longer an issue. although large in total count, the individual members of such libraries are usually large in terms of size and molecular weight. they are mostly in the range of typical drug-size molecules, having been synthesized in other drug development projects. however, if they turn out to be a micromolar screening hit, they still require optimization to improve their affinity towards the target protein by two or three orders of magnitude. this has to be accomplished while maintaining reasonable molecular size and appropriate absorption, distribution, metabolism and excretion properties. in consequence, it involves stripping down to a scarcely decorated core skeleton while subsequently building up this core again with novel well-tailored side chains. this is a challenging task, and is often difficult to achieve. experience from drug optimization programs has shown that small core fragments ('privileged templates' or 'fragments') known to bind with significant affinity are ideal starting points for further optimization [ , ] . accordingly, the compounds selected for vs should leave some room for optimization, thus matching the range of socalled leadlike or fragment-like molecules [ ] . verdonk et al. [ ] have presented a very insightful discussion on approaches to be taken when constructing databases for vs, and have suggested that the methods involved should be validated (e.g. by assessing the enrichment rates of known binders). for this purpose, in a vs campaign, known binders are pooled with the sample set of screening candidates to assess how well the known binders 'enrich' during the course of the screening process. such considerations are important for the development of new methods; however, in an actual drug development project, at the end of the day, the medicinal chemist will ask for novel leads that are worthwhile pursuing with synthesis and optimization. impressive enrichment rates of known actives will not be convincing. in light of these considerations, what is a suitable compound collection? some general criteria have to be matched either by inhouse proprietary compounds or by substances offered by commercial suppliers. compounds can be validated with respect to drug likeness considering lipinski's rule of five, or lead likeness applying more stringent criteria (as defined by the rule of three) [ ] [ ] [ ] . recently, martin tested lipinski's frequently applied rule with respect to experimentally determined bioavailability in the rat [ ] . this study showed that the rule of five has predictive ability for neutral and positively charged molecules; however, anions obey different rules, and the size of their polar surface area confers some predictive power. similarly to the considerations concerning the druggability of binding sites, the criteria for bioavailability are multifactorial. irwin and shoichet took the initiative to set up the freely available database zinc with validated compounds for vs [ ] . it is built from d compound information, generates d coordinates and curates, if possible, from stereo-and regioisomeric ambiguities. multiple states with respect to protonation, charges and tautomers are enumerated. however, as described for proteins, the properties of compounds might be altered upon protein binding. insoluble, reactive and aggregating compounds are not represented in the database. the rule of five is a straightforward filter to discard compounds with putatively undesired properties from the screening sample. depending on the strategy pursued in the subsequent vs campaign, multiple conformers can be precalculated and stored as separate entries in the database. limiting the search sample to purchasable compounds in vs is a pragmatic approach because screening hypotheses can be tested rapidly [ , ] . however, vs can also scan over virtual compound libraries, and synthesis can be postponed to a later stage, considering only the most promising hits. reymond's group attempted to generate a database of all possible organic molecules up to da under the constraints of defined chemical stability and synthetic feasibility [ ] . this database contains . million entries. it is possible that such a sample could be used for fragment screening. for larger compounds, exhaustive sampling of possible molecular skeletons will end up in a combinatorial explosion with too many possible solutions. however, proper design criteria, defined by the architecture of the binding site used in vs as the target, might guide the generation of target-tailored virtual libraries for vs. in particular, considering the criteria of combinatorial chemistry and parallel synthesis, such vs strategies can actually help to synthesize only the most promising entries of a large virtual combinatorial library. principally, two strategies can be followed in a vs campaign: forward or backward filtering of hits obtained by docking. the most crucial step in vs is the docking of candidate molecules to the target protein [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in forward filtering, various criteria are used to reduce the initial data sample, which might comprise several millions of test compounds, to the several hundred or thousand most promising candidates to be docked [ ] . in backward filtering, all entries from the data sample are docked to the target protein, and filter criteria are subsequently applied to rank the generated docking solutions. nowadays, the speed of computers is no longer the limiting factor in selecting the strategy. the forward technique requires fewer computational resources. this is mainly because of the fact that at each hierarchical filtering drug discovery today volume , numbers / july step, a significant amount of the original data sample is discarded. however, with a decreasing number of compounds, the filtering becomes computationally increasingly demanding and sophisticated. flexible docking is the computationally most intensive step of all; thus, the fewer candidates to be considered here, the more effort can be spent in controlling, validating and assessing docking results. this is clearly an advantage of this strategy. forward filtering eliminates compounds initially according to simple descriptors such as molecular weight, number of rotatable bonds, lipophilicity (usually expressed by the logarithm of the partition coefficient, log p) or crude shape descriptors, such as the ellipticity of the overall structure. subsequently, information about the receptor's binding site is exploited. once a hotspot analysis of the most likely anchoring positions in the binding pocket has been performed, a protein-based pharmacophore can be derived [ ] . this pharmacophore sets the constraints for the minimal requirement of functional groups to be matched by putative ligands (e.g. number of hydrogen bond donors, acceptors or hydrophobic groups). molecules satisfying such criteria can be retrieved by any database engine capable of a functional group substructure search. once the topographical arrangement of the protein-based pharmacophore has been incorporated into the search and the remaining candidates are requested to match this pattern, the study can be further focused. unity (unity chemical information software, version . , tripos) and catalyst [ ] are prototypes of such database engines supporting this screening step. an alternative tool is featuretrees, which can retrieve molecules of similar topology in feature space [ ] . in this context, 'features' are considered as being similar types of functional groups or molecular building blocks. similarity can be considered as a further filter criterion in vs. for example, if candidate molecules are requested to match with a predefined protein-based pharmacophore, this condition already involves a selection in terms of similarity criteria; however, they are regarded very generally. furthermore, information about known binders can be used for filtering, although the danger then exists that, owing to biased filters and preconceived concepts, some unexpected and novel chemistry is discarded during the early filtering steps. finally, docking is pursued, usually considering only - % of the initial sample collection. an advantage of the forward filtering approach is that it enables more elaborate docking protocols to be performed -for example, taking multiple protein conformational states into account or reflecting a protein-based pharmacophore as a restraint in docking. most importantly, this hierarchical filtering strategy enables the tracking of the performance at the various filter levels by human intervention, and, in particular, visual inspection of docking solutions remains feasible. backwards filtering starts with high-throughput docking and analyses the generated docking modes as subsequent steps. this approach is especially challenging because docking returns multiple solutions for most candidate molecules. strongly discriminative and reliable scoring functions must be available for analysing the computed results in an automated fashion because visual inspection for the large number of diverse compounds to be examined is hardly feasible. however, it is questionable whether the existing scoring functions are reliable enough to succeed with such heroic demands (see below). nevertheless, to proceed with the multiple docking solutions from a high-throughput run, the generated docking modes can be filtered with respect to achieved matching of the protein-based pharmacophore, contact complementarity of protein and ligand surfaces or the remaining residual unoccupied voids along the protein-ligand interface. because nature probably avoids gaps in molecular assemblies, the latter criterion could be a powerful indicator for irrelevant binding modes or the putative accommodation of interstitial water molecules. docking is the crucial step in vs. the seminal program dock, originally described in by kuntz et al. [ ] , has evolved as the first vs tool. later, other programs were successfully applied in vs, such as gold [ ] , flexx [ ] , glide [ , ] or autodock [ ] , to name the most popular prototypes. these have been recently reviewed [ ] [ ] [ ] [ ] [ ] [ ] [ ] . all docking tools follow slightly different concepts. this might give individual programs a particular advantage in one task with respect to another -for example, incorporating aspects such as flexibility of ligand and receptor or restraining the docking search engine to particular regions in configuration space (e.g. mapping a protein-based pharmacophore) [ ] . however, all docking tools are still far from perfect. an even more challenging, but carelessly disregarded, aspect of docking is the appropriate consideration of water molecules. as indicated above, water molecules are involved in binding in about two-thirds of the known protein-ligand complexes. however, most docking tools ignore them simply because conclusive concepts of how to consider them correctly are missing. if structural evidence is given, preplacement of water molecules in a docking run is a feasible strategy [ ] . the docking tool slide [ ] treats preplaced water molecules in a way that still enables their replacement by ligand atoms in docking. the particle concept in flexx [ ] enables the placement of water molecules 'on the fly' during the course of the generation of individual docking solutions. in the docking tool gold, water molecules can be switched on or off, and can spin around their principal axes to achieve good contacts with a docked ligand [ ] . recently, the popular docking tools dock and flexx have been equipped with features that enable docking on a preconceived pharmacophore or property distributions [ , ] . consideration of such criteria will drive docking solutions especially into regions either frequently trapped by other bound ligands or featured by complementary analytical tools as being particularly relevant for binding. the program autodock [ ] performs docking on a precalculated grid, storing potential values from any sort of interaction field [ ] . in the original implementation, lennard-jones and coulomb potential values are used. sotriffer et al. [ ] replaced these by knowledge-based potentials, originally implemented into drugscore. the latter potentials have proven to be powerful for discriminating, and rank among multiple ligand poses. the potential grid approach in autodock also enables one to average across the fields produced by various protein frames, so that the conformational degrees of freedom of a protein can be considered. in addition, adapted fields optimized with respect to the binding properties of some known actives in the comparative molecular field-type approach 'adaptation of fields for molecular comparison' (afmoc) [ ] can be used as target potential values in autodock [ ] . the latter docking tool performs multiple stochastic searches on the potential hypersurface; accordingly, the frequency of occurrence of certain docking solutions can be used as an additional figure of merit for their relevance [ ] . the issue of an optimal ligand size for screening has been addressed above. the complexity of the docking problem increases with the size of the ligand and its number of rotatable bonds. thus, smaller molecules in the typical range of ligand fragments should be simpler to dock. perplexingly, present experience indicates the opposite to be the case. fragments are easily scattered over a binding site by docking; reliably successful docking can only occur if the binding site itself is restricted in size and shows dimensions similar to the fragments [ ] . interestingly, experimental approaches show the opposite: small molecular fragments (> da) usually populate, in a well-defined manner, in a very limited number of sites in binding pockets [ , ] . enrichment rates to control the achievements of virtual screening vs runs are usually monitored and validated by comparing the performance of a set of known actives with a large number of 'randomly' picked compounds. the actives are pooled with the random entries. all compounds are submitted to the selected vs protocol, and the performance ranks of the known actives with respect to the remaining pool are converted into enrichment plots. such a process is essential for keeping control over the performance and achievements of vs. however, the choice of the random compound library is crucial and can strongly affect the enrichments obtained [ ] . accordingly, it has to be examined whether the pooled, randomly picked decoy structures are actually nonbinders. in a real-life scenario, it should be noted that merging known actives with a set of candidate ligands should result in a gradually declining enrichment rate at the subsequent hierarchical filter steps because novel actives, retrieved by vs, will populate in prominent ranks and dilute the set of predefined known actives. independent of the actual vs strategy applied, docking and subsequent scoring of the suggested solutions is the key performancedetermining factor in vs. the discriminatory power of the applied scoring function is of utmost importance for ranking, and hopefully enriching, potentially active binders at the top of the list of docking solutions. in consequence, a myriad of scoring functions has been developed over the past few years [ ] [ ] [ ] . approaches have been taken not simply to rely on one single function but to take on board the consensus picture of several scoring schemes [ , ] . to characterize the binding affinity of putative lead candidates experimentally, the binding constant or its inverse, the dissociation constant, is determined (or approximated by values such as the ic ). if we assume that the basic rules of equilibrium thermodynamics can be applied, we can define, according to the law of mass, an equilibrium constant that describes the formation of a protein-ligand complex. this equilibrium constant is logarithmically related to the gibbs free energy, comprising both an enthal-pic and entropic contribution. whereas the former relates to energetic features, the latter concerns configurational and ordering phenomena. the entropic contribution estimates how the energy content of the system is distributed over internal and external molecular degrees of freedom. up until now, three strategies have been followed to predict binding affinities on the basis of a given protein-ligand binding geometry. the most rigorous and theoretically most solid approaches are first-principle methods [ ] . using quantum mechanics or computationally less demanding (however approximate) force fields, the partition function of a system is computed and the free energy differences between the bound and unbound state are determined. with the increasing speed of computers, such methods are becoming more accurate and obtaining a growing relevance for scoring [ ] . however, screening large samples of docked solutions to estimate binding affinities is still far beyond tractability. nevertheless, first-principle methods do not need any calibration or training in experimentally determined affinity data; thus, they will not suffer from inherent experimental shortcomings or accuracy limits. this differs from the other two approaches described in the following section, the regression-and knowledgebased scoring functions, which are based on empirical concepts [ ] . regression-based approaches assume additivity of individual terms considered in a master equation to describe the total gibbs free energy of binding. in this context, a 'term' can reflect any physicochemical property of relevance for the protein-ligand binding process -for example, the number of charged or uncharged hydrogen bonds, the size of the polar or nonpolar surface portion, the number of rotatable bonds or the enthalpy required to desolvate either the ligand or the protein, among others. they are assumed to be independent from each other, and their individual contribution in reproducing the known affinities of some training set ligands is extracted by regression analysis, partial least-squares analysis or neural networks [ ] . however, independence of the 'terms' is unlikely, and fair to strong correlations between terms are probable. in the correlation analysis, this fact might point out that another, at first glance surprising, property pops up as the best explanation. however, it might be the case that the latter property is not on its own essential in a physical sense, but is highly correlated with another property that is actually crucial. we recently parameterized a new regression-based scoring function on the basis of a large sample of crystal structures with known affinities for the bound ligands. depending on the composition of the training set, different terms are found to be relevant in the analysis. this clearly shows that such empirical scoring functions reflect a best fit with respect to the training set used but they rarely achieve generality. an alternative to regression-based approaches are knowledgebased scoring functions [ ] . these evaluate the occurrence fre- quencies of some properties of interest -for example, the mutual distance between particular atom types found across the proteinligand interface. the sample distributions describe occurrence probabilities and can be compared with a statistical mean reference situation. any deviations from this average can be translated using some type of mathematical relationship into statistical preferences that can be used to determine the geometry of protein-ligand complexes. conceptionally, the knowledge-based functions appear to be more general because no master equation with preconceived 'terms' is required. however, the data selected to derive the function, and the definition of atom types, together with many adjustable parameters needed to actually establish the method, also attenuates the generality of this approach. to estimate binding constants, both the regression-and knowledge-based scoring functions require experimental affinity data for internal calibration. thus, their prediction accuracy can never be better than the precision by which binding data can be measured. it is interesting to note that the estimated standard deviations of such empirical scoring functions are reduced if data for a selected number of targets, determined in one laboratory based on assay data recorded under strictly conserved conditions, are used or if broad-range data covering many targets are considered based on assay data determined in various laboratories. the relative differences in binding data within a series of compounds can be determined precisely -usually much better than across data from various systems for which the comparison has to be performed on an absolute scale. in consequence, the standard deviations of predicted binding affinities of presently available functions range between . and . logarithmic units in binding affinity. this range matches the experimental accuracy achieved for binding data across training sets of growing heterogeneity covering a broad spectrum of targets. in our experience, knowledge-based scoring functions are better able to extract binding geometries generated by a docking program that closely approximate the experimentally confirmed binding mode from a sample set of decoy placements. by contrast, regression-based scoring functions are better in the actual affinity prediction, provided that a fairly accurate binding geometry is given. unlike the professed opinion that in docking the geometry problem has been resolved to a sufficient extent, and that the scoring problem remains an open question [ , ] , it appears that both are intimately related. we recently developed a knowledge-based scoring function based on accurate contact data from small molecule crystal data [ ] . this function reliably recognizes the experimentally determined binding mode found in a crystal structure among a set of decoy poses. it appears selfevident that the scoring problem can only be alleviated if more relevant, near-native binding poses are produced by docking programs and reliably recognized by a scoring function. accordingly, it appears advisable to drive docking solutions as close as possible to the native geometries -that is, as they would show up in a corresponding crystal structure. this can also be achieved by optimizing them towards the near-native situation. at best, this involves minimization with respect to the function used for scoring. as a disadvantage, this process would be computationally demanding. binding affinity: a sufficiently well understood property? any discussion of scoring and ranking must ask the question as to how well we understand the target value gibbs free energy. is it advisable to focus scoring on free energy or would it be better to treat enthalpy and entropy as separate terms in the scoring [ ] ? there is a mutual compensation between enthalpy and entropy owing to the fact that both entities scatter over much larger ranges than does the free energy itself. considering the binding of a ligand to a protein, enthalpy (dh) and entropy (-tds) can easily scatter over a - -fold larger range than the spread of gibbs free energy (dg). the mutual compensation of enthalpy and entropy can even be found across closely related molecules [ ] (figure ). furthermore, many physicochemical phenomena of relevance for the binding process are not yet fully understood and, therefore, have not yet been correctly incorporated into scoring functions (e.g. the role of water, change in protonation states or an appropriate consideration of entropy). interestingly, microcalorimetry indicates that with increasing temperature, the protein-ligand binding process becomes more exothermic and entropically less favourable [ , ] . because this observation applies to all targets, it suggests that general phenomena are involved which are not yet understood at a molecular level. despite our present deficiencies in understanding the physics of the binding process, scoring still works satisfactorily -most likely because we consider the binding of ligands to a protein on a relative scale to each other. accordingly, any unappreciated phenomena, similar across all complexes in the analysis, will simply be cancelled out. for example, as mentioned above, one approach for reflecting protein flexibility applies parallel docking into several rigid binding-competent conformers of the protein. the disadvantage of this strategy is the fact that additional degrees of scoring are created: what discriminates the scoring against different protein conformers? cancellation of unreflected internal protein energy contributions is no longer certain. studies have shown that a special scoring scheme or protocol is required [ , ] . because dramatic energy differences between low-energy conformers are unlikely, a modulated pocket size for the different protein conformers might require individual scoring of the altered desolvation properties of the binding site. required accuracy of scoring: enrichment or hit identification? vs is used to enrich putative actives from a large sample set of test ligands. accordingly, the desired accuracy of scoring depends on whether, at first glance, prioritization of the sample set for testing is anticipated or whether putative actives are expected among the top ten or of a hit list. the latter requires very powerful discrimination of actives over inactive decoy binders. present scoring functions have been optimized to discriminate for a particular ligand decoy binding mode from near-native ones. the discrimination of binders from decoy nonbinders still remains as major challenge for vs protocols [ ] . at worst, in these cases the above-mentioned poorly understood contributions to binding become overwhelmingly important. perhaps the consideration of similarity criteria in the search [ , , , [ ] [ ] [ ] enables the problem to be alleviated to some degree because this drives the search towards more closely related ligands for which some of the disregarded effects in scoring cancel themselves out. ultimate proof of concept: crystal structure analysis of vs hits only rarely, the crystal structure of detected vs hits has been determined and subsequently published. such case studies have recently been reviewed by shoichet [ ] . cases have been described for which vs has correctly predicted the subsequently found binding mode. other examples point to deficiencies arising from the superficially understood phenomena described above. finally, it has also been reported that vs inappropriately identified an active compound because binding actually occurs in a totally different region of the protein surface. vs has been established as a powerful alternative and complement to hts. when performed optimally, impressive hit rates have been reported, which have been significantly higher (by a factor of - [ ] ) than those for hts. comparative studies of hts and vs indicate that the methods can capture alternative and complementary ligands. undoubtedly, vs is not yet a fully mature technology following a well-established process line. few of the foundations of proteinligand recognition are understood well enough to be deployed in a large scale, multi-compound effort such as that commonly undertaken in vs. this calls for further indepth research. in particular, protein flexibility and induced-fit adaptations, the role of water in solvation, desolvation and ligand binding, and the electrostatics involved, including changes in protonation states and an appropriate consideration of entropic changes, will need to be better understood to improve the hit retrieval rates in vs. frequently, experimental work performed in parallel or as a follow-up to a vs campaign provides a whole bunch of unexpected results pointing towards manifold deficiencies of the concepts applied. many more experimental studies are required. nevertheless, vs has proven successful and as a valuable alternative to hts, in particular if it is used as a tool to support and complement hit discovery. compared with hts, it is significantly cheaper and faster to use. it can be easily rerun under modified conditions -for example, if additional information about the target protein under consideration becomes available or novel filter criteria are taken into account. it is interesting to note that an experienced modeller or medicinal chemist can often figure out whether a particular binding pocket appears druggable or a certain molecule obeys the rules of drug likeness; however, putting such knowledge into computer algorithms makes the multifactorial nature of these rules apparent, complicating attempts to generalize them. the same multifactorial nature holds true for docking and scoring. it is therefore highly advisable to refrain from fully automated strategies in vs. experience and human intervention are of the utmost importance for keeping control over the various filter steps in a vs run. used in such a way, vs can be successful; in particular, if information about molecular similarity is considered in terms of generic physicochemical properties and not simply as chemical formulae. con- drug discovery today volume , numbers / july similar ligands decompose differently into enthalpic and entropic binding contributions. crystal structures of two closely related thrombin inhibitors bearing a cyclopentyl or cyclohexyl moiety as terminal substituent to accommodate the s /s pocket of the catalytic site (surface of the binding pocket is indicated in blue). whereas the five-membered ring (left) gives rise to a well-defined difference in electron density (white 'chicken-wire' contouring), the six-membered ring (right) cannot be assigned to any density (see inside white circles). it is likely that the latter fragment shows enhanced residual mobility and is scattered around several conformational states. interestingly, this deviating behaviour is well reflected in the thermodynamic properties (centre). both compounds exhibit the same free energy of binding (dg, blue columns). however, the cyclohexyl derivative (right) with the enhanced residual mobility is entropically (-tds, red columns) more favoured than the 'less-well clamped' five-ring derivative (left). the latter experiences better enthalpic contributions (dh, green columns) to binding [ ] . sidering similarity concepts takes the risk that highly diverse molecules remain undetected; however, it probably makes the searches simpler because many parameters that actually matter in vs, and which are not properly considered, simply cancel each other out in a relative comparison. considering all of the mentioned limitations, and taking molecular similarity as some kind of work-around to evade existing shortcomings, a critical reviewer might raise the question of whether, under such restrained conditions, vs can really contribute something novel that an experienced medicinal chemist would not have thought about. only successful examples can convince. we applied vs to a trnamodifying enzyme for which the inhibitor replaces either guanine or preq (a precursor to the modified base queuine) as a substrate in the enzyme recognition pocket ( figure ). initial vs searches retrieved structures that, admittedly, an experienced medicinal chemist might also have suggested as being 'substrate-like'. however, a follow-up vs screen in which several water molecules were allowed to be replaced in the binding site suggested a cyclic urea structure with a different orientation in the binding pocket [ ] . synthetically, this lead appeared tempting and presently serves as a starting point for a new series of compounds ( figure ). this unbiased approach, resulting, in chemical terms, in an unexpected lead, demonstrates that vs can make important contributions to drug discovery, providing some unexpected candidates. nevertheless, there is still a long way to go until it becomes an established tool for routine lead discovery. interestingly, nowadays, most aspects of contemporary drug discovery are optimized towards high throughput; by contrast, our current increase in the knowledge and understanding of protein-ligand recognition principles is still proceeding at a low-output rate. drug discovery today volume , numbers / july reviews an unexpected ligand skeleton from virtual screening. virtual screening (vs) has been used to search for putative inhibitors of t-rna guanine transglycosylase [ ] . initial hits such as - (lower left box), which were followed up by chemical synthesis, showed structural similarity with the natural substrates of this enzyme guanine, preq and preq (upper box). these searches were based on the protein-based pharmacophore shown on the left (contours for hydrogen bond acceptor group) and in figure . an experienced medicinal chemist could possibly have come up with similar suggestions for potential leads such as - . however, in a second vs campaign we focused on the replacement of two water molecules at the lower right rim of the binding pocket of t-rna guanine transglycosylase (right, contours for hydrogen bond acceptor group [ ] ). this screen suggested as a potential hit. its cyclic urea-type skeleton is distinct in its structure and binding mode from any known natural substrate and it can serve as a synthetically easily accessible lead. several derivatives (e.g. - ) have been synthesized and show a reasonable structure-activity relationship (stengl et al., unpublished) . it is unlikely that this novel lead structure would have been suggested by comparative substrate considerations. this example underlines the power of vs as an alternative source for novel lead discovery. www.drugdiscoverytoday.com reviews foundation review trends in development cycles a new grammar for drug discovery how many leads from hts? comment: how many leads from hts? chemical feature-based pharmacophores and virtual library screening for discovery of new leads virtual screening to enrich hit lists from high-throughput screening: a case study on small-molecule inhibitors of angiogenin molecular docking and high-throughput screening for novel inhibitors of protein tyrosine phosphatase- b comparing performance of computational tools for combinatorial library design inhibitors of dihydrodipicolinate reductase, a key enzyme of the diaminopimelate pathway of mycobacterium tuberculosis high throughput screening identifies novel inhibitors of escherichia coli dihydrofolate reductase that are competitive with dihydrofolate experimental screening of dihydrofolate reductase yields a ''test set'' of , small molecules for a computational data-mining and docking competition mcmaster university data-mining and docking competition: computational models on the catwalk evaluating the high-throughput screening computations screening for dihydrofolate reductase inhibitors using molprint d, a fast fragment-based method employing the naïve bayesian classifier: limitations of the descriptor and the importance of balanced chemistry in training and test sets virtual ligand screening against escherichia coli dihydrofolate reductase: improving docking enrichment using physics-based methods here be dragons: docking and screening in an uncharted region of chemical space a geometric approach to macromolecule-ligand interactions docking flexible ligands to macromolecular receptors by molecular shape structure-based design of nonpeptide inhibitors specific for the human immunodeficiency virus protease structure of a nonpeptide inhibitor complexed with hiv- protease. developing a cycle of structure-based drug design rational design of potent, bioavailable, nonpeptide cyclic ureas as hiv protease inhibitors structure-based virtual screening: an overview virtual screening in structure-based drug design. mini rev virtual screening of chemical libraries integration of virtual screening into the drug discovery process virtual screening in lead discovery and optimization virtual screening methods that complement hts recent development and application of virtual screening in drug discovery: an overview docking and scoring in virtual screening for drug discovery: methods and applications hit and lead generation: beyond high-throughput screening success stories of computer-aided design validation of an empirical rna-ligand scoring function for fast flexible docking using ribodock identification of ligands for rna targets via structurebased virtual screening: hiv- tar the druggable genome druggability indices for protein targets derived from nmr-based screening data comparative protein structure modelling all are not equal: a benchmark of different homology modelling programs utility of homology models in the drug discovery process protein-based virtual screening of chemical databases. ii. are homology models of g-protein coupled receptors suitable targets? successful virtual screening for a submicromolar antagonist of the neurokinin- receptor based on a ligand-supported homology model structure-based drug discovery using gpcr homology modeling: successful virtual screening for antagonists of the alpha a adrenergic receptor docking ligands onto binding site representations derived from proteins built by homology modelling ligand-supported homology modelling of protein binding sites using knowledge-based potentials implications of protein flexibility for drug discovery molecular docking to ensembles of protein structures ligand docking to proteins with discrete side-chain flexibility flexe: efficient molecular docking considering protein structure variations testing a flexible-receptor docking algorithm in a model binding site accommodating protein flexibility in computational drug design incorporating protein flexibility in structure-based drug discovery: using hiv- protease as a test case probing flexibility and ''induced-fit'' phenomena in aldose reductase by comparative crystal structure analysis and molecular dynamics simulations unveiling the full potential of flexible receptor docking using multiple crystallographic structures information decay in molecular docking screens against holo, apo, and modeled conformations of enzymes expect the unexpected or caveat for drug designers: multiple structure determinations using aldose reductase crystals treated under varying conditions ph-dependent binding modes observed in trypsin crystals: lessons for structure-based drug design understanding protein-ligand interactions: the price of protein flexibility zz made ez: influence of inhibitor configuration on enzyme selectivity isothermal titration calorimetry and differential scanning calorimetry as complementary tools to investigate the energetics of biomolecular recognition approaches to the description and prediction of binding affinity of small-molecule ligands to macromolecular receptors reconstructing the binding site of factor xa in trypsin reveals ligand-induced structural plasticity predicting binding modes, binding affinities and ''hot spots'' for protein-ligand complexes using a knowledge-based scoring function a computational procedure for determining energetically favorable binding sites on biologically important macromolecules superstar: a knowledge based approach for identifying interaction sites in proteins knowledge-based scoring function to predict proteinligand interactions relibase: design and development of a database for comprehensive analysis of protein-ligand interactions utilising structural knowledge in drug design strategies: applications using relibase zinc-a free database of commercially available compounds for virtual screening the art and practice of structure-based drug design: a molecular modelling perspective fragment-based drug discovery fragment-based lead discovery current trends in lead discovery: are we looking for the appropriate properties? virtual screening using protein-ligand docking: avoiding artificial enrichment experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings a rule-of-three for fragment-based lead discovery admet in silico modelling: towards prediction paradise? a bioavailability score drug-like annotation and duplicate analysis of a -supplier chemical database totalling . million compounds virtual exploration of the small-molecule chemical universe below daltons lead discovery using molecular docking small molecule docking and scoring high-throughput docking for lead generation docking: successes and challenges high-throughput docking as a source of novel drug leads comparing protein-ligand docking programs is difficult detailed comparison of current docking and scoring methods on systems of pharmaceutical relevance target-based scoring approaches and expert systems in structure-based virtual screening successful virtual screening for novel inhibitors of human carbonic anhydrase: strategy and experimental confirmation virtual screening for submicromolar leads of trna-guanine transglycosylase based on a new unexpected binding mode detected by crystal structure analysis pharmacophore modeling and three-dimensional database searching for drug design using catalyst feature trees: a new molecular similarity measure based on tree matching development and validation of a genetic algorithm for flexible docking a fast flexible docking method using an incremental construction algorithm glide: a new approach for rapid, accurate docking and scoring. . method and assessment of docking accuracy glide: a new approach for rapid, accurate docking and scoring. . enrichment factors in database screening automated docking using a lamarckian genetic algorithm and an empirical binding free energy function high-throughput docking for lead generation principles of docking: an overview of search algorithms and a guide to scoring functions molecular recognition and docking algorithms comparison of automated docking programs as virtual screening tools binding site characteristics in structurebased virtual screening: evaluation of current docking tools comparative evaluation of eight docking tools for docking and virtual screening accuracy a detailed comparison of current docking and scoring methods on systems of pharmaceutical relevance pharmacophore-based molecular docking to account for ligand flexibility virtual screening with solvation and ligandinduced complementarity the particle concept: placing discrete water molecules during protein-ligand docking predictions modeling water molecules in protein-ligand docking using gold flexible docking under pharmacophore type constraints similarity-driven flexible ligand docking automated docking to multiple target structures: incorporation of protein mobility and structural water heterogeneity in autodock docking into knowledge-based potential fields: a comparative evaluation of drugscore drugscore meets comfa: adaptation of fields for molecular comparison (afmoc) or how to tailor knowledge-based pair-potentials to a particular protein improving binding mode predictions by docking into protein-specifically adapted potential fields comparative docking studies on ligand binding to the multispecific antibodies ige-la and ige-lb detailed analysis of scoring functions for virtual screening comparative evaluation of scoring functions for molecular docking assessing scoring functions for protein-ligand interactions consensus scoring: a method for obtaining improved hit rates from docking databases of three-dimensional structures into proteins how does consensus scoring work for virtual library screening? an idealized computer experiment large-scale validation of a quantum mechanics based scoring function: predicting the binding affinity and the binding mode of a diverse set of protein-ligand complexes evaluation of the casp docking section chris lipinski discusses life and chemistry after the rule of five drugscore(csd)-knowledge-based scoring function derived from small molecule crystal data with superior recognition rate of nearnative ligand poses and better affinity prediction structural parameterization of the binding enthalpy of small ligands library design based on privileged scaffolds through docking and direct design in the protein binding pocket. abstracts of papers decoys for docking crystallographic study of inhibitors of trna-guanine transglycosylase suggests a new structure-based pharmacophore for virtual screening you can access endeavour online on sciencedirect, where you'll find book reviews, editorial comment and a collection of beautifully illustrated articles on the history of science. featuring: waxworks and the performance of anatomy in mid-eighteenth-century italy by l. dacome representing revolution: icons of industrialization by p. fara myths about moths: a study in contrasts by d.w. rudge the origins of research into the origins of life by i. fry in search of the sea monster by the author is grateful to current and former members of his research group for their joint efforts in developing tools and strategies for virtual screening and in performing the experimental proof of concept. this article was written during a very stimulating sabbatical at the university of california, san francisco, usa, and many discussions with the author's host, brian shoichet, and his colleagues and research group are kindly acknowledged. endless bus rides on muni, san francisco, are appreciated for providing the opportunity to do the required reading for this review. the livingstone story and the industrial revolution by l. dritsas endeavour is available on sciencedirect, www.sciencedirect.com key: cord- -qah pk authors: ekins, s; mestres, j; testa, b title: in silico pharmacology for drug discovery: applications to targets and beyond date: - - journal: british journal of pharmacology doi: . /sj.bjp. sha: doc_id: cord_uid: qah pk computational (in silico) methods have been developed and widely applied to pharmacology hypothesis development and testing. these in silico methods include databases, quantitative structure-activity relationships, similarity searching, pharmacophores, homology models and other molecular modeling, machine learning, data mining, network analysis tools and data analysis tools that use a computer. such methods have seen frequent use in the discovery and optimization of novel molecules with affinity to a target, the clarification of absorption, distribution, metabolism, excretion and toxicity properties as well as physicochemical characterization. the first part of this review discussed the methods that have been used for virtual ligand and target-based screening and profiling to predict biological activity. the aim of this second part of the review is to illustrate some of the varied applications of in silico methods for pharmacology in terms of the targets addressed. we will also discuss some of the advantages and disadvantages of in silico methods with respect to in vitro and in vivo methods for pharmacology research. our conclusion is that the in silico pharmacology paradigm is ongoing and presents a rich array of opportunities that will assist in expediating the discovery of new targets, and ultimately lead to compounds with predicted biological activity for these novel targets. the first part of this review (ekins et al., ) has briefly described the history and development of a field that can be globally referred to as in silico pharmacology. this included the development of methods and databases, quantitative structure-activity relationships (qsars), similarity searching, pharmacophores, homology models and other molecular modelling, machine learning, data mining, network analysis and data analysis tools that all use a computer. we have also previously introduced how some of these methods can be used for virtual ligand-and target-based screening and virtual affinity profiling. in this second part of the review, we will greatly expand on the applications of these methods to many different target proteins and complex properties, and discuss the pharmacological space covered by some of these in silico efforts. in the process, we will detail the success of in silico methods at identifying new pharmacologically active molecules for many targets and highlight the resulting enrichment factors when screening active druglike databases. we will finally discuss some of the advantages and disadvantages of in silico methods with respect to in vitro and in vivo methods for pharmacology research. the applicability of computational approaches to ligand and target space in which a lead molecule against one gene family member is used for another similar target (termed chemogenomics) (morphy et al., ; sharom et al., ) , will be discussed thoroughly in an upcoming review in this journal from didier rognan (personal communication) and will be only briefly addressed here. however, there have been several attempts to establish relationships between molecular structure and broad biological activity and effects that should be considered (see also section . . in ekins et al. ( ) ) (kauvar et al., (kauvar et al., , b kauvar and laborde, a) . for example, the work of fliri et al. ( b) presented the biological spectra for a cross-section of the proteome. using hierarchical clustering of the spectra similarity enabled a relationship between structure and bioactivity to be constructed. this work was extended to identify agonist and antagonist profiles at various receptors, correctly classifying similar functional activity in the absence of drug target information (fliri et al., c) . interestingly, using ic data as affinity fingerprints did not identify functional activity similarities between molecules as this approach was suggested to introduce a pharmacophoric bias (fliri et al., c) . a similar probabilistic approach has also been applied by the same authors to link adverse effects for drugs (obtained from the drug labelling information) with biological spectra. for instance, clustering molecules by side effect profile showed that similar molecules had overlapping profiles, in the same way that they had similar biological spectra, linking preclinical with clinical effects (fliri et al., a) . this work offers the intriguing possibility of predicting a biospectra profile, possible functional activity and a side effect profile for a new molecule based on similarity alone. however, confidence in this approach would be greatly enhanced by further prospective testing with a large test set of drug-like molecules not used to generate the underlying signature database. a second group also from pfizer presented a global mapping of pharmacological space and in particular focused on a polypharmacology network of molecules with activity against multiple proteins (paolini et al., ) . they have additionally generated bayesian binary models (for molecules active at o mm or inactive) for targets using over molecules with biological data (from their in-house collection and the literature), suggesting that they would be useful for predicting primary pharmacology. assessment of approved oral drugs in two-dimensional ( d) molecular property space (molecular weight versus clogp) showed that many of them had clogp and mw . in spite of this, their associated targets were potentially druggable but had yet to realize their potential (paolini et al., ) . perhaps this work needs to be combined with that of fliri and others for its true potential to be realized, to enable simultaneous understanding and prediction of target, proteomic, functional activity and side effects. a recent analysis using molecular d descriptors followed by principal component (pca) of over anticancer molecules representing cancer medicinal chemistry space, showed that they populated a different space broader than hit-like space and orally available drug-like space. this would indicate that in order to find molecules for anticancer targets in commercially available databases, different rules are required other than those widely used for drug-likeness, as they may unfortunately filter out possible clinical candidates (lloyd et al., ) . methods to predict the potential biological targets for molecules from just chemical structure have been attempted by using different approaches to those already described above. for example, one study used probabilistic neural networks with atom-type descriptors to classify molecules from the mdl drug data reports (mddr) database with activity against one of the seven targets (g protein-coupled receptors (gpcrs), kinases, enzymes, nuclear hormone receptors and zinc peptidases) with excellent training, testing and prediction statistics (niwa, ) . twenty-one targets related to depression were selected and molecules from the mddr database were used to create support vector machine (svm) classification models from atom-type descriptors (lepp et al., ) . these models had satisfactory predictions and recall values between and %, the molecules recovered being on average of low molecular weight (o ) and some were active against more than one model. it was suggested that general svm filters would be useful for virtual screening owing to their speed. others have used similarity searching of the mddr database against small numbers of reference inhibitors for several different targets and were able to show variable enrichment factors that were greater than random (hert et al., ) . the structure-based alternative to understanding small molecule-protein interactions is to flexibly dock molecules into multiple proteins. a representative of this inverse docking approach is invdock, which was recently applied for identifying potential adverse reactions using a database of proteins related to toxicities (dart). this method has been recently demonstrated with marketed anti-hiv drugs resulting in reasonable accuracy against the dna polymerase beta and dna topoisomerase i (ji et al., ) . the public availability of data on drugs and drug-like molecules may make the analyses described above possible for scientists outside the private sector. for example, chemical repositories such as drugbank (http://redpoll. pharmacy.ualberta.ca/drugbank/) (wishart et al., ) , pubchem (http://pubchem.ncbi.nlm.nih.gov/), kidb (http:// kidb.bioc.cwru.edu/) (roth et al., ; strachan et al., ) and others consist of a wealth of target and small molecule data that can be mined and used for computational pharmacology approaches. although much of the in silico pharmacology research to date has been focused on human targets, many of these databases contain data from other species that would also be useful for understanding species differences and promoting discovery of molecules for animal healthcare as well as assisting in understanding the significance of toxicological findings for chemicals released into the environment. to exhaustively describe all of the proteins that have been computationally modelled under the auspices of in silico pharmacology would be impossible in the confines of this review. therefore, we will briefly overview the types of proteins that have been modelled and the methods used (see below and table ). in addition, we will focus on and describe particular pharmacological applications with regard to virtual screening where novel ligands have been identified. the reader is highly encouraged to study an extensive review of success stories in computer-aided design, which covers a large number of proteins that have been targets for all manner of in silico methods (kubinyi, ) , as well as other reviews that have dealt with the successes of individual methods (fujita, ; kurogi and guner, a; guner et al., ) . as described previously, computational approaches for drug discovery and development may have more impact if integrated (swaan and ekins, ) and we have previously attempted to show that computational methods have been broadly applied to virtually all important proteins in absorption, distribution, metabolism, excretion and toxicity (adme/tox) (ekins and swaan, b) . the qaim of this paper is to provide an up-to-date review of all proteins and protein families addressed through current state-of-the-art in silico pharmacology methods. drug target examples. enzymes: the ubiquitin regulatory pathway, in which ubiquitin is conjugated and deconjugated with substrate proteins, represents a source of many potential targets for modulation of cancer and other diseases santo et al. ( ) abbreviations: ampa, a-amino- -hydroxy- -methyl- -isoxazole propionate; cox, cyclooxygenase; cyp, cytochrome p ; hiv- , human immunodeficiency virus; lox, lipoxygenase. in silico pharmacology for drug discovery s ekins et al (wong et al., ) . the recent crystal structure of a mammalian de-ubiquitinating enzyme hausp, which specifically de-ubiquitinates the ubiquitinated p protein, may also assist in drug development despite the peptidic nature of its substrate (hu et al., ) . novel non-peptidic inhibitors of the protease ubiquitin isopeptidase, which not only de-ubiquitinates p but other general ubiquitinated proteins as well, were discovered recently using a simple pharmacophore-based search of the national cancer institute (nci) database (mullally et al., ; mullally and fitzpatrick, ) . these inhibitors had ic values in the low micromolar range and caused cell death independent of the tumour suppressor p , which is mutated in greater than % of all cancers (hence, p inhibition per se may not represent an optimal target for modulation). the ubiquitin isopeptidase inhibitors shikoccin, dibenzylideneacetone, curcumin and the more recently described punaglandins from coral indicate that a sterically accessible a,b-unsaturated ketone is essential for bioactivity (verbitski et al., ) . all these molecules represent valuable leads for further chemical optimization. aromatase (cytochrome p (cyp) ) is a validated target for breast cancer. a ligand-based pharmacophore was generated with three non-steroidal inhibitors. this model could recognize known inhibitors from an in-house library and was further refined by the addition of molecular shape. the model was further used to search the nci database and molecules were scored with a quantitative catalyst hypo-refine (accelrys inc., san diego, ca, usa) model generated with molecules. the hits were also filtered with other pharmacophores for toxicity-related proteins, before testing. two out of the three compounds were ultimately found to be micromolar inhibitors (schuster et al., ) . a structure-based catalyst pharmacophore was developed for acetylcholine esterase, which was subsequently used to search a natural product database. the strategy identified scopoletin and scopolin as hits and were later shown to have moderate in vivo activity (rollinger et al., ) . the same database was also screened against cyclooxygenase (cox)- and cox- structure-based pharmacophores, leading to the identification of known cox inhibitors. these represent examples where a combination of ethnopharmacological and computational approaches may aid drug discovery (rollinger et al., ) . a combined ligand-based and structure-based approach was taken to gaining structural insights into the human -lipoxygenase (lox). a catalyst qualitative hiphop model was created with different molecules that resulted in a five-feature pharmacophore. a homology model of the enzyme was based on two soybean lox enzymes and one rabbit lox enzyme. molecular docking was then used to update and refine the pharmacophore to a four-feature model that could also be visualized in the homology model of -lox. as a result of these models, amino-acid residues in the binding site were suggested as targets for site-directed mutagenesis while virtual screening with the pharmacophore had suggested compounds with a phenylthiourea or pyrimidine- -carboxylate group for testing (charlier et al., ) . homology models for the human -lox and -lox have also been used with the flexible ligand docking programme glide (schrödinger inc.) to perform virtual screening of compounds. out of compounds tested, had inhibitory activity and several were in the low micromolar range (kenyon et al., ) . more than years of research on renin have not been enough to deliver a marketed drug that inhibits this enzyme. in spite of this, renin remains an attractive yet elusive target for hypertension (fisher and hollenberg, ; stanton, ) . in this respect, application of structure-based design leads to the identification of new non-peptidic inhibitors of human renin. these molecules include aliskiren (rahuel et al., ; torres et al., ) , piperidines, including ro- oefner et al., ; vieira et al., ) , and related , -disubstituted piperidines (marki et al., ) . interestingly, these piperidines bind to and stabilize a different conformer of the protein termed 'open renin' (bursavich and rich, ) , whereas aliskiren binds to 'closed renin'. since these latter structure-based design efforts, there have been remarkably very few published attempts at computer-aided design of novel renin inhibitors. a single early qsar was derived for a series of chainmodified peptide analogues of angiotensinogen. the activity of these molecules was found to correlate with kier's firstorder molecular connectivity index descriptor and molecular weight but not with lipophilicity as measured by logp (khadikar et al., ) . another computational method for renin drug discovery used the de novo design software growmol, which could apparently regenerate , -disubstituted piperidines in % of the grown structures (bursavich and rich, ). an attempt to use a catalyst pharmacophore to discover new renin inhibitors was described in the early s (van drie, ) . several novel molecules from the pomona database (an early three-dimensional ( d) molecule database) were found that mapped to a renin pharmacophore but apparently were not tested in vitro. more recently, a ligandfit docking study with a crystal structure of the 'open renin' form was able to detect known inhibitors seeded in a library of compounds within the top . % when using a consensus scoring function. four examples of high-scoring compounds that were not tested as inhibitors fulfilled the pharmacophore derived from the x-ray data, consisting of four hydrophobes, a hydrogen bond donor or positive ionizable feature as well as excluded volumes (krovat and langer, ) . another study has used similarity searching of the mddr database (for over compounds) using renin inhibitors and was able to produce enrichment factors that were -fold greater than random (hert et al., ) . genetic algorithms have also been used for class discrimination between renin inhibitors and noninhibitors in a subset of the mddr using a small number of interpretable descriptors. among them, amide bond count, molecular weight and hydrogen bond donor counts were found to be much higher in renin inhibitors (ganguly et al., ) . the recent publications on novel renin inhibitors represent a considerable amount of new information that could be used for further qsar model development and database searching efforts in order to derive novel starting scaffolds for optimization. cathepsin d is an aspartic protease found mainly in lysosomes, which may have a role in b-amyloid precursor protein release and hence may well be a target for in silico pharmacology for drug discovery s ekins et al alzheimer's disease. cathepsin d may also be elevated in breast cancer and ovarian cancer hence a means to modulate this activity could be beneficial in these diseases. there has been a brief overview of cathepsin d in a comprehensive review of protease inhibitors (leung et al., ) . a combination of a structure-based design algorithm and combinatorial chemistry has been successfully applied to finding novel molecules for cathepsin d in the nanomolar range (kick et al., ) . structures based on pepstatin (a . pm inhibitor (baldwin et al., ) ) yielded a - % hit rate. these molecules were tested in vitro using hippocampal slices and were shown to block the formation of hyperphosphorylated tau fragments (bi et al., ) . there have been relatively few computational studies to date on cathepsin d and other related aspartic proteases such as renin and b-secretase. one study has used molecular dynamics and free energy analyses (mm-pbsa) of cathepsin d inhibitor interactions to suggest new substitutions that may improve binding (huo et al., ) . a genetic algorithmbased de novo design tool, adapt has also been used to rediscover active cathepsin d molecules, by placing key fragments in the correct positions (pegg et al., ) . computational models may aid in the selection of novel ligands for protease inhibition that are non-peptidic and selective. using the structural features of eight published inhibitors for cathepsin d (huo et al., ) , a five-feature pharmacophore was derived consisting of three hydrophobes and two hydrogen bond acceptors (r ¼ . ). this pharmacophore was used to search a molecule database and selected molecules out of present. in contrast, a similarity search at the % level using chemfinder (cambridgesoft, cambridge, ma, usa) suggested different molecules. all of these were selected for testing in vitro. the pharmacophore produced four hits ( % hit rate) and the similarity search generated five hits ( % hit rate), where at least one replicate showed greater than % inhibition (ekins et al., a) . in silico evaluation of the adme properties for all active compounds estimated that the molecules would be well absorbed, although some were predicted to have solubility and cyp d inhibition problems. pharmacophore-and structure-based approaches have been used to optimize an acyl urea hit for human glycogen phosphorylase. a catalyst hypogen five-feature pharmacophore was developed and used to guide further analogue synthesis. these compounds showed a good correlation with prediction (r ¼ . ). an x-ray structure for one molecule was used to confirm the predicted binding conformation. ultimately, a comparative molecular field analysis (comfa) model was generated with all molecules synthesized and was found to be complementary to the x-ray structure. the outcome of this study was a molecule with good cellular activity that could inhibit blood glucose levels in vivo in rat (klabunde et al., ) . the human sirtuin type , a target for controlling aging and some cancers, deacetylates a-tubulin and has been crystallized at high resolution. this structure has been used for docking the maybridge database and returned a small hit list from which compounds were tested and showed activity at the micromolar level (tervo et al., ) . catechol o-methyltransferase is a target for parkinson's disease and there is currently a crystal structure of the enzyme that has been used to generate a homology model of the human enzyme. this model was used to dock with flexx software several catechins from tea and understand the structure-activity relationship (sar) for these molecules and their metabolites, which had been tested in vitro. ultimately, the combination of in vitro and computational work indicated that the galloyl group on catechins, the distance between lys on the enzyme, and the reacting catecholic hydroxy group were important for inhibition . kinases: the kinases represent an attractive family of over targets for the pharmaceutical industry, with several drugs approved recently. kinase space has been mapped using selectivity data for small molecules to create a chemogenomic dendrogram for kinases that showed the highly homologous kinases to be inhibited similarly by small molecules (vieth et al., ) . virtual screening methods have been applied quite widely for kinases to date (fischer, ) . the structure-based design method has produced new potent inhibitors of cdk starting from the highly similar apo cdk and the positioning of olomoucine. a few aminoacid residues were mutated to conform to the cdk sequence. macromodel was used to energy minimize molecules in the atp pocket and visual inspection suggested points for molecular modification on the ligand. very quickly, design efforts guided ligand optimization to improve activity from . mm to nm (furet et al., ) . a more recent cdk /cyclin b homology model was also used to manually dock ligands, which enabled progression from alsterpaullone with an ic of nm to a derivative with an ic of . nm (kunick et al., ) . a structure-based in silico screening method was pursued for the syk c-terminal sh domain using dock to find low molecular weight fragments for each binding site with millimolar binding affinity. the fragments were then linked to result in molecules in the - mm range, which is a starting point for further lead optimization (niimi et al., ) . a pseudoreceptor model was built with a set of epidermal growth factor receptor (egfr) tyrosine kinase inhibitors with the flexible atom receptor model method. the top models created had high r and q and were also validated with a six-molecule test set. the pseudoreceptor was also in accord with a crystal structure of cdk (peng et al., ) . virtual screening using dock with the crystal structure of the lck sh domain was used to screen two million commercially available molecules. extensive filtering was required to result in a manageable hit list using molecular weight and diversity. out of compounds tested in vitro, were inhibitory at mm, while had activities of and mm. fluorescence titrations of some of these compounds suggested the k d values were in the low micromolar range (huang et al., ) . the same group also took a similar approach to discover inhibitors of erk by screening compounds computationally and testing in vitro of them (hancock et al., ) . five of these molecules inhibited cell in silico pharmacology for drug discovery s ekins et al proliferation and two were shown by fluorescence titration to bind erk with k d values, which were in the low micromolar range. in both cases, docking of the active molecules suggested orientations for verification by x-ray crystallography (hancock et al., ) . the ligand scout method was used with bcr-abl tyrosine kinase to find sti- (imatinib, gleevec) in a single and multiple conformation database (wolber and langer, ) . a structurally related three-substituted benzamidine derivative of sti- was suggested by structure-based design and when manually docked into the binding site and energy minimized, it was shown to form favourable interactions with a hydrophobic pocket. ck and pkd are part of the cop signalosome and can control stability of p and c-jun, which are important for tumour development. curcumin, besides being an inhibitor of ubiquitin isopeptidase (mullally et al., ; mullally and fitzpatrick, ) and activator protein- (tsuchida et al., ) , also inhibits ck and pkd. using curcumin and emodin as reference structures against which a database of over a million molecules was screened by means of d and d similarity searches retrieved molecules. among them, seven possessed inhibitory activity. for example, piceatannol was more potent than curcumin against both ck and pkd, with ic values of . and . mm, respectively (fullbeck et al., ) . obviously, these examples suggest there has been some success in finding active molecules for kinases, but interestingly in few of these studies is selectivity toward other kinases accounted for. ultimately, for therapeutic success activity toward several kinases (but selectivity toward others) may be required. drug-metabolizing enzymes and transporters: mathematical models describing quantitative structure-metabolism relationships were pioneered by hansch et al. ( ) using small sets of similar molecules and a few molecular descriptors. later, lewis and co-workers provided many qsar and homology models for the individual human cyps (lewis, ) . as more sophisticated computational modelling tools became available, we have seen a growth in the number of available models (de groot and ekins, b; de graaf et al., ; de groot, ) and the size of the data sets they encompass. some more recent methods are also incorporating water molecules into the binding sites when docking molecules into these enzymes and these may be important as hydrogen bond mediators with the binding site amino acids (lill et al., ) . docking methods can also be useful for suggesting novel metabolites for drugs. a recent example used a homology model of cyp d and docked metoclopramide as well as other drugs to show a good correlation between ic and docking score r ¼ . . a novel aromatic n-hydroxy metabolite was suggested as the major metabolite and confirmed in vitro. now that several crystal structures of the mammalian cyps are available, they have been found to compare quite favourably to the prior computational models (rowland et al., ) . however, for some enzymes like cyp a , where there is both ligand and protein promiscuity, there may be difficulty in making reliable predictions with some computational approaches such as docking with the available crystal structures (ekroos and sjogren, ) . hence, multiple pharmacophores or models may be necessary for this and other enzymes (ekins et al., a, b) , as it has been indicated by others more recently (mao et al., ) . the udp-glucuronosyltransferases are a class of versatile enzymes involved in the elimination of drugs by catalysing the conjugation of glucuronic acid to substrates bearing a suitable functional group, so called phase ii enzymes. there have been numerous qsar and pharmacophore models that have been generated with relatively small data sets for rat and human enzymes. the pharmacophores for the human ugt a , ugt a and ugt a all have in common two hydrophobes and a glucuronidation feature, while ugt a has an additional hydrogen bond acceptor feature sorich et al., ) . sulfotransferases, a second class of conjugating enzymes, have been crystallized (dajani et al., ; gamage et al., ) and a qsar method has also been used to predict substrate affinity to sult a (dajani et al., ) . to the best of our knowledge, computational models for other isozymes have not been developed. in general, conjugating enzymes have generally been infrequently targeted for in silico models. perhaps because of a paucity of in vitro data and limited diversity of molecules tested, they have been less widely applied in industry. the computational modelling of drug transporters has been thoroughly reviewed by numerous groups (zhang et al., a, b; chang and swaan, ) and will not be addressed here in detail. various transporter models have also been applied to database searching to discover substrates and inhibitors pleban et al., ; chang et al., b) and increase the efficiency of in vitro screening (chang et al., a) or enrichment over random screening. a pharmacophore model of the na þ /d-glucose co-transporter found in renal proximal tubules was derived indirectly using phlorizin analogues with the disco programme to superpose molecules. this enabled an estimate of the size of the binding site to be obtained. in contrast to more recent studies with transporter pharmacophores, this model was not tested or used for database searching (wielert-badt et al., ) . receptors: there are more than different families of receptors that are present in the plasma membrane, altogether representing over proteins of the receptorome (strachan et al., ) . receptors have been widely used as drug targets and they have a wide array of potential ligands. however, it should be noted that to date we have only characterized and found agonists and antagonists for a small percentage of the receptorome. the a-amino- -hydroxy- -methyl- -isoxazole propionate receptor is central to many central nervous system (cns) pathologies and ligands have been synthesized as anticonvulsants and neuroprotectants. there is currently no d structure information and therefore a four-point catalyst hiphop pharmacophore was developed with antagonists. this was then used to search the maybridge database and select eight compounds for testing of which six of these were found to be active in vivo as anticonvulsants (barreca et al., ) . serotonin plays a role in many physiological systems, from the cns to the intestinal wall. along with its many receptors, it has a major developmental function regulating cardiovascular morphogenesis. the -ht receptor family are g protein-coupled -transmembrane spanning receptors with -ht b expressed in cardiovascular, gut, brain tissues, as well as human carcinoid tumors (nebigil et al., ) . in recent years, this receptor has been implicated in the valvular heart disease defects caused by the now banned 'fen-phen' treatment of patients. the primary metabolite, norfenfluramine, potently stimulates -ht b (fitzgerald et al., ; rothman et al., ) . computational modelling of this receptor has been limited to date. a traditional qsar study used a small number of tetrahydro-b-carboline derivatives as antagonists of the rat -ht b contractile receptor in the rat stomach fundus (singh and kumar, ) . a d-qsar with grid-golpe using (aminoalkyl)benzo and heterocycloalkanones as antagonists of the human receptor resulted in very poor model statistics, possibly owing to the limited range of activity measured and the fact that the data corresponded to a functional response that is likely more complex (brea et al., ) . neither of these models was validated with external predictions. on the basis of bacteriorhodopsin and rhodopsin, homology models for the mouse and human -ht b receptor have been combined with site-directed mutagenesis. the bacteriorhodopsin structure provided more reliable models, which confirmed an aromatic box hypothesis for ligand interaction along transmembrane domains , , with serotonin (manivet et al., ) . a more recent -ht b homology model based on the rhodopsin-based model of the rat -ht a was used to determine the sites of interaction for norfenfluramine following molecular dynamics simulations. site-directed mutagenesis showed that val . was implicated in highaffinity binding through van der waals interactions and the ligand methyl groups (setola et al., ) . there is certainly an opportunity to develop further qsar models for this receptor in order to rapidly screen libraries of molecules to identify undesirable potent inhibitors. the serotonin -ht a receptor has been frequently modelled. for example, a conformational study of four ligands defined a pharmacophore of the antagonist site using sybyl (hibert et al., ) . the model resulting from such an active analogue approach was used in molecule design and predicted molecule stereospecificity. more recently, a series of over homology models were iteratively created based on the crystal structure of the bovine rhodopsin that were in turn tuned by flexx docking of known ligands. the final model was used in a virtual screening simulation that was enriched with inhibitors, compared with random selection and from this the authors suggested its utility for a real virtual screen (nowak et al., ) . a homology model of the -ht a receptor has also been used with dock to screen a library of compounds seeded with -ht a ligands. ninety percent of these active compounds were ranked in the top compounds (becker et al., ) , representing a significant enrichment. the same model was used to screen a library of vendor compounds and select for testing, of which had activities below mm, one possessing nm affinity. structure-based in silico optimization was then performed to improve selectivity with other gpcrs and optimize the pharmacokinetic (pk) profile. however, as this proceeded, the molecules were found to have affinity for the human ether a-go-go-related gene (herg), and this was subsequently computationally assessed using a homology model that pointed to adjusting the hydrophobicity. the resulting clinical candidate had good target and antitarget selectivity and backup compounds were selected in the same way (becker et al., ) . another early computer-aided pharmacophore generated with sybyl using a set of selective and non-selective analogues was used to design agonists for -ht d as antimigraine agents with selectivity against -ht a (linked to undesirable changes in blood pressure) (glen et al., ) . a range of typical and atypical antipsychotics bind to the -ht receptor. based on the structure of bovine rhodopsin, homology models of the human and rodent -ht receptors were constructed and used to dock ligands that were known to exhibit species differences in binding (hirst et al., ) . following sequence alignment, amino-acid residues were identified for mutation and the rationalization of these mutations and their effects on ligand binding were obtained from the docking studies. the models generated were in good agreement with the in vitro data and could be used for further molecule design. this study was a good example where computational, molecular biology and traditional pharmacology methods were combined (hirst et al., ) . the na þ , k þ -atpase is a receptor for cardiotonic steroids, which in turn inhibit the atpase and cation transport and have ionotropic actions. although the effects of digitalis have been known for hundreds of years, a molecular understanding has remained absent until recently. a homology model was generated with the serca a crystal structure and tested with nine cardiac glycosides (keenan et al., ) . the model was also mutated to mimic the rat receptor and showed how oubain would orient differently in these models, perhaps explaining the species difference in affinity. these models also suggested amino acids that could be experimentally mutated to validate the hypothesis for the binding site identification, although this has yet to be tested. the dopamine receptors have been implicated in parkinson's disease and schizophrenia. unfortunately, no crystal structure is currently available and thus the search for new antagonists has used qsar models. a set of compounds was used with four different qsar methods (comfa, simulated annealing-partial least square (pls), k-nearest neighbours (knn) and svm), and training as well as testing statistics were generated. svm and knn models were also used to mine compound databases of over molecules that resulted in consensus hits. five of these hits were known to bind the receptor and were not in the training set, while other suggested hits did not contain the catechol group normally seen in most dopamine inhibitors (oloff et al., ) . the a a receptor is a target for controlling vascular tone and therefore useful for antihypertensive agents. a novel approach for ligand-based screening called multiple feature tree (mtree) describes the training set molecules as a feature tree descriptor derived from a topological molecular graph that is then aligned in a pairwise fashion (hessler et al., ) . a set of six antagonists was used to derive a model with this method and was compared with a catalyst pharmacophore model. both approaches identified a central positive ionizable feature flanked by hydrophobic regions at either end. these two methods were compared for their ability to rank a database of over molecules. within the top % of the database, mtree had an enrichment factor that was over twice that obtained with catalyst (hessler et al., ) . nuclear receptors: nuclear receptors constitute a family of ligand-activated transcription factors of paramount importance for the pharmaceutical industry since many of its members are often considered as double-edged swords (shi, ) . on the one hand, because of their important regulatory role in a variety of biological processes, mutations in nuclear receptors are associated with many common human diseases such as cancer, diabetes and osteoporosis and thus, they are also considered highly relevant therapeutic targets. on the other hand, nuclear receptors act also as regulators of some the cyp enzymes responsible for the metabolism of pharmaceutically relevant molecules, as well as transporters that can mediate drug efflux, and thus they are also regarded as potential therapeutic antitargets (off-targets). examples of the use of target-based virtual screening to identify novel small molecule modulators of nuclear receptors have been recently reported. using the available structure of the oestrogen receptor subtype a (era) in its antagonist conformation, a homology model of the retinoic acid receptor a (rara) was constructed (schapira et al., ) . using this homology model, virtual screening of a compound library lead to the identification of two novel rara antagonists in the micromolar range. the same approach was later applied to discover novel and diverse micromolar antagonists of the thyroid hormone receptor (schapira et al., ) . by means of a procedure designed particularly to select compounds fitting onto the lxxll peptide-binding surface of the oestrogen receptor, novel era antagonists were identified (shao et al., ) . since poor displacement of b-estradiol was observed in the er-ligand competition assay, these compounds may represent new classes of era antagonists, with the potential to provide an alternative to current anti-oestrogen therapies. the discovery of three low micromolar hits for erb displaying over -fold binding selectivity with respect to era was also recently reported using database screening (zhao and brinton, ) . a final example reports the identification and optimization of a novel family of peroxisome proliferator-activated receptors-g partial agonists based upon pyrazol- -ylbenzenesulfonamide after employing structure-based virtual screening, with good selectivity profile against the other subtypes of the same nuclear receptor group . ion channels: therapeutically important channels include voltage-gated ion channels for potassium, sodium and calcium that are present in the outer membrane of many different cells such as those responsible for the electrical excitability and signalling in nerve and muscle cells (terlau and stuhmer, ) . these represent validated therapeutic targets for anaesthesia, cns and cardiovascular diseases (kang et al., ) . a recent review has discussed the various qsar methods such as pharmacophores, comfa, svm, d-qsar, genetic programming, self organizing maps and recursive partitioning that have been applied to most ion channels (aronov et al., ) in the absence of crystal structures. to date l-type calcium channels and herg appear to have been the most extensively studied channels in this regard. in contrast, there are far fewer examples of computational models for the sodium channel. these three classes of ion channels have been studied as they represent either therapeutic targets or antitargets to be avoided. for example, one of many models for the herg potassium channel has compared three different methods with the same set of molecules for training and a test set. recursive partitioning, sammon maps and kohonen maps were used with atom path lengths . the average classification quality was high for both training and test selections. the sammon mapping technique outperformed the kohonen maps in classification of compounds from the external test set. the quantitative predictions for recursive partitioning could be filtered using a tanimoto similarity to remove molecules that were markedly different to the training set (willett, ) . the path length descriptors can also be used to visualize the similarity of the molecules in the whole training set (figure a ). in addition, a subset of molecules can also be compared, with those highlighted in blue representing close neighbours and those in red being more distant (figure b) . transcription factors: a cyclic decapeptide with activity against the ap- transcription factor was used to derive a d pharmacophore to which low energy conformations of non-peptidic compounds were compared. new -thia- azaspiro [ , ] decane and benzophenone derivatives with activity in binding and cell-based assays were discovered as ap- inhibitors in a lead hopping approach (tsuchida et al., ) . antibacterials: twenty deoxythymidine monophosphate analogues were used along with docking to generate a pharmacophore for mycobacterium tuberculosis thymidine monophosphosphate kinase inhibitors with the catalyst software. a final model was used to screen a large database spiked with known inhibitors. the model was suggested to have an enrichment factor of , which is highly significant. in addition, the model was used to rapidly screen half a million compounds in an effort to discover new inhibitors (gopalakrishnan et al., ) . antivirals: neuroamidase is a major surface protein in influenza virus. a structure-based approach was used to generate catalyst pharmacophores and these in turn were used for a database search and aided the discovery of known inhibitors. the hit lists were also very selective (steindl and langer, ) . human rhinovirus c protease is an antirhinitis target. a structure-based pharmacophore was developed initially around ag but this proved too restrictive. a second pharmacophore was developed from seven peptidic inhibitors using the catalyst hiphop method. this hypothesis was useful in searching the world drug index database to retrieve in silico pharmacology for drug discovery s ekins et al compounds with known antiviral activity and several novel compounds were selected from other databases with good fits to the pharmacophore, indicative that they would be worth testing although these ultimate testing validation data were not presented (steindl et al., a) . human rhinovirus coat protein is another target for antirhinitis. a combined pharmacophore, docking approach and pca-based clustering was used. a pharmacophore was generated from the structure and shape of a known inhibitor and tested for its ability to find known inhibitors in a database. ultimately, after screening the maybridge database, compounds were suggested that were then docked and scored. six compounds were tested and found to inhibit viral growth. however, the majority of them were found to be cytotoxic or had poor solubility (steindl et al., b) . the ligand scout approach was tested on the rhinovirus serotype and was able to find known inhibitors in the pdb (wolber and langer, ) . the sars coronavirus c-like proteinase has been addressed as a potential drug design target. a homology model was built and chemical databases were docked into it. a pharmacophore model and drug-like rules were used to narrow the hit list. forty compounds were tested and three were found with micromolar activity, the best being calmidazolium at mm (liu et al., ) , perhaps a starting point for further optimization. a pharmacophore has also been developed to predict the hepatitis c virus rna-dependent rna polymerase inhibition of diketo acid derivatives. a catalyst hypogen model was figure (a) a distance matrix plot of the molecule herg training set showing in general that the molecules are globally dissimilar as the plot is primarily red . (b) a distance matrix plot of a subset of the training set to show molecules similar to astemizole. blue represents close molecules and red represents distant molecules based on the chemtree pathlength descriptors (see colour scale). in silico pharmacology for drug discovery s ekins et al derived with molecules with activities over three log orders to result in a five-feature pharmacophore model. this was in turn tested with compounds from the same data set as well as nine diketo acid derivatives, for which the predicted and experimental data were in good agreement (di santo et al., ) . other therapeutic targets: the integrin vla- (a b ) is a target for autoimmune and inflammatory diseases such as asthma and rheumatoid arthritis. the search for antagonists has included using a catalyst pharmacophore derived from the x-ray crystal structure of a peptidic inhibitor (singh et al., b) . this was used to search a virtual database of compounds that could be made with reagents from the available chemicals directory. twelve compounds were then selected and synthesized, with resulting activities in the range between . nm and mm. hence, a peptide was used to derive non-peptide inhibitors that were active in vivo. a second study by the same group used comfa with a set of antagonists with activity from to nm to generate a model with good internal validation statistics that was subsequently used to indicate favourable regions for molecule substituent changes (singh et al., a) . it is unclear whether the comfa model was also successful for design of further molecules. it is possible to use approved drugs as a starting point for drug discovery for other diseases. for example, the list of world health organization essential drugs has been searched to try to find leads for prion diseases using d tanimoto similarity or d searching with known inhibitors. this work to date has suggested compounds, yet they appear not to have been tested, so the approach has not been completely validated (lorenzen et al., ) . protein-protein interactions are key components of cellular signalling cascades, the selective interruption of which would represent a sought after therapeutic mechanism to modulate various diseases (tesmer, ) . however, such pharmacological targets have been difficult for in silico methods to derive small molecule inhibitors owing to generally quite shallow binding sites. the g-protein gbg complex can regulate a number of signalling proteins via protein-protein interactions. the search for small molecules to interfere with the gbg-protein-protein interaction has been targeted using flexx docking and consensus scoring of molecules from the nci diversity set database (bonacci et al., ) . after testing compounds as inhibitors of the gb g -sirk peptide, nine compounds were identified with ic values from nm to mm. further substructure searching was used to identify similar compounds to one of the most potent inhibitors to build a sar. these efforts may eventually lead to more potent lead compounds. up to this point, we have generally considered in silico pharmacology models that essentially relate to a single target protein and either the discovery of molecules as agonists, antagonists or with other biological activity after database searching and in vitro testing or following searching of databases seeded with molecules of known activity for the target. however, there are many complex properties that have been modelled in silico and these will be briefly discussed here. it should also be pointed out that while several physicochemical properties such as clogp and water solubility have been extensively studied, the training sets for these models are in the s or tens of thousands of molecules, while other complex properties have generally used much smaller training sets in the range of hundreds of molecules. for example, a measure of molecule clearance would be indicative of elimination half-life that would naturally be of value for selecting candidates. the intrinsic clearance has therefore been used as a measure of the enzyme activity toward a compound and this may involve multiple enzymes. some of the earliest models for this property includes a comfa model of the cyp-mediated metabolism of chlorinated volatile organic compounds, likely representative of cyp e (waller et al., ) . a more generic set of molecules with clearance data derived from human hepatocytes has been used to predict human in vivo clearance using multiple linear regression, pca, pls, neural networks with leave-oneout cross-validation (schneider et al., ) . microsomal and hepatocyte clearance data sets have also been used separately to generate catalyst pharmacophores, which were then tested by predicting the opposing data set. this method assumes there are some pharmacophore features intrinsic to the molecules that dictate intrinsic clearance (ekins and obach, ) . a second complex property is the volume of distribution that is a function of the extent of drug partitioning into tissue versus plasma and there have been several attempts at modelling this property (lombardo et al., (lombardo et al., , . this property, along with the plasma half-life, determines the appropriate dose of a drug. for example, diverse drugs from the literature were used with eight molecular descriptors with sammon and kohonen mapping methods. these models appeared to classify correctly % of the compounds (balakin et al., ) . recently, a set of drugs with literature volume of distribution at steady-state data was used with a mixture discriminant analysis-random forest method and molecular descriptors to generate a predictive model. this model was tested with molecules, resulting in a geometric mean fold error of . , which was comparable to the values for other predictions for this property from animal, in vitro, or other methods (lombardo et al., ) . a third property, the plasma half-life determined by numerous adme properties has also been modelled with sammon and kohonen maps using data for drugs from the literature and four molecular descriptors. like the previously described volume of distribution models, these models appeared to classify correctly % of the compounds (balakin et al., ) . a fourth complex property is renal clearance, which assumes the excretion of the unchanged drug that takes place only by this route, hence this represents a method of monitoring the proportion of drug metabolized. in one set of published qsar models, molecules were used with volsurf or molconn-z descriptors. the models were tested with molecules and one using soft independent modelling of class analogies and molconn-z descriptors obtained % correct classification between the two classes ( - and - %) (doddareddy et al., ) . a fifth example of a complex property is the proteinligand interaction and appropriate scoring functions for which several methods have been developed such as force fields, empirical and knowledge-based approaches (see also ekins et al., ) . these are important in computational structure-based design methods for assessing virtual candidate molecules to select those that are likely to bind a protein with highest affinity (shimada, ) . recently, a kernel partial least squares (k-plss) qsar approach has been used along with a genetic algorithm feature selection method for the distance-dependent atom pair descriptors from the or small molecule training sets with binding affinity data and the proteins they bind to. bootstrapping, scrambling the data and external test sets were used to test the models (deng et al., ) . in essence, such k-pls qsar models across many proteins perhaps isolate the key molecular descriptors that relate to the highest affinity interactions. it will be interesting to see whether such models can continue to be generated with the much larger binding affinity data sets that are now available. a final example of a complex property is the v max of an enzyme that has been modelled on a few occasions (hirashima et al., ; mager et al., ; ghafourian and rashidi, ; sipila and taskinen, ) . this value will depend on the properties of the compound in question and will be influenced by the steric properties of the active site as well as the ease of expulsion of the leaving group from the active site. balakin et al. ( ) , have recently used neural network methods to model the v max data for n-dealkylation mediated by cyp d and cyp a , using whole molecules, centroid of the reaction and leaving group-related descriptors. these models were also used to predict small sets of molecules not included in training. ultimately, many other reactions and the evaluation of other enzymes will be necessary. similarly, larger test sets are required for all the above complex property models to provide further confidence in the models in terms of their utility and applicability. uses of in silico pharmacology we propose a general schema for in silico pharmacology, which is shown in figure . this demonstrates some of the key roles of the computational technologies that can assist pharmacology. these roles include finding new antagonists or agonists for a target using an array of methods either in the absence or presence of a structure for the target. computational methods may also aid in understanding the underlying biology using network/pathways based on annotated data (signalling cascades), determining the connectivity of drug as a network with targets to understand selectivity, integration with other models for pk/pd (pharmacodynamic) and ultimately the emergence of systems in silico pharmacology. obviously, we have taken more of a pharmaceutical bias in this review but we would argue these methods are equally amenable and should be considered to discover new chemical probes for the academic pharmacologist as opposed to lead molecules for optimization to become drugs. some of the advantages of in silico pharmacology and in silico methods in general are the reduction in the number of molecules made and tested through database searching to find inhibitors or substrates, increased speed of experiments through reliable prediction of most pharmaceutical properties from molecule structure alone and ultimately reductions in animal and reagent use. we must however consider the multiple optimization of numerous predicted properties, possibly either weighting in silico pharmacology models by importance (or confidence in the model and or data), as well as data set size and diversity. similarly, we should consider the disadvantages of in silico pharmacology methods as protein flexibility, molecule conformation and promiscuity all hinder accurate predictions. for example, even with the recent availability of crystal structures for several mammalian drug-metabolizing enzymes, there is still considerable difficulty in reliable metabolism predictions. our focus thus far has been on the creation of many in silico pharmacology models for human properties, yet as pharmacology uses animals for much in vivo testing and subcellular preparations from several species for in vitro experiments, we need models from other species both to understand differences as well as enable better scaling between them. a widely discussed disadvantage of in silico methods is the applicability of the model, which will now be discussed further. defining in silico model applicability domain some of the in silico pharmacology methods that can be used have similar limitations to models used in other areas, such as those for predicting physicochemical and adme/tox properties. for example, models may be generated with a narrow homologous series of pharmacologically relevant molecules (local model) or a structurally diverse range of molecules (global model). these two approaches have their pros and cons, respectively. the applicability domain of the local model may be much narrower than for the global model such that changing to a new chemical series will result in prediction failure. however, global models may also fail if the predicted molecule falls far enough away from representative molecules in the training set. these limitations are particularly specific to qsar models. from many of the in silico pharmacology model examples described above, the qsar models are generally local in nature and this will limit lead hopping to new structural series, whereas global models may be more useful for this feature. several papers have described the applicability domain of models and methods in considerable detail (dimitrov et al., ; tetko et al., ) to calculate this property. molecular similarity to training set compounds may be a reliable measure for prediction quality (sheridan et al., ) as demonstrated for a herg model . to our knowledge, there has not been a specific analysis of the applicability domain specifically for in silico pharmacology models (other than for those examples described above) to the same degree as there has been for physicochemical properties like solubility and logp. the applicability domain of pharmacophore models have not been addressed either as the focus has primarily been on statistical qsar methods. as we shift toward hybrid or meta-computational methods (that integrate several modelling approaches and algorithms) for predicting from molecular structure the possible physicochemical and pharmacological properties, then these could be used to provide prediction confidence by consensus. the docking methods with homology models for certain proteins of pharmacological interest could be used alongside qsar or pharmacophore models if these are also available. there have been numerous occasions in the study of drug-metabolizing enzymes were qsar and homology models have been combined or used to validate each other (de groot et al., a; de graaf et al., ; de groot, ) . drug metabolism is a good example as several simultaneous outcomes (for example, metabolites) often occur, a condition not normally found in other pharmacological assays where a single set of conditions yields a single outcome. it is here that the classification into specific ('local') and comprehensive ('global') methods finds its clearest use (see figure ) , with local methods being applicable to simple biological systems such as a single enzyme or a single enzymatic activity (testa and krämer, ) . the production of regioselective metabolites (for example, hydroxylation to a phenol and an alcohol) is usually predictable from such methods, but that of different routes (for example, oxidation versus glucuronidation) is not. this is where global algorithms (that is, applicable to versatile biological systems) are most useful in their potential capacity to encompass all or most metabolic reactions and offer predictions, which are much closer to the in vivo situation. it is readily apparent that in a minority of papers we have found that computational approaches have resulted in predicted lead compounds for testing without the authors providing further experimental verification of biological activity (krovat and langer, ; langer et al., ; steindl and langer, ; gopalakrishnan et al., ; lorenzen et al., ; steindl et al., a; amin and welsh, ) . this is an interesting observation as for many years computational studies were generally performed after synthesis of molecules, and essentially provided illustrative pictures and explanation of the data. now it appears we are seeing a shift in the other direction as predictions are published for pharmacological activity without apparently requiring in vitro or in vivo experimental verification, as long as the models themselves are validated in some manner. as the models may only have a limited prediction domain so perhaps in future we will see some discussion of the predicted molecules and their distance from the training set or some other measure of how far the predictions can be extended. many of the molecules identified by virtual screening techniques have not been tested in vitro to ensure that they are not false positives that may actually be involved in molecule aggregation. these types of molecules have been termed so-called 'promiscuous inhibitors', occurring as micromolar inhibitors of several proteins mcgovern and shoichet, ; seidler et al., ) . a preliminary computational model was developed to help identify these potential promiscuous inhibitors (seidler et al., ) . from reviewing the literature, we suggest it would be worth researchers either implementing filters for 'promiscuous inhibitors' or performing rigorous experimental verification of their predicted bioactive molecules to rule out this possibility. . it would certainly be very useful to know the existence of difficult targets for modelling with different methods, as this apparently is a process of trial and error for each investigator currently. in summary, in this and the accompanying review (ekins et al., ) , we have presented our interpretation of in silico pharmacology and described how the field has developed so far and is used for: discovery of molecules that bind to many different targets and display bioactivity, prediction of complex properties and the understanding of the underlying metabolic and network interactions. while we have not explicitly discussed pk/pd, whole organ, cell or disease simulations in this review, we recognize they too are an important component of the computer-aided drug design approach (noble and colatsky, ; gomeni et al., ; kansal, ) and may be more widely integrated with other in silico pharmacology methods described previously . the brief history of in silico pharmacology has taken perhaps a rather predictable route with computational models applied to many of the most important biological targets where they have the capacity to be used to search large databases and quickly suggest molecules for testing. many of the examples we have presented have demonstrated significant enrichments over random selection of molecules and so far these have been the most plentiful types of metrics that are routinely used to validate in silico models. the future of in silico pharmacology may be somewhat difficult to predict. while we are seeing a closer interaction between computational and in vitro approaches to date, will we see a similar relationship with in vivo studies in the future? more broadly, will in silico pharmacology ever be able to replace entirely experimental approaches in vitro and even in vivo, as some animal rights activists want us to believe? the answer here can only be a clear and resounding 'no' (at least in the near future), for two irrefutable reasons. first, biological entities are nonlinear systems showing 'chaotic behaviour'. as such, there is no relation between the magnitude of the input and the magnitude of the output, with even the most minuscule differences between initial conditions rapidly translating into major differences in the output. and second, no computer programme, however 'complex and systemslike', will ever be able to fully model the complexity of biological systems. indeed, and in the formulation of the mathematician gregory chaitin, biological systems are algorithmically incompressible, meaning that they cannot be modelled fully by an algorithm shorter than themselves. in the meantime, in silico pharmacology will likely become more complex requiring some degree of integration of models, as we are seeing in the combined metabolism modelling approaches (figure ) . ultimately, to have a much broader impact, the in silico tools will need to become a part of every pharmacologist's tool kit and this will require training in modelling and informatics, alongside the in vivo, in vitro and molecular skills. this should provide a realistic appreciation of what the different in silico methods can and cannot be expected to do with regard to the pharmacologists aim of discovering new therapeutics. in silico pharmacology for drug discovery s ekins et al a preliminary in silico lead series of -phthalimidinoglutaric acid analogues designed as mmp- inhibitors applications of qsar methods to ion channels. in: ekins s (ed) computational toxicology: risk assessment for pharmaceutical and environmental chemicals quantitative structure-metabolism relationship modeling of the metabolic n-dealkylation rates comprehensive computational assessment of adme properties using mapping techniques crystal structures of native and inhibited forms of human cathepsin d: implications for lysosomal targeting and drug design pharmacophore modeling as an efficient tool in the discovery of novel noncompetitive ampa receptor antagonists an integrated in silico d model-driven discovery of a novel, potent, and selective amidosulfonamide -ht a agonist (prx- ) for the treatment of anxiety and depression novel cathepsin d inhibitors block the formation of hyperphosphorylated tau fragments in hippocampus differential targeting of gbetagamma-subunit signaling with small molecules new serotonin -ht( a), -ht( b), and -ht( c) receptor antagonists: synthesis, pharmacology, d-qsar, and molecular modeling of (aminoalkyl)benzo and heterocycloalkanones designing non-peptide peptidomimetics in the st century: inhibitors targeting conformational ensembles developing a dynamic pharmacophore model for hiv- integrase rapid identification of p-glycoprotein substrates and inhibitors pharmacophorebased discovery of ligands for drug transporters computational approaches to modeling drug transporters structural insights into human -lipoxygenase inhibition: combined ligand-based and target-based approach inhibition of human liver catechol-o-methyltransferase by tea catechins and their metabolites: structure-activity relationship and molecular-modeling studies x-ray crystal structure of human dopamine sulfotransferase, sult a cytochrome p in silico: an integrative modeling approach designing better drugs: predicting cytochrome p metabolism development of a combined protein and pharmacophore model for cytochrome p c pharmacophore modeling of cytochromes p generation of predictive pharmacophore models for ccr antagonists: study with piperidine-and piperazine-based compounds as a new class of hiv- entry inhibitors predicting proteinligand binding affinities using novel geometrical descriptors and machine-learning methods simple but highly effective three-dimensional chemical-feature-based pharmacophore model for diketo acid derivatives as hepatitis c virus rna-dependent rna polymerase inhibitors a stepwise approach for defining the applicability domain of sar and qsar models in silico renal clearance model using classical volsurf approach molecular docking and highthroughput screening for novel inhibitors of protein tyrosine phosphatase- b insights for human ether-a-go-go-related gene potassium channel inhibition using recursive partitioning, kohonen and sammon mapping techniques applying computational and in vitro approaches to lead selection three and four dimensional-quantitative structure activity relationship analyses of cyp a inhibitors three dimensional quantitative structure activity relationship ( d-qsar) analysis of cyp a substrates in silico pharmacology for drug discovery: methods for virtual ligand screening and profiling techniques: application of systems biology to absorption, distribution, metabolism, excretion, and toxicity three dimensional-quantitative structure activity relationship computational approaches of prediction of human in vitro intrinsic clearance development of computational models for enzymes, transporters, channels and receptors relevant to adme/tox structural basis for ligand promiscuity in cytochrome p a the design of drug candidate molecules as selective inhibitors of therapeutically relevant protein kinases is there a future for renin inhibitors possible role of valvular serotonin -ht( b) receptors in the cardiopathy associated with fenfluramine analysis of drug-induced effect patterns to link structure and side effects of medicines biological spectra analysis: linking biological activity profiles to molecular structure biospectra analysis: model proteome characterizations for linking molecular structure and biological response identification of nonpeptidic urotensin ii receptor antagonists by virtual screening based on a pharmacophore model derived from structure-activity relationships and nuclear magnetic resonance studies on urotensin ii recent success stories leading to commercializable bioactive compounds with the aid of traditional qsar procedures novel curcumin-and emodin-related compounds identified by in silico d/ d conformer screening induce apoptosis in tumor cells structure-based design of potent cdk inhibitors derived from olomoucine structure of a human carcinogenconverting enzyme, sult a introducing the consensus modeling concept in genetic algorithms: application to interpretable discriminant analysis quantitative study of the structural requirements of phthalazine/quinazoline derivatives for interaction with human liver aldehyde oxidase computer-aided design and synthesis of -substituted tryptamines and their pharmacology at the -ht d receptor: discovery of compounds with potential anti-migraine properties computerassisted drug development (cadd): an emerging technology for designing first-time-in-man and proof-of-concept studies from preclinical experiments a virtual screening approach for thymidine monophosphate kinase inhibitors as antitubercular agents based on docking and pharmacophore models combining structurebased drug design and pharmacophores piperidine-renin inhibitors compounds with improved physicochemical properties pharmacophore modeling and three dimensional database searching for drug design using catalyst: recent advances identification of novel extracellular signal-regulated kinase docking domain inhibitors structure-activity correlations in the metabolism of drugs comparison of fingerprint-based methods for virtual screening using multiple bioactive reference structures multiple-ligand-based virtual screening: methods and applications of the mtree approach graphics computer-aided receptor mapping as a predictive tool for drug design: development of potent, selective, and stereospecific ligands for the -ht a receptor quantitative structure-activity studies of octopaminergic -(arylimino)thiazolidines and oxazolidines against the nervous system of periplaneta americana l differences in the central nervous system distribution and pharmacology of the mouse -hydroxytryptamine- receptor compared with rat and human receptors investigated by radioligand binding, site-directed mutagenesis, and molecular modeling crystal structure of a ubp-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde identification of non-phosphate-containing small molecular weight inhibitors of the tyrosine kinase p lck sh domain via in silico screening against the py þ binding site molecular dynamics and free energy analyses of cathepsin d-inhibitor interactions: insight into structure-based ligand design in silico search of putative adverse drug reaction related proteins as a potential tool for facilitating drug adverse effect prediction identification of novel farnesyl protein transferase inhibitors using three-dimensional searching methods interactions of a series of fluoroquinolone antibacterial drugs with the human cardiac k þ channel herg modeling approaches to type diabetes predicting ligand binding to proteins by affinity fingerprinting the diversity challenge in combinatorial chemistry protein affinity map of chemical space elucidation of the na þ , k þ -atpase digitalis binding site novel human lipoxygenase inhibitors discovered using virtual screening with homology models qsar studies on , -dithiole- -thiones: modeling of lipophilicity, quinone reductase specific activity, and production of growth hormone structure-based design and combinatorial chemistry yield low nanomolar inhibitors of cathepsin d acyl ureas as human liver glycogen phosphorylase inhibitors for the treatment of type diabetes development of novel edg antagonists using a d database search and their structure-activity relationships impact of scoring functions on enrichment in docking-based virtual screening: an application study on renin inhibitors computer applications in pharmaceutical research and development structure-aided optimization of kinase inhibitors derived from alsterpaullone pharmacophore modeling and threedimensional database searching for drug design using catalyst discovery of novel mesangial cell proliferation three-dimensional database searching method lead identification for modulators of multidrug resistance based on in silico screening with a pharmacophoric feature model screening for new antidepressant leads of multiple activities by support vector machines protease inhibitors: current status and future prospects on the recognition of mammalian microsomal cytochrome p substrates and their characteristics prediction of small-molecule binding to cytochrome p a : flexible docking combined with multidimensional qsar virtual screening of novel noncovalent inhibitors for sars-cov c-like proteinase oncology exploration: chartering cancer medicinal chemistry space a hybrid mixture discriminant analysis-random forest computational model for the prediction of volume of distribution of drugs in human prediction of human volume of distribution values for neutral and basic drugs. . extended data set and leave-class-out statistics prediction of volume of distribution values in humans for neutral and basic drugs using physicochemical measurements and plasma protein binding in silico screening of drug databases for tse inhibitors structure-based drug design of a novel family of ppargamma partial agonists: virtual screening, x-ray crystallography, and in vitro/in vivo biological activities judging models in qsar-and lfe-like studies if there are no replications: correlation of dipeptidyl peptidase iv hydrolytic activities of l-alanyl-l-alanine phenylamides the serotonin binding site of human and murine -ht b receptors: molecular modeling and site-directed mutagenesis qsar modeling of in vitro inhibition of cytochrome p a piperidine renin inhibitors: from leads to drugs a common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening kinase inhibitors: not just for kinases anymore from magic bullets to designed multiple ligands pharmacophore model for novel inhibitors of ubiquitin isopeptidases that induce p -independent cell death cyclopentenone prostaglandins of the j series inhibit the ubiquitin isopeptidase activity of the proteasome pathway serotonin b receptor is required for heart development hiv- integrase pharmacophore: discovery of inhibitors through three-dimensional database searching design and synthesis of non-peptidic inhibitors for the syk c-terminal sh domain based on structure-based in-silico screening prediction of biological targets using probabilistic neural networks and atom-type descriptors a return to rational drug discovery: computer-based models of cells, organs and systems in drug target identification homology modeling of the serotonin -ht a receptor using automated docking of bioactive compounds with defined geometry computational tools for the analysis and visualization of multiple protein-ligand complexes renin inhibition by substituted piperidines: a novel paradigm for the inhibition of monomeric aspartic proteinases? application of validated qsar models of d dopaminergic antagonists for database mining global mapping of pharmacological space a genetic algorithm for structure-based de novo design d-qsar and receptor modeling of tyrosine kinase inhibitors with flexible atom receptor model (flarm) targeting drug-efflux pumps -a pharmacoinformatic approach structure-based drug design: the discovery of novel nonpeptide orally active inhibitors of human renin discovering cox-inhibiting constituents of morus root bark: activity-guided versus computer-aided methods acetylcholinesterase inhibitory activity of scopolin and scopoletin discovered by virtual screening of natural products screening the receptorome to discover the molecular targets for plant-derived psychoactive compounds: a novel approach for cns drug discovery evidence for possible involvement of -ht( b) receptors in the cardiac valvulopathy associated with fenfluramine and other serotonergic medications crystal structure of human cytochrome p d rational discovery of novel nuclear hormone receptor antagonists combining in vitro and in vivo pharmacokinetic data for prediction of hepatic drug clearance in humans by artificial neural networks and multivariate statistical techniques pharmacophore modeling and in silico screening for new p (aromatase) inhibitors in silico pharmacology for drug discovery s ekins et al identification and prediction of promiscuous aggregating inhibitors among known drugs molecular determinants for the interaction of the valvulopathic anorexigen norfenfluramine with the -ht b receptor identification of novel estrogen receptor alpha antagonists from large networks to small molecules similarity to molecules in the training set is a good discriminator for prediction accuracy in qsar orphan nuclear receptors, excellent targets of drug discovery the challenges of making useful protein-ligand free energy predictions for drug discovery. in: ekins s (ed) computer applications in pharmaceutical research and development d qsar (comfa) of a series of potent and highly selective vla- antagonists identification of potent and novel alpha beta antagonists using in silico screening quantitative structure-activity relationship study on tetrahydro-beta-carboline antagonists of the serotonin b ( ht b) contractile receptor in the rat stomach fundus comfa modeling of human catechol o-methyltransferase enzyme kinetics development of biologically active compounds by combining d qsar and structure-based design methods towards integrated adme prediction: past, present and future directions for modelling metabolism by udp-glucuronosyltransferases multiple pharmacophores for the investigation of human udp-glucuronosyltransferase isoform substrate selectivity evaluation of a novel shape-based computational filter for lead evolution: application to thrombin inhibitors potential of renin inhibition in cardiovascular disease human rhinovirus c protease: generation of pharmacophore models for peptidic and nonpeptidic inhibitors and their application in virtual screening influenza virus neuraminidase inhibitors: generation and comparison of structure-based and common feature pharmacophore hypotheses and their application in virtual screening pharmacophore modeling, docking, and principal component analysis based clustering: combined computer-assisted approaches to identify new inhibitors of the human rhinovirus coat protein screening the receptorome: an efficient approach for drug discovery and target validation reengineering the pharmaceutical industry by crash-testing molecules structure and function of voltage-gated ion channels an in silico approach to discovering novel inhibitors of human sirtuin type hitting the hot spots of cell signaling cascades the biochemistry of drug metabolism -an introduction. part : principles and overview can we estimate the accuracy of adme-tox predictions? solution structure of cnerg (ergtoxin), a herg specific scorpion toxin discovery of nonpeptidic small-molecule ap- inhibitors: lead hopping based on a three-dimensional pharmacophore model protein-structure-based drug discovery of renin inhibitors punaglandins, chlorinated prostaglandins, function as potent michael receptors to inhibit ubiquitin isopeptidase activity substituted piperidines-highly potent renin inhibitors due to induced fit adaption of the active site kinomics-structural biology and chemogenomics of kinase inhibitors and targets modeling the cytochrome p -mediated metabolism of chlorinated volatile organic compounds the discovery of novel, structurally diverse protein kinase c agonists through computer d-database pharmacophore search. molecular modeling studies probing the conformation of the sugar transport inhibitor phlorizin by d-nmr, molecular dynamics studies, and pharmacophore analysis similarity-based approaches to virtual screening drugbank: a comprehensive resource for in silico drug discovery and exploration ligandscout: -d pharmacophores derived from protein-bound ligands and their use as virtual screening filters drug discovery in the ubiquitin regulatory pathway in silico prediction of drug binding to cyp d : identification of a new metabolite of metoclopramide structural biology and function of solute transporters: implications for identifying and designing substrates modeling of active transport systems structure-based virtual screening for plant-based erbeta-selective ligands as potential preventative therapy against age-related neurodegenerative diseases acknowledgements se gratefully acknowledges dr cheng chang and dr peter w swaan (university of maryland), dr konstantin v balakin (chemical diversity inc.) for in silico pharmacology collaborations over the past several years and dr hugo kubinyi for his insightful efforts in tabulating the successful applications of in silico approaches, which was inspirational. gold-enhelix inc. graciously provided chemtree. se kindly acknowledges dr maggie az hupcey for her support. jm acknowledges the research funding provided by the spanish ministerio de educació n y ciencia (project reference bio - ) and the instituto de salud carlos iii. owing to limited space, it was not possible to cite all in silico pharmacology-related papers, our sincere apologies to those omitted. the authors state no conflict of interest. key: cord- -d qr xg authors: buehler, markus j. title: nanomechanical sonification of the -ncov coronavirus spike protein through a materiomusical approach date: - - journal: nan doi: nan sha: doc_id: cord_uid: d qr xg proteins are key building blocks of virtually all life, providing the material foundation of spider silk, cells, and hair, but also offering other functions from enzymes to drugs, and pathogens like viruses. based on a nanomechanical analysis of the structure and motions of atoms and molecules at multiple scales, we report sonified versions of the coronavirus spike protein of the pathogen of covid- , -ncov. the audio signal, created using a novel nanomechanical sonification method, features an overlay of the vibrational signatures of the protein's primary, secondary and higher-order structures. presenting musical encoding in two versions - one in the amino-acid scale and one based on equal temperament tuning - the method allows for expressing protein structures in audible space, offering novel avenues to represent, analyze and design architectural features across length- and time-scales. we further report a hierarchical frequency spectrum analysis of five distinct protein structures, which offer insights into how genetic mutations, and the binding of the virus spike protein to the human ace cell receptor directly influence the audio. applications of the approach may include the development of de novo antibodies by designing protein sequences that match, through melodic counterpoints, the binding sites in the spike protein. other applications of audible coding of matter include material design by manipulating sound, detecting mutations, and offering a way to reach out to broader communities to explain the physics of proteins. it also forms a physics-based compositional technique to create new art, referred to as materiomusic, which is akin to finding a new palette of colors for a painter. here, the nanomechanical structure of matter, reflected in an oscillatory framework, presents a new palette for sound generation, and can complement or support human creativity. proteins are the building blocks of virtually all life, providing the material foundation of spider silk, cells, and hair, but also offering other countless functions from enzymes to drugs, and pathogens such as viruses. we cannot see small nanoscopic objects like proteins or other molecules that make up virtually all living matter including our cells, tissues, as well as pathogens such as viruses. here we apply a computational algorithm to a recently reported protein structure, which allows us to make protein material manifestation audible, by rendering molecular structure information such as vibrational spectra, secondary structure, and folding geometry into a complex audio signal [ ] [ ] [ ] [ ] [ ] . protein molecules consist of naturally occurring amino acid building blocks that are assembled into hierarchical structures across various length-scales [ ] [ ] [ ] . proteins are usually classified by their structural organization, from primary structure (also known as the sequence of amino acids), secondary structure (local folded geometry: e.g., beta-sheet or alpha-helix), as well as tertiary and quaternary structure (overall folded arrangement). more broadly, the folded structure of proteins, encoded by the sequence of dna, determines material function and failure in the context of disease or injury [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this biomaterial organization is reminiscent of the way by which musical compositions can be described, starting from basic harmonic waves (for instance a sine wave), different timbres (overlaying harmonic waves modulated in pitch, volume, and other measures, over time), melodies, chords and multiple chords and melodies played together and against each other. the integrated perspective on all aspects of these assemblies, processed simultaneously through the human auditory system, provides an efficient approach for our brains to understand complex data structures in soundwhat humans commonly perceive as "music" [ ] . in sonification, we exploit this skill, and use sonification to translate complex data into audible sounds [ , ] . this article focuses on an analysis of the nanomechanical vibrational spectrum of a virus spike protein that is the pathogen of covid- , which appears on the surface of the virus as outward facing molecular "spikes", which are critical of the virus' entry into host cells [ ] . figure depicts a rendering of this protein structure, revealing its characteristic spike shape, and also outlines the method used to translate the structure in audible signals based on its vibrational patterning. the plan of this article is as follows. we begin with a brief review of materials and methods, introducing to approaches used here, and then present specifics of the sonification examples. we discuss the nanomechanical foundation for the approach, and also review basic elements of the musical choices. sonification is a method to translate data structures into audible signals. here, we use a particular approach of sonification termed materiomusic, to use the actual vibrations and structures of molecules to create audible materials that be used for analysis and design. this method has been used in earlier work to translate from material to music and from music to material, due to its reversible and unique mapping [ , ] . the main focus of this paper is on the protein structure with protein data bank (pdb) id vsb. it features three distinct protein chains that are folded into the virus spike protein, consisting of , residues each ( figure shows an overview of the structure [ ] ). a protein structure is built up from one-dimensional chains of amino acids. the basis to mapping each of the amino acids onto a unique musical tone is the distinct vibrational spectrum of the molecules as explained in these references: [ , ] . in fact, since each of the amino acids has a unique vibrational spectrum, we may distinguish each of them by its unique sound, or timbre, which is reflected in the overlay of all modal frequencies. since the eigenmodes are orthogonal, energy does not spill from one mode to another, which implies that they are a fundamental way to understand the vibrational spectrum and sounding of a molecule or assembly. since each amino acid features a distinct audible expression, defined by their unique profile of molecular vibrations, we can assume that they can be assigned a particular musical "note" or tone. in this framework, the primary sequence of amino acids defines the note sequence played, which is augmented by the other components [ , ] the next higher level of organization of a protein is the secondary structure, reflecting the local organization of the d chain into helices or sheets (or other structures). we incorporate information about the secondary structure associated with each amino acid in the translation step by affecting the duration and volume of notes. during the translation process from protein to music we use the dssp algorithm to compute the secondary structure from the protein geometry file and sequence [ , ] . as presented in earlier work [ , ] the method of transpositional equivalence is used to scale the frequencies to the audible range, however, while maintaining the accurate ratio of frequencies in the signals generated. in addition to mapping primary and secondary structure, we realize the overall vibrational motions of the molecule by incorporating their normal mode frequencies into an audio signal, using the method described in [ ] . to obtain the normal modes, we use the anisotropic network model [ ] approach, and compute the spectra of frequencies. the data is then imported in the max device [ , ] , and sounds are rendered using ableton live [ ] , forming the basis for the secondary signal. in sound generation, both signalsthe primary and secondary, as well as audio generated by the overall vibrations are overlaid and played together, creating a multi-dimensional image of the protein's structure, true to its accurate reflection of vibrational spectra. sidechain compression is used in some examples to module the volume of the secondary signal based on the level of the primary signal. this renders a dynamic interplay of the two sound sources. in the approach described in section . , all sounds generated are based on the spectrum of the actual molecular vibrations. this, however, poses challenges in matching it with conventional tuning systems. while the sound of amino acids closely resembles that of bells, another approach that can be used is to map each of the amino acids onto a unique tone in a particular musical scale, such as a dorian mode [ , ] as used in the example described in this article. other possible scale choices could be a major or minor scale, or variations thereof, whereas notes are picked in ascending order to describe each of the amino acids. the note assignments are made in the order tyr, asn, leu, met, glu, pro, trp, arg, gln, his, phe, ser, lys, val, asp, thr, ile, cys, ala, gly, following an ordering based on their lowest vibrational frequency from small to large. as in the other approach described in section . , the next level of organization of a protein is the secondary structure, reflecting the local organization of the d chain into helices or sheets, or potentially other structures. we incorporate information about the secondary structure associated with each amino acid in the translation step by affecting the duration and volume of notes, as described in detail in [ , ] . the format used to represent the distance correlation matrix depicted in figure a the concept of rapidly played note insertions, represented musically through strummed notes similar as in a guitar [ ] . this approach is further explained in figure , which shows the folding of a protein into a three-dimensional structure. to encode the information that points i and j are neighbors in the folded state, we insert a sequence segment around point j at point i. similarly, a sequence segment around point i is inserted at point j, creating a symmetric representation that reflects the geometric fact that points i and j are neighbors. see figure d for the geometry in the folded state, as a visual example of how the coding is implemented in a musical score, in figure e . furthermore, additional notes are inserted and played simultaneously, where the volume of the notes is modulated based on the distance of the amino acid inserted from the reference amino acid in the primary sequence, and reflecting the distance correlation matrix shown figure . these reflect the same sequence patterns of the insertions, where a choice can be made about how long the overlaid fragments are. unlike the insertions described in the previous section, the insertions now added are played on top of the primary notes reflecting the primary and secondary structure of the protein. the resulting complex overlapping melodies represent a kind of counterpoint argument in musicthe concept where notes are played against notessuch as used widely by j.s. bach [ , ] . counterpoint is a powerful means to manifest references of closeness in a time sequence coded manifestation of signals. it is also the basis for pleasantness of music, whereas a balance of repetition and variation is a foundational structural element. this universality, reminiscent of the universality in proteins represented by secondary structure motifs (such as alpha-helices of beta-sheets), or repetitions of sequence patterns within a protein design is common across nature. importantly, the concept a more generalized counterpoint argument represents physical closeness in musical space in an indirect manner. similar concepts of counterpunctual approaches are used in many other fields of communication and art, where referencing conceptsalbeit distortedis a powerful way to represent connections in a coded form. for instance, allegories in art can realize connections to multiple touchpoints and associations [ ] . these and other concepts are quite similar to the natural structure of proteins, where one residue may be close to many others, as can be directly confirmed in the distance correlation matrix. moreover, such associations are also reminiscent to other structures, such as the hierarchical organization of neurons in the brain, as visualized in figure b [ ]. through the transformations described above, a total number of , , notes are generated in the raw musical coding, reflecting the hierarchical structure of the protein in musical space. after rendering the sound using max patches [ , ] and ableton live [ ] , spectral analysis is performance using sonic visualizer [ ] , and the audio files processed using audacity . . [ ] . we specifically focus on the melodic spectrogram, which offers a visual representation of how the energy in different frequency bands changes over time. it provides an analytical tool to assess the content of an audio signal, which can complement or validated audible inspection. to show the potential application to distinguish protein structures through sound, we depict a spectral comparison of the overall vibrational spectrum of proteins with protein data bank (pdb) [ ] identification code vsb and i in figure . figure a shows their normal mode vibrational frequencies, and the figure b a spectrogram analysis of the vibrational signal generated of these proteins. representative audio signals are included as supplementary material (comparison_ i - vsb.wav), where the distinction is clearly audible, between distinct protein conformations (the order of tones is as depicted in figure b to d, repeated twice overall, representing a total of tones). this example illustrates how the audible system is capable to quickly discern differences in the spectrum, and hence, the vibrational context of a protein structure. this method to assess vibrational changes can be useful to investigate the impacts of sequence changes, or to screen for effects across a very large number of protein data. figure c shows the normal mode vibrational frequencies of three protein complexescomparing scenarios of the virus spike protein attached ( pdb id m ) with proteins without the virus spike protein (pdb ids m and m d) (for a detailed discussion of these protein confirmations, see [ ] ). figure d shows the frequency spectrum of the audio signal. it can be clearly recognized how the attachment of the virus spike protein with the human cell receptor changes the frequency spectrum, and it can also be clearly heard. you can hear the transition between the attached state vs. the unattached state between the third and fourth tone, respectively. notably, when looking at the frequency spectrum, the binding of the virus to the ace receptor leads to a lowering of the frequency spectrum. coding for the multiple structural levels, the audio file coronavirus_ vsb_aa-scale_excerpt.wav represents a sonification of the novel coronavirus pathogen, with pdb id vsb. a spectral analysis of the piece is depicted in figure a . the file coronavirus_ vsb_aa-scale-complex_excerpt.wav represents a more complex version of the original score, with added musical ornamentation, expressing creative choices. as can be determined, the raw structure of the original completely algorithmic composition is expanded upon, through added musical experimentation, realizing a more complex exploration of the audible content. the total length of the piece is around minutes. what you hear is a multi-layered algorithmic composition featuring both the vibrational spectrum of the entire protein (expressed in sound and rhythmic elements), the sequence and folding of amino acids that compose the virus spike structure, as well as interwoven melodiesforming counterpoint music -reflecting the complex hierarchical intersecting geometry of the protein. the audio file coronavirus_ vsb_chromatic-scale_excerpt.wav represents a sonification of the novel coronavirus pathogen, with pdb id vsb. a spectral analysis of the entire piece is depicted in figure b . the techniques used to realize a musical composition include a variety of approaches: • the primary sequence, all overlaid notes, and all insertions are played as, and rendered as lead sound using physical modeling of a koto (a japanese harp instrument). the use of physical modeling allows us to directly generate sound using a physical basis, accounting for a true reflection of the way by which vibrating strings generate sound. • overlaid notes, coded through the simultaneous excitation of the sounds of amino acid residues in close vicinity, are played through arpeggios. an arpeggio is a way to play the notes of a chord in succession, rather than sounding them simultaneously. this approach enables to create a flowing expression of the complex chord progressions generated through the protein structure, to offer a contrast to the simultaneously played notes. • a variety of classical instruments are used in the realization of these scores, and artistic choices are executed to vary the relative loudness of one over other instruments. the realization of the musical score includes a total number of independent musical instruments, including: koto (several), harp (several), strings, strings played pizzicato, celli, solo violin, clarinet (several), oboe, flutes (several), trumpet, fluegelhorn, and percussion. the total length of the piece is around : . this analysis presented two approaches to sonify a large protein structure, to provide access to structural and potentially functional information through sound. it provides not only access to a truthful reflection of protein structures via sound, but also offers intriguing new ideas for musical composition. when applied in this context, human composers may intersect with the compositions generated by alone, forming a new axis of intersection across scales and species. the focus on proteins is motivated since they are the most abundant material component of all living things: especially their motion, structure and failure in the context of both normal physiological function and disease is a foundational question that transcends academic disciplines and can be a footing to explore fundamental nanomechanical questions. we focused on developing a model for the vibrational spectrum of the amino acid building blocks of proteins, an elementary structure from which materials in living systems are built on. this concept is broadly important as at the nanoscale observation, all structures continuously move, reflecting the fact that they are tiny objects excited by thermal energy and set in motion to undergo large deformations as is the case in injury or disease, or due to disruptive mutations. the approach allowed us to extract new musical compositions of the coronavirus spike protein as one way to represent nature's concept of hierarchy as a paradigm to create function from universal building blocks, and exploring the vibrational complexity of a large protein design. this sort of bio-inspired music is an expansion of bio-inspired materials design, and can offer new design ideas for geometric and structural patterning. indeed, the translation from various hierarchical systems into one another poses a paradigm to understand the emergence of properties in materials, sound, and related systems. the use of sonification and comparisons of music generated by proteinsas a core physical material building block of lifewith conventional compositions may shed light into the unique features of the effects of music and sound and interactions with matter and life, including the brain and other non-human forms of life, as demonstrated in earlier work of sonifications of spider webs [ ] . the music and its pleasing nature, especially the realization in the dorian mode (coronavirus_ vsb_chromatic-scale_excerpt.wav), is a metaphor for this nature of the virus to deceive the host and exploit it for its own multiplication. a virus' genome hijacks the host cell's protein manufacturing machinery, and forces it to replicate the viral genome and produce viral proteins to make new viruses from it. in light of this nature of viruses, the work explores the fine line between the emergent beauty of life and death as opposite poles, an aspect also critically important for nanomaterials and human health. as one listens to the protein we find that the intricate design results in interesting and actually pleasing, relaxing sounds. the reason for this is that the virus spike protein represents a complex, beautiful design (see also figure ). of course, the intricate protein principles at the basis of the virus do not at all convey the deadly impacts this particularly protein is having on the world. from another vantage point, the pleasing aspect of the music shows the deceiving nature of the virus when it enters the cells by binding to exterior receptors, in order to hijacks our body to replicate. the pleasant sounds represent an analogy for the nature of the virus to deceive the host and exploit it for its own multiplication, emerging as the residue after the transactional infections that diminish or kill the host. a related important aspect of materiomusical coding is the agency of singular eventsin which case, albeit a building block in a system may be small, its impact on the greater context of the overall system behavior is outsized. as in many musical compositions, a single note or sound can be a focal point of a composition (such as a accidentals, or out-of-scale notes or chords in music). in materials science, a pair of atoms may be subject to singular forces near a crack tip [ ] . in many systems, a complex structure of a composition of building blocks may solely exist for the purpose of a single, unique, singular note, one of the significant deep foundational questions of many forms of art. specifically in the context of music, when played alone that note would not have particular exposition. it is only in the context of the whole piece that it stands out. in proteins, we can see a similarity to this singular perspective in point mutations, where certain changessuch as point mutationsare particularly relevant. applications of the approach could include the development of antibodies by designing protein sequences that match, through melodic counterpoints, the binding sites in the spike protein, to identify sequences that create strong adhesion for the interruption of disease progression through protein-based drugs. the novel coronavirus is believed to enter human cells via the ace receptor on the exterior of cells [ , ] . ace is an enzyme attached to the outer surface of cells found in our lungs, heart, arteries, kidney, and intestines (whereas one of its natural role is blood pressure regulation). one possible engineering task could be to create a protein molecule, through counterpoint composition, that binds more easily to the virus spike, hence shutting down the pathway of infection. an analysis of the mechanism of interaction between ace and the spike protein can perhaps provide useful insights, and the intersection of these two proteins could be subject of future experimentation in musical space. not only could this be tackled using human writing, but could also be performed using ai models, as demonstrated in earlier work [ , ] . the data depicted in figure c clearly demonstrated that the binding of the virus spike protein to the human cell leads to an audible change in the sounds generated. other aspects by which vibrations of the virus proteins play an important role include our understanding of the virus stability as temperatures increase, for example. vibrational spectra may also tell us why the sars-cov- spike gravitates toward human cells more than other viruses. further applications of materiomusical coding of matter include de novo material design by manipulating sound, detecting mutations, and offering a way to reach out to broader communities to explain the science of nanomaterials such as proteins. it also forms a physicsbased compositional technique to create new art, referred to as materiomusic, which can be explained akin to finding a new palette of colors for a painter. here, the nanomechanical structure of matter, reflected in an oscillatory framework, presents a new palette for sound generation. a summary of the new palette of sounds, specifically the amino acid tones, are summarized graphically in figure . the bio-nano-science of molecular motions can hence offer several avenues of future investigation and applications in the interrelated fields of science, art and engineering. [ ] , and visualized using a sequence analysis tool [ ] . the analysis shows that the protein features % helical and % beta sheet content. the image provides a direct visualization of the various secondary structure motifs that make up the protein, which in turn fold into complex shapes. all this information is incorporated into the musical expression of the protein. the plot in panel a shows amino acid residues that are close to each other in brighter red colors, providing an overall image of the protein's d folded structure. panel b depicts the connectome as reported in [ ] , and rendered via the neuprint server [ ] , showing the similarity of folding concepts in the neural network design in the brain. panel c shows a plot with only the closest neighbors highlighted, with distances less than . a. panel d depicts, schematically, how the overall folding geometry is coded in that way. the inlay depicts the folding dynamics of a beta-sheet protein from an initial elongated unstructured protein. panel e shows an example how two sequence patterns are assembled in the overlapped form, creating note-against-note musical content. analysis of the vibrational and sound spectrum of over , protein structures and application in sonification a self-consistent sonification method to translate amino acid sequences into musical compositions and application in protein design using ai reoccurring patterns in hierarchical protein materials and music: the power of analogies materials by design -a perspective from atoms to structures mater. impuls. natur, acatec ultrathin free-standing bombyx mori silk nanofibril membranes deformation and failure of protein materials in physiologically extreme conditions and disease silk-its mysteries, how it is made, and how it is used materials by design: merging proteins and music materiomics: an -omics approach to biomaterials research nature's hierarchical materials predictive modelling-based design and experiments for synthesis and spinning of bioinspired silk fibres a series of pdb related databases for everyday needs structure and stability of the lamin a tail domain and hgps mutant evaluating hierarchical structure in music annotations sounds interesting: can sonification help us design new proteins? cryo-em structure of the -ncov spike in the prefusion conformation a self-consistent sonification method to translate amino acid sequences into musical compositions and application in protein design using artificial intelligence sonification based de novo protein design using artificial intelligence, structure prediction and analysis using molecular modeling e-msd: an integrated data resource for bioinformatics dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features the anisotropic network model web server at (anm . ) the structure of atonal music formalized music: thought and mathematics in composition, notes a geometry of music : harmony and counterpoint in the extended common practice the craft of tonal counterpoint allegory : the theory of a symbolic mode a connectome of the adult drosophila central brain sonic visualiser audacity multi-track audio editor and recorder the protein data bank structural basis for the recognition of the sars-cov- by full-length human ace imaging and analysis of a three-dimensional spider web architecture atomistic modeling of materials failure on the nature of the transparent teeth of the deep-sea dragonfish neuprint: analysis tools for em connectomics pre-fusion structure of a human coronavirus spike protein acknowledgements: this work was supported by mit cast via a grant from the mellon foundation, with additional support from onr (grant # n - - - ) and nih u eb . • comparison_ i - vsb.wav: comparison of the overall molecular frequency spectrum of two distinct spike proteins from two different coronaviruses, calculated by a normal mode analysis. the analysis of the spectral content is shown in figure . https://soundcloud.com/user- /comparison- i - vsb • coronavirus_ vsb_aa-scale_excerpt.wav: sonification of the novel coronavirus pathogen, with pdb id vsb (excerpt). key: cord- -q f el authors: farhadi, tayebeh; hashemian, seyed mohammadreza title: computer-aided design of amino acid-based therapeutics: a review date: - - journal: drug des devel ther doi: . /dddt.s sha: doc_id: cord_uid: q f el during the last two decades, the pharmaceutical industry has progressed from detecting small molecules to designing biologic-based therapeutics. amino acid-based drugs are a group of biologic-based therapeutics that can effectively combat the diseases caused by drug resistance or molecular deficiency. computational techniques play a key role to design and develop the amino acid-based therapeutics such as proteins, peptides and peptidomimetics. in this study, it was attempted to discuss the various elements for computational design of amino acid-based therapeutics. protein design seeks to identify the properties of amino acid sequences that fold to predetermined structures with desirable structural and functional characteristics. peptide drugs occupy a middle space between proteins and small molecules and it is hoped that they can target “undruggable” intracellular protein–protein interactions. peptidomimetics, the compounds that mimic the biologic characteristics of peptides, present refined pharmacokinetic properties compared to the original peptides. here, the elaborated techniques that are developed to characterize the amino acid sequences consistent with a specific structure and allow protein design are discussed. moreover, the key principles and recent advances in currently introduced computational techniques for rational peptide design are spotlighted. the most advanced computational techniques developed to design novel peptidomimetics are also summarized. different diseases may be caused by pathogens or malfunctioning organs, and using therapeutic agents to heal them has an old recorded history. small molecules are conventional therapeutic candidates that can be easily synthesized and administered. however, many of these small molecules are not specific to their targets and may lead to side effects. moreover, a number of diseases are caused due to deficiency in a specific protein or enzyme. thus, they can be treated using biologically based therapies that are able to recognize a specific target within crowded cells. under the biologic conditions, some macromolecules such as proteins and peptides are optimized to recognize specific targets. therefore, they can override the shortcomings of small molecules. recently, pharmaceutical scientists have shown interest in engineering amino acid-based therapeutics such as proteins, peptides and peptidomimetics. [ ] [ ] [ ] theoretical and experimental techniques can predict the structure and folding of amino acid sequences and provide an insight into how structure and function are encoded in the sequence. such predictions may be valuable to interpret genomic information and many life processes. moreover, engineering of novel proteins or redesigning the existing proteins has opened the ways to achieve novel biologic macromolecules with desirable therapeutic functions. protein sequences comprise tens to thousands of amino acids. besides, the backbone and side chain degrees of freedom lead to a large number of configurations for a single amino acid sequence. protein design techniques give minimal frustration through precise identification of sequences and their characteristics. [ ] [ ] [ ] [ ] considering energy landscape theory, the adequately minimal frustration in natural proteins occurs when their native state is adequately low in energy. the de novo design of a sequence is difficult because there are huge numbers of possible sequences: n for n-residue proteins with only natural amino acids. peptide design should incorporate computational approaches. it can benefit from searching the more advanced fields used for small molecules and protein design. however, the straightforward adoption of computational approaches employed to small-molecule and protein design has not be accepted as a reasonable solution to the peptide design problem. [ ] [ ] [ ] in the peptide drug design, the conformational space accessible to peptides challenges the small-molecule computational approaches. besides, the necessity for nonstandard amino acids and various cyclization chemistries challenges the available tools for protein modeling. furthermore, the aggregation of peptide drugs during production or storage can be an unavoidable problem in the peptide design procedure. rational design of a peptide ligand is also challenging because of the elusive affinity and intrinsic flexibility of peptides. peptide-focused in silico methods have been increasingly developed to make testable predictions and refine design hypotheses. consequently, the peptide-focused approaches decrease the chemical spaces of theoretical peptides to more acceptable focused "drug-like" spaces and reduce the problems associated with aggregation and flexibility. , for the discussions that follow, peptides can be defined as relatively small ( - residues) polymers of amino acids. in physiological conditions, several problems such as degradation by specific or nonspecific peptidases may limit the clinical application of natural peptides. moreover, the promiscuity of peptides for their receptors emerges from high degrees of conformational flexibility that can cause undesirable side effects. besides, some properties of therapeutic peptides, such as high molecular mass and low chemical stability, can result in a weak pharmacokinetic profile. therefore, peptidomimetic design can be a valuable solution to circumvent some of undesirable properties of therapeutic peptides. , in the biologic environment, peptidomimetics can mimic the biologic activity of parent peptides with the advantages of improving both pharmacokinetic and pharmacodynamic properties including bioavailability, selectivity, efficacy and stability. a wide range of peptidomimetics have been introduced, such as those isolated as natural products, synthesized from novel scaffolds, designed based on x-ray crystallographic data and predicted to mimic the biologic manner of natural peptides. using hierarchical strategies, it is possible to change a peptide into mimic derivatives with lower undesirable properties of the origin peptide. over the past years, computational methods have been developed to discover peptidomimetics. in a part of this review, novel computational methods introduced for peptidomimetic design have been summarized. peptidomimetics can be categorized as follows: peptide backbone mimetics (type ), functional mimetics (type ) and topographical mimetics (type ). the first generation of peptidomimetics (type ) mimics the local topography of amide bond. it includes amide bond isosteres, pyrrolinones or short fragments of secondary structure, such as beta-turns. such mimetics generally match the peptide backbone atom-for-atom, and comprise chemical groups that also mimic the functionality of the natural side chains of amino acids. a number of prosperous instances of type peptidomimetics have been reported. the second type of peptidomimetics is described as functional mimetics or type mimetics, which include small, non-peptide compounds that are able to identify the biologic targets of their parent peptide. at first, they were assumed to be conservative structural analogs of parent peptides. however, using site-directed mutagenesis, their binding sites to biologic targets were investigated. the results indicated that type peptidomimetics routinely bind to protein sites that are different from those selected by the original peptide. therefore, type mimetics maintain the ability to interfere with the peptide-protein interaction process without the necessity to mimic the structure of the natural peptide. type peptidomimetics reveal the best conception of peptidomimetics. they consist of the necessary chemical groups that act as topographical mimetics and contain novel chemical scaffolds that are unrelated to natural peptides. here, theoretical and computational techniques to design proteins, peptides and peptidomimetics are reviewed. however, the current review does not deeply highlight the computational aspects of amino acid-based therapeutic design, but only discusses the methods used to design the mentioned therapeutics. figure summarizes the key concepts presented in this study. as some examples, the structures of aldesleukin, leuprolide and spaglumic acid, important amino acid-based therapeutics approved by the us food and drug administration (fda), are shown in figure a computer-aided design of amino acid-based therapeutics figure a ) and leuprolide (pdb id: yy ; figure b ) were obtained from the protein data bank (pdb; http://www. rcsb.org/) and visualized by pymol tool. the structure of spaglumic acid was retrieved (in mol format) from pub-chem database (https://pubchem.ncbi.nlm.nih.gov/) with the pubchem id ( figure c ) and visualized using pymol. aldesleukin, a lymphokine, is a recombinant protein used to treat adults with metastatic renal cell carcinoma (https://www.drugbank.ca/drugs/db ). leuprolide, a synthetic nine-residue peptide analog of gonadotropin releasing hormone, is used to treat advanced prostate cancer (https://www.drugbank.ca/drugs/db ). spaglumic acid is used in allergic conditions such as allergic conjunctivitis. the drug belongs to a class of peptidomimetics known as hybrid peptides. hybrid peptides contain at least two dissimilar types of amino acids (alpha, beta, gamma or delta) linked to each other via a peptide bond (https://www.drugbank.ca/ drugs/db ). in the current study, all fda-approved therapeutics (in ) were retrieved from drugbank (https://www.drugbank. ca/biotech_drugs) and an analysis was conducted to compare their percentages. protein-based therapies, gene or nucleic acid-based therapies, vaccines, allergenics and cell transplant therapies made up . %, . %, . %, . % and . % of total approved therapeutics, respectively. small-molecule drugs made up . % of the approved therapeutics ( figure ). computational designing of proteins can be classified as follows: ) template-based designing in which three-dimensional ( d) farhadi and hashemian structure of a predefined template is adapted to design a sequence and ) de novo designing in which the amino acids' arrangement is changed to generate both sequence and d structure of a completely novel protein. the problem of predicting the fold of an unknown sequence could be solved by utilizing templates. since the fold is unaltered, the backbone atoms are directly located on this framework. moreover, to generate a functional protein, the side chains that can effectively stabilize the structure are added to the backbone. , routine concerns and methods for template-based protein design are reviewed below. selecting the template (scaffold) protein the template (also named as scaffold protein) contains a group of backbone atom coordinates. the coordinates can be retrieved from an available x-ray crystal structure or cautiously from a nuclear magnetic resonance (nmr) structure. computer-aided design of amino acid-based therapeutics fixing the backbone decreases the computational complication, but it may inhibit the main chain modifications to adjust sequence alternation. backbone flexibility can generate designed functionalities over the protein's normal function. the backbone flexibility is introduced through incorporating other closely associated conformations to an existing structure. [ ] [ ] [ ] recently, new functionalities were effectively introduced into the tim-barrel topology. this fold has been detected as one of the most shared structures in distinct protein superfamilies. sequence search and characterization in a design procedure, a protein sequence is selected such that it meets the energetic and geometric constraints established by the chosen fold. sequence search techniques sample different sequences and estimate their energies to gain the one owing the minimum energy. in order to identify the sequences subject to an objective function or a specific energy, a diverse strategies including optimization and probabilistic approaches have been developed. optimization processes may recognize candidate sequences using stochastic or deterministic methods. probabilistic approaches focus on characterizing the sequence space probabilistically. deterministic methods: to achieve a sequence folded into a global minimum energy conformation, deterministic methods search the whole sequence space and identify the global optima. , these methods include dead-end elimination (dee), self-consistent mean field, graph decomposition and linear programming. stochastic algorithms search the sequence space in an exploratory manner. these algorithms include monte carlo algorithms (simulated annealing), graph search methods and genetic algorithms. some of the most commonly used methods are discussed below. dee has been considered as a thorough search algorithm. to find and remove sequence-rotameric positions that are not portions of the global minimum energy conformation, dee compares two amino acid rotamers and removes the one with greater interaction energy. interaction energies are computed for each rotamer of the test amino acid, along with all rotamers of every other amino acid. the situation is repetitively examined for total amino acid states as well as their rotamers until it no longer holds true. , expanding the sequence length increases the combinatorial complication of dee exponentially. therefore, to design sequences of amino acids or larger, application of dee may be restricted. details of the theorem are explained elsewhere. , stochastic search algorithms: as mentioned before, deterministic approaches are perfect to design proteins with small sizes, but show the applied disadvantages with extension of sequence size. stochastic or heuristic methods are valuable to design large proteins. the most widely used method for protein design includes monte carlo sampling. , monte carlo method samples positions of complicated proteins in a way related to a selected probability distribution such as boltzmann distribution. boltzmann distribution specially weighs low-energy configurations. the monte carlo algorithm performs iterative series of calculations. at the primary step of each search, a partially accidental test sequence is generated, and its energy is calculated via a physical potential. during the primary step, both rotamer state and amino acid identity are adjusted and an efficient temperature controls the probable energy alterations. in the next step, named simulated annealing, the temperature gradually decreases and permits favorable sampling of lowerenergy configurations. multiple independent calculations are carried out to converge the system to a global minimum. , for more explanation about the theorems and details of the formulation of the probability distribution and weights, readers are referred to study previous reports. , probabilistic approach: probabilistic approaches are frequently employed when thorough information is not accessible for protein design. in a probabilistic approach, sitespecific amino acid probabilities may be utilized, rather than particular sequences. the procedure is partially motivated by the uncertainties to find sequences consistent with a specific structure. briefly, the backbone atoms are fixed or greatly constrained, side chain conformations are discretely handled, energy functions are estimated and solvation is handled by simple models. however, in order to offer valuable sequence information for design experiments and to find structurally significant amino acids, probabilistic techniques leverage structural characteristics of interatomic interactions. generally, monte carlo methods give a probabilistic sampling of sequences. , in addition, an entropy-based formalism has been defined to predict amino acid probabilities for a certain backbone structure. , the method employs concepts from statistical thermodynamics to assess the sitespecific probabilities. to address the whole space of existing compositions, the theory is not restricted by the computational enumeration and sampling. large protein structures with . variable residues can be supplied simply. sampling sequence space to generate conformations the chemical variability of a sequence and the number of various amino acids permitted at each position are defined as "degrees of freedom for each amino acid". moreover, each of the natural residues search the whole sequence space. drug design, development and therapy : submit your manuscript | www.dovepress.com to decrease the degrees of freedom for each amino acid and searching the sequence space, diverse approaches such as hydrophobic patterning have been proposed. monomers can be used to probe a protein structure and improve its function, other than the naturally occurring amino acids. sampling of side chain conformational space to form conformations side chain conformations are typically consistent with the energy minima of molecular potentials and can be obtained from a structural database. rotamer statuses are related to the repeatedly detected values of dihedral angles in the side chain of each amino acid. for example, the simplest amino acids including alanine and glycine have only one rotamer status, while the bigger amino acids have . diverse rotamer statuses. a variety of rotamer libraries including backbonedependent, secondary structure-dependent and backboneindependent libraries have been developed for protein design. , by using a rotamer library, one can discretize a meaningful state space to decrease the computational difficulty. rotamer libraries can be extended beyond the natural amino acids. the effective rotamers can model cofactors, ligands, water and posttranslational modifications. for example, to improve the modeling of protein-protein interactions and model water within proteins interiors, the structurally definite water molecules can be inserted as a solvated rotamer library. energy functions have been employed to quantify sequencestructure compatibilities. they include linear associations of hydrogen bonds made by backbone atoms, repulsion among atoms, hydrophobic attraction among non-polar groups and electrostatic interactions among sequential neighbors. the sequence of a protein is selected so that it can adjust the energetic and geometric constraints enforced by the favorite fold. constraints typically contain several intramolecular interactions such as van der waals, hydrophobic, polar and electrostatic interactions, as well as hydrogen bonds. generally, by using a scoring function, it is possible that energetic contributions of the mentioned parameters are taken into account. , , de novo design: designing the sequence and d structure through assembly of proteins fragments , or secondarystructure elements, , novel structures can be modeled de novo. in the design procedures, the backbone coordinates are generally constrained. summary and important findings of some proteins designed using computational approach including a retroaldol enzyme, the kemp elimination enzyme, a novel βαβ protein, a redesigned procarboxypeptidase, a novel α/β protein structure and the top are shown in table . peptide design methods have been categorized as ligand-and target-based design methods. in the ligand-based designing procedure, information derived from peptides is used to design novel therapeutic peptides. in the target-based method, information derived from target proteins is specifically utilized. typically, a hybrid approach including both ligand-and target-based design is utilized. ligand-based peptide design the ligand-based design has been classified as follows: ) sequence-based, ) property-based and ) conformationbased design. sequence-based approach uses the information of conserved regions and analyzes the multiple sequence alignments. this method is directed by the hypothesis that conserved regions are functionally and structurally significant. computational tools allow the ligand-based peptide design, although they lag behind bioinformatics strategies developed for protein designing. recently, using a method based on a pam matrix, the relationship between a series of collagen peptides and antiangiogenic activity including proliferation, migration and adhesion was analyzed. the pam matrix captured information of mutation rates among all pairs of amino acids. based on the results, regions at the c and n termini of the peptides were detected to be significant for an ideal activity and suggested as two distinct binding sites. the approach showed the potential worth of the sequence-based peptide design. in another report, a computational platform called sarvision was developed to support sequence-based design. sarvision signifies an important step for peptide sequence/activity relationship (sar) analysis. moreover, it pools the improved visualization abilities with advanced sequence/activity analysis. compared to small molecules, property-based design methods for peptides are in the early stages of development. in a recent study, the Δg decomposition per residue and the physicochemical characteristics of amino acids, such as hydrophilicity, hydrophobicity and volume, were used computer-aided design of amino acid-based therapeutics to model peptide binding to targets of interest. , finally, a model was built to estimate peptide Δg values for binding to the class i major histocompatibility complex (mhc) protein hla-a* . furthermore, in a wide range of studies, antimicrobial peptides were successfully analyzed by using the property-based approach. for example, a machinelearning method was employed to design novel antimicrobial peptides. the victory of the property-based methods with antimicrobial peptides may be explained by the fact that the desired biologic activity of membrane disruption is relatively nonspecific. in the case of conformation-based peptide design, computational techniques were developed to predict the conformational ensembles or structure of peptides and analyze the sars. , pep-fold is an online tool used to predict the d structures of peptides of length - residues. a remarkable suggestion from the data is that pep-fold seems to solve the conformational sampling problem. , in order to search conformational spaces of a peptide, long timescale molecular dynamic simulations have been employed. , besides, quantum mechanical calculations are promising to address the scoring deficiency in the peptide conformational examination. apparently, to affect the peptide design processes positively, improving the major theoretical and technical issues is necessary before such computationally sophisticated and costly procedures. conformation of a peptide may be modeled to generate a d pharmacophore hypothesis. a certain pharmacophore hypothesis is useful to determine the adme/tox activities or particular potencies of a peptide. for example, screening of a peptide library was jointed to generate a pharmacophore hypothesis to identify potent agonists of melanocortin- receptor isoforms. a combinatorial tetrapeptide library was screened, and sar and ligand-derived pharmacophore templates were generated. the pharmacophore hypothesis was proposed to allow continuous attempts in the rational design of melanocortin receptor molecules. target-based peptide design compared to ligand-based peptide design, target-based design appears to be in a more improved level. targetbased design is initiated with the computer-aided survey of a ligand-bound or unbound protein target to recognize its potential binding sites, prospective specificity surfaces and other pharmacologic activity elements. the phase is generally followed by an in silico design phase where computational methods perform, refine and evaluate peptide design ideas. some recently developed computational methods for targetbased peptide design are reviewed below. recently, an increase in the number of protein-peptide d structures deposited in the pdb has assisted to search the molecular mechanism and structural basis of peptide recognition and binding. information of crystal structures of protein-peptide complexes can improve our knowledge of the farhadi and hashemian chemical forces involved in the binding and special modes of binding. dynamic data of the complexes can be partially extracted from the solution nmr structures deposited in the pdb. to record the structures and functions of various protein-peptide complexes, the experimentally resolved structure data were gathered, annotated and analyzed, and several distinctive databases such as pepx, pepbind and peptiddb were generated. the pepx database, derived from the pdb, comprises unique protein-peptide interface collections. the pepbind database contains , proteinpeptide complex structures from the pdb. peptiddb is a curated database of protein-peptide complexes. the abundance of the structural information specifically on monomeric proteins could be gathered to design proteinpeptide interactions with no requirement for their sequence homology. protein-peptide docking precise docking of a highly flexible peptide is a major challenge. traditional docking protocols, such as autodock, vina , and moe-dock, developed for docking of small molecules, were also used to dock a peptide to a protein receptor. however, comparative studies revealed that these techniques would face failure if the docked peptides were . residues long. therefore, development of peptide-focused docking protocols is very important. other protein-protein docking tools such as z-dock and hex have been used for the computational peptide design in some studies. below, details of recently developed peptide-focused docking approaches are discussed. first, heuristic evolution procedures were applied to search the large conformational space of linear peptides before the binding. however, these procedures were not efficient and their use was limited. then, a scheme based on conformational sampling became common in the peptide docking. besides, several illustrative approaches were proposed to balance between the accuracy and efficacy of the flexible peptide docking. in this aspect, docscheme, dynadock and pepspec were integrated to online userfriendly interfaces and introduced. recently, pepcrawler and flexpepdock were developed as the peptide docking tools. it is reported that flexpepdock has sub-angstrom accuracy in reproducing the crystal structures of protein-peptide complexes. all of the flexpepdock-based methods assume previous information about the peptide-binding site. anchordock, a recently described algorithm, allows powerful blind docking calculations through relaxing the constraint. the program predicts anchoring origins on a protein surface. following recognition of the anchoring origins, an assumed peptide conformation is refined using an anchor-constrained molecular dynamic process. haddock, a well-known protein-protein docking tool, has been recently expanded to run the flexible peptide-protein docking. to handle a docking procedure, haddock uses ambiguous interaction restraints based on the experimental information about intermolecular interactions. this rigid body peptide docking is followed through a flexiblesimulated annealing process. the novel haddock strategy initiates docking computations from an ensemble of three dissimilar peptide conformations (eg, α-helix, extended and polyproline-ii) that are high informative inputs. cabs-dock is a recently introduced protein-peptide docking tool and runs a primary docking procedure whose outcomes can be refined by other tools such as flexpepdock. in the primary phase of the procedure, random conformations of a peptide are predicted and located around the protein target of interest. the process is followed by replica exchange monte carlo dynamics. subsequently, models are selected for the last optimization using the modeller tool to gain accurate scoring and ranking poses. , galaxypepdock was developed to use experimentally resolved protein-peptide structures for running the template-based docking pooled by flexible energy-based optimization. atomistic simulation atomistic monte carlo and molecular dynamics simulations are accurate, but they are meticulous techniques to investigate peptide-protein binding interactions. these techniques can also detect the thermodynamic profile and trajectory included in protein-peptide identification. these methods predict the association among conformations of a peptide in solution or protein. in a study, in order to describe the binding of a decapeptide to the cognate sh receptor, a long-term molecular dynamic simulation was used and a two-state model was built. in the first step, a relatively quick diffusion phase, nonspecific encounter complexes were generated and stabilized by using electrostatic energy. the secondary step was a slow modification phase, in which the water molecules were emptied out from the space between the peptide ligand and the receptor. in another report, by using monte carlo method, the mentioned two-state model was verified to trace some oligopeptide routes for binding to various pdz (post synaptic density protein, drosophila disc large tumor suppressor, and zonula occludens- protein) domains. drug design, development and therapy : submit your manuscript | www.dovepress.com computer-aided design of amino acid-based therapeutics the affinity of bh peptides to bcl- protein was investigated, and results showed the higher affinity of bound peptides occurred when the corresponding peptides were in a lower degree of disorder in unbound states and vice versa. these results showed that the highly structured peptides could increase their affinity through reducing the entropic loss associated with the binding. overall, in addition to the electrostatic and hydrophobic forces, protein-peptide interactions can be affected by the entropic effect and conformational flexibility that could be willingly examined with atomistic simulations. very recently, using a fast molecular dynamics simulation, the energetic and dynamic features of protein-peptide interactions were studied. in most cases, the native binding sites and native-like postures of protein-peptide complexes were recapitulated. additional investigation showed that insertion of motility and flexibility in the simulation could meaningfully advance the correctness of protein-peptide binding prediction. peptide affinity prediction most features of computational peptide design are based on the accuracy and efficacy of affinity prediction. hence, the fast and reliable prediction of peptide-protein affinity is significant for rational peptide design. in this aspect, two categories of prediction algorithms including sequence-and structure-based approaches were developed. the sequencebased method uses the information derived from primary polypeptide sequences to approximate and evaluate the standards of the binding affinity. the structure-based process takes the information derived from d structures of proteinpeptide complexes to predict the binding affinity. at the sequence level, the quantitative structure-activity relationships (qsars) have been widely utilized to forecast the binding affinity of peptides and conclude the biologic function. to model the statistical correlation between sequence patterns and biologic activities of experimentally assessed peptides, machine-learning methods such as partial least squares (pls), artificial neural networks (ann) and support vector machine (svm) have been used. the obtained correlations have been used to infer experimentally undetermined peptides. the relationship between the biologic activity and molecular structure is an important issue in biology and biochemistry. qsar is a well-established method employed in pharmaceutical chemistry and has become a standard tool for drug discovery. however, the predictive capacity of qsar techniques is generally weaker than statistics-based approaches. therefore, a combination of the qsar method with a statistic-based technique may bring out the best in each other and can be a trend in future developments of drug discovery. at the structural level, numerous reports on affinity prediction have addressed the mhc-binding peptides. plentiful mhc-peptide complex structure records have been deposited in the pdb. the significance of domain-peptide recognition has been recently illustrated in the metabolic pathway and cell signaling. to predict the protein-peptide binding potency, a number of strict theories were suggested based on the potential free energy perturbation. the theories computed the alteration of free energies upon the interaction between phosphor-tyrosine-tetra-peptide (pyeei) and human lck sh domain. furthermore, to obtain a deep insight into the structural and energetic aspects of peptide recognition by the sh domain, a number of molecular modeling experiments such as homology modeling, molecular docking and mechanism dynamics were used. peptide array strategies confirmed that some peptide candidates may be potent binders of the abl sh domain. very recently, an approach including quantum mechanics/molecular mechanics, semiempirical poisson-boltzmann/surface area and empirical conformational free energy analysis was developed to quantitatively illustrate the energetic contributions involved in the affinity losing of pdz domain and oppa protein to their peptide ligands. , de novo peptide design recently, in order to de novo target-based peptide design, two remarkable methodologies including the vital method and an approach developed by bhattacherjee and wallin were introduced. the vital method pools verterbi algorithm with autodock to design peptides for the binding sites of a target. the "bhattacherjee and wallin" approach explores both peptide sequence and conformational space around a protein target at the same time. this approach was tested on three dissimilar peptide-protein domains to assess its ability. a brief list of the existing computational resources employed in peptide design is presented in table . in recent years, some computational methods have been proposed to design peptidomimetics. these methods can be classified based on their specificity to translate peptides to peptidomimetics. to select the best method, drug design, development and therapy : submit your manuscript | www.dovepress.com farhadi and hashemian awareness about the structure of peptide-protein complexes is important. , herein, recently introduced methods for computer-aided design of peptidomimetics are presented. growmol is a combinatorial algorithm employed in the peptidomimetics design. growmol searches a variety of probable ligands for the binding sites of a target protein and produces molecules with the chemical and steric complementarity for the d structure of binding sites. this method was used to generate peptidomimetic inhibitors of thermolysin, hiv protease and pepsin. by using the x-ray crystal structures of pepstatin-pepsin complexes, growmol predicted therapeutic peptidomimetics against the aspartic proteases. the algorithm created some cyclic inhibitors bridging the side chains of cysteine residues in the pl and p inhibitor subsites. the binding modes were checked using x-ray crystallography. , ludi is another interesting software referring to the de novo methodology. by using natural and non-natural amino acids as building blocks, the software designed peptidomimetics against renin, thermolysin and elastase. conformational flexibility of each novel peptidomimetic was searched through sampling the multiple conformers of each amino acid. peptide-driven pharmacophoric hypothesis is the most perceptive computational technique discovered in the peptidomimetics design. the method is especially useful when the x-ray structures of protein-protein complexes exist. the main idea is to adapt the hot spot concept into the associated pharmacophoric feature concept. with a pharmacophorebased virtual screening process, this strategy can determine novel type mimetics. in fact, the side chains of each amino acid can be simply categorized based on the conventional pharmacophoric characteristics, such as hydrogen bond donors and acceptors, aromatic ring and charged and hydrophobic centers. for example, in a report, pharmacophore model directed synthesis of the non-peptide analogs of a cationic antimicrobial peptide identified an anti-staphylococcal activity. to make a pharmacophore hypothesis, a model of rna iii-inhibiting peptide (rip), a well-known heptapeptide inhibitor of the staphylococcal pathogenesis, was utilized. through the virtual screening of , commercially available small molecules based on the rip-based pharmacophore, hamamelitannin was discovered as a non-peptide mimetic of rip. hamamelitannin is a tannin derivate extracted from hamamelis virginiana. , in another study, two rounds of in silico screening were performed to discover potential peptidomimetics able to mimic a cyclic peptide (cyclo- [cpfvktqlc] ) that is known to bind the anb integrin receptor. at the end of the process, the most potent representatives were at least , times better than the original cyclopeptide (around mm). in a prosperous instance, virtual screening was done by using multi-conformational forms of a large commercial library. a target-based pharmacophoric model mapped the cd -binding site on hiv- gp . the pharmacophore hypothesis was made based on a homology model of the protein cavity. in a cell-based assay, two of the top scoring molecules were detected as micromolar inhibitors of hiv- replication. computer-aided design of amino acid-based therapeutics the pharmacophore-based screening was used to find the novel alzheimer's therapeutics as mimetics of neurotrophins. the therapeutic utilization of neurotrophins might be restricted because of several deficiencies such as its reduced central nervous system penetration, decreased stability and potency to enhance neuronal death through interaction with the p ntr receptor. the mimetism of particular nerve growth factor domains could inhibit neuronal death. peptidomimetics of the loop and loop domains of nerve growth factor can prevent neuronal death induced by p ntr-dependent and trk-related signaling. in another study, a full-computational pharmacophorebased approach assessed the fda-approved drugs as valuable candidates to inhibit protein-protein interactions. peptide structures were designated in terms of pharmacophores and searched against the fda-approved drugs to detect same molecules. the top ranking drug matches contained several nuclear receptor ligands and matched allosterically to the binding site on the target protein. the top ranking drug matches were docked to the peptide-binding site. the majority of the top-ranking matches presented a negative free energy change upon binding that was comparable to the standard peptide. geometry similarity method geometry similarity methods create a geometric similarity between non-peptide templates and peptide patches. in a study, the supermimic tool was developed to recognize peptide mimetics. in the program, a complex library of peptidomimetics composed of several protein structure libraries has been deposited. moreover, supermimic includes the d-peptides, synthetic components (reported as betaturn or gamma-turn mimetics) and peptidomimetic ligands obtained from the pdb. in the program, the searching process allows scanning a library of small molecules that mimic the tertiary structure of a query peptide followed by scanning of a protein library where a query for small molecule can adopt into the backbone. , sequence-based method recently, a method has been developed to rank peptide compound matches that are limited to short linear motifs in proteins and compounds with amino acid substituents. the algorithm allows mapping the side chain-like substituents on every compound of a large chemical library. the complete molecule can be signified by a short sequence, and each fragment in the molecule can be represented as a distinct letter abbreviation. a cross-search between the pubchem database (about . million molecules) and a non-redundant collection of , peptides obtained from pdb demonstrated that the algorithm can be useful for high-throughput measurements. to recognize a true positive, the method explored identified protein motifs against the national cancer institute developmental therapeutic program compound database. in another study, the similarity of amino acid motifs to compounds web server was developed to ease screening of identified motif structures against bioactive compound databases. the methodology was reported to be efficient since the compound databases were preprocessed to maximize the accessible data, and the necessary input data was minimal. in similarity of amino acid motifs to compounds, motif matching can be full or partial that may decrease or enhance the number of potential mimetics, respectively. using a novel search algorithm, the web service can perform a fast screening of known or putative motifs against ready compound libraries. the classified results can be examined by linking to appropriate databases. , fragment-based method replacement with partial ligand alternatives through computational enrichment is a fragment-based approach. by using structures of peptide-bound proteins as design anchors, the program can computationally find a non-peptide mimetic for specific determinants of known peptide ligands. hybrid peptide-driven shape and pharmacophoric method development and application of strategies for pharmacophore modeling indicate that the medicinal chemistry community has broadly accepted the intuitive nature of the pharmacophore concept. besides, shape complementarity has been identified as a significant element in the molecular identification between ligands and their targets. in virtual screening efforts, using the pharmacophore-and shape-based techniques distinctly may increase the rate of false-positive results. therefore, incorporating both pharmacophore-and shape-matching techniques into one program can potentially diminish the rate of false positives. recently, to discover novel peptidomimetics, a weboriented virtual screening tool named pepmmsmimic was developed to pool the conventional pharmacophore matching with shape complementarity. a library of million conformers were extracted from . million commercially available chemicals and gathered in the mmsinc database. the database was used as a skeleton to develop farhadi and hashemian pepmmsmimic. in the pepmmsmimic interface, the d structure of a protein-bound peptide is used as an input. then, chemical structures able to mimic the pharmacophore and shape similarity of the original peptide are proposed to involve in the protein-protein recognition. a list of in silico methods used to design potential peptidomimetics along with their strengths and weaknesses is presented in table . overall, design and development of therapeutics are tedious, expensive and time-consuming procedures. therefore, using modern approaches including computer-aided design methods can lessen the examination phase, price and failure of therapeutics discovery. computational methods used to design amino acid-based therapeutics can increase the range of available biotherapeutics. benefiting from the dramatic advance in bioinformatics, computational tools can be used to find and develop therapeutic proteins, peptides and peptidomimetics. , moreover, using the computational tools decrease the cost of therapeutics development, from concept to market, by up to %. however, in the computational protein designing, there are some challenges such as our inadequate knowledge of folding and physical forces that stabilize protein structures. moreover, sequences and local structures have many degrees of freedom that can complicate the sequence search. therefore, there is a requirement for effective methods to find sequences related to a particular structure and measure essential protein folding criteria. overall, in silico design of amino acid-based therapeutics includes many challenges that should be removed to improve the overall performance of the design processes. for example, although structure determination of all disease-related proteins through crystallography and nmr is a laborious task, it is necessary to gather much structural information of peptide-protein interactions. besides, development of vigorous algorithms to calculate protein-protein binding energies is essential. the estimation of binding constant between two macromolecules with an appropriate speedaccuracy tradeoff needs millisecond scale molecular dynamics. moreover, understanding of both protein-protein and protein-peptidomimetics recognition processes in a molecular level can be improved using higher accurate force fields such as quantum mechanical polarizable force. in recent years, there are growing examples on the approval of monoclonal antibodies (therapeutic antibodies) by the fda for treatment of various diseases. this important area of amino acid-based therapeutics has been covered in more depth elsewhere. , for more explanation about the theorems and details of antibody informatics for drug discovery as well as the computer-aided antibody design, readers are referred to study previous reports. , the authors report no conflicts of interest in this work. computer-aided design of amino acid-based therapeutics what is the future of targeted therapy in rheumatology: biologics or small molecules protein therapeutics: a summary and pharmacological classification in silico methods for design of biological therapeutics constructing novel chimeric dna vaccine against salmonella enterica based on sopb and groel proteins: an in silico approach in silico phylogenetic analysis of vibrio cholera isolates based on three housekeeping genes designing of complex multi-epitope peptide vaccine based on omps of klebsiella pneumoniae: an in silico approach theoretical and computational protein design annu evaluation of in silico protein secondary structure prediction methods by employing statistical techniques inhibition of mycobacterial cyp enzyme by sesamin and β-sitosterol: an in silico and in vitro study theory of protein folding: the energy landscape perspective toward an outline of the topography of a realistic protein-folding funnel artificial diiron enzymes with a de novo designed four-helix bundle structure computer-enabled peptide drug design: principles, methods, applications and future directions docking small peptides remains a great challenge: an assessment using autodock vina empirical estimation of local dielectric constants: toward atomistic design of collagen mimetic peptides recent work in the development and application of protein-peptide docking rational design of peptide drugs: avoiding aggregation computational peptidology: a new and promising approach to therapeutic peptide design strategies employed in the design and optimization of synthetic antimicrobial peptide amphiphiles with enhanced therapeutic potentials multifaceted roles of disulfide bonds. peptides as therapeutics peptidomimetics, a synthetic tool of drug discovery an in silico pipeline for the design of peptidomimetic proteinprotein interaction inhibitors (order no. ) natural products as sources of new drugs over the last years diversity-oriented synthesis of macrocyclic peptidomimetics structure-based design, synthesis, and biological evaluation of peptidomimetic sars-cov clpro inhibitors advances in amino acid mimetics and peptidomimetics a hierarchical approach to peptidomimetic design mimicking peptides… in silico peptidomimetic design rational design for peptide drugs peptidomimetics as a cutting edge tool for advanced healthcare an unusual functional group interaction and its potential to reproduce steric and electrostatic features of the transition states of peptidolysis low molecular weight, non-peptide fibrinogen receptor antagonists neurotrophin small molecule mimetics: candidate therapeutic agents for neurological disorders design of peptides, proteins, and peptidomimetics in chi space molecular technology. designing proteins and peptides molecular engineering: an approach to the development of general capabilities for molecular manipulation x-ray versus nmr structures as templates for computational protein design high-resolution protein design with backbone freedom prediction of protein-protein interface sequence diversity using flexible backbone computational protein design backbone flexibility in computational protein design de novo computational design of retro-aldol enzymes one fold with many functions: the evolutionary relationships between tim barrel families based on their sequences, structures and functions search and sampling in structural bioinformatics the dead-end elimination theorem and its use in protein side-chain positioning application of a self-consistent mean field theory to predict protein sidechains conformation and estimate their conformational entropy design of protein-interaction specificity gives selective bzip-binding peptides computational methods for protein design and protein sequence variability: biased monte carlo and replica exchange exploring the conformational space of protein side chains using dead-end elimination and the a* algorithm side-chain and backbone flexibility in protein core design dead-end elimination with a polarizable force field repacks pcna structures improved prediction of protein side-chain conformations with scwrl trading accuracy for speed: a quantitative comparison of search algorithms in protein sequence design using self-consistent fields to bias monte carlo methods with applications to designing and sampling protein sequences computational design and characterization of a monomeric helical dinuclear metalloprotein statistical theory of combinatorial libraries of folding proteins: energetic discrimination of a target structure achieving stability and conformational specificity in designed proteins via binary patterning photophysics of a fluorescent non-natural amino acid: p-cyanophenylalanine an expanded eukaryotic genetic code a "solvated rotamer" approach to modeling watermediated hydrogen bonds at protein-protein interfaces rotamer libraries in the st century improved side-chain prediction accuracy using an ab initio potential energy function and a very large rotamer library potential energy functions for protein design de novo design of foldable proteins with smooth folding funnel: automated negative design and experimental verification assembly of protein tertiary structures from fragments with similar local sequences using simulated annealing and bayesian scoring functions structure by design: from single proteins and their building blocks to nanostructures computational de novo design and characterization of a four-helix bundle protein that selectively binds a nonbiological cofactor using α-helical coiled coils to design nanostructured metalloporphyrin arrays kemp elimination catalysts by computational enzyme design de novo design of a βαβ motif high-resolution structural and thermodynamic analysis of extreme stabilization of human procarboxypeptidase by computational protein design design of a novel globular protein fold with atomic-level accuracy novel peptide-specific quantitative structure activity relationship (qsar) analysis applied to collagen iv peptides with antiangiogenic activity development of an informatics platform for therapeutic protein and peptide analytics two-level qsar network ( l-qsar) for peptide inhibitor design based on amino acid properties and sequence positions recent development of peptide drugs and advance on theory and methodology of peptide inhibitor design predicting the affinity of epitope-peptides with class i mhc molecule hla-a* : an application of amino acid-based peptide prediction a brief overview of antimicrobial peptides containing unnatural amino acids and ligand-based approaches for peptide ligands machine learning assisted design of highly active peptides for drug discovery pep-fold: an updated de novo structure prediction server for both linear and disulfide bonded cyclic peptides in silico predictions of d structures of linear and cyclic peptides with natural and nonproteinogenic residues long-timescale molecular dynamics simulations of protein structure and function how fastfolding proteins fold bond distances in polypeptide backbones depend on the local conformation identification of tetrapeptides from a mixture based positional scanning library that can restore nm full agonist function of the l p, i t, i s, a v, c y, and c r human melanocortin- polymorphic receptors (hmc rs) the protein data bank protein design with fragment databases pepbind: a comprehensive database and computational tool for analysis of protein-peptide interactions the structural basis of peptide-protein binding strategies protein-peptide interactions adopt the same structural motifs as monomeric protein folds highly flexible protein-peptide docking using cabs-dock computer-aided design of amino acid-based therapeutics virtual screening for potential inhibitors of ctx-m- protein of klebsiella pneumoniae in silico panning for a non-competitive peptide inhibitor comparative evaluation of eight docking tools for docking and virtual screening accuracy in silico designing of peptide inhibitors against pregnane x receptor: the novel candidates to control drug metabolism computation of the binding of fully flexible peptides to proteins with flexible side chains a flexible docking procedure for the exploration of peptide binding selectivity to known structures and homology models of pdz domains dynadock: a new molecular dynamics-based algorithm for protein-peptide docking including receptor flexibility structure-based prediction of proteinpeptide specificity in rosetta pepcrawler: a fast rrt-based algorithm for high-resolution refinement and binding-affinity estimation of peptide inhibitors rosetta flexpepdock ab-initio: simultaneous folding, docking and refinement of peptides onto their receptors sub-angstrom modeling of complexes between flexible peptides and globular proteins anchordock: blind and flexible anchordriven peptide docking a unified conformational selection and induced fit approach to proteinpeptide docking cabs-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site galaxypepdock: a proteinpeptide docking tool based on interaction similarity and energy optimization predicting peptide structures in native proteins from physical simulations of fragments mechanism of fast peptide recognition by sh domains binding free energy landscape of domain peptide interactions molecular dynamics simulations of pro-apoptotic bh peptide helices in aqueous medium: relationship between helix stability and their binding affinities to the anti-apoptotic protein bcl-xl structural and dynamic determinants of protein-peptide recognition quantitative sequenceactivity model (qsam): applying qsar strategy to model and predict bioactivity and function of peptides, proteins and nucleic acids recent advances in qsar and their applications in predicting the activities of chemical molecules, peptides and proteins for drug design comprehensive comparison of eight statistical modelling methods used in quantitative structure retention relationship studies for liquid chromatographic retention times of peptides generated by protease digestion of the escherichia coli proteome prediction of mhc-peptide binding: a systematic and comprehensive overview domain mediated protein interaction prediction: from genome to network calculation of absolute protein-ligand binding free energy from computer simulations prediction of binding affinities between the human amphiphysin- sh domain and its peptide ligands using homology modeling, molecular dynamics and molecular field analysis characterization of domain-peptide interaction interface: a generic structure-based model to decipher the binding specificity of sh domains why oppa protein can bind sequence-independent peptides? a combination of qm/mm, pb/sa, and structure-based qsar analyses characterization of pdz domain-peptide interactions using an integrated protocol of qm/mm, pb/sa, and cfea analyses computational design of peptide ligands exploring protein-peptide binding specificity through computational peptide screening multiple highly diverse structures complementary to enzyme binding sites: results of extensive application of a de novo design method incorporating combinatorial growth transformation of peptides into non-peptides. synthesis of computer-generated enzyme inhibitors towards the automatic design of synthetically accessible protein ligands: peptides, amides and peptidomimetics structure-based pharmacophores for virtual screening antimicrobial activity of small β-peptidomimetics based on the pharmacophore model of short cationic antimicrobial peptides small molecule inhibitors of hantavirus infection a dynamic target-based pharmacophoric model mapping the cd binding site on hiv- gp to identify new inhibitors of gp -cd protein-protein interactions alzheimer's therapeutics approved drug mimics of short peptide ligands from protein interaction motifs supermimic-fitting peptide mimetics into protein structures identification of potential small molecule peptidomimetics similar to motifs in proteins drug design, development and therapy web server to identify similarity of amino acid motifs to compounds (saamco) replace: a strategy for iterative design of cyclin-binding groove inhibitors swimming into peptidomimetic chemical space using pepmmsmimic mmsinc: a large-scale chemoinformatics database computational drug discovery developability assessment as an early de-risking tool for biopharmaceutical development antibody informatics for drug discovery computer-aided antibody design tumorhope: a database of tumor homing peptides drug-permeability and transporter assays in caco- and mdck cell lines pepx: a structural database of non-redundant protein-peptide complexes rosetta flexpepdock web server -high resolution modeling of peptide-protein interactions pdock: a new technique for rapid and accurate docking of peptide ligands to major histocompatibility complexes predicting peptide binding sites on protein surfaces by clustering chemical interactions protein-peptide complex prediction through fragment interaction patterns pep-sitefinder: a tool for the blind identification of peptide binding sites on protein surfaces vital: viterbi algorithm for de novo peptide design drug design, development and therapy : submit your manuscript | www.dovepress.com submit your manuscript here: http://www.dovepress.com/drug-design-development-and-therapy-journal drug design, development and therapy is an international, peerreviewed open-access journal that spans the spectrum of drug design and development through to clinical applications. clinical outcomes, patient safety, and programs for the development and effective, safe, and sustained use of medicines are the features of the journal, which has also been accepted for indexing on pubmed central. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/testimonials.php to read real quotes from published authors. key: cord- -jibmg ch authors: dunbar, r. i. m. title: structure and function in human and primate social networks: implications for diffusion, network stability and health date: - - journal: proc math phys eng sci doi: . /rspa. . sha: doc_id: cord_uid: jibmg ch the human social world is orders of magnitude smaller than our highly urbanized world might lead us to suppose. in addition, human social networks have a very distinct fractal structure similar to that observed in other primates. in part, this reflects a cognitive constraint, and in part a time constraint, on the capacity for interaction. structured networks of this kind have a significant effect on the rates of transmission of both disease and information. because the cognitive mechanism underpinning network structure is based on trust, internal and external threats that undermine trust or constrain interaction inevitably result in the fragmentation and restructuring of networks. in contexts where network sizes are smaller, this is likely to have significant impacts on psychological and physical health risks. the processes whereby contagious diseases or information propagate through communities are directly affected by the way these communities are structured. this has been shown to be the case in primates [ ] [ ] [ ] and has been well studied in humans in the form of epidemiological [ ] and information diffusion (opinion dynamics or voter) models [ ] . the ising phase state model (originally developed to describe the magnetic dipole moments of atomic spin in ferromagnetism) has been the workhorse of most of these models and of many of the models currently used to calculate the value of the r-number (or reproduction rate) used to drive current covid- management strategies. most early models were mean field models that assumed panmixia. however, human social networks are highly structured and small world: most people interact with only a very small number of individuals whose identities remain relatively stable over time. when it became apparent that the structure of networks could dramatically affect the flow of information (or infections) through networks [ , ] , structure began to be incorporated into epidemiological models [ ] [ ] [ ] [ ] [ ] . many of the best current models are 'compartmental models' which represent structure by the fact that a community consists of households or other small population units [ , ] . in effect, these use spatial structure as a proxy for social structure, which has the advantage of ensuring that the models compute easily. in reality, of course, it is people's interactions with each other that give rise to the spatial structure represented by households. while it is true that most (but not all) individuals see and interact with household or family members more often than with anyone else, in fact this dichotomizes what is in reality a continuum of interaction that flows out in ripples from each individual. these ripples create social layers of gradually declining amplitude that spread through the local community well beyond the household. my aim in this paper is to examine the social and psychological processes that underpin natural human sociality in order to better understand how these affect both network structure and the way information or diseases propagate through them. like all monkeys and apes, humans live in stable social groups characterized by small, highly structured networks. individuals do not interact with, let alone meet, everyone else in their social group on a regular basis: a very high proportion of their interactions are confined to a very small subset of individuals. these relationships are sometimes described as having a 'bonded' quality: regular social partners appear to be fixated on each other [ , ] . the mechanisms that underpin these relationships have important consequences for the dynamics of these networks. i will first briefly review evidence on the size and structure of the human social world. i will then explain how the cognitive and behavioural mechanisms that underpin friendships in all primates give rise to the particular form that human networks have. finally, i explore some of the consequences of this for information and disease propagation in networks, and how networks respond to external threats. humans have lived in settlements only for the past years or so, with mega-cities and nation states being at all common only within the last few hundred years. prior to that, our entire evolutionary history was dominated by very small-scale societies of the kind still found in contemporary hunter-gatherers. our personal social worlds still reflect that long evolutionary history, even when they are embedded in connurbations numbering tens of millions of people. table summarizes the sizes of egocentric personal social networks estimated in a wide variety of contexts ranging from xmas card distribution lists (identifying all household members) to the number of friends on facebook, with sample sizes varying between and a million individuals. the mean network size varies across the range - , with an overall mean of approximately . table also lists a number of studies that have estimated community size in a variety of pre-industrial societies as well as some contemporary contexts where it is possible to define a personalized community within which most people know each other on a personal level. these include the size of hunter-gatherer communities, historical european villages from the eleventh to the eighteenth centuries, self-contained historical communes, academic subdisciplines (defined as all those who pay attention to each other's publications) and internet communities. the average community sizes range between and , many with very large sample sizes, with an overall mean of approximately . christmas card distribution list . [ ] . [ ] . the value of approximately as a natural grouping size for humans was, in fact, originally predicted from an equation relating social group size to relative neocortex size in primates before this empirical evidence became available [ ] . this prediction had a % confidence interval of - , very close to the observed variance in the data. in primates as a whole (but not other birds and mammals), social group size is a function of neocortex volume, and especially the more frontal neocortex regions (the social brain hypothesis [ ] ). in the last decade, neuroimaging studies of both humans [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and monkeys [ , ] indicate that the relationship between personal social networks (indexed in many different ways) and brain size also applies within species at the level of the individual as well as between species. the social brain relationship arises because primates are unusual in that they live in relatively large, stable, bonded social groups [ ] . in contrast with the more casual groups (i.e. herds, flocks) of most mammals and birds, the problem with bonded groups is that they are, in effect, a version of the coupled oscillator problem. if animals' foraging and resting schedules get out of synchrony, . . some individuals will drift away when others go to rest, resulting in the fragmentation of the group [ , ] . individuals have to be willing to accept short-term costs (mainly in relation to the scheduling of foraging) in order to gain greater long-term benefits (protection from predators by staying together). maintaining spatial coherence through time is cognitively difficult. it depends on two key psychological competences that appear to be unique to the anthropoid primates: the ability to inhibit prepotent actions (a prepotent response is the tendency to take a small immediate reward in preference to waiting for a larger future reward) and the capacity to mentalize. inhibition depends on the volume of the brain's frontal pole [ ] , while mentalizing depends on a dedicated neural circuit known as the theory of mind network (also known as the default mode neural network) that integrates processing units in the brain's prefrontal, parietal and temporal lobes [ ] , supplemented by connections with the limbic system [ , ] . the frontal pole is unique to the anthropoid primates [ ] ; the default mode network that underpins mentalizing is also common to both humans and monkeys [ ] . maintaining group cohesion is not simply a memory problem (though it is commonly misunderstood as such). rather, it is one of predicting others' future behaviour under different conditions (e.g. knowing how others will respond to one's own actions) and being able to gauge the future reliability (trustworthiness) of other individuals [ , ] . this is much more costly in terms of both neural activity and neural recruitment than simple factual recall [ ] . in humans, the number of friends is directly correlated with mentalizing skills [ , ] , and mentalizing skills are, in turn, correlated with the volumetric size of the brain's default mode neural network [ , ] . the latter relationship has recently been shown to extend to primates as a whole [ ] . social networks have generally been viewed from two different perspectives. network analysts with a statistical physics background have tended to view them top-down as macroscopic phenomena (i.e. from above looking down on the spatial distribution of a population of nodes), whereas sociologists have tended to view them from below as egocentric networks (the individual's experience of that population). on the whole, the first group have tended to focus on large-scale patterns in very large networks, often with an emphasis on triadic closure (heider's structural balance theory [ ] ) as the glue that gives structure to a network; the second have focused on the micro-structure of individual's personal social networks, often focusing on the inner core of intimate friendships immediately beyond the simple triad. an important finding from the second approach has been that networks actually consist of a series of layers that correspond to relationships of different quality [ , , ] . seen from an egocentric point of view, the frequency with which an individual contacts the members of their network does not follow a conventional power law distribution but, on closer inspection, contains a series of plateaux. cluster analyses of very large datasets invariably reveal that these personal networks contain four layers within them (figure ). this gives the network a layered structure, where individual alters in a given layer are contacted with more or less similar frequency and there is a sharp drop-off in contact frequencies to the next layer. it turns out that, while there is some individual variation, these layers have quite characteristic sizes. moreover, when counted cumulatively, they have a very distinct scaling ratio: each layer is approximately three times the size of the layer immediately inside it (figure ). this layered structure in figure (referred to as a dunbar graph [ ] ) has been identified, with virtually the same numerical layer values, in surveys of network size, the calling patterns in national cellphone databases, science co-author networks and the frequencies of reciprocated postings in both facebook and twitter (table ). each layer seems to correspond to a very specific frequency of interaction (figure ), and these frequencies are remarkably consistent across media [ ] , suggesting both that they are hardwired and that communication media are substitutable. one way this structure might arise would be if the basal layer of five people represented, for example, a family or household, such that the next layer of consists of three families with an especially close relationship, and the -layer beyond that consisted of three of these trios. this [ ] . (b) optimal number of clusters identified by k-means clustering algorithm in three online datasets. reproduced from [ ] . pattern, however, is likely to reflect small-scale traditional communities; in more mobile, postindustrial societies, figure can arise simply a consequence of the patterns of interaction between individuals and need have no family-based (or spatial) underpinning to it at all. it is notable, nonetheless, that the same patterns emerge in either case, suggesting that there is an underlying universal constraint on how networks are constructed. this same pattern, with the same layer sizes, has also been identified in a number of topdown analyses of the social (or spatial) organization of human populations (table ), including hunter-gatherers, the size distribution of irish bronze age stone circles (as estimates of local population distribution), the sizes of residential trailer parks, the structure of modern armies, the size of communities of practice in the business world and even the patterns of alliance formation in massive online multiplayer games (moms). this pattern, with the same scaling ratio, has also been noted in the political organization of a large sample of historical city states [ ] . in fact, this layered structure with a consistent scaling ratio was first noted in an analysis of huntergatherer societies in the early s by johnson [ ] , who suggested that it was a 'span on control' management solution in response to internal stresses created as grouping size increases. more surprisingly, perhaps, these same numbers reappear in both the distribution of primate social group sizes [ ] and in the layered structure of groups for those mammals that live in multilevel social systems (mainly baboons, chimpanzees, elephants and dolphins) [ , ] (table ) . animal societies with stable groups do not extend beyond the -layer, but all the internal layers are present. the fact that these numbers are so consistent across so wide a range of species perhaps suggests that they may be the outcome of natural scaling effects due to the structural stresses that social groups incur as they grow in size in response to ecological demands. as a result, social evolution in primates [ ] occurs as a result of a stepwise increase in group size [ ] achieved by bolting together several basal subgroups to create successive layers rather than through a continuous adjustment of group sizes as in most birds and mammals. primate species achieve larger groups by delaying group fission that would normally act as a nonlinear oscillator to keep group size within a defined range around the local mean [ ] [ ] [ ] . the process thus seems to behave more like a series of phase transitions triggered by a natural fractionation process. although, in humans, there is remarkably little variation in both overall network size and layer sizes across samples, irrespective of sample size, sample origin and cluster detection algorithm, nonetheless within populations, there is considerable variation between individuals (figure a). some of this variation is due to sex (women tend to have larger inner layers than men [ but smaller outer layers [ ] ), some due to age (network and layer sizes are an inverted-jshaped function of age, peaking in the s- s [ , , ] ) and personality (extroverts have larger networks at all layer levels than introverts [ ] [ ] [ ] [ ] ). in addition, all human social networks are divided into two components, family and friends. although in small-scale societies, virtually everyone in your network is a member of your extended family, in the post-industrial world with our lower birth rates, the typical network is split roughly : between family and friends [ , , ] . however, it still seems that preference is given to family: those who come from large extended families have fewer friends [ , ] . in effect, family and friends form two largely separate subnetworks that are interleaved through the layers of the network, with a roughly even split in the two innermost -and -layers, friends predominating in the middle -layer and family predominating in the outer -layer [ ] . the latter seems to reflect the fact that friends are more costly to maintain in terms of time investment than family members [ ] , and hence survive less well in the outermost layer (see below). conventional top-down networks tend to focus on the degree of individual ego's, usually with some kind of cut-off to define how many primary contacts an individual has. irrespective of where the cut-off is taken to be, these relationships tend to be viewed as one-of-a-kind. dunbar graphs, by contrast, recognize that individuals have relationships of different quality with many individuals, which might be viewed as primary, secondary, tertiary, etc. relationships. the first will usually be a subset of the second. in this section, i provide a brief explanation of how the primate bonding process works. the main purpose is to stress that it is both complex and time-consuming. this will help explain some of the patterns we will meet in the following two sections where i discuss network dynamics and their consequences. primate social groups are implicit social contracts designed to ensure protection from predators and, secondarily, rival conspecifics through group augmentation selection effects [ ] . group-living is often mistaken for a cooperation problem, but it is in fact a coordination problem. cooperation problems invariably involve a public goods dilemma (cooperators pay an upfront cost), whereas a coordination problem does not (you are either in the group or not, and everyone pays the same simultaneous cost) [ ] . the problem animals have to solve is how to maintain group stability (i.e. coordination) in the face of the stresses that derive from living in close proximity [ , , ] which would otherwise cause the members to disperse (as happens in herd-and flock-forming species [ , ] . the primate solution to this problem is bonded relationships, since this ensures that individuals will maintain behavioural synchrony and stay together as a group. in primates, relationships are built up by social grooming [ ] . grooming is an exceptionally time-costly activity, with some species devoting as much as one-fifth of their entire day to this seemingly trivial activity [ ] . grooming creates a sense of reciprocity, obligation and trust (operationally, a form of bayesian belief in the honesty and reliability of an alter's future behaviour [ ] ). the layered structure of human social networks is a consequence of how we choose to distribute our social time around the members (figures and ). in both monkeys [ ] and humans [ , ] , the quality of a relationship (indexed by its likelihood of producing prosocial support when it is needed) depends directly on the time invested in it. however, the time available for social interaction is severely limited [ , , ] , and this forces individuals to make trade-offs between the benefits offered by relationships of different strength and the costs of maintaining those relationships. the process involved is a dual process mechanism that involves two separate mechanisms acting in parallel. one is a psychopharmacological mechanism acting at the emotional (raw feels) level that is mediated by the way social grooming triggers the brain's endorphin system (part of the pain management system); the other is the cognitive element that forms the core of the social brain itself [ ] . social grooming is often assumed to have a purely hygienic function. while it certainly has this effect, in primates it has been coopted to form a far more important function in social bonding. the action of leafing through the fur during grooming triggers the endorphin system in the brain [ , ] via a specialized neural system, the afferent c-tactile (or ct) fibres [ ] . these are highly specialized nerves that respond only to light, slow stroking at approximately . cm s − (the speed of hand movements in grooming), and nothing else [ ] . endorphin activation and uptake in the brain creates an opioid-like sense of relaxation, contentment and warmth [ , ] that seems to provide a psychopharmacological platform for bondedness [ , [ ] [ ] [ ] off which the second process, a cognitive sense of trust and obligation, is built. endorphins have a relatively short half-life (around . h), and so the system needs constant activation via grooming to maintain the requisite bonding levels, thereby making the system very time-costly. physical touch in the form of stroking and casual touch continues to form an important part of human social interaction and yields exactly the same endorphin effect [ ] as it does in primate grooming. however, physical contact has an intimacy that limits it mainly to the inner layers of our networks [ , ] . moreover, it is physically impossible to groom with more than one individual at a time with the same focused attention that seems to be important in its execution. this, combined with the constraints on time and the minimum time required to maintain a working relationship, ultimately places a limit on the number of relationships an animal can bond with using only this mechanism. in primates, this limits group size to about individuals (the upper limit on average species group size in primates [ ] ). groups larger than this are prone to fragmentation and ultimately to fission [ ] . in order to be able to increase group size, humans discovered how to exploit other behaviours that also trigger the endorphin system in a way that is, in effect, a form of grooming-at-adistance. these include laughter [ ] , singing [ ] , dancing [ ] , emotional storytelling [ ] , and communal eating [ , ] and drinking (of alcohol) [ ] , all of which trigger the endorphin system and do so incrementally when done in synchrony [ , ] . because they do not involve direct physical contact, more individuals can be 'groomed' simultaneously, thereby allowing a form of time-sharing that makes it possible to reach more individuals and so increase group size. the second component of this system is a cognitive mechanism. it centres around the knowledge of other individuals that can be built up by being in close contact. evolutionary studies of cooperation tend to view relationships as a form of debt-logging. such knowledge would not necessarily require frequent interaction since that can be done by third-party observation. rather, interacting closely with others allows an individual to get to know them well enough to predict their behaviour-to know that they really will come to your aid when you really need them, not because they owe you a favour but because they have a sense of obligation and commitment to you. in effect, it creates a sense of trust that acts as a rapid, intuitive (albeit imperfect) cue of reliability. in humans, this has been augmented by a capacity to build a more detailed 'picture' of another individual through conversation in a way that short circuits the need to invest excessive amounts of time in getting to know them. in other words, we can form a near-instantaneous impression of a stranger and use that as the basis for decisions about whether or not to engage with them. we do this by evaluating an individual's status on a set of largely exogenous cultural dimensions known as the seven pillars of friendship [ ] . the seven pillars are: language (or, better still, dialect), place of origin, educational trajectory, hobbies and interests, worldview (religious/moral/political views), musical tastes and sense of humour. the more of these we share in common with someone, the stronger the relationship between us will be and the more altruistic we will be to each other [ ] . this 'birds of a feather flock together' phenomenon is termed homophily [ ] . the seven pillars are cues of membership of the same community. in small-scale societies, they would identify an extended kinship group, where kin selection (the 'kinship premium' [ ] ) provides an additional guarantee of trustworthiness [ ] . in effect, they function as a cultural totem pole at the centre of the metaphorical village green on which the community can hang its hats-an emotional consensus of who we are as a community and how we came to be that way, a way of building up mutual trust. in the contemporary context, it still identifies your (now reduced) kin group, but it also seems to identify the small community where you spent your formative years-the period when you acquired your sense of who you are and what community you belong to. this is the community whose mores and behaviour you understand in a very intuitive way, and it is this understanding that determines your sense of how well you can trust its members. homophily in friendships has also been documented in respect of a number of endogenous traits, including gender [ , , , ] , ethnicity [ ] and personality [ , ] . gender has a particularly strong effect: approximately % of men's social networks consist of men, and approximately % of women's networks consist of women, with most of the opposite sex alters in both cases being extended family members. one reason for this is that the two sexes' style of social interaction is very different. women's relationships are more dyadic, serviced mainly by conversation and involve significantly more physical touch, whereas men's relationships are more group-based, typically involve some form of activity (sports, hobbies, social drinking) rather than conversation, and make much less use of haptic contact [ , , ] . men's friendships are also typically less intense, more casual and more substitutable than women's: women will often try to keep core friendships going long after one of them has moved away (e.g. through e-mail or facebook), whereas men simply tend to find someone else to replace the absent individual(s) in a rather out-of-sight-out-of-mind fashion. homophily enables interactions (and hence relationships) to 'flow', both because those involved 'understand' each other better and because they share strategic interests. between them, these emotional and cognitive components create the intensity of bonds that act as the glue to bind individuals together. human, and primate, friendships are often described in terms of two core dimensions: being close (desire for spatial proximity) and feeling close (emotional proximity) [ ] . between them, these ensure that bonded individuals stay together so that they are on hand when support is needed, thereby ensuring group cohesion through a drag effect on other dyads created by the ties in the network. since bonding, and the time investment this is based on, is in principle a continuum, this will not, of itself, give rise to the layered structure of a dunbar graph. to produce this, two more key components are needed: a constraint on time and the fact that friendships provide different kinds of benefits. the next section explains how this comes about. two approaches have been used to understand why social networks might have the layered structures they do. one has been to use agent-based models to reverse engineer the conditions under which the intersection between the time costs of relationships and the benefits that these provide give rise to structured networks. the other has been to solve the problem analytically as an optimal investment decision problem. in many ways, these represent top-down and bottom-up approaches, with the first focusing on the macro-level exogenous conditions that produce layered networks, and the second focusing on the micro-decisions that individuals have to make within these environments when deciding whom to interact with. building on the time budget models of dunbar et al. [ ] , sutcliffe et al. [ ] conceived the problem as the outcome of trying to satisfy two competing goals (foraging and socializing, where socializing provides a key non-foraging ecological benefit such as protection against internal or external threats) in a time-constrained environment. the aim was to identify which combination of strategies and cost/benefit regimes reproduced the exact layer structure of human social networks (for these purposes, taken as the , and layers) when agents try to maximize fitness (with fitness being the additive sum of five proximate components that reward social strategies differentially). in the model, agents armed with different strategic preferences for investing in close versus distant ties interacted with each other to form alliances that provided access to different resources. in each run, the population initially consisted of equal numbers of agents pursuing each of three investment preference strategies. following the conventional design for models of this kind, at the end of each round (generation) the % of agents with the lowest fitness were removed (died) and were replaced by offspring produced by the top % (duplicating the parent's strategy), thereby allowing the size of the population to remain constant but its composition to evolve. the model was allowed to run until the distribution of strategy types reached an equilibrium (typically cycles). the outcomes from greater than runs (in which weightings on the fitness functions were systematically varied) were sorted into clusters using k-mean cluster analysis with quantitative fit to the dunbar numbers as the criterion. numerically, strategies that favour having just a few strong ties dominate the fitness landscape, yielding the kinds of small monogamous or harem-based societies that are widely common in mammals. the second most common strategy was the one where agents have no preferences for forming close ties of any kind and instead form large, anonymous herds with no internal structure similar to those characteristic of herd-forming mammals. by contrast, multilevel social structures of the kind found in many primates, and especially those with the specific layer sizes of human social networks, were extremely rare (accounting for less than % of runs). they occurred only under a very limited set of circumstances, namely when a capacity for high investment in social time (i.e. sufficient spare time over that needed for foraging), preferential social interaction strategies, high mortality risk and steep reproductive differentials between individuals coincided. these are all traits characteristic of primates. the alternative approach considers the investment decisions themselves. tamarit et al. [ ] developed a one-parameter bayesian statistical urn model in which individuals choose to invest their limited social capital in relationships that provide different benefits (identified here as network layers). the model seeks to optimize the distribution of effort across relationship types where s k is the cost of a relationship in layer k∈ , . . . r, k is the number of alters assigned to layer k (with l = Σl k ), s is the total amount of resource (time) available, n is the total population size and µ is the lagrange multiplier associated with the constraint imposed by total resources. when the cost (i.e. the time investment that has to be made in a relationship) is a monotonic negative function of layer (as in figure ), this yields layered structures of exactly the kind found in human social networks-few close ties and many weak ones (the lower curve in figure ). however, it turned out that the structure of the network inverts (more close ties and few distant ties) when the lagrange multiplier falls below µ = (upper curve in figure ). one context in which this might happen is when the available community of alters to choose from is small, and so there is spare resource capacity per available alter. a comparison of migrant versus host communities in spain revealed that there is indeed a phase transition between conventionally structured networks (layers with a concave structure, i.e. a few close friends, more intermediate friends and many casual friends) to inverted networks (convex structures, in which ego has more close friends, some intermediate friends and only a few casual friends) at µ ≈ [ ] . this had not been noted previously because in the normal population, most individuals fall in the µ > region; the very small number falling in the µ < region had simply been viewed as statistical error. however, most migrants, who typically have fewer social opportunities, fall in the µ < region and so have inverted (convex) networks as a result, with only a small number having standard (i.e. concave) networks. this seems to suggest that, when surplus time is available, people prefer to invest more heavily in their closest ties rather than distributing their effort more evenly because the emotional and practical support offered by these inner layer ties is more valuable to them. this may explain the structure of primate groups where the inner core of strong ties typically represents a larger proportion of group size in species that live in smaller groups. the fact that the scaling ratio is consistently approximately in human (and primate) social networks raises the possibility that heider's structural balance triads might explain the layered structuring. this possibility was considered by klimek et al. [ ] who studied an ising-type coevolutionary voter model in a context where there are several social functions (say, friendship and business alliances) such that there are separate linked networks where most individuals occupy positions in each network. they showed that when the networks (i.e. functions) vary in rewiring probability (slow versus fast turnover in relationships), the single large network will eventually undergo a phase transition known as shattered fragmentation in which the community fractures into a large number of small subnetworks (or cliques). this happens only when one of the rewiring frequencies reaches a critically high level. when klimek et al. examined data from the pardus online mom game world, they found that the slow rewiring network (friendship) produced a weakly multimodal right-skewed, fattailed distribution with modal group sizes at - and approximately players with a few very large super-communities centred around or members. by contrast, the two fast rewiring networks (in this context, trading and communication functions) both underwent fragmentation into a large number of smaller subnetworks, with a single peak in group size at approximately in both cases, just as the model predicts. when the three networks were projected onto a single multidimensional mapping, very distinct peaks emerged in the distribution of group sizes in both model and data at approximately , and , much as we find in table . this seems to suggest that when triadic closure is a criterion for relationship stability and there is more than one criterion by which individuals can form ties, layering emerges naturally in networks through self-organizing fragmentation effects. so far, we have considered hierarchically inclusive layer structures. in these, the whole population is contained in the lowest layer, and the higher layers are created by successively bolting together, layer by layer, the groupings that make up each lower layer rather in the way military units are structured [ ] . most social networks seem to work like this. however, layers can also arise when some individuals are allocated positions of status, so that the members of the community are distributed across different layers with most individuals in the base layer and a few individuals in one or more higher layers. networks of this kind are characteristic of management structures and the kinds of social hierarchies found in feudal societies. layered structures of this kind seem to emerge rather easily when individuals differ in their willingness to compromise. dávid-barrett & dunbar [ ] used an agent-based model to investigate the processes of group coordination when a community has to converge on an agreed compass direction (a proxy for any communal action or opinion that has the advantage of allowing up to different values to be held rather than just two as in more conventional ising models), but one group member is so convinced they have the right answer that they refuse to compromise. if agents can assign weightings to each other on the basis of some preference criterion, however arbitrary, a layered structure emerges with an 'elite' subgroup that acts, in effect, as a management clique. multilevel structures of this kind have the advantage that they increase the speed with which decisions are adopted. multilayer networks are optimal when the costs associated with maintaining relationships, combined with the costs of information flow, are high. in such cases, a social hierarchy can be adaptive: when the hierarchy is steep, information needs to traverse fewer relationships (shorter path lengths), either because the elite effectively act as bridges between lower level groups (distributed management) or because the elite imposes its decisions on the individuals in the lower strata (dictatorial management). falling communication costs lead to a less steep hierarchy when socially useful information is evenly distributed, but to a steeper hierarchy when socially useful information is unevenly distributed. in human social networks, the layers have very characteristic interaction frequencies with ego ( figure ) . approximately % of all social effort (whether indexed as the frequency or duration of interaction) is directed to the five individuals in the closest layer, with another % devoted to the remaining members of the second layer. thus, % of social time is devoted to just people. comparable results have been reported from large-scale surveys in the uk [ ] and in china [ ] . this will inevitably affect the rate with which information, innovations or disease propagate through a network. however, network structure can speed up or slow down the rate of propagation, depending on the precise nature of the social processes involved. in a very early ( ) analysis of this, we used boyd & richerson's [ ] mean field ising model of cultural transmission to study what happens when these kinds of models are applied to structured networks [ ] . in the model, individuals acquire their information from n cultural parents, each of whom can differ in their cultural state. the model was run with a population of agents mapped as nodes on a * lattice wrapped on a torus so as to prevent edge effects. structure was imposed by allowing nodes to interact only with their eight closest neighbours on the lattice. on a regular lattice, these consist of two distinct sets: direct contacts (the four adjacent nodes on the main diagonals) and indirect contacts (the four corner nodes that can only be reached indirectly through the four adjacent nodes). in effect, these are equivalent to friends and friends-of-friends. at each generation, a node can change its cultural variant either by mutation or by imitation from one of its neighbouring nodes, with transition probabilities determined by a three-element vector specifying node-specific values of the boyd-richerson cultural inheritance bias functions (one reflecting the self-mutation rate, the other two the transmission, or copying, rates from the four 'friends' and the four 'friends-of-friends', with the proviso that all three sum to ). when the spatial constraint is dropped and everyone is allowed to interact with everyone else, the model replicates exactly the findings of the boyd-richerson [ ] cultural inheritance model. the population evolves to full penetrance by a mutant cultural variant initially seeded at just one node (i.e. with a probability of occurrence of just . ) in - generations. with spatial (or social) constraints in place, however, two important effects emerge. first, depending on the steepness of the inheritance bias functions, - % of mutant seedings went extinct before achieving full penetrance, apparently because they often became trapped in eddies at particular locations and could not break out before going extinct. in those runs where the mutant achieved full penetrance (i.e. all nodes became mutants), the time to penetrance was - generations for the same set of transmission biases. in other words, the mutant trait took far longer to spread through the population. once again, the time taken to break out of local eddies was the main reason for the much slower penetrance. the difference between these runs and those where the mutant went extinct depended on the balance between the stochastic rates at which new 'infected' clusters were created and died out. if a local extinction occurred early in the system's evolution when few mutant clusters had become established, global extinction was more likely (the classic small population extinction risk phenomenon in conservation biology [ ] ). changing population size or the number of cultural 'parents' had a quantitative effect, but did not change the basic pattern. the cumulative probability asymptotes at a network size of approximately , but the optimum number of alters is (identified by the point at which the curve begins to decelerate, defined by e − of the way down from the asymptote) since after this, the benefits of increasing network size diminish exponentially. reproduced from [ ] . (b) effect on reachability of removing nodes in different layers of egocentric twitter graphs: the larger the effect, the greater the disruption on information flow. the horizontal dotted lines demarcate /eth from the asymptote, and the vertical dotted line the optimal group size for information diffusion. data from [ ] . penetrance was slower if there were fewer cultural 'parents', for example, or if the population size was larger. to explore the rate at which information flows through a community, dunbar [ ] modelled the likelihood that ego (at the centre of the network) would hear about a novel innovation via direct face-to-face contact for communities of different size ( , , , , , and individuals), given the layer-specific rates of contact shown in figure (extrapolated out to the layer). the probability, p i , of hearing about an innovation seeded somewhere in a network with i layers is the conjoint probability of encountering any given individual in a network layer of size n i and the likelihood that any one of these individuals would have the trait in question (i.e. be infected), summed across all layers where n i is the number of individuals in the ith annulus (network layer), r k is the likelihood that any one of them will have the trait (here taken to be constant, and equivalent to r k = . ), c i [f f] is the likelihood of contacting any given individual face-to-face. figure a plots the results. the probability of acquiring information reaches an asymptotic value at a community size of approximately , with no further gain in the likelihood of hearing about an innovation as community size increases beyond this. the optimal community size for information transmission can be identified by the inflection point (the point at which the marginal gain begins to diminish). with a graph of this form, this occurs at the value on the x-axis when the asymptotic value on the y-axis is reduced by /e. this is at a community size of exactly . the gains to be had by increasing community size beyond approximately diminish exponentially and become trivial beyond a community size of approximately individuals. this was later confirmed by arnaboldi et al. [ ] who modelled information diffusion in actual twitter networks. figure importantly, and contrary to granovetter's [ ] well-known claim, it seems that it is the inner layers (stronger ties) that may have most impact on the likelihood of acquiring information by diffusion, not the outermost layer (weak ties). the outermost -layer (which is disproportionately populated by distant kin [ , ] ) presumably serves some other function such as a mutual support network [ ] . this finding appears to conflict with earlier analyses [ , ] that have emphasized the importance of weak links (long-range connections) in the rate at which infections or information are propagated in networks. the issue, however, hinges on which layers are counted as strong (short-range) and which weak (long-range). previous analyses, including granovetter himself, have tended to be unspecific on this point. if what he had in mind as weak ties was the -layer, then his claim holds; if he was thinking of the -or even layer, then it seems he was wrong. even so, it seems that the information value of -layer ties is considerably less than that of alters in the -and -layers, who are also likely to be considered more trustworthy sources of information. nonetheless, granovetter might still be right if either of two conditions hold. one is that the analyses considered only ego acquiring information by direct personal contact; the models did not consider the impact of upward information flow through the network from the source of the information towards ego. the other is that granovetter might have been right for the wrong reason: the function of networks is not information flow (or acquisition) but the provision of direct functional support such as protection against external threats or sources of economic support (a view which would accord better with the view of primate social systems elaborated in § ). in other words, as is the case in primate social systems, information flow is a consequence of network structure, not its driving force in terms of evolutionary selection [ ] . it may, nonetheless, be that the -layer provides the principal access channels to the global network beyond the individual's primary personal social sphere. this is suggested by an analysis that used m-polynomials derived from chemical graph theory to integrate dunbar graphs into the milgram small world 'six degrees of separation' phenomenon. the capacity to reach an unknown remote individual in - links is only possible if, at each step in the chain, the message-holder can access a large number of alters in their personal network [ ] . however, this analysis only considered versus network contacts, and significantly over-engineers the solution. further work is needed to explore the optimal network size and structure for transmission in more detail. a moot point, of course, is whether the capacity to send letters to a remote stranger is ever of any real functional value to us, or simply an amusing but unimportant by-product of the way personal networks interface with each other in global networks. one complicating aspect of real social networks not usually considered in these models is the fact that social subnetworks are characterized by a high level of homophily, especially in the inner layers. in other words, people's friends tend to resemble them on an array of social and cultural dimensions [ , , ] . analysing a large ( million users) cellphone dataset, miritello et al. [ ] differentiated, on the basis of calling patterns, two distinct phenotypes: 'social keepers' who focused their calls on a small, stable cohort of contacts (introverts?) and 'social explorers' who had a larger number of contacts with high churn (social butterflies, or extraverts?). each tended to exhibit a strong preference for its own phenotype. assuming that phone contact rates mirror face-to-face contact rates (as, in fact, seems to be the case [ , , ] ), explorers were more likely to contact an infected individual because they were more wide-ranging in their social contacts. keepers remained buffered by their small social radius for longer. this reinforces the suggestion made earlier that innovations frequently go extinct in structured networks because they get trapped in eddies created by network structuring and risk going extinct before they can escape into the wider world. the role of extraverts in facilitating information flow was also noted by lu et al. [ , ] in a study of networks parametrized by personality-specific contact rates from the community studied by [ ] . they found that information flow was more efficient if the network consisted of two distinct phenotypes (in this case, actual introvert and extravert personality types) than if all community members were of the same phenotype. in large part, this was because extraverts (those who opted to prioritize quantity over quality of relationships) acted as bridges between subnetworks, thereby allowing information to flow more easily through the network as a whole. much of the focus in network dynamics has been on disease propagation. in most models, networks are assumed to remain essentially static in structure over time. this may not always be the case, since network structure may itself respond to both internal threats (stress or deception) and external threats (such as disease or exploitation or attack by outsiders). this is because threats such as disease or exploitation cause a breakdown in trust and trust is, as we saw, central to the structure of social networks. other factors that might cause networks to restructure themselves include a reduction in the time available for social interaction, access to a sufficiently large population to allow choice [ ] or a change in the proportion of phenotypes (sex, personality or family size) when these behave differently. methods for studying networks that change dynamically through time have been developed [ ] , although in practice these typically reflect past change rather than how networks are likely to respond to future challenges. here, my concern is with how networks might change as a consequence of the internal and external forces acting on them. because of the way relationships are serviced in social networks ( § ), a reduction in time devoted to a tie results in an inexorable decline in the emotional strength of a tie, at least for friendships ( figure ) . note, however, that family ties appear to be quite robust in the face of lack of opportunity to interact. figure suggests that this effect happens within a matter of a few months (see also [ , ] ). saramäki et al. [ ] reported a turnover of approximately % in the network membership of young adults over an -month period after they had moved away from their home town, most of which occurred in the first nine months. a similar effect will occur when there is a terminal breakdown in a relationship. these seem to occur with a frequency of about % of relationships per year, though it is clear that some people are more prone to relationship breakdown than others [ ] . most such breakdowns occur because of a breakdown in trust [ , ] . although almost never considered in models of network dynamics, the division between family and friends can have significant consequences for the dynamics of networks, especially when comparing natural fertility (no contraception) with demographic transition fertility regimes (those that actively practise contraception). friendships require significantly more time investment than family relationships to maintain at a constant emotional intensity, especially so in the outer layers [ , ] , and because of this are more likely to fade (and to do so rather quickly) if contact is reduced [ ] ( figure ). family relationships, on the other hand, are more forgiving of breaches of trust and underinvestment. in addition, when family relationships breakdown, they are apt to fracture catastrophically and irreparably [ ] , creating structural holes. by contrast, most friendships die quietly as a result of reduced contact, in many cases because they are simply replaced by alternatives. this probably means that, when a network is under threat, friendship ties are more likely to be lost than family ties. this would seem to be born out by casual observation of the response to the covid- lockdown: virtual zoom-based family gatherings seem to be much more common than friendship-based meetings. under normal circumstances, the gaps left by the loss of a tie following a relationship breakdown are filled by moving someone up from a lower layer or by adding an entirely new person into the network from outside. saramäki et al. [ ] noted that, when this happens, the new occupant of the vacated slot is contacted with exactly the same frequency as the previous occupant, irrespective of who they are. it seems that individuals have a distinctive social fingerprint, and this fingerprint is very stable across time [ ] . however, if the opportunity for social interaction is restricted, or there is widespread breakdown in the level of trust (as when many people cease to adhere to social rules, or a culture of deception or antisocial behaviour evolves), then the inevitable response is for networks to fragment as individuals increasingly withdraw from casual contacts and focus their attention on those whom they trust most (normally the alters in the innermost layers of their network). iñiguez et al. [ ] and barrio et al. [ ] modelled the effect of two kinds of deception (selfish lies versus white lies) on network structure. selfish lies are those that benefit the liar, while white lies are those that benefit either the addressee or the relationship between the liar and the addressee (e.g. 'likes' on digital media). these two phenotypes differ radically in the effect they have on the relationship between the individuals concerned: the first will cause a reduction in the frequency of contact resulting in a fragmentation of the network, whereas the second often reinforces network cohesion. if networks shrink sufficiently under stress, they may invert (figure ). there is indirect evidence for this in the effect that parasite load has on the size of communities in tribal societies: these decline in size the closer they are to the equator (the tropics being the main hotspot for the evolution of new diseases), and this correlates in turn with a corresponding increase in the number of languages and religions, both of which restrict community size [ , ] . at high latitudes, where parasite loads tend to be low and less stable climates make long-range cooperation an advantage, community sizes are large, people speak fewer languages and religions tend to have wider geographical coverage, which, between them, will result in more extensive global networks [ , ] . similar effects have been noted in financial networks, where network structure between institutions that trade with each other also depends on trust. there has been considerable interest in how network structure might influence the consequences of contagion effects when financial institutions collapse. network structure can affect how shocks spread through networks of banks, giving rise to default cascades in ways not dissimilar to the way diseases propagate through human social networks. although well-connected banks may be buffered against shocks because of the way the effects are diluted [ ] [ ] [ ] much as a well-connected individuals may be buffered against social stresses, a loss of trust between institutions invariably results in the contraction of networks, associated with more conservative trading decisions and a greater reluctance to lend [ ] in ways reminiscent of social networks fragmenting in the face of a loss of trust. if effective network size (i.e. the number of ties an individual has) is reduced as a result of such effects, more serious consequences may follow at the level of the individual for health, wellbeing and even longevity. smaller social networks are correlated with increasing social isolation and loneliness, and loneliness in turn has a dramatic effect on morbidity and mortality rates. there is now considerable evidence that the number and quality of close friendships that an individual has directly affects their health, wellbeing, happiness, capacity to recover from surgery and major illness, and their even longevity (reviewed in [ , ] ), as well as their engagement with, and trust in, the wider community within which they live [ , ] . indeed, the effects of social interaction can even outweigh more conventional medical concerns (obesity, diet, exercise, medication, alcohol consumption, local air quality, etc.) as a predictor of mortality [ ] . most epidemiological studies have focused on close friends, but there is evidence that the size of the extended family can have an important beneficial effect, especially on children's morbidity and mortality risks [ ] . these findings are mirrored by evidence from primates: the size of an individual's intimate social circle has a direct impact on its fertility, survival, how quickly it recovers from injury, and ultimately its biological fitness [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it is worth noting that dunbar graphs, with their basis in trust, have been used to develop online 'secret handshake' security algorithms for use in pervasive technology (e.g. safebook [ , ] ). pervasive technology aims to replace cellphone masts by using the phones themselves as waystations for transmitting a call between sender and addressee. the principal problem this gives rise to is trust: a phone needs to be able to trust that the incoming phone is not intent on accessing its information for malicious purposes. safebook stores the phone owner's seven pillars as a vector which can then be compared with the equivalent vector from the incoming phone. a criterion can be set as to how many 'pillars' must match for another phone to be considered trustworthy. the dunbar graph has also been used to develop a bot-detection algorithm by comparing a node's network size and shape with that of a real human (i.e. a dunbar graph): this algorithm out-performs all other currently available bot-detection algorithms [ ] . we might ask what effects we might expect in the light of this from the lockdowns imposed by most countries in in response to covid- . i anticipate four likely effects. one is that if lockdown continues for more than about three months, we may expect to see a weakening of existing friendships, especially in groups like the elderly whose network sizes are already in age-dependent decline. since older people find it more difficult to make new friends, an increased level of social isolation and loneliness is likely to result, with consequent increases in the diseases of old age, including dementia and alzheimer. second, we may expect to see an increased effort to recontact old friends, in particular, immediately after lockdown is lifted. we already see evidence for this in telephone call patterns: if there is a longer than normal gap before an alter is called again, the next call is significantly longer than average as though attempting to repair the damage to relationship quality [ ] . third, the weakening of friendship quality can be expected (i) to make subsequent meetings a little awkward because both parties will be a little unsure of how they now stand when meeting up again and (ii) to result in some churn in networks where new friendships developed through street-based covid community groups are seen as more valuable (and more accessible) than some previous lower rank friendships. finally, we may expect the fear of coronavirus contagion (an external threat) to result in a reduction in the frequency with which some individuals (notably introverts and the psychologically more cautious) visit locations where they would come into casual contact with people they do not know. they may also reduce frequencies of contact with low-rank friends, and perhaps even distant family members, whose behaviour (and hence infection risk) they cannot monitor easily. this is likely to result in more inverted networks, as well as networks focused mainly on people who are more accessible. although this effect will weaken gradually with time, and network patterns are likely to return to pre-covid patterns within - months, some friendship ties may have been sufficiently weakened to slip over the threshold into the (acquaintances) layer. my aim in this paper has been to introduce a rather different perspective on the social structure of communities than that normally considered in disease propagation models, and to explain the forces that underpin real-world social networks. i have presented evidence to suggest that human social networks are very much smaller and more highly structured than we usually assume. in addition, network size and composition can vary considerably across individuals as a function of sex, age, personality, local reproductive patterns and the size of the accessible population. while casual contacts might be important for the spread of highly infectious diseases [ social time is actually devoted to no more than individuals and this will slow down rates of transmission if physical contact or repeated exposure to the same individual is required for successful infection. both external and internal threats can destabilize network ties by affecting the level of trust, 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heinz, dirk w title: structures and diseases date: journal: nat struct mol biol doi: . /nsmb - sha: doc_id: cord_uid: hydy v structural biology is making significant contributions toward an understanding of molecular constituents and mechanisms underlying human diseases at an atomic resolution, as discussed at the international murnau conference on structural biology of disease mechanisms held in september in murnau, germany. from its very beginning, biostructural research has not only provided tremendous breakthroughs in basic biological processes but also significantly contributed to the understanding of the molecular mechanisms underlying human disease. this was first exemplified when max perutz and colleagues elucidated the molecular pathology of human hemoglobin mutations leading to sickle cell anemia . more recently, three-dimensional structures of many human and pathogen proteins have served to guide drug design, with an increasing number of potential target structures determined in the context of structural genomics . beyond singleprotein approaches, structural biology is now en route toward a high-resolution picture of the cell, assessing protein-protein and protein-nucleic acid complexes at steadily increasing levels of complexity. it is clear that an ever more integrated understanding of the molecular mechanisms of human disease will emerge from this path, although a major challenge lies in translating such knowledge into therapeutic strategies. in early september , about structural biologists and biochemists met in the picturesque town of murnau, located near staffelsee lake in the bavarian alpine upland, to reflect on these questions and discuss recent biostructural data on the molecular determinants of human diseases, including microbial and viral infections, protein misfolding diseases, cancer and metabolic disorders. these topics were addressed during five scientific sessions, two poster sessions and ample time for discussions. the scientific part of the meeting was complemented by a traditional bavarian-style social program. the meeting started out with the "murnau lecture" held by wim hol (university of washington, seattle), who gave an impressive overview of the mechanisms of maturation and activity of cholera toxin. with the example of the medical structural genomics of pathogenic protozoa (msgpp) program, he also demonstrated the power of integrating structural biology and biophysical screening approaches to quickly obtain new lead structures for potential drug targets . the first session, on infectious diseases, was opened by gabriel waksman (university college london and birkbeck college), who investigates the assembly process of escherichia coli p pili, structures crucial for bacterial virulence. combining structural and biophysical methods, waksman and colleagues have shown that the donor strand-exchange mechanism of the p pilus assembly proceeds through a 'zip in-zip out' process, involving a transient intermediate complex with a key role in subunit ordering and biogenesis termination , . this approach also led to the characterization of pilicides, small-molecule inhibitors of pilus formation that interrupt the interactions between the chaperone and the usher , a noncooperative event in the cooperative assembly process. another surface structure involved in virulence of gram-negative pathogens such as shigella flexneri and yersinia pestis is the type iii secretion system (t ss). using em, x-ray crystallography and molecular modeling, steven johnson (university of oxford) presented the first near-atomic model of a t ss needle and models of the proteins associated with the needle tip , with the structural basis for the regulation of t ss assembly under investigation. erec stebbins (rockefeller university) presented a wealth of structural data on the mechanisms of action of t ss effectors, such as the yersinia protein kinase a (ypka), a guanine nucleotide dissociation inhibitor (gdi) for rac gtpase that disrupts the actin cytoskeleton of the host cell . deshmukh gopaul (institute pasteur) presented data on integron integrases, enzymes that mediate recombination between short symmetric dna sequences and are thus involved in genetic information exchange between bacteria. the structure of such an enzyme from vibrio cholerae bound to dna shows that it recognizes dna structure rather than a specific sequence . by exploring cell-wall biosynthesis, gunther kern and gautam sanyal (astrazeneca) showed that glutamate racemases are suitable targets for narrow-spectrum antimicrobial agents, which are sorely needed against hospitalacquired infections . hartmut niemann (helmholtz centre for infection research and university of bielefeld) showed how the protein inlb from listeria monocytogenes exploits the signaling pathways of the receptor tyrosine kinase and protooncogene met to promote bacterial uptake by the host cell. they find that inlb functionally mimics the natural ligand hepatocyte growth factor/scatter factor (hgf/sf), albeit binding met at a different domain. the structure also provides insight into the activation mechanism of met, an important cancer drug target . stephen matthews (imperial college london) presented the latest results on host-cell recognition by the protozoan parasite toxoplasma gondii, which secretes microneme proteins (mics) to attach to and penetrate host cells . starting the session on viral diseases, rolf hilgenfeld (university of lübeck) reviewed the work from his laboratory on proteases of rna viruses, such as severe acute respiratory syndrome (sars) coronavirus and coxsackievirus b , and also highlighted recent structural data on falcipain- from plasmodium falciparum, discussing implications for the design of active-site directed and allosteric inhibitors for these cysteine proteases . young do kwon from peter kwong's group (us national institutes of health) shifted the focus to hiv- , whose ability to evade the human immune system is a major obstacle for vaccine development. the binding site for human receptor cd on viral gp is accessible, but most antibodies directed to this site are not neutralizing. kwon compared the structure of gp in complex with non-neutralizing antibody f and with neutralizing antibody b (ref. ; fig. ), revealing that, upon cd binding, a hydrophobic surface in gp becomes exposed, to which the non-neutralizing antibody f binds. dennis bamford (university of helsinki) explored the architectural principles of capsids from viruses that infect various hosts from bacteria to humans to suggest that early cells were infected with many different viruses and only a limited number of folds have been selected to assemble viable virus coats . eloise mastrangelo from martino bolognesi's group (university of milan) presented work on the ns proteasehelicase from kunjin virus, a flavivirus affecting livestock and man. small-angle x-ray scattering (saxs) data reveal that domain rearrangements upon rna binding may explain the unwinding efficiency of ns (ref. ) . winfried weissenhorn (european molecular biology laboratory grenoble) presented the three-dimensional structure of the rabies virus nucleoprotein-rna complex at . -Å resolution, revealing how these viruses compact and protect their genomes . various pathogens are efficiently cleared from the bloodstream by the complement system, an important part of innate immunity. the complement protein c in its activated form (c b), the subject of recent hot debate among the protein crystallographic community, binds to pathogens and tags them for phagocytosis via the complement receptors (crig). christian wiesmann (genentech) presented the structure of the c b-crig complex, revealing the dramatic structural rearrangements that take place during complement activation . wiesmann and colleagues further demonstrated that crig inhibits alternative pathway convertases, a finding with implications for the development of therapeutics targeting the complement system, which is involved in various human diseases. the session on protein misfolding was started out by roland riek (salk institute), who recently returned to the swiss federal institute of technology (eth, zurich). combining solid-state nmr, em and hydrogen-deuterium exchange methods, riek has identified fibrillogenic sequences in various proteins, including the amyloid peptide aβ - implicated in alzheimer's disease . studies with aβ - mutants figure the site on hiv- gp (gray) of interaction with the cd receptor (yellow) represents a conserved accessible surface on hiv- , and many commonly elicited antibodies compete with cd for binding to gp . however, most of these are weakly neutralizing and relatively impotent against primary hiv- isolates. one exception is the b antibody: whereas all of the cd binding site ligands seem to have an extended loop tipped by a hydrophobic residue (red), b recognizes gp in a slightly different way than cd does. these structures might help design an immunogen that is able to elicit more b -like antibodies. figure indicate that the toxicity of aβ - relates to the morphology of the aggregate formed. riek also discussed the use of amyloid fibrils in nanotechnology as a deposit form for the slow release of bioactive peptides. marcus fändrich (leibniz institute, jena) reported the three-dimensional em reconstruction of an aβ - amyloid fibril in collaboration with niko grigorieff (brandeis university). at -Å resolution, the presented em map deviates significantly from previous models, with an entirely different fibril cross section. on the basis of fibril classification results, fändrich also demonstrated that aβ - fibrils can be vastly heterogeneous, which is important when considering the biological or structural properties of a given amyloid sample. luigi vitagliano (l' istituto di biostrutture e bioimmagini and consiglio nazionale delle ricerche, naples) presented insights on fibril models from molecular dynamics calculations, starting from the crystal structure of hexa-or heptapeptides from eisenberg's laboratory (see below) and showing that the minimally stable oligomer was a pentamer of hexapeptides, with one peptide significantly shielded by the other four . christian betzel (university of hamburg) presented work on aggregated prion proteins, using a cellfree assay that converts cellular prp into an isoform similar to its infectious form, prpsc, by mimicking oxidative stress, with saxs analysis providing structural insights into the mechanism of oligomerization . the session was concluded by a superb presentation by david eisenberg (university of california los angeles), who gave a brief overview of early fibril models derived from cross-β-diffraction images before moving on to recent work on amyloidogenic peptides. on the basis of about crystal structures of amyloid peptides determined in the eisenberg laboratory , computational approaches were developed to predict the amyloid propensity of peptide stretches in proteins, identifying lvealyl from human insulin as a potentially fibrillogenic sequence. in summary, this session gave a good overview of the recent achievements in this field. although it might be a long way until a drug against the disease discovered by aloys alzheimer is developed and available to patients, it is truly remarkable how much has been learned about a process that a few years ago was the subject of wild speculations. alan fersht (university of cambridge) opened the session on cancer, reporting on the tumor-suppressor protein p , which is inactive in about half of all human cancers, in many cases because of mutations that lower the thermal stability of the core domain to below body temperature. fersht's group has designed compounds to rescue the function of such p mutants as a potential cancer therapeutic strategy , with the crystal structures of oncogenic p mutants revealing cavities that could be appropriate targets for drugs designed to chemically rescue p function. finally, the combination of saxs, em and nmr studies, together with previous crystallographic data, resulted in the complete architecture of the p tetramer, which includes large intrinsically unstructured regions . holger rehmann (university medical centre utrecht) reported on the structural basis of the regulation of the guanine nucleotide exchange factor epac by camp . alfred wittinghofer (max-planck institute, dortmund) has applied the detailed understanding of the gtpase reaction of ras to devise potential strategies for cancer therapy. wittinghofer's group found a small molecule of undisclosed structure that could induce activity of an oncogenic inactive ras variant in vitro . guillermo montoya (spanish national cancer center, madrid) reported the molecular basis of substrate recognition by polo-like kinase (plk ) and its implications for centrosomal localization . claus kuhn (gene center, munich) reported on the functional architecture of yeast rna polymerase i, which carries out the synthesis of ribosomal rna, a question that was unraveled by combining cryo-em, x-ray crystallography and homology modeling. biochemical and genetic experiments nicely complemented the structural data, showing how rna polymerase i deviates in structure and function from rna polymerase ii . finally, titia sixma (netherlands cancer institute, amsterdam), out of a collaboration with andrea pichler (medical university vienna) and frauke melchior (university of göttingen), reported the crystal structure of a sumoylated e , which showed that sumoylation affects the e enzymatic activity by modulating e 's interaction with e and that recognition of the sumoylation site depends on the surrounding structure of the site. finally, she showed that sumo can bind to different sites on ubc , depending on whether a covalent thioester-linked complex, a modified lysine or a non-covalent complex is formed . the final session started with structures of extracellular receptors involved in metabolic control and cardiovascular disease. michael lawrence (walter and eliza hall institute) presented the . -Å crystal structure of the insulin receptor ectodomain in complex with four fabs and an insulin mimetic peptide , thereby providing the first view of the spatial arrangement of low-and high-affinity insulin binding sites. günter fritz (university of konstanz) presented the unpublished structure of the ligand binding domain of rage, a multiligand receptor for advanced glycation end products, s proteins, hmgb and amyloid-β, whose activation is key to numerous chronic diseases such as diabetes, inflammation, arteriosclerosis and neurodegeneration, making it a potential therapeutic target , . armin ruf (hoffmann-la roche) provided a view on the structure-guided design of two newly identified classes of pparα/γ dual agonists whose profile seems well suited for addressing both hyperglycemia as well as the enhanced cardiovascular risk of diabetic patients . annalisa pastore (medical research council, london) shifted the focus of the session to rare diseases, with her recent findings on the anomalous expansion of polyglutamine motifs as a basis for neurodegenerative misfolding diseases . antti haapalainen (university of oulu) presented structural and functional studies of the human mitochondrial acetoacteyl-coa thiolase t , whose loss-of-function mutations result in severe ketoacidosis . markus wahl (max-planck institute, göttingen) combined structural and functional studies in search of a molecular basis for retinitis pigmentosa (rp). the data support a model in which prp , a spliceosomal factor whose mutations cause the severe rp form, serves as a scaffold for the assembly of other factors such as the dead box protein brr and gtpase snu , and show that viable rp -related mutations weaken but do not abolish these interactions . ryota kuroki (japan atomic energy agency) presented recent structural studies on the complex of human granulocyte colony-stimulating factor (gcsf), a cytokine used for treatment of granulopenia, with its receptor. although various stochiometries had been proposed for this assembly, kuroki's work underscores the relevance of the : complex, a result that is in line with thermodynamic and mutational analyses . roger williams (medical research council, cambridge) closed the session with a comprehensive overview of recent structural results on the cellular escrt machinery , which mediates the trafficking of monoubiquitinated proteins to lysosomes via the multivesicular budding (mvb) pathway. this process has a role in the downregulation of cell-surface receptors and the budding of hiv and other retroviruses. williams discussed the molecular understanding reached for many components of the mvb pathway, their molecular assemblies and the sorting signal ubiquitin. major challenges remain in understanding the higher-level molecular organization of the escrt lattice together with a molecular mechanism for vesicle budding. solution of these challenges will require the application of hybrid methods to close the resolution gap between x-ray structures and current em reconstructions. in summary, the murnau conference featured presentations and posters on many molecular aspects underlying human disease. several of the presentations demonstrate how an advanced molecular understanding of disease-relevant factors can open new strategies for both the design of interfering small molecules and screening procedures for such compounds. in other cases, the complexity of supramolecular assemblies solved by structural methods seems to overwhelm our current ability to translate the emerging higher-order molecular view into concrete options for therapeutic intervention. as demonstrated by hol, waksman, fersht, wittinghofer, riek and many others, this step can come within reach only when structural biology research is tightly integrated with biophysical, biochemical and cellular studies. this strategy of integrating functional and structural studies of ever more complex cellular processes in search of new therapeutic entry points was reflected in many presentations and discussions at murnau. for the upcoming meeting in , it may be of interest to focus on the interface of structural and chemical biology, where structural information is used to reveal the action mechanism of new bioactive compounds for improved intervention strategies . structural characterization of β-sheeted oligomers formed on the pathway of oxidative prion protein aggregation in vitro key: cord- -josb pi authors: kumaraswamy, priyadharshini; sethuraman, swaminathan; yakhmi, jatinder vir; krishnan, uma maheswari title: hierarchical self-assembled peptide nano-ensembles date: - - journal: handbook of nanomaterials properties doi: . / - - - - _ sha: doc_id: cord_uid: josb pi a variety of peptides can be self-assembled, i.e. self-organized spontaneously, into large and complex hierarchical structures, reproducibly by regulating a range of parameters that can be environment driven, process driven, or peptide driven. these supramolecular peptide aggregates yield different shapes and structures like nanofibers, nanotubes, nanobelts, nanowires, nanotapes, and micelles. these peptide nanostructures represent a category of materials that bridge biotechnology and nanotechnology and are found suitable not only for biomedical applications such as tissue engineering and drug delivery but also in nanoelectronics. self-assembly is defined as a process where individual components form organized structures via specific and local interactions without any external intervention [ ] . molecular self-assembly is a spontaneous process where the molecular components organize into ordered structures through non-covalent interactions such as van der waals, hydrophobic, capillary forces, electrostatic forces, or hydrogen bonds [ ] . although these interactions are relatively weak when compared to covalent bonds, they form reasonably stable higher-order structures through self-assembly due to the additive effect of these secondary forces. in other words, these self-assembled structures are thermodynamically more stable due to lower values of gibbs free energy when compared to that of the individual components (building blocks). since the underlying interactions are rather weak, any external stimulus can alter the self-assembled structures. however, once the stimulus is removed, they can revert back to their original structure. molecular self-assembly is ubiquitous in nature, and it has evolved in many areas including chemical synthesis, nanotechnology, polymer science and materials science, and engineering [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the molecular level self-assembly is a typical example of the 'bottom-up' approach where molecules in the sub-nm range come together to form assemblies that are in nm or bit larger in dimensions [ , ] . numerous self-assembling systems ranging from diand tri-block copolymers, complex dna structures, simple and complex proteins, and peptides have been developed [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . complex and intricate monodisperse structures can be obtained through self-assembly with high precision and reproducibility. biomolecules possess an inherent ability to form hierarchical self-assemblies in aqueous medium. biomolecules such as proteins, deoxyribonucleic acid, and lipids have been widely investigated for their self-assembling properties [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in fact, these three biomolecules form the 'molecular trinity' of biomolecular self-assembly. peptide systems have been especially popular self-assembling systems due to the large number of structures that can be generated by slight modification of the number and nature of amino acid residues in the sequence [ , , ] . the stability, ease of synthesis, and controlled self-assembly regulated by various physicochemical parameters have resulted in the popularity of selfassembled peptide systems [ ] . since peptide self-assembly is a bottom-up process where amino acids form the building blocks, it is easy to introduce functionalities on the carboxyl or amine terminal groups, opening up the possibilities of a wide range of chemical interactions leading to specific functions. though peptides containing naturally occurring l-amino acids have been widely investigated for their self-assembling characteristics, d-amino acid containing peptide systems have also been explored due to their stability against proteases [ ] . self-assembling peptides may vary in the number of amino acids starting from to as high as . the simplest building block reported thus far is the dipeptide (diphenylalanine -ff) from the core recognition motif of alzheimer's amyloid beta peptide [ ] . this dipeptide is reported to form different structures based on the ph that is employed (fig. . ). for instance, at a ph lower than the isoelectric point of the peptide, it forms nanofibrils, whereas at a ph higher than its isoelectric point, the peptide forms nanotubes [ ] . despite the numerous advantages of self-assembling peptides, there are several challenges associated with their use in biomedical applications, which include problems related to processability, control of size, functionalization, and stability in aqueous media [ ] . for example, biosensing platforms employing self-assembling peptides require electric contacts between the selfassembled nanostructures and transducers, which become tedious due to the small dimensions involved. however, with the advancement in micro-and nanofabrication techniques, such problems are being overcome, paving the way for use of peptide nanostructures in molecular electronics. another impediment relates to the low conductivity of the self-assembled peptide nanostructures, which limits their use in sensing and diagnosis. however, by the introduction of conductive polymers, enzymes, and metallic particles, the electrical current conductivity can be enhanced [ , ] . the self-assembly process is influenced by many factors that can be grouped into any of the three categories, namely, environment-driven factors, substrate-driven factors, and peptide-driven factors. the ph, temperature, solvent, nature of ions, and ionic strength are factors that influence the self-assembly process. the ph of the medium alters the charge status on the peptide and hence the electrostatic forces between the peptide molecules. for instance, the peptide stviie forms beta-sheets when its net charge is + , whereas in its zwitterionic state, it forms random coils and it exists as a mixture of random coils and beta-sheets when the net charge is À . this is because when the net charge is zero as in the zwitterionic form, the packing of the peptides could happen in many ways leading to an amorphous structure. the presence of a net charge gives directionality to the associations as well as determines the distance between the peptide chains [ ] . the charge distribution in the peptide also influences the self-assembled structures formed. for example, when the peptides eak -i (aeakaeakaeakaeak), eak -ii (aeaeakakaeaeakak), and eak -iv (aeaeaeaeakakakak) were self-assembled, it was found that eak -i and eak -ii formed fibrillar assemblies, while eak -iv formed globular structures between ph . and . and fibrillar structures at other ph due to the neutralization of charges in the beta-sheet structures formed, which promotes aggregation at ph away from the neutral ph [ ] . the nature of anion also was found to influence the structures formed by the eak -ii peptide in the presence of cu + ions. while so caused formation of nanofibers, the monovalent cland no caused formation of short fibrils with a mixture of alpha helix and random coils. this is due to the ability of the divalent sulfate anions to act as an electrostatic bridge between two lysine residues unlike the monovalent ions. higher ionic strength of the medium contributes the shielding of the electrostatic charges on the ionizable groups present in the peptide sequence, thereby altering the critical aggregation concentration as well as the ph required for association or dissociation of the self-assembled structure [ ] . factors like the solvent polarity, surface tension, hydrogen bond-forming ability, and dielectric constant influence the peptide self-assembly and strength of the self-assembled structures. introduction of methanol as cosolvent contributed to the formation of nanofibers of diphenylalanine (ff) on a glass substrate. this was attributed to the high hydrogen bond donor and acceptor property of methanol that promoted formation of highly crystalline nanofibers [ ] . on increasing the methanol content, solvation of the peptide molecules occurred, which prevented aggregation of the solvated peptide. it was also observed that organic cosolvents with higher surface tension contributed to reduction in fiber dimensions to the nm range. the dielectric constant of the solvent has been found to influence the peptide substrate binding affinities [ ] . the surface tension, hydrophobicity, and surface texture of the substrate influence the self-assembly process. hydrophobic substrates promote better spreading of peptide sequences that have greater number of hydrophobic residues. surface topography, on the other hand, directs the orientation as well as fiber dimensions. the dipeptide ff was found to self-assemble into nanofibers with a well-spread morphology on poly(vinyl chloride), whereas in silicon, which had a periodic rough texture, finer fibers were observed along with vertically aligned hollow nanotubes of larger dimensions suggesting that the rough morphology retards the stacking interactions between the peptide molecules [ ] . peptide-driven factors that direct self-assembly are the number and nature of amino acid residues in the sequence, the isoelectric point, and the peptide concentration [ ] . aromatic residues led to the formation of rigid structures that possessed nanotape or nanoribbon morphology [ ] . reduction in the surface tension of the peptide molecule can lead to the formation of globular assemblies instead of fibrillar structures as observed with the peptide eak -iv at neutral ph. peptide aggregates are formed above a particular concentration known as critical aggregation concentration (cac), which in turn is dependent on the peptide sequence. below the cac, the seeding and nucleation occur, while above cac, the aggregated ensembles are discernible [ ] . in the case of surfactant-like peptide amphiphiles, if the surfactant number is between ⅓ and ½, then cylindrical micelles and nanofibers are observed ( fig. . ). however, if the surfactant number is between ½ and , then bilayer formation occurs. in the case of micelle-forming peptides, increase in the intermolecular cross-links has been found to reduce the curvature leading to the formation of cylindrical micelles as observed in the hexadecyl-modified peptide sequence ccccggg phosphoserine-rgd [ ] . peptides that self-assemble are amphiphilic and are classified based on their nature of self-assembly. peptide lego systems consist of both hydrophilic and hydrophobic residues that form beta-sheet structures and well-defined nanofiber matrices with an average pore size of - nm in aqueous solution. these peptides are termed as molecular lego peptides as they possess alternating charged and hydrophobic amino acids like the pegs and holes of lego blocks. for example, in the peptide rad -i, the sequence is radaradaradarada, where r (arginine) is a cationic amino acid and d (aspartate) is an anionic amino acid. these oppositely charged amino acid residues are separated by a hydrophobic amino acid residue, alanine. the charge status of the peptide sequence will therefore be represented as (+ À + À + À + À + À + À + À + À), and such sequences are referred to as modulus i peptides. similarly, modulus ii (++À À++À À) and modulus iii (+++À À À+++À À À) have also been reported based on their charge pattern. these peptides spontaneously form nanofibers of nm length in the presence of cations of alkaline earth metals due to electrostatic forces. since ionic interactions are involved in the self-assembly process, the molecular lego peptides readily form hydrogels [ ] . the hexadecapeptide dar -iv with the peptide sequence dadadadarararara has similar amino acid residues as rad, with the only difference that the sequence of the charged amino acid residues varies. this difference is markedly reflected in the self-assembled structures formed by the two peptides. the self-assembled structures formed by dar -iv can transform from an alpha helix to a beta-sheet depending on the ph, ionic strength, and temperature i does not form alpha helical structures under any condition. this is because in the case of rad -i, if the peptide assumes an alpha helical structure, the positively charged side groups in arginine (r) will be repelled by the positively charged n-terminus, and the anionic aspartate (d) will experience electrostatic repulsion from the like-charged carboxylate in the c-terminus. hence, it always remains in the beta-sheet form. in the case of dar -iv, the negatively charged aspartate will stabilize the positive n-terminus, and the positively charged arginine will exhibit electrostatic attraction with the negative c-terminus when it adopts an alpha helical form. thus the nature, number, and sequence of amino acids are critical parameters that determine the type of self-assembled structures that can be formed by peptides [ ] . the molecular lego peptides are also known as ionic self-complementary peptides due to their pattern of electrostatic association that contribute to their stability. zhang and his coworkers identified the first molecular lego peptide eak- from a z-dna binding protein zuotin from yeast [ ] . the ionic self-complementary peptides initially form beta-sheets, which later form a fibrous network, progressively by undergoing sol-gel transition. the substitution of a basic amino acid with another basic amino acid (for instance r with k) or an acidic amino acid with another acidic amino acid (e.g. d with e) does not bring about significant changes in the self-assembly pattern. however, substitution of an acidic amino acid with a basic amino acid and vice versa was found to alter the self-assembly pattern. such structures were found to form beta-sheets but did not form higher-order structures. substitution of the alanine residues with more hydrophobic residues such as leucine, valine, and isoleucine accelerates the self-assembly process [ ] . surfactant-like peptides self-assemble either into nanotubes or nanovesicles [ ] [ ] [ ] [ ] (fig. . ). they are termed as surfactant-like due to the presence of a hydrophilic head comprising charged amino acids (lysine, arginine, glutamic acid, aspartic acid, etc.) and a hydrophobic segment comprising nonpolar amino acids (alanine, leucine, valine, etc.). intermolecular hydrogen bonding plays a major role in determining the structures formed by the self-assembly of these peptides. vauthey et al. were the first to design a self-assembling peptide that can self-assemble into nanotubes and nanovesicles [ ] . some common examples of surfactant-like peptides include a d, v d, v d , l d , and g d . these peptides initially self-assemble to form a bilayer, which later undergo further associations to form nanotubes. the nature of the amino acids in the sequence has an important role in dictating the type of self-assembled structures formed. the peptide sequences a k and a d both formed nanotubes often exhibiting twisted tape or fibrillar morphology. a heptapeptide, namely, ac-gavilrr-nh , formed donut-like ring structures [ ] . peptides like v d , v dvd, and v d v have been reported to form fibers, tapes, and twisted ribbons rather than nanotubes. the differences in the self-assembled structures formed could be attributed to the variations in the packing density of the peptide aggregates [ ] . the number of hydrophobic amino acids in each surfactant-like peptide also influences the final self-assembled structure. three peptides a k, a k, and a k, with different hydrophobic chain lengths were investigated for their self-assembling properties [ ] . while a k formed stacked bilayers, a k formed nanofibers and a k formed nanorods. the absence of higher-order structures in a k peptide was attributed to the absence of measurable critical aggregation concentration (cac), probably due to the short hydrophobic segment. several surfactant-like peptides found in nature also exhibit similar self-assembling characteristics to form vesicular structures that may be relevant to prebiotic enclosures that sequester enzymes from their environment [ ] . the lipid-like peptides are a subtype of surfactant-like peptides and consist of a hydrophilic head group and a tunable hydrophobic tail. though the lipid-like peptides possess different composition, sequence, and packing, they share several similarities with phospholipids that self-assemble to form lipid bilayers. the length of the peptide is about . nm, which is comparable to natural phospholipids. both systems self-assemble in water to form nanovesicles with an average diameter of - nm. a point of distinction between the two systems is in the nature of association between the individual components. in phospholipids, the acyl chains in the hydrophobic tails compactly pack together to displace water molecules from the interior and hydrophobic forces drive this process, which impedes formation of hydrogen bonds. however, in the case of lipid-like peptides, in addition to the hydrophobic tail packing, intermolecular hydrogen bonds are formed in the backbone. the presence of charged side chains in these peptides confers ph sensitivity as well as responsiveness to change in the ionic strength of the medium. carpet peptides also known as molecular paint peptides were first developed by zhang et al. [ ] . these peptides can undergo self-assembly and form monolayers, a few nanometers thick on a surface. these peptides thus act as a carpet for the attachment of cells or they can trap other molecules, thereby providing molecular recognition ( fig. . ). these peptides consist of three segments. the first segment or head contains ligands that serve as molecular recognition motifs for cell surface receptors. the middle segment serves as a linker that allows the head to interact at a distance away from the surface and also provides certain degree of flexibility to the peptide structure. the last segment or tail enables covalent binding with the surface. these peptides are widely used to study cell-cell communication. the peptide sequence rgdaaaaac is a typical example of a molecular paint peptide [ ] . the rgd segment serves as a recognition motif for the cell surface receptors integrins and hence can promote cell adhesion. the five alanine residues (aaaaa) serve as linkers, while the lone cysteine residue can enable anchoring of the peptide to gold substrates through its sulfhydryl group. similarly, the tetradecapeptide radsradsaaaaac that also possesses a ligand (rads) for cell recognition has been developed for painting gold surfaces [ ] . the switch peptides possess a unique ability to transform its molecular structure in response to environmental stimuli. for example, the hexadecapeptide dar -iv can form beta-sheet structures at ambient temperatures but transforms to an alpha helix when the temperature or ph of the system is modified. this suggests that secondary structures of sequences flanked by the negative charges on n-terminus and positive charges on c-terminus may undergo drastic changes if the ph and temperature are changed. these peptides were converted to electronically responsive structures through incorporation of metal nanocrystals [ ] . an undecapeptide with the sequence ac-qqrfqwqfeqq-nh was found to self-assemble into structures with progressively increasing order -tapes, ribbons, fibrils, and finally fibers [ ] (fig. . ) . the side chains of the glutamine (q) residues involve in hydrogen bonding and promote formation of b-sheets. the arginine (r) and glutamate (e) residues facilitate electrostatic interactions with the complementary countercharges on the neighboring chains leading to stabilization of antiparallel b-sheets forming a tape-like structure. the phenylalanine (f) and tryptophan (w) residues contribute to hydrophobic forces that drive the formation of ribbons. in the ribbon-like morphology, two tapes associate face-to-face stabilized by the hydrophobic forces leading to a twist. at higher concentrations of the peptide, the ribbons stack together to form fibrils. the substitution of the glutamine residues with glutamate (e) in the peptide sequence induces ph responsiveness in the peptide. at acidic ph (< ), the glutamate residue is protonated and hence will exhibit associative interactions promoting the existence of a nematic phase. as the ph is increased, the glutamate residues get deprotonated, and hence greater repulsive forces are introduced leading to transformation of the nematic phase to an isotropic fluid phase. thus the peptide acts as a molecular switch in response to ph changes by transforming reversibly between nematic and isotropic fluid phases. the design of cyclic peptides, whose dimensions and assembly could be tailored as desired, was inspired from the tubular pores formed by the tobacco mosaic virus [ ] . ghadiri and his coworkers were the first to report the self-assembly of a rationally designed cyclic octapeptide cyclo(l-gln-d-ala-l-glu-d-ala-) [ ] . these cyclic peptides have alternating d-and l-amino acids, which interact through intermolecular hydrogen bonding to form an array of self-assembled nanotubes with an internal diameter of - Å (fig. . ). the diameter of the tube depends on the number of amino acid residues forming the cyclic peptide. at alkaline ph, the carboxylate groups of glutamate residues become negatively charged as a result of deprotonation and prevent stacking associations due to strong electrostatic repulsive forces. at acidic ph, the carboxylate groups become protonated and hence favor association through extensive hydrogen bonding between the amide carbonyl and -nh-groups in the backbone. each cyclic peptide forms a flat ring that stacked over one another and is stabilized by hydrogen bonding resulting in a hollow nanotube. the side chains of the amino acids face the exterior of the tube to minimize steric repulsions. these side chains can also be functionalized to incorporate desired properties to the nanotubes. the cyclic octapeptide lanreotide nh -(d)naphthylalanine-cys-tyr-(d) trp-lys-val-cys-thr-conh , an analogue of somatostatin , also self-assembled into tubular structures with a diameter of nm and length running to several microns [ ] . this category of peptides represents a hybrid molecule formed from oligonucleotides and amino acids. the sequence of both components influences the nature of self-assembly. gour et al. have reported the self-assembly of a nucleopeptide formed by grafting the dipeptide ff to the -mer oligonucleotide with sequence ctctctctcttt [ ] . the diphenylalanine (ff) is part of the core recognition motif of the amyloid peptide and self-assembles to fibrillar structures in its pristine state. however, the nucleopeptide formed using this peptide motif self-assembled to spherical structures, which may be attributed to the hydrogen bonding interactions and amphiphilicity of this hybrid molecule. li et al. had developed nucleopeptides that self-assemble to form supramolecular hydrogels using the dipeptide ff conjugated to a nucleobase (a, g, t, or c). these nucleopeptides served as hydrogelators forming entangled nanofibers in water and could lead to many interesting biomedical applications [ , ] . peptide amphiphiles comprise of a hydrophilic head and hydrophobic tail that self-assemble in aqueous solution to form well-defined nanostructures like peptide bilayers, micelles, nanotubes, nanorods, and nanovesicles [ ] [ ] [ ] . these molecules can be chemically modified easily to tailor their properties for specific applications such as cell adhesion and internalization. the mechanistic insights into the self-assembly of peptide amphiphiles using v d as a model have suggested that the peptide amphiphile initially self-assembles into a bilayer and then into a cyclic vesicular form that undergoes stacking to form nanotubes. it has also been suggested that higher-order structures could be obtained by interconnection of these tubes through three-way junctions [ ] . most of the peptide amphiphiles form betasheet containing nanofibrils that can be induced either by addition of divalent salts or by altering the ph of the solution. the divalent cations form an ion bridge leading to stronger intra-and interfibrillar associations. experiments have revealed a high degree of solvation in the interior of the self-assembled structures formed by peptide amphiphiles [ ] . incorporation of a cysteine residue in a peptide sequence promotes reversible cross-linking of the peptide leading to modification of the stiffness of the peptide chain. another strategy to impart amphiphilic character to the peptide sequence is to incorporate a fatty acyl chain to the n-terminus of a peptide sequence. the acyl chain contributes to the hydrophobic character to the peptide amphiphile. using an elegant set of experiments, lowik et al. demonstrated the influence of alkyl chain length on the self-assembly of the peptide ganpnaag [ ] . the peptide molecules modified with c , c , and c acyl chains self-assembled into random coils independent of temperature, while those containing c and c acyl chains underwent a transition from b-sheets to random coils on increasing the temperature. increasing the acyl chain length contributes to enhanced hydrophobicity leading to differences in the thermal stability. in a seminal work, hartgerink et al. developed a peptide amphiphile with four distinct domains that self-assembled into cylindrical micelles in aqueous solution [ ] . the n-terminus of the peptide sequence cys-cys-cys-cys-gly-gly-gly-phosphoser-arg-gly-asp was modified with a -carbon alkyl chain that forms the hydrophobic component. the cysteine residues contribute to the formation of b-sheets and stabilize the self-assembled structure by covalent capture where superstructures are transformed into a supramolecule through covalent bonding. the disulphide bridges formed due to the oxidation of the cysteine residues confer rigidity to the supramolecular structure. further, stabilization of the structure is provided through extensive hydrogen bonding. the glycine-rich segment forms the flexible spacer domain. the phosphoserine residue confers charge and hence ph responsiveness to the sequence, while the rgd serves as a recognition motif for cell adhesion. this peptide amphiphile associated at acidic ph and dissociated at alkaline ph ( fig. . ). similar analogues have now been developed for many biological applications. reverse peptide amphiphiles like c o-veve with a free n-terminus were prepared using unnatural amino acid (ornithine, o) modified with a fatty acid chain and were mixed with conventional peptide amphiphiles containing free c-terminus. such de novo designed peptide amphiphiles formed nanobelts [ ] . a twisted nanoribbon morphology was observed when the cell adhesion motif rgd was incorporated in the c-terminus (c o-vevegrgd). apart from normal method of peptide synthesis, recombinant dna techniques have also been employed to produce two amphiphilic peptides, namely, ac-a v l wg -cooh and ac-a v l wg -cooh, which self-assembled into nanovesicles [ ] . a bolaamphiphilic peptide consists of two hydrophilic terminals linked through a hydrophobic segment. this class of peptides derives its name from the south american hunting weapon that consists of two balls linked by a string. stupp and his coworkers reported the self-assembly of a bolaamphiphilic peptide consisting of glycylglycine (gg) residues at either end linked through a -carbon acyl chain [ ] ( fig. . ) . the bolaamphiphilic peptides were ph responsive and formed helical ribbon-like structures at alkaline ph. the hydrophobicity of the acyl chain in the middle influences the twist in the structure so as to minimize contact with the polar environment. at acidic ph, the peptide self-assembles to form nanotubes presumably due to the additional hydrogen bonds formed through the protonated carboxylic groups of the amino acid residues. crick, in , first reported coiled-coil structures, commonly seen in many proteins. the major driving force is the hydrophobic interaction among the helices and a typical coiled structure consists of - left-handed alpha helices containing seven amino acid residues (heptad), each wrapped around each other to form a supercoil. according to the peptide velcro (pv) hypothesis, there are three major requirements for formation of such structures [ ] . the first and fourth residues must be hydrophobic to facilitate dimerization of the peptide chains along one face of the helix. the length of the hydrophobic side chain dictates the formation of dimers, trimers, or tetramers. increasing hydrophobicity stabilizes the self-assembled structure through van der waals' and hydrophobic interactions. the fifth and seventh amino acid residues should have charge to promote electrostatic interactions between the peptide chains. in order to facilitate attractive associations, it is important to have an acidic and a basic amino acid residue at these positions. leucine zipper proteins and cartilage oligomeric matrix proteins exhibit such type of coiled-coil structures. a right-handed alpha helical coiled-coil structure has also been identified in tetrabrachion, a protein from the bacterial staphylococcus marinus. this structure contains undecapeptide helices that possess a core filled with water, thereby exhibiting a different packing pattern with the hydrophobic and hydrophilic residues in the first, fourth, and eighth positions falling on the same face [ ] . apart from peptide velcro where one strand of acidic amino acid residues mingles with other strand of basic residues to form a parallel heterodimer, ryadnov et al. have designed belts and braces where two peptides are bound together by the third peptide of opposite charge. these belt and braces were used as a template to form colloidal gold particles and were commonly referred as peptide-mediated nanoparticle assembly [ ] . other types of self-assembled peptide systems include amphiphilic peptides in beta strand conformation which self-assemble into twisted tapes, helical dipolar peptides that undergo conformational change between a helix and beta-sheet similar to molecular switch, and surface binding peptides that form monolayers that are covalently bound to a surface. table . gives a list of some of the major peptide systems investigated for their self-assembling properties. formation of nanotubes and vesicles [ ] v k, v k , v k adsorption at air/water interface used for dna immobilization [ ] v k , l k , a k, v h, v k, h v , kv formation of nanotubes and vesicles [ ] ac-a d-cooh and ac-a k-cooh determination of critical aggregation concentration (cac) of particles formed during self-assembly [ ] mixtures of ac-a d-oh and ac-a k-nh formation of twisted fibrils [ ] ac-ga vilrr-nh formation of 'nanodonut' structures [ ] correlation of secondary structure with the morphology of nanostructures formed [ ] influence of sequence and purity on self-assembly [ ] a k, a k, a k determination of cmc, self-assembled structures, correlation with its antibacterial activity [ ] a k elucidation of nanotube structure and its mechanism of formation [ , ] ac-a v l wg -cooh and ac-a v l wg -cooh formation of vesicles [ ] chol-h r , chol-h r (chol denotes cholesterol) vehicles for delivering genes [ ] a h k , a h k , and h k (non-amphiphilic control) vehicles for delivering genes [ ] ac-(af) vehicles for delivering genes [ ] chol-g r tat, tat ¼ ygrkkrrqrrr antimicrobial activity [ ] a d and a k stabilization of g-protein-coupled receptor bovine rhodopsin against denaturation [ , ] ac-v r -nh , ac-v k -nh , ac-a k-nh , ac-i k -nh , ac-a k-oh, da -nh , ac-v d -nh , ac-a d-oh, ka -nh stabilization of protein complex photosystem-i and enhancement of activity [ ] where sl denotes the spin label acetamidoproxyl micelle aggregation and formation of particles and beads [ ] the formation of nanofibers can be promoted by peptides that have an ability to form beta-sheets. presence of branched amino acid residues confers an ability to transform from a a-helical structure to a b-sheet depending on the nature of the medium [ ] . the propensity of beta-sheet forming ability of peptide sequences can be retarded by introduction of proline residues at the n-and c-terminals. as proline lacks hydrogen bond-forming ability owing to its planar ring structure, it restricts the expansion of the beta-sheet network. this results in formation of straight nanofibers of about - nm in diameter and several mm long. the fine structure will show striations arising due to packing of tightly coiled alpha helical structures, especially if phenylalanine was one of the amino acid residues in the sequence. this is due to additional aromatic interactions contributed by phenylalanine. if phenylalanine was substituted by aliphatic hydrophobic residue such as isoleucine, the straight fibers formed revealed tape-like inner structures. attempts to functionalize these nanofibers to impart biorecognition have been made using biotin conjugation at their n-terminus [ ] . these biotin terminals can be used to tether molecules linked to anti-biotin molecules. however, such functionalization strategies resulted in a loss of the supramolecular assembly formed by the peptide. three main forces, namely, hydrophobic, hydrogen bonding, and coulombic forces, are mostly involved in controlling the self-assembly process [ ] . a combination of hydrogen bonding, p-p interactions, and van der waals' interactions promotes formation of columnar, disc-like aggregates, while hydrophobic effects and p-p stacking favor formation of d sheets and rectangular aggregates. cross-linking between peptide chains leads to rigid rods, while cross-linking in micellar assemblies leads to reduction curvature, thereby forming cylindrical micelles. rod-shaped structures can further stack to form columnar assemblies as their elongated, anisotropic geometry permits their preferential alignment along one spatial direction [ ] . hartgerink had employed three main design principles for customizing the peptide nanostructures that could be formed from cyclic peptide sequences [ , ] . the cyclic peptide should contain only eight amino acid residues as shorter sequences lead to strained amide backbone, while longer sequences will be flexible. steric interactions between the side chain and the backbone of the heterochiral alignment are prevented by designing the register of the stack in such a way that the rings will align with homochiral residues as their neighbors. side chain-side chain interactions are manipulated using glutamine residues that play a major role in intra-and intermolecular hydrogen bonding interactions. incorporation of non-peptide moieties into a self-assembled ensemble can lead to emergence of novel properties. for instance, the poor conductivity of self-assembled peptide nanostructures, which limits their use in sensing and diagnosis, can be overcome by the introduction of conductive polymers, enzymes, and metallic particles. alignment and positioning of the peptide nanostructures on a solid surface can be achieved through appropriate chemical modification of the substrate surface or peptide nanostructures, or both. atomic force microscopy (afm), dielectrophoresis, or optical tweezers have been employed for making appropriate connection between the self-assembled peptide nanostructures and transducers [ , ] . sedman et al. have used afm as a thermomechanical lithographic tool to create indents and trenches in the self-assembled nanotubes formed by diphenylalanine and dinaphthylalanine, thus utilizing them as nanobarcodes [ ] . reches and gazit have also tried manipulation of the peptide nanostructures using magnetic forces. apart from modification, functionalization of the peptide nanostructures has been carried out by rica and coworkers using dielectrophoresis (dep) for the incorporation of antibody-functionalized peptide nanotube on top of gold electrodes for the development of label-free pathogen detection chip [ ] . schnarr et al. have created coiled-coil heterotrimeric assembly by employing electrostatic forces, and this molecular self-assembly is driven by the hydrophobic forces, while the building blocks were matched via electrostatic interactions [ ] . many attempts have been made to improve stability of the self-assembled ensembles through cross-linking or enhancing associative forces. recently, selfassembled polymeric vesicles with enhanced stability, specificity, and tunability were formed from amphiphilic block copolymers with alternating hydrophilic and hydrophobic segments [ ] . introduction of a polypeptide chain in this amphiphilic block copolymer results in the formation of peptosomes [ , ] . the peptide segments are mostly associated with the hydrophobic segments resulting in the self-assembly. the peptide wnvfdflivigsiidvilse derived from the calcium channel forming protein caivs exhibits adhesive properties and thereby enhanced stability [ ] due to the cohesive forces between the chains that are responsible for driving their association even in the absence of water. gudlur et al. had designed two amphiphilic peptides with an oligolysine main chain (k ). the aand e-amino groups were both modified with either the hydrophobic nonapeptide flivigsii (h ) or the hydrophobic pentapeptide flivi (h ). these amphipathic lipid-like peptides when mixed in equimolar quantities (h h ) spontaneously self-assembled to form vesicular structures in an aqueous medium. differential scanning calorimetry studies on the h h peptide vesicles indicated good thermal stability over a wide range of temperature, and no alteration in the structure was observed [ ] . a peptide derived from human elastin consisting of hydrophobic repeats pgvgva along with the cross-linking regions composed of polyalanine interspersed with lysine [ ] formed highly insoluble nanofibers upon incubation at c. once fibers were formed, the side chain of lysine is converted to an aldehyde by lysyl oxidase enzyme, which then reacts with neighboring primary amines to form dehydrolysinonorleucine that then forms desmosine cross-links. these cross-linked fibers formed from a small peptide fragment of elastin exhibit good mechanical properties like resilience and strain at breaking point. covalent capture is a recently developed strategy that integrates the design and synthesis features offered by non-covalent self-assembly along with structural integrity. this approach involves covalent bond formation for stabilizing the self-assembled supramolecular ensembles without significantly affecting their structure. the formation of covalent bonds can occur before or after self-assembly. the order of self-assembly and the covalent bond formation play an important role in determining the physical structure of the aggregate formed. if the covalent bond is formed before self-assembly, either there will be low yield or the desired molecular aggregate will not be formed. however, if the covalent bond is formed after self-assembly, the pre-organization of reacting species happens, and the covalent bond formation is also enhanced. bilgicer et al. have employed the covalent capture method to dimerize two coiled peptides -one containing a leucine residue and the other an unnatural amino acid hexafluoro leucine at the same specific site [ ] . the dimerization of the two peptide sequences containing the natural leucine and unnatural fluorinated leucine hydrophobic residues was achieved by incubating them in a glutathione buffer. characterization of the self-assembled peptide structures for their electrical, physical, and chemical properties is essential to determine their potential in applications. a wide range of characterization tools have been employed to elicit such information on the self-assembled structures (table . ). this effort is intensifying as newer customized protocols become available with time to evaluate the performance and properties of novel peptide ensembles. different types of microscopic tools have been used to visualize the self-assemblies formed by peptides. apart from forming nanostructures, self-assembled peptides also form micrometer scale structures that can be analyzed using polarized light, epifluorescence, and confocal microscopy [ ] . while polarized light microscopy deals with birefringence and is employed to investigate the behavior of light-crystalline phases, epifluorescence and confocal microscopy are specific for the peptidic structures involving the fluorophores. polarized light microscopy is used to identify various mesophases like nematic, cholesteric, and cubic involved in the lyotropic behavior of the nanostructures formed from the amino acid units. epifluorescence microscopy is a technique that is widely applied to biological systems. the sample is irradiated with electromagnetic radiation of a particular wavelength known as excitation wavelength and the longer wavelength that is emitted from the sample is then detected. in epifluorescence microscopy, both excitation and observation of the emission occur from above ('epi') the sample. this technique has been applied to detect both intrinsic fluorescence of the peptide structures and the emission from fluorophores linked to the peptide chains. the topography of the self-assembled structures formed by two amphiphilic peptidolipids derived from the - residues of the amyloid beta peptide (c -iiglm-oh and c -iiglm-nh ) was observed using this technique [ ] . studies on the association between the ff nanotubes and pyrenyl derivatives have revealed that the final structure and its photophysical response are found to be dependent on the ph and the fluorophore concentration. when the pyrenyl concentration is low and when the ph is less or equal to , the structures formed are shorter and thinner, while at higher peptide concentrations and at alkaline ph, the fibrils formed are thicker [ ] . these findings confirm that the final structure is due to the balance between electrostatic and hydrophobic forces. at lower ph, protonation of carboxyl groups of either pyrenyl chromophore or ff molecules occurs, and hence electrostatic interactions become weakened, while the aromatic p stacking between the aromatic rings of the pyrenyl structure dominates. at neutral ph, though the amino groups are protonated, the carboxyl groups of ff and pyrenyl determination of presence of beta-sheet structures. employed in studying the mechanism of amyloid fibril formation surface plasmon resonance spectroscopy atomic force microscopy investigation of the kinetics of self-assembly, conduct structure manipulation, determine the size and morphology of the nanostructures formed during the self-assembly diffraction studies nanofiber alignment and determination of cross b-sheet structures electron microscopy morphology of self-assembled structures gel electrophoresis determination of monomers and its subsequent polymerization into dimers, tetramers, and oligomers circular dichroism determination of secondary structure and transition between secondary structures during self-assembly process fourier transform infrared spectroscopy determination of beta-sheet structures are not protonated, and hence only weak induced dipole interactions are favored. at alkaline ph, both carboxyl and amino groups are deprotonated, and hence electrostatic forces cannot compete with the hydrophobic forces resulting in side-chain contacts to form thicker fibrils [ ] . thioflavin t (tht) is a fluorescent molecule that binds to beta-sheet structures of peptide assemblies. the binding results in a red shift in the emission of thioflavin t from to nm. the presence of aromatic residues that contribute to p-p stacking interactions of the aromatic rings in tht with the peptide causes a change in the charge distribution of tht in its excited state causing the red shift [ ] . the rigidity of the peptide structure and its flatness are additional factors that contribute to tht binding. one of the limitations of tht is its poor solubility in aqueous solvents. to overcome this issue, a sulfonated analogue thioflavin s (ths) has been introduced with sulfonated groups [ ] . congo red is yet another fluorescent probe that has exhibited selective binding to the beta-sheets of amyloid and amyloid-like fibrils [ ] . confocal laser scanning microscopes (clsms) provide high in-plane resolutions by restricting entry of out-of-focus light by employing a pinhole for illumination of a sample, thus making it an attractive tool for investigating peptide self-assemblies. they are mainly used in d reconstruction of images recorded at different slices along the z direction. the imaging of the peptide nanostructures formed has been accomplished by using confocal laser scanning microscopy (clsm). ff microtubes labeled with rhodamine b were imaged using clsm [ ] , and this technique has been used to identify the hydrophobic-rich and hydrophilic-rich regions by labeling ff structures with two fluorescence dyes, namely, rhodamine and phthalocyanine. since rhodamine is relatively hydrophilic, it was localized in the inner core of the peptide assembly, which consists of hydrophilic clusters. phthalocyanine was found in the hydrophobic external wall of ff nanostructures. the clsm enabled visualization of both the hydrophilic and hydrophobic clusters by reconstructing the images in d. similarly, the distribution of the peptide amphiphiles c -vvvaaaggklakklakklakklak and c -vvvaaakkk in a hyaluronic acid membrane was imaged using clsm [ ] . the peptide amphiphiles were modified with a fluorophore at the n-terminus to enable imaging. the z-sectioning served to understand the localization of the peptide self-assemblies within the membrane. the effect of the nanofibers formed by the self-assembly of glucagon-like peptide (glp- ) mimetic peptide amphiphiles on the cell viability and proliferation of rat insulinoma cells were investigated using clsm. the clsm technique has been extensively used as a powerful tool to investigate cell morphology, migration, and proliferation. the influence of a self-assembled scaffold formed from an ionic complementary peptide modified with biorecognition motifs on the cell morphology, spreading, and migration was investigated using clsm. the results indicated that the designer peptides modified with cell adhesion motif from osteopontin and signaling motif from osteogenic signaling peptide promoted excellent growth and proliferation of osteoblasts. scanning probe microscopies, especially the atomic force microscopy (afm), have been widely used to investigate the geometry of the self-assembled nanostructures, to measure their conductivity, and to determine the young's modulus and thermal stability of the structures under dry conditions. the afm contains a probe attached to a flexible cantilever, and as the probe moves over the sample at a preset rate, the force of interactions between the probe tip and the sample surface is measured, and the topography of the sample is constructed. the deflections in the cantilever are recorded by monitoring the reflection of a laser beam focused on the cantilever and recorded through a photosensitive photodiode. this technique offers atomic level resolution and can be a very valuable tool in research on self-assembled structures. the probe tip, generally in the range of nm, can be conducting or nonconducting and can be functionalized to investigate specific interactions. the material of the probe and its geometry are vital in determining its performance. the data acquisition in afm can be made in the contact mode or noncontact mode or tapping mode. one of the challenges involved in using afm technique is to study peptide self-assembly in solution as it requires sufficient adhesion with the substrate. generally, afm had been extensively employed to study the morphology of the aggregates formed by the self-assembly of peptides. chaudhary et al. had investigated the propensity of two sequences derived from the amyloid tau protein ac-vqivyk-amide and ac-qivyk-amide to form beta-sheets in the presence of different solvents using afm [ ] . the results revealed that the ac-vqivyk-amide existed both as alpha helices and beta-sheets and the nature of the solvent was a key determinant of the form in which the peptide aggregates existed. the formation of the supramolecular assemblies by the peptide can also be monitored in a time-dependent manner using the afm probe. time-lapse liquid imaging of amyloid fibrils has been employed for kinetic studies on the rate of formation as well as morphological changes introduced in the amyloid peptide during the aggregation process under various self-assembling conditions [ ] . for the determination of young's modulus, the afm tip is positioned on the top of the structure and pressed. from the force-distance curves, the young's modulus can be directly derived using the theoretical model proposed by niu et al. [ ] . however, this model has a limitation since it depends on the type of structure that can be assumed. for example, if it is a hollow structure rather than a solid, then the young's modulus calculated using this model will exhibit significant deviations from the actual value. knowles et al. have used another approach to understand the rigidity and mechanical strength of the self-assembled amyloid fibrils on mica substrate [ ] . the topography of more than fibrils was imaged using afm and the shape fluctuations were used to compute the bending rigidity (c b ) of the fibrils. the cross-sectional moments of inertia (i) were computed for each fibril based on their height measured using afm. the young's modulus (y) was then computed as y ¼ c b /i. these values range between and gpa for most protein fibrils. the young's modulus for the completely self-assembled amyloid fibrils falls in the range and gpa. the elastic modulus of the peptide assemblies can be further dissected into contributions from the backbone as well as the side chains, i.e. y ¼ y bb + y sc where y bb and y sc are contributions from the peptide backbone and side chains, respectively. the computed results suggest that the contribution of the backbone interactions towards the elastic modulus is more than twice that of the side chains. in the case of amyloid fibrils, the contribution of y bb is about %. it is also postulated that peptide structures with moduli greater than gpa have significant contributions from the involvement of side chains in hydrogen bonding. in the absence of significant intermolecular hydrogen bonding in the peptide structures, the surface tension arising due to the hydrophobic and hydrophilic residues can also be employed to determine the young's modulus using the relation y ¼ g/h where g is the surface tension and h is the inter-sheet spacing with values in the range of and Å . for the observation of thermal stability of the nanostructures under dry conditions, the afm tip is positioned above the structures and the position of the tip is monitored with increase in temperature. at a particular temperature, as the structure degenerates, the tip will move downward which gives an indication of the maximum temperature beyond which the nanostructure will lose its stability. transthyretin fibrils have been examined for their stability after prolonged exposure to high temperatures using afm, and it was found that the thermal stability of the pre-fibrillar aggregates was poor when compared to the fibrillar assemblies [ ] . electron microscopic techniques, which include scanning and transmission electron microscopy, are commonly employed tools for imaging self-assembled structures. focused ion beam milling techniques were also recently employed to characterize the nanostructures. scanning electron microscopy (sem) is usually used to determine the geometry of the nanostructure. however, the presence of any defects or cavities in the structure can be better visualized using transmission electron microscopy (tem) where high-energy electrons pass through the nanostructures and the final image is reconstructed by mapping the intensities of electrons from each point in the sample. the more conducting regions in the sample will therefore appear dark when compared with regions with some resistance to the passage of electrons. the imaging is usually done in ultrahigh vacuum of the order of - pa. the main advantages of electron microscopy techniques when employed for visualizing the morphology of the peptide structures are ease of implementation, high magnification, resolution, and its ability to image a range of dimensions ranging from sub-nanometric to micrometric scales. these prospects are possible due to the smaller wavelength of electrons employed when compared to the visible light. the scanning electron microscopic technique when applied to nonconducting samples such as peptide nanostructures requires a thin coating of an inert metal such as platinum or gold to enable generation of the image. otherwise, the electrons will remain on the surface of the sample making the visualization of the finer structures on the sample impossible. in the case of transmission electron microscopy, there is a restriction in the thickness of the sample that can be imaged. samples less than . mm alone can be imaged using transmission electron microscopy, as thicker samples will not permit transmission of the electrons. it is not advisable to use accelerating voltages beyond kv in scanning electron microscopy for analyzing peptide nanostructures. similarly, in transmission electron microscopy, very high voltages will lead to damage of the peptide structures. apart from giving information on the morphology of the aggregates, the dimensions of the individual self-assembled structures can also be obtained from the electron microscopy techniques. the thermal stability of the structure can also be investigated using sem where after exposing the nanostructure to particular temperature, the structures can be imaged to observe any deformations postexposure to high temperature. the strength of the structure can be determined using sem combined with fib source (fib -focused ion beam). the nanostructure is placed inside fib sem and the time taken to mill the sample is analyzed [ ] . by comparing the time with the standard materials, the stability of the structure can be determined. the incorporation of metallic compounds in the peptide assemblies can be imaged using tem. for instance, tem was employed to determine the presence of cuo within the nanotubes due to the enhanced contrast provided by the metallic compounds [ ] . recently modified ff peptide nanotubes were used for the determination of the neurotransmitter dopamine. the ff nanotubes were modified with cyclic-tetrameric copper (ii) species containing the ligand ( -imidazolyl)-ethylene- -amino- -ethylpyridine [cu (apyhist) ] + (apyhist refers to the ligand -( h-imidazol- -yl)-n-( -(pyridin- -yl)ethylidene)ethanamine) in nafion membrane on a glass carbon electrode. the morphology of the modified tubes imaged using scanning electron microscopy revealed tubular structures of thickness around - nm. the deposition of the nafion membrane on the nanotubes was also clearly distinguished [ ] . circular dichroism (cd) is a technique that is applied to optically active chiral molecules such as proteins and peptides. it is based on the principle of differential absorption of right and left circularly polarized light by the chiral molecule. cd spectroscopy is an invaluable tool to identify the secondary structures adopted by a peptide during the self-assembly process. the exact location of the alpha helices or beta-sheets or random coils can be identified by this technique, and it provides information on the structural transformations that can occur during the self-assembly process under different conditions. hauser et al. have used this technique to show that the self-assembly process proceeds through structural transition that may occur in three possible steps based on the peptide concentration [ ] . the peptide monomers interact via antiparallel pairing which is followed by a structural transition to a-helical conformation. the peptide pairs assemble to form fibers and condense to form fibrils. the assembly of the peptide monomers serves as the nucleation step, which then proceeds to form fibrils via nucleation-dependent polymerization mechanism. the assembly of peptide monomers, however, requires a transition from random coil to a-helical conformation. though it is reported that short peptides of - amino acids cannot form a-helical conformation, it is indeed possible above a threshold concentration. the cd spectra also demonstrated that at low concentrations, the peptides were stable up to c and changed its random coiled structure as the temperature is increased from c to c. this change was reversed on cooling. however, once the fibril is formed, the peptide ensembles adopt b-turn structures, and no reversal in conformation is observed upon changing the temperature. circular dichroism has also been widely used to study the kinetics involved in the formation of a peptide network. it was used to show that the beta-sheet structures progressively increase with concomitant reduction in helical coils in peptides with a propensity to form fibers [ ] . zhang et al. had reported that the ionic complementary peptide eak formed a macroscopic membranous structure due to extensive beta-sheet formation [ ] . the membrane formation propensity was retarded in the dodecapeptide eak , while the octapeptide eak did not form membranous structure. investigations with cd spectroscopy revealed that the eak formed beta-sheets extensively, while eak had both alpha helix and beta-sheets and eak had only random coils. similarly cd spectroscopic studies on the self-assembly of kfe (fkfefkfe) revealed the presence of left-handed double helical beta-sheets that could represent a new category of molecular materials. the properties of the peptide bond can be studied using x-rays. linus pauling and robert corey were the first to report the length of c-n bond in the peptide link. they also found that the peptide bond is planar, i.e. all the four atoms in the peptide bond are located in the same plane and the a-carbon atoms attached to the c and n are in trans conformation. the structural and functional relationships in the fibrous protein in wool were established using x-ray diffraction patterns by william astbury [ ] . x-ray diffraction has been now employed to determine the crystal structure of any newly synthesized peptide. it has also been used to calculate the bond distances and bond angles between the moieties in the peptide. based on the torsion angle measurements, it is also possible to predict the secondary structure conformation adopted by the peptide. moreover, the forces that stabilize the crystal structure such as van der waals and hydrogen bonding interactions can also be predicted. x-ray diffraction technique is also employed to determine the ultrastructural organization found in self-assembled peptide structures. nanofibrillar arrangement results in characteristic diffraction patterns that reveal the presence of cross b-sheet structures. the meridional and equatorial reflections have been used to identify the amyloid structure. the spacing between the hydrogen-bonded b-strands results in meridional reflection at . - . Å , and the distance between the b-sheets contributes to an equatorial reflection at and Å [ ] . the main challenge in employing x-ray diffraction techniques is that it is difficult to obtain pure crystals of many self-assembled structures. the x-ray data is usually obtained using a beam wavelength of . Å and an extremely small beam size of about mm. generally, the crystals need to be cooled to about k for data recording. localized radiation damage to the crystals could be avoided by illuminating the peptide crystals at different locations. due to the high degree of complexity involved in understanding the mechanism of self-assembly, various theoretical and computational methods have also been employed. these techniques not only help in understanding the mechanism but also to design the new sequences with the selected properties for nanobiotechnological applications. in particular, molecular dynamic simulations have been used to monitor the dynamics and investigate the influence of mutations and solvent effects in the conformational transition from alpha helices into beta-sheet structures [ ] . various algorithms and theories have been developed to investigate the properties of complex biomolecular systems at different levels. due to the high degree of complexity involved along with the small time scales, coarsegrained models were used to follow the actual process [ ] . however, in this model, fine atomic details are neglected and only relevant degrees of freedom of peptide molecule are retained. activation relaxation technique is a subcategory of the coarse-grained model, where the interactions among several peptide chains were simulated ab initio with no bias in original orientation and conformation. molecular dynamics studies have now been employed to address peptide self-assembly [ ] . these simulations have certain advantages such as ability to mimic the actual self-assembly of the peptide residues, reveal the structural information at an atomic level of the peptide, and provide a dynamic molecular model at atomic level of ordered assembly. however, there are certain limitations associated with this technique, too, such as a limited resolution, strong dependency of the quality of predictions on the size of the peptide, and the limited number of peptide residues for which such predictions could be made. several computational approaches have been used to predict the aggregation propensity of the proteins. for example, a computer program named tango considers the secondary structure and the desolvation penalty of the residues for calculating the aggregation propensity [ ] . all the techniques discussed thus far are used to characterize the nanostructures in dry conditions. however, stability of the structures differs when they are analyzed in wet conditions. to determine the stability of the nanostructures in wet conditions, they are submerged in aqueous solution, and the concentration of monomers is monitored with time using high-performance liquid chromatography (hplc). the increase in the concentration of monomers with time is an indication that the nanostructures are dissolving in the medium and are, therefore, not stable. high-performance liquid chromatography has also been employed to identify impurities associated with the peptide after synthesis [ ] . microrheometry technique is widely used to perform measurement on weak hydrogels without affecting their structural components. the measurements are very fast, and the experiments are not affected by external factors as they carried out in a closed chamber. recently, multiple particle-tracking microrheology experiments were used to follow the hydrogel formation by the peptide kfe [ ] . fourier transform infrared spectroscopy (ftir) analysis reveals the presence of parallel or antiparallel beta-sheets and nature of hydrogen bonds in the self-assembled structures. differential scanning calorimetry (dsc) and thermogravimetric analysis (tga) are used to determine the effect of temperature on the self-assembled structures. while dsc gives information on the phase transition temperatures of the peptide assembly, tga provides information on the degradation profile of the assemblies. crystallographic studies have also been carried out to understand the mechanism of self-organization. biocompatibility and immunogenicity of the selfassembled peptide structures have to be evaluated before their use in biological systems. both in vitro and in vivo studies need to be carried out to evaluate the biocompatibility of a peptide assembly. the self-assembled structure fabricated using the fmoc-diphenylalanine peptide was used as a scaffold to grow chinese hamster ovary (cho) cells. the cell viability analyzed using mtt assay showed that % of the cells were viable, suggesting that the peptide scaffold is biocompatible. more in-depth biocompatibility and immunogenicity assessment is however necessary to eliminate any potential risk of using these structures for biomedical applications [ ] . the unique reproducible structures obtained through self-assembly of peptide sequences have many interesting applications in biological and nonbiological fields. figure . depicts multiple applications that can arise from a cyclic peptide. a few of them are highlighted in the following sections. peptide systems that form nanofibrils similar to those formed by the amyloid beta peptide have been investigated extensively to understand the mechanism of formation of amyloid fibrils. amyloid fibril formation has been implicated in a wide range of degenerative diseases like alzheimer's, parkinson's, type ii diabetes, and other prion-related diseases [ ] [ ] [ ] [ ] . the switch peptides have also been employed to find information about the interactions between the various proteins involved in the pathology of protein conformation diseases like scrapie, kuru, huntington's, parkinson's, and alzheimer's disease [ , ] . amyloid fibrils are formed by various peptides such as the full-length human islet amyloid polypeptide (hiapp), nfgsvq peptide fragment from medin, and ff peptide from alzheimer's amyloid beta peptide. nfgail a hexapeptide fragment from islet amyloid polypeptide is reported to form well-ordered amyloid fibrils which exactly mimics those formed by the parent peptide [ ] . two active amyloidogenic peptides, namely, nflvh fragment of hiapp and nfgsvq fragment derived from aortic medial amyloid, also formed fibrils. another peptide from the human calcitonin, namely, nh -dfnkf-cooh, is also reported to form amyloid fibrils similar to those formed by the parent protein [ ] . a short truncated tetrapeptide, namely, nh -dfnk-cooh, also formed fibrils, which clearly shows there is no correlation between hydrophobicity and amyloidogenic potential since most of the short peptides are relatively hydrophilic. these results were further supported by johansson et al., who studied charged tetrapeptides and proved hydrophobicity is not sufficient for fibril formation [ ] . peptide legos (rada, eak), peptide amphiphiles, bolaamphiphiles, peptide conjugates, ionic self-complementary peptides, and long-acting gonadotropin-releasing hormone have all been reported to self-assemble into amyloid fibril structure. many peptides derived from the alzheimer's amyloid beta peptide also been shown to form fibrils. these include aaklvff, klvffae, ßaßaklvff, yyklvffc, ffklvff-peg, ffklvff, ac-klvffae-nh , ff, and yyklvff-peg [ ] [ ] [ ] [ ] . it has been concluded that alternating binary patterns and the peptides with high beta-sheet propensity are prone to form amyloid structures. gazit and coworkers have identified that aromatic amino acids play an important role in amyloid fibril formation through p-p stacking interactions [ ] . custom-made peptides used as model systems should possess strong electrostatic binding with the negatively charged lipid membranes and should exhibit structural transition from random coil to beta-sheet on binding to lipid membranes which initiates the association and the formation of oligomers and larger aggregates. apart from utilizing the self-assembling peptides to understand the pathological mechanism, it is also used to understand the functions and mechanisms of assembly of proteins like collagen. tobacco mosaic virus (tmv) is also a form of supramolecular structure assembled from a single strand of mrna along with many copies of identical coat proteins, which organize to form rod-like shape. this tmv has been used by schlick et al. for the construction of nanoscale materials without disrupting its self-assembly [ ] . peptide molecules also have the ability to self-organize at the air-water interface, which are widely used for mineralization studies because of its resemblance to insoluble proteins found in nature. self-assembling peptides have gained considerable interest in the field of tissue engineering because functional tissue recovery has been observed in brain and heart lesions [ , ] . one common example is the incorporation of cell adhesion motif rgd in the peptide sequence, which improves the cell adhesion onto the self-assembled structures formed by peptide amphiphiles. collagen-mimetic peptide amphiphiles, which self-assemble into nanofibers that exactly mimic the structural and biological properties of the native collagen, have been widely used in tissue regeneration as natural collagen is immunogenic and difficult to process [ ] . molecular lego peptides, such as rad -i and eak , have been used as scaffolds for neurite outgrowth, hepatocyte regeneration, etc. [ ] . chondrocytes encapsulated in a peptide scaffold using a self-assembled peptide with the sequence (ac-kldlkldlkldl-nh ) exhibited excellent phenotype and secreted its own growth factors within weeks of culture [ ] . peptide nanovesicles have been employed as drug carriers and it is believed that these nanovesicles enter the cells via endocytosis mechanism and thus can deliver drugs, genes, etc. [ ] . hydrogelating self-assembling fibers (hsafs) designed by woolfson and group using coiled-coil assemblies had limited use as a drug delivery system due to the limitation of rapid drug release. however, this was overcome by incorporating an anionic polyelectrolyte in the cationic peptide [ ] . ghosh et al. designed a smart self-assembling peptide amphiphile that transforms from a linear or spherical form to fibrous form upon altering the ph for drug delivery and in vivo imaging applications [ ] . the nanotubes formed by cyclic peptides comprising d-and l-amino acids serve as ion channels and form pores in the membranes leading to disruption of the cell architecture. as d-amino acids are taken up preferentially by microbes, these systems can function as effective antimicrobial agents which kill the microorganisms through formation of pores in the cell membrane, thereby causing osmotic collapse [ ] . mackay et al. have created a chimeric polypeptide consisting of an elastin-like polypeptide (elp) fragment and a short cysteine-rich fragment for the treatment of cancer [ ] . the drug conjugated with the cysteine residue drives the self-assembly that finally forms a drug-rich core and a hydrophilic peptide corona. instead of a drug, the self-assembly can also be triggered by linking the hydrophobic cholesterol moieties with hydrophilic cell penetrating peptides consisting of six arginine residues. these peptide nanoparticles have antimicrobial properties and were shown to effectively terminate the bacterial growth in the infected brains of the rabbits. beta-sheet-rich peptide nanofibers functionalized with b-cell and t-cell epitopes have been developed for immunization [ ] . these functionalized nanofibers developed antibodies in mice when injected with saline, and the levels were comparable with those injected with complete freund's adjuvant. protection against the beta-sheet-rich peptide nanofibers lasted for a year and the antibody response is t-cell dependent. recently, self-assembling peptide/protein nanoparticles have been used as antigen display systems for the development of vaccines. for example, a fragment of the surface protein of severe acute respiratory syndrome coronavirus (sars-cov) was incorporated in a self-assembling peptide nanofiber in the native trimeric coiled conformation [ ] . antibody formation was elicited when these peptide structures were injected into the mice, and the antibodies were conformation specific as determined by qualitative enzyme-linked immunosorbent assay. peptide nanofibers have been extensively investigated as the next-generation biosensors where they are used both as a fabrication material and also as a component in the final system (biofet) [ ] . nanotubes coated with proteins, nanocrystals, and metalloporphyrins by hydrogen bonding have been employed as chemical sensors. these nanotubes also served to improve the catalytic activity of the enzymes like lipase. apart from using ff nanotubes as a template for the fabrication of nanowires, they have also been used for biosensing where ff nanotubes are deposited on the surface of screen-printed graphite and gold electrodes for improving the sensitivity. it is also reported that these electrodes exhibit greater sensitivity compared to those electrodes modified with carbon nanotubes. the possible reason may be that the ff nanotubes increase the functional surface area of the electrode. electrical and magnetic fields have been used to align ff nanotubes. apart from this, patterning of the ff tubes has been carried out using inkjet technology, machined by thermomechanical lithography via atomic force microscopy, manipulated and immobilized using dielectrophoresis, and arranged on the surfaces by low electron irradiation [ ] . nanotubes are also used in the detection of pathogens, neurotoxins, glucose, ethanol, and hydrogen peroxide and as an immunosensor [ ] . peptide nanofibers have been reported for the detection of copper, dopamine, yersinia pestis, glucose, etc. [ , [ ] [ ] (fig. . ). amyloid-forming peptides are used in the field of nanoelectronics as nanowires. for example, ff nanotubes are used as a template for silver nanowires of diameter of approximately nm. switch peptides find application in molecular electronics by serving as a nanoswitch [ ] . ryu et al. developed photoluminescent peptide nanotubes by incorporating luminescent complexes composed of photosensitizers like salicylic acid. kasotakis et al. employed self-assembled peptides as a scaffold for the introduction of metal-binding residues at specific locations within the structure [ ] . an octapeptide from the fiber protein of adenovirus was used to design cysteine-containing octapeptides that can bind to silver, gold, and platinum nanoparticles. these metal-decorated fibers were employed in photodynamic therapy and also used in the development of surface-enhanced raman-scattering biosensors for detecting dna. lipid-like peptides find application in solubilizing, stabilizing, and crystallizing membrane proteins, used for drug formulations and also as model systems for studying protein conformational diseases [ ] . velcro peptides have been mainly used to study cell-cell communication and cell behavior [ ] . ff nanotubes are used as an etching mask for the fabrication of silicon nanowires. it reduces fabrication time, cost, and use of aggressive chemicals. it is also possible to scale up these arrays of ff nanotubes by vapor deposition methods. self-assembling peptide nanostructures have also been used as photosynthetic devices, and the peptide ac-klvffae-nh is reported to form nanotubes. precise ordering of strong chromophores along the inner and outer walls of the nanotubes enables utilization of this peptide structure as nanoscale antennas and photosynthetic device [ ] . the peptide nanofibers can also serve as a template for the growth of inorganic materials such as silver, gold, platinum, cobalt, nickel, and various semiconducting materials (fig. . ). an in-depth understanding of the molecular self-assembling mechanism of peptides and various stabilizing forces associated with the overall stability of the nanostructures formed by them is essential to design novel applications using these peptides. various classes of self-assembling peptides have been developed that have led to emergence of novel applications in the fields of tissue engineering, drug delivery, electronics, etc. different characterization tools have been designed to decipher the structural and functional aspects of the self-assembled peptide structures. the field of self-assembly continues to expand and has opened up new vistas for further research. synthesis of a tubular polymer from threaded cyclodextrins self-assembly of peptide-based colloids containing lipophilic nanocrystals transition of cationic dipeptide nanotubes into vesicles and oligonucleotide delivery novel electrochemical biosensing platform using self-assembled peptide nanotubes self-assembly and application of diphenylalanine based nanostructures peptide and protein building blocks for synthetic biology: from programming biomolecules to self-organized biomolecular systems emerging biological materials through molecular self-assembly fabrication of novel materials through molecular self-assembly design of molecular biological materials. using peptide motifs building from bottom up: fabrication of molecular materials using peptide construction motifs construction of biologically active protein molecular architecture using self-assembling peptide-amphiphiles induction of protein-like molecular architecture by self-assembly directed self-assembly: expectations and achievements designer self-assembling peptide nanomaterials self-assembly of chiral dna nanotubes design and characterization of programmable dna nanotubes lipid tubules: a paradigm for molecularly engineering structures chiral self-assembly of nanotubules and ribbons from phospholipid mixtures hydrogen-bonded sugar-alcohol trimmers as hexadentate silicon chelators in aqueous solution carbohydrate nanotubes biological and biomimetic materials biomimetism and bioinspiration as tools for the design of innovative materials and systems biomimetic systems for hydroxyapatite mineralization inspired by bone and enamel challenges and breakthroughs in recent research on self-assembly patterning surfaces with functional polymers organogels as scaffolds for excitation energy transfer and light harvesting microcapsules containing a biomolecular motor for atp biosynthesis self-assembled peptide nanostructures: the design of molecular building blocks and their technological utilization designing peptide based nanomaterials tuning the mechanical and bioresponsive properties of peptide-amphiphile nanofiber networks self-assembling organic nanotubes based on a novel cyclic peptide architecture role of p-p stacking in the self-assembly of amyloid fibrils self-assembly of peptides: influence of substrate, ph and medium on the formation of supramolecular assemblies self-assembled peptide nanostructures for biomedical applications: advantages and challenges, biomaterials science and engineering, prof. rosario pignatello (ed) manipulation of self-assembly amyloid peptide nanotubes by dielectrophoresis zuotin, a putative z-dna binding protein in saccharomyces cerevisiae de novo designed peptide-based amyloid fibrils rational molecular design of stimulus-responsive supramolecular hydrogels based on dipeptides morphology control of one-dimensional peptide nanostructures sequence dependence of kinetics and morphology of collagen model peptide self-assembly into higher order structures structures of helical beta-tapes and twisted ribbons: the role of side-chain interactions on twist and bend behavior concentration effect on the aggregation of a self-assembling oligopeptide self-assembly of nanodonut structure from a cone-shaped designer lipid-like peptide surfactant molecular designer self-assembling peptides dipoles of the alpha-helix and beta-sheet: their role in protein folding self-assembly at all scales molecular self-assembly of surfactant-like peptides to form nanotubes and nanovesicles self-assembly of surfactant-like peptides with variable glycine tails to form nanotubes and nanovesicles positively charged surfactant-like peptides self-assemble into nanostructures self-assembling behavior of designer lipid-like peptides self-assembly of peptide surfactants hydrophobic region induced transitions in self-assembled peptide nanostructures ) ß structures of alternating polypeptides and their possible prebiotic significance biological surface engineering: a simple system for cell pattern formation context-dependent secondary structure formation of a designed protein sequence conformational behavior of ionic self-complementary peptides peptide-based stimuli-responsive biomaterials high-resolution conformation of gramicidin a in a lipid bilayer by solid-state nmr self-assembling peptide nanotubes biomimetic organization: octapeptide self-assembly into nanotubes of viral capsid like dimension self-assembling dna-peptide hybrids:morphological consequences of oligopeptide grafting to a pathogenic amyloid fibrils forming dipeptide ) ß-peptide helix bundles long-range interactions stabilize the fold of a non-natural oligomer integration of photosynthetic protein molecular complexes in solid-state electronic devices self-assembling peptide detergents stabilize isolated photosystem i dynamic behaviors of lipid-like self-assembling peptide a d and a k nanotubes understanding self-assembled amphiphilic peptide supramolecular structures from primary structure helix propensity tuning secondary structure and self-assembly of amphiphilic peptides self-assembly and mineralization of peptide-amphiphile nanofibers self-assembly of giant peptide nanobelts self-assembly of recombinant amphiphilic oligopeptides into vesicles label-free pathogen detection with sensor chips assembled from peptide nanotubes tetrabrachion: a filamentous archaebacterial surface protein assembly of unusual structure and extreme stability belt and braces: a peptide-based linker system of de novo design interfacial dynamic adsorption and structure of molecular layers of peptide surfactants synergistic effect and hierarchical nanostructure formation in mixing two designer lipid-like peptide surfactants peptide nanotube nematic phase structure of single-wall peptide nanotubes: in situ flow aligning x-ray diffraction cationic micelles self-assembled from cholesterol-conjugated oligopeptides as an efficient gene delivery vector self-assembled cationic peptide nanoparticles capable of inducing efficient gene expression in vitro self-assembled oligopeptide nanostructures for co-delivery of drug and gene with synergistic therapeutic effect a class of cationic triblock amphiphilic oligopeptides as efficient gene delivery vectors self-assembled cationic peptide nanoparticles as an efficient antimicrobial agent designer peptide surfactants stabilize functional photosystem-i membrane complex in aqueous solution for extended time reversible peptide particle formation using a mini amino acid sequence micro and nano techniques for the handling of biological samples biological and chemical decoration of peptide nanostructures via biotin-avidin interaction novel polymers: molecular to nanoscale order in three-dimensions supramolecular honeycomb and columnar assemblies formed by self-assembly of coil-rod-coil molecules with a conjugated rod segment electrostatic force microscopy of self-assembled peptide structures qualitative mapping of structurally different dipeptide nanotubes thermomechanical manipulation of aromatic peptide nanotubes the colloidal domain: where physics, chemistry, biology and technology meet biomimetic surfactants: biosurfactants peptide nanovesicles formed by the self-assembly of branched amphiphilic peptides sequence and structure determinants for the self-aggregation of recombinant polypeptides modeled after human elastin engineering protein specificity: gene manipulation with semisynthetic nucleases secondary structure nucleation in peptides. transition metal ion stabilized alpha-helices programmed self-sorting of coiled coils with leucine and hexafluoroleucine cores microscopy tools for investigating nano-to-mesoscale peptide assemblies peptidolipid as binding site of acetylcholinesterase: molecular recognition of paraoxon in langmuir films influence of ph and pyrenyl on the structural and morphological control of peptide nanotubes bioorganic chemistry neurotoxic effects of thioflavin s-positive amyloid deposits in transgenic mice and alzheimer's disease is congo red an amyloid-specific dye? rhodamine b and rhodamine as reference substances for fluorescence quantum yield measurements a bioactive self-assembled membrane to promote angiogenesis morphology of self-assembled structures formed by short peptides from the amyloidogenic protein tau depends on the solvent in which the peptides are dissolved time-lapse atomic force microscopy in the characterization of amyloid-like fibril assembly and oligomeric intermediates using the bending beam model to estimate the elasticity of diphenylalanine nanotubes nanostructured films from hierarchical self-assembly of amyloidogenic proteins transthyretin fibrillogenesis entails the assembly of monomers: a molecular model for in vitro assembled transthyretin amyloid-like fibrils self-assembled peptide nanotubes as an etching material for the rapid fabrication of silicon wires confined conversion of cus nanowires to cuo nanotubes by annealinginduced diffusion in nanochannels electrochemical determination of dopamine based on selfassembled peptide nanostructure natural tri-to hexapeptides self-assemble in water to amyloid beta-type fiber aggregates by unexpected alpha-helical intermediate structures william thomas astbury - hierarchical self-assembly of tjernberg peptide at nanoscale conformational transition of amyloid beta-peptide connecting macroscopic observables and microscopic assembly events in amyloid formation using coarse grained simulations peptide self-assembly at the nanoscale: a challenging target for computational and experimental biotechnology prediction of aggregation rate and aggregation-prone segments in polypeptide sequences stability of diphenylalanine peptide nanotubes in solution molecular dynamics simulation of the α-helix to β-sheet transition in coiled protein filaments: evidence for a critical filament length scale rigid self-assembled hydrogel composed of a modified aromatic dipeptide from the globular to the fibrous state: protein structure and structural conversion in amyloid formation amyloid fibrillogenesis: themes and variations protein misfolding and disease; protein refolding and therapy the "correctly-folded" state of proteins: is it a metastable state? direct conversion of an oligopeptide from a ß-sheet to an a-helix: a model for amyloid formation identification of a penta-and hexapeptide of islet amyloid polypeptide (iapp) with amyloidogenic and cytotoxic properties conformational transitions and fibrillation mechanism of human calcitonin as studied by high-resolution solid-state c nmr charge attraction and beta propensity are necessary for amyloid fibril formation from tetrapeptides self-assembly of a modified amyloid peptide fragment: ph responsiveness and nematic phase formation controlling amyloid growth in multiple dimensions templating molecular arrays in amyloid's crossbeta grooves effect of peg crystallization on the self-assembly of peg/peptide copolymers containing amyloid peptide fragments molecular self-assembly of peptide nanostructures: mechanism of association and potential uses puramatrix: self-assembling peptide nanofiber scaffolds rational design and application of responsive alpha-helical peptide hydrogels lipid-like self-assembling peptides production of self-assembling biomaterials for tissue engineering light harvesting antenna on an amyloid scaffold dual-surface modification of the tobacco mosaic virus neural stem cells encapsulated in a functionalized self-assembling peptide hydrogel for brain tissue engineering fine-tuning the ph trigger of self-assembly artificial transmembrane ion channels from self-assembling peptide nanotubes self-assembling chimeric polypeptidedoxorubicin conjugate nanoparticles that abolish tumours after a single injection a novel vaccine using nanoparticle platform to present immunogenic m e against avian influenza infection peptide nanoparticles as novel immunogens: design and analysis of a prototypic severe acute respiratory syndrome vaccine development of an electrochemical metal-ion biosensor using self-assembled peptide nanofibrils self-assembled diphenylalanine nanowires for cellular studies and sensor applications an auto-biotinylated bifunctional protein nanowire for ultra-sensitive molecular biosensing remote electronic control of dna hybridization through inductive coupling to an attached metal nanocrystal antenna design of metal-binding sites onto self-assembled peptide fibrils self-assembling peptide-based nanostructures for regenerative medicine self-assembly of collagen-mimetic peptide amphiphiles into biofunctional nanofiber self-assembling functionalized nanopeptides for immediate hemostasis and accelerative liver tissue regeneration key: cord- - eu v d authors: chastain, michael; tinoco, ignacio title: structural elements in rna date: - - journal: prog nucleic acid res mol biol doi: . /s - ( ) - sha: doc_id: cord_uid: eu v d this chapter describes the rna structural characteristics that have emerged so far. folded rna molecules are stabilized by a variety of interactions, the most prevalent of which are stacking and hydrogen bonding between bases. many interactions among backbone atoms also occur in the structure of trna, although they are often ignored when considering rna structure because they are not as well-characterized as interactions among bases. backbone interactions include hydrogen bonding and the stacking of sugar or phosphate groups with bases or with other sugar and phosphate groups. the interactions found in a three-dimensional rna structure can be divided into two categories: secondary interactions and tertiary interactions. this division is useful for several reasons. secondary structures are routinely determined by a combination of techniques discussed in chapter, whereas tertiary interactions are more difficult to determine. computer algorithms that generate rna structures can search completely through possible secondary structures, but the inclusion of tertiary interactions makes a complete search of possible structures impractical for rna molecules even as small as trna. the division of rna structure into building blocks consisting of secondary or tertiary interactions makes it easier to describe rna structures. in those cases in which rna studies are incomplete, the studies of dna are described with the rationalization that rna structures may be analogous to dna structures, or that the techniques used to study dna could be applied to the analogous rna structures. the chapter focuses on the aspects of rna structure that affect the three-dimensional shape of rna and that affect its ability to interact with other molecules. . for example, protein synthesis is reduced in prokaryotic systems if the nucleotides within the shine-dalgarno region upstream from the initiation codon form a stable structure, since these nucleotides must pair with the ' end of the ribosome to initiate translation ( ) ( ) ( ) ( ) . the structure of the mrna for topoisomerase gene in bacteriophage t has a more dramatic effect on protein synthesis ( ) . a sequence of nucleotides in the mrna is skipped by the ribosome during translation so that amino acids corresponding to these nucleotides are not incorporated into the protein. different aspects of rna structure influence biological processes. the three-dimensional structure of self-splicing rnas arranges functional groups to catalyze specific phosphodiester bond cleavage and formation. the global folded structure of an mrna is less likely to regulate translation since the ribosome unfolds the mrna as it is translated; however, the local structure of the mrna can influence the ability of the ribosome to bind and translate the mrna. the increased interest in rna function has led to a corresponding interest in rna structure. two problems have slowed the characterization of rna structure. one is that techniques for synthesizing rna were limited in their ability to generate large quantities of many rna sequences. the second problem is that biological rna molecules, with the exception of trna, have not formed crystals suitable for x-ray diffraction. the development of enzymatic ( ) and chemical ( , ) methods for synthesizing large amounts of rna oligonucleotides, plus the development of new techniques for determining rna structure, have now led to the characterization of several rna molecules. here we describe the rna structural characteristics that have emerged so far. in those cases in which rna studies are incomplete, studies of dna are described with the rationalization that rna structures may be analogous to dna structures, or that the techniques used to study dna could be applied to the analogous rna structures. we focus on aspects of rna structure that affect the three-dimensional shape of rna and that affect its ability to interact with other molecules. folded rna molecules are stabilized by a variety of interactions, the most prevalent of which are stacking and hydrogen bonding between bases. watson-crick base-pairing is usually thought of first, but the importance of base stacking is seen in the crystal structure of trnaphe, where of the bases are stacked ( ) . many interactions between backbone atoms also occur in the structure of trna, although they are often ignored when considering rna structure since they are not as well-characterized as interactions be-tween bases. backbone interactions include hydrogen bonding and stacking of sugar or phosphate groups with bases or with other sugar and phosphate groups. the interactions found in a three-dimensional rna structure can be divided into two categories: secondary interactions and tertiary interactions. this division is useful for several reasons. secondary structures are routinely determined by a combination of techniques discussed in section , whereas tertiary interactions are more difficult to determine. computer algorithms that generate rna structures can search completely through possible secondary structures, but inclusion of tertiary interactions makes a complete search of possible structures impractical for rna molecules even as small as trna. finally, the division of rna structure into building blocks consisting of secondary or tertiary interactions makes it easier to describe rna structures. to distinguish tertiary interactions from secondary structure, the sequence of the rna is drawn on a plane; the backbone forms a continuous closed boundary if a line is drawn joining the ' end to the ' end. hydrogen bonding between bases in the sequence may be depicted by lines joining the bases; these lines must remain within the closed boundary. a secondary structure can be represented without any lines crossing. tertiary interactions occur when lines cross; this is called chord crossing. secondary and tertiary structures of trnaphe are shown in fig. . the secondary structure of rna consists of duplex and loop regions that can be divided into six different types, as shown in fig. : duplexes, single- stranded regions, hairpins, internal loops or bubbles, bulge loops or bulges, and junctions. duplex rna forms a right-handed double helix stabilized by stacking between adjoining bases and by hydrogen bonds between bases on opposite, antiparallel strands. the conformations of double-helical regions in rna have been determined by x-ray diffraction studies of fibers and single crystals ( - ). the structures are all similar to that found for dna fibers at low humidity; the geometry of these helices is termed "a-form." proton nmr measurements on a variety of rna oligonucleotides in solution are also consistent with a-form geometry ( , - ). the a form of duplex rna differs in several ways from dna duplexes, which typically have b-form geometry in aqueous solution. the riboses in aform rna adopt a '-endo conformation, whereas b-form dna deoxyriboses are in the '-endo conformation; with the usual phosphodiester backbone angles, the distance between phosphorus atoms in rna is . a, compared to a distance of . , in b-form dna ( ) . the base-pairs in a-form helices are tilted with respect to the helix axis and displaced from it by about a; this causes the minor groove in rna to be wide and shallow, and the major groove to be very narrow and deep (fig. ) . in crystals, the a-form rna helix has bp per turn, as opposed to bp per turn of b-form dna helix. in both rna and dna, the helices wind tighter in aqueous solution. the helical repeat of rna in solution is reported ( ) as . bp per turn or as . bp ( a), as compared to . bp per turn for dna ( - ). although there are local variations in conformation depending on base sequence, rna duplex regions are, in general, in standard a-form geometry. unusual sequences may adopt other conformations. for example, z-rna is formed by alternating c-g base-pairs in the presence of high salt concentrations ( , ) . single-stranded regions consist of unpaired nucleotides at the ' or ' end or between duplex regions of an rna secondary structure. the conformation of these nucleotides differs from that of nucleotides in loop regions of secondary structure, since the ends of loop regions such as hairpins are constrained by the secondary structure. in the absence of tertiary interactions to constrain single-stranded regions, these regions are assumed to be roughly ordered by base stacking in a helical geometry similar to the structures of single-stranded rnas that have no potential for base-pairing ( ) . a hairpin consists of a duplex bridged by a loop of unpaired nucleotides. hairpin loops are known to bind proteins, form tertiary interactions, and serve as nucleation sites for rna folding. the conformation of the backbone in hairpin loops must differ from the conformation of the backbone in helical regions in order to reverse the direction of the rna strand. the goal of studies on hairpin loops is to understand how the conformation of the loop nucleotides depends on the size and sequence of the loop. the smallest loop capable of bridging a duplex was originally thought to be three nucleotides ( ), but there is growing evidence that in some sequences, two unpaired nucleotides suffice to form hairpins in both rna ( ) and dna ( ) . thermodynamic studies of hairpins with loop sequences (u),, (c),, or (a), (n ranging from to ) showed that loops containing four or five nucleotides are the most stable ( fig. ) . u, and g, form a reverse wobble u*g pair (shown in fig. ) with the guanosine in the syn conformation. this leaves a loop of only two unpaired nucleotides: u and c,. the u base stacks on the sugar of c,, and the c, base stacks on the us base. the c, amino proton is close to a ' phosphate oxygen of us, implying the formation of a base-phosphate hydrogen bond. the presence of this hydrogen bond is consistent with the . kcal/mol destabilization of the loop (in mm buffer, ph . , at °c) when the cytidine at position is changed to uridine. nmr studies of the u, mutant show little change in loop conformation. g, is still syn, and most of the chemical shifts are the same as in the original molecule. the destabilization can be explained by the replacement of the favorable contact between the c, amino and a ' phosphate oxygen of u, by an unfavorable interaction of the u, keto with the phosphate oxygen. the loop is also destabilized by switching the closing base-pair from c .gg to g cg ( ); n m r studies have not yet been done to try to understand this effect. n m r studies of three hairpins with small loops indicate that the conformation of the sugar-phosphate backbone throughout the loop is very different from a-form geometry. in the uucg loop just described, u and c, have downfield region of the spectrum, indicating non-a-form backbone conformations. in the case of the hairpin with loop sequence uuu, the deviation from a-form geometry extended bp into the stem region. we suspect that changes in the sugar-phosphate backbone, including '-endo sugar puckers, are a general property of small hairpin loops. a-form geometry is preserved in portions of larger hairpin loops. as proposed prior to the crystal structure determination ( ), five of the seven nucleotides in the anticodon loop of yeast trnaphe continue stacking on the ' strand ( ) . the stacking of the anticodon bases in a-form helix geometry leaves these nucleotides accessible for base-pairing with the mrna. the five stacked nucleotides are followed by a turn in the sugar-phosphate backbone created by a change in the phosphodiester torsion angles between g, and u, . the turn structure is stabilized by a hydrogen bond between the imino proton of u, , and a ' phosphate oxygen of a, , , as well as stacking between the phosphate of g, and the base of u . one-dimensional nmr studies of the anticodon loop of yeast trnaphe showed that the structure in solution is consistent with the crystal structure described ( ), although two-dimensional nmr methods are needed to give unambiguous assignments. a hairpin that occurs in wheat germ -s rrna containing a loop of nucleotides has been studied by nmr ( , ) . a-form stacking does continue into the loop from the stem, but, unlike the anticodon loop, the stacking continues for several nucleotides along the ' strand. the loop is also stabilized by c-u mismatch hydrogen bonding (see fig. ) between the first two loop nucleotides. the hairpins studied so far show that the stability of a hairpin loop changes with different loop sequences and sizes. the structures of the trna anticodon loop and the uucg loop suggest that the specific loop sequences adopt conformations that are more stable because they contain more hydrogen bonding and stacking interactions-particularly interactions with the sugar-phosphate backbone. it has been suggested ( ) that loop nucleotides stacked in a-form geometry along the ' strand are a common feature of rna hairpins. the examples discussed here show that there is a range of loop structures. the anticodon loop contains nucleotides stacked along the ' strand, but the hairpin from -s rrna has nucleotides stacked along the ' strand. in the three small hairpin loops-uucg, ucu, and uuu-the backbone angles of each loop nucleotide differ significantly from a-form geometry. the conformations of the nucleotides in these small loops suggest that these nucleotides are not very accessible for base-pairing with other regions of rna. five of the nucleotides in the anticodon loop, on the other hand, are stacked in normal a-form geometry which facilitates pairing with other nucleotides. the structures of hairpin loops determine how the loop nucleotides can interact with regions of the same rna molecule, with other rna molecules, and with proteins. further studies of rna hairpins are needed to determine more about the interactions that stabilize hairpin loops. helical regions are stabilized by hydrogen bonding and stacking between bases. the nucleotides in hairpin loops are stabilized by hydrogen bonding between base protons and phosphate oxygens, and by stacking between bases, sugars, and phosphates. the effect that the closing base-pair of the stem has on the stability and structure of hairpin loops must be determined. bulge loops (or bulges) are defined as unpaired nucleotides on one strand of a double-stranded region; the other strand has continuous base-pairing. bulges range in size from one to many nucleotides. the main structural questions of interest are: ( ) does one unpaired base intercalate into the helix, or is it extrahelical, with the base-pairs on either side stacked on each other? ( ) how much does a bulge bend or kink the helix? ( ) what is the effect of very large bulge loops? do the surrounding base-pairs break to form an internal loop instead of a bulge? the local conformation of single-base bulges in several dna oligonucleotides has been studied, but there has been very little work on rna bulges. the equilibrium between a nucleotide bulge intercalating in or looping out of the helix depends on temperature, the identity of the bulge nucleotide, and the sequence of base-pairs in the duplex surrounding the bulge. this equilibrium is demonstrated by nmr studies on dna oligonucleotides containing a thymidine bulge that loops out of the helix at °c and intercalates into the helix at °c when located between two guanosines, whereas a thymidine bulge located between two cytidines remains looped out of the helix independent of the temperature ( ). nmr studies on an rna duplex with a uridine bulge between two guanosines show that it loops out of the helix ( ). the local conformation of nucleotides in bulge loops containing more than one nucleotide have not been studied by high-resolution structural techniques. determining the conformation of these loops in rna is important for understanding how these nucleotides interact with other elements of secondary structure to form tertiary interactions and for understanding how proteins bind to bulge loops. the chemical reactivity of bulge loops in rrna ( ) has been studied. a bulge loop containing six nucleotides appears to exist without breaking the base-pairs on either side, since these pairs are protected from chemical modification. some of the nucleotides within the bulge loop were also protected from modification, which suggests either that the loop nucleotides form a structure involving stacking and hydrogen bonding, or that the nucleotides form tertiary interactions. bulge loops can affect the long-range structure of nucleic acid helices by creating a bend in the double helix. bending has been detected by the altered mobility in nondenaturing gel electrophoresis for both rna and dna helices containing bulge loops ( - ). bulge loops in dna or rna helices containing five adenosines were found to alter the gel mobility more than bulge loops containing five thymidines or uridines ( , ), which shows that the structure of the bend depends on the identity of the bulge nucleotides. the bending was also shown to be affected by the base-pairs surrounding the bulge loop. one of the assumptions made when studying rna secondary structure is that the structure is independent of the surrounding conformations. for example, a short region of duplex rna is assumed to be the same whether it occurs next to a hairpin or next to a junction. several studies suggest that bulge nucleotides affect the structure of the duplex surrounding them for several base-pairs. thermodynamic studies showed that the free energies of bulge loops containing adenosines were kcal/mol more stable (at °c) for the sequences gcg(a),gcgacgccgca than for gcg(a),guca-gaccgca. helices containing dangling nucleotides were more destabilized by bulge loops than were helices without dangling nucleotides ( ) . nuclease v, (ec . . . or . . . ) cutting in duplex regions containing bulges and intercalated ethidium showed that ethidium intercalation into a double helix is affected for several base-pairs around a bulge nucleotide ( ) . a distortion in the duplex region near an adenosine bulge (in boldface) in the sequence d [cgcgaaatttacgcg] , was observed by nmr. the presence of a phosphate resonance in the downfield region of the spectrum indicated an unusual backbone conformation. the downfield resonance was assigned to the ' phosphate of the guanosine one nucleotide removed from the bulge ( ). thus, the distortions due to bulges are not necessarily localized at the bulge, but may extend into the surrounding duplex region. many questions remain about bulge loop structures. bulge loops bend rna, but a correlation between the number and sequence of bases in the bulge and the extent of bending has not been established. does a single base bulge that is looped out of the helix create a bend? bulge loops containing more than one nucleotide have not been structurally characterized at high resolution. the structures of these loops determine the ability of bulge loop nucleotides to form tertiary pairs. finally, the effect bulge loops have on the surrounding duplex structures must be determined. two apposed nucleotides that cannot form a watson-crick pair are called mismatches. the two mismatched bases can engage in some other form of hydrogen-bonding, or they can form an open loop of two nonbonded nucleotides. figure shows some of the hydrogen-bonding schemes proposed for mismatches with two hydrogen bonds, although mismatches containing only one hydrogen bond have been found in trna ( ). mismatches occur frequently in proposed secondary structures. the stability, the contribution of the surrounding sequences to the stability, the hydrogen bonding, and the effect on the sugar-phosphate backbone of the various mismatches has not been determined systematically in rnas. the most-characterized mismatch is the wobble g*u pair (fig. ) , which forms two hydrogen bonds and is virtually as stable as an a-u pair ( ) . crystal structures of trnaphe and trnaasp containing g.u mismatches show that they are usually incorporated into the helix without creating distortions in the backbone. however, one of the g*u pairs in the crystal structure of yeast trnaasp does have an irregularity in the backbone. two of the sugar-phosphate backbone angles (a and y in fig. ) at the g-u pair in the anticodon stem are in the trans conformation instead of the normal gauche conformation found in helical regions ( ). the distortion in the helix backbone is presumably caused by the difference in the width of a mismatch pair (c '-cl' distance) versus that of watson-crick base-pairs. the difference in width could also explain why g-u mismatches are slightly less stable when they are in the middle of a watson-crick region of a helix than at the end ( ) and why many of the mismatches in proposed rrna secondary structures are found at helix termini ( , ) . a change in backbone conformation may be a recurring feature of mismatches, although these distortions seem to depend on the sequence surrounding the mismatch. the hairpin gcga,uu,(ucu)g,&,,cgcc has been studied by nmr ( ) . it has a stem of bp, including a$ *c and u,.g,, mismatches. interproton distances by nmr were consistent with a-form stem geometry, even at the mismatches. the a + .c pair forms by protonation (at ph . ) of the adenine at the imino nitrogen, and is proposed to result in a pair containing two hydrogen bonds whose geometry is very similar to that of a g.u pair (fig. ) . the free energy of formation of the a + c pair, is kcal/mol less stable than an a*u pair at °c. two hydrogen bonds form in the g-a mismatch (fig. ) found at the end of the anticodon helix in trnaphe. g-a mismatches occur frequently at helix termini in rrna ( ), and hydrogen bonding in g*a mismatches has been proposed (on the basis of chemical modification) to occur in several internal loops in rrna ( , ) . a symmetrical rna duplex containing two g.a mismatches (in boldface) and ' dangling guanosines, [gcgagcg],, is . kcal/mol more stable at °c than predicted by the nearest-neighbor parameters, suggesting that g*a mismatches from stable structures ( ). more data are available on mismatches in dna than in rna. the gel mobilities of dna restriction fragments containing all possible mismatches are unchanged compared to a fully watson-crick helix; this shows that mismatches do not cause significant bends in the helix axis ( ). thermodynamic studies on mismatches in the dna oligonucleotides dcaaaxaaag + dgtttytttc showed that mismatches involving guanine (gat, g-g, g-a) are the most stable, and mismatches involving cytosine ( c c , c*a) are the least stable ( ). internal loops contain three or more nucleotides not capable of forming watson-crick base-pairs; there is at least one unpaired nucleotide on each strand. internal loops containing equal numbers of unpaired nucleotides on each strand are symmetrical, and those containing unequal numbers are asymmetrical. comparison of computer-predicted secondary structures to phylogenetically derived ones suggests that asymmetrical internal loops are thermodynamically less stable than symmetrical ones results). the structure of the asymmetrical internal loop which contains four nucleotides on one strand and five on the other (fig. ) is very different from the structure of the symmetrical one, despite the addition of only one nucleotide. nmr studies of the asymmetrical loop show that it closes with the guanosine on the longer strand looped out of the helix and hydrogen-bond formation in at least one of the mismatches in the loop. a closed structure for this loop was also proposed on the basis of chemical modification (m), although the guanosine thought to be extrahelical on the basis of nmr was proposed to be involved in a g-a pair (fig. ) . determining where bends occur in rna structures is important for understanding which regions of secondary structure are close together in the tertiary structure. it has been proposed in a model of the -s rrna structure that a large bend occurs in a six-nucleotide symmetrical internal loop ( ), but the effect of internal loops on the sugar-phosphate backbone in rna has not been studied experimentally. gel-mobility studies show that symmetrical internal loops of six or ten adenosines or thymidines in dna bend the helix axis very much less than bulge loops ( ). the structures of internal loops in rna remain poorly defined. the influence of loop sequence and size on the ability of internal loops to close by the formation of non-watson-crick hydrogen bonding must be determined. the accessibility of nucleotides in internal loops to tertiary pairing depends on the loop conformation. extensive stacking of the bases within open internal loops may facilitate tertiary pairirig, whereas bases in closed internal loops would be unavailable. finally, the effects of symmetrical and asymmetrical internal loops on the sugar-phosphate backbone must be explored: do bends occur at internal loops? junctions, or multibranched loops, contain three or more double-helical regions with a variable number of unpaired nucleotides where the helical regions come together. junction regions are important because helical regions can stack coaxially at these regions, and because the alignment of helical regions at junctions gives these regions characteristic shapes. the only rna junction whose conformation has been established is the four-stem junction in trna. for each trna for which a crystal structure has been determined, the acceptor stem stacks coaxially on the t stem, and the d stem stacks on the anticodon stem forming two long helical regions. these two helical regions are oriented roughly perpendicular to each other, cresting the overall l shape of the molecule. several of the unpaired nucleotides in the junction are involved in tertiary interactions, which are discussed in section . an example of an rna junction whose conformation plays a role in protein binding is found in the -s rrna from x. zueuis. this three-stem junction contains four unpaired nucleotides critical for binding transcription factor iiia. mutations of the unpaired nucleotides in the junction greatly reduce protein binding ( ), even though the protein does not directly contact these nucleotides ( ). the implication of these studies is that the unpaired nucleotides in the junction act as a hinge controlling how the helical regions stack on each other, thus determining the overall three-dimensional conformation of the -s rrna. the conformation of a four-stem dna junction, a model of recombination intermediates, has been determined ( ) . the conformation of the fourstem dna junction was determined by measuring fluorescence energy transfer between acceptor and donor groups located on the four different helical regions. the four stems of the junction stack coaxially in pairs to form two long helical regions oriented in an x shape. the difference between the shapes of four stem-junctions in trna and in the four-stem dna junction could be caused by the presence of unpaired nucleotides in the junction of trna. on the basis of the trna structure, -s rrna studies, and the dna four-stem junction structure, it seems to be a general feature of junction regions that different stems stack coaxially to form longer helical regions. coaxial stacking between helical regions in the rrna has been proposed on the basis of phylogenetic comparison ( ) . one criterion for deciding whether two helical regions stack is that their combined length in basepairs is conserved between different organisms, although the lengths of individual helices are not conserved. it has also been suggested ( ) that the helical regions separated by the fewest unpaired nucleotides will stack coaxially. both of these methods for predicting coaxial stacking predict more coaxially stacked helical regions than are found in a -s rrna model based on chemical crosslinking ( ). experimental studies must be done to determine which factors, such as the number of stems and unpaired nucleotides in the junction, govern the stacking of different helices. in addition to stacking helical regions together, junction regions act as hinges that orient the different helical regions in space. the four-stem junction in trnas adopt an l shape and the four-stem dna junction adopts an x shape. the conformations of more rna junctions should be characterized in order to learn the various shapes that junctions can adopt. junction regions appear to constitute catalytic rna sites. the hammerhead self-cleaving rna region is coded for by newt satellite dna ( ) and is found in plant viruses ( ), virusoids ( ), and viroids ( ). the consensus secondary structure for the catalytic domain contains a three-stem junction containing unpaired nucleotides. most of the unpaired nucleotides of the junction region are conserved (fig. reaction ( , ). the specific cleavage takes place between one of the unpaired nucleotides and a stem region (fig. ) ; a ', '-cyclic phosphate and a '-oh group are formed ( ). the catalytic activity must involve the conformation of the unpaired nucleotides in the junction since these comprise almost all of the required nucleotides. growing evidence implies that the unpaired nucleotides in a five-stem junction of the rrna form the peptidyl transferase site of the ribosome, where peptide bonds are formed when amino acids are transferred from trnas to the nascent protein ( ) . chemical "footprinting" experiments have established that unpaired nucleotides in this junction are protected from chemical modification when trna is bound to the peptidyl transferase site ( ). direct chemical crosslinking of the trna to the five-stem junction has also been seen ( , ) . the examples of the hammerhead catalytic domain and the peptidyl transferase junction of -s rrna illustrate the importance of determining the conformation of unpaired nucleotides in junction regions. not only do the conformations of these nucleotides have a great impact on the threedimensional structure by orienting the stem regions that meet at the junction, but also these nucleotides can be positioned to catalyze specific reactions. two techniques that are commonly used either separately or together to predict rna secondary structure are phylogenetic comparison ( , ) and thermodynamic stability ( , ). phylogenetic comparison is the method currently used as the standard for determining secondary structure. the underlying principle is that mutations that do not alter function will be preserved. since function is assumed to depend on structure, the preservation of a structure between different organisms, despite changes in the base sequence, is good evidence for the structure's existence. phylogenetic comparison requires that rna sequences from several different organisms be known. the sequences are first aligned and then searched for regions capable of base-pairing. since helical regions are maintained if g*c pairs are replaced by a*u pairs or vice versa, covariance of nucleotides establishes which regions are involved in base-pairing and which are not. a helix is usually considered to exist if two or more covariations are found in it ( , ) . in practice, organisms whose primary sequences differ by - % give the best results with phylogenetic comparison ( ). sequences that are too dissimilar are difficult to align, and sequences that are too similar do not have enough compensatory base changes to establish the existence of helical regions. a limitation of the phylogenetic method is that it cannot provide any information about regions of secondary structure that contain conserved nucleotides. thus, phylogenetic methods tend to predict fewer helices than actually exist in the molecule. thermodynamic stabilities are used routinely to predict secondary structure. computer algorithms predict structures by calculating the free energies for all possible base-pairing schemes and finding the secondary structure of lowest free energy. computer algorithms that find several different secondary structures whose calculated free energies are close to the lowest free energy ( , ) are important for several reasons. one reason is that the calculated energies are based on incomplete experimental data and thus have significant uncertainties. another reason is that biological rna molecules begin folding as they are synthesized; they could become kinetically trapped in a structure that is not the structure of lowest energy. furthermore, tertiary interactions may stabilize a secondary structure that is not calculated to have the lowest free energy. the most fundamental reason for calculating alternate secondary structures is that biological rna molecules may not form a single secondary structure, but may instead have several structures in equilibrium. this has been suggested, for example, for the ciz gene mrna of bacteriophage x ( ) . free energies are calculated from experimentally determined parameters by the computer algorithms using a nearest-neighbor model. since stacking interactions are short-range, it is reasonable to assume that the free energy of a helical region depends on the sequence of dinucleotide steps it contains ( ). this nearest-neighbor model has been tested for short duplexes with different sequences, but with the same set of dinucleotide steps; with only a few exceptions, the average agreement between the predicted and measured free energies is within % ( ). the free energies of loop regions (hairpins, internal loops, and bulges) are more difficult to quantitate. originally, loop free energy was assumed to depend only on the number of unbonded nucleotides within the loop. this was based on free energies of loop formation of a small set of molecules with limited sequence variation ( , ) . there are now examples of loop regions whose free energy of formation depends markedly on the sequence of nucleotides within the loop. for example, the hairpin loop uucg discussed above is much more stable than would be predicted by the current freeenergy parameters. so is the internal loop containing two g-a mismatches (in boldface) formed by the duplex with ' dangling guanosines [gcgagcg],. as more thermodynamic data are collected for loop regions, more accurate formulas for predicting the free energy of loop regions will become possible. junction regions present bigger problems than other loop regions. no thermodynamic data have been measured for the free energy of junction formation in rna. the present computer programs typically assume that the free energy of a junction region depends on the number of stems and the number of unpaired nucleotide within the junction ( ) . the values of the parameters are empirically derived to best fit the structures of rna sequences whose secondary structures are well established. despite the problems with free energy of loop formation, current computer algorithms do remarkably well in predicting rna secondary structure. the lowest free-energy structures calculated by the zuker program for over molecules predicted % of the helices deduced from phylogeny ( ). furthermore, the best structure within % of the lowest free energy predicted % of the helices correctly. the number of correctly predicted helices will surely increase as more free energies are measured for loop formation. in practice, both phylogenetic comparison and computer algorithms are used to predict secondary structure. if only a few sequences that are not homologous enough to align for phylogenetic comparison are available, they can be folded with computer algorithms. a model secondary structure can be determined by searching for a common secondary structure among the various suboptimal foldings generated for the different sequences ( ) . determining a secondary structure by either method is usually an iterative process. the secondary structure model is refined as more sequences are determined or as the model is compared to the results of chemical and enzymatic modification procedures that map the accessibilities of different nucleotides. rna molecules fold into compact structures stabilized by tertiary interactions not included in the secondary structure. secondary and tertiary interactions are distinguished in terms of chord crossing (fig. ) . tertiary interactions are considered separately from secondary structure, since the formation of tertiary interactions depends not only on the nucleotides that form the tertiary interaction but also on the rest of the secondary structure. for example, the conformation about the four-stem junction in trna brings the t loop and the d loop close together; this allows tertiary pairing between nucleotides in these loops. the nucleotides in the anticodon loop cannot interact with either of the other loops, since the anticodon loop is fixed at one end of the molecule by the junction conformation. the biological functions of rna molecules-in particular, catalytic behavior-are determined by their three-dimensional structures. characterizing all of the tertiary interactions that occur in an rna molecule does not completely describe the three-dimensional structure, but tertiary interactions do indicate regions of the secondary structure that are close together in space. most of what we know about tertiary interactions comes from a few trna crystal structures, but we are beginning to learn more about tertiary interactions from a variety of methods (discussed in section iv) to study an increasing number of rna molecules. the goal is to find recurring tertiary elements: specific arrangements of secondary structure elements stabilized by tertiary contacts. here we discuss the three types of tertiary interactions so far characterized: tertiary base-pairing, single-strand-helix interactions, and helix-helix interactions. a. tertiary base-pairing nucleotides that are unpaired in the secondary structure of an rna molecule can form tertiary contacts by hydrogen bonding to other nucleotides unpaired in the secondary structure. in general, this could occur between any of the secondary structure regions containing unpaired nucleotides (single-stranded regions, hairpin loops, bulge loops, internal loops, and junction loops). all of these interactions were originally termed knots or pseudoknots ( ) ; here, we use the term "pseudoknot" to describe only the structure in which nucleotides in a loop (hairpin, internal, or bulge) pair with nucleotides in a single-stranded region. different types of pseudoknots are shown in fig. ( ) . pseudoknots have been found in an increasing number of biological sys- tems since their discovery at the ' end of several plant rnas ( , ) . the viral rnas were recognized by trna-specific enzymes, but the secondary structures predicted for these sequences did not include an amino-acid acceptor stem. the formation of a pseudoknot allows these molecules to fold into a tertiary structure functionally similar to trna, even though the secondary structures are different. pseudoknot formation also enhances frameshifting during translation in the coronavirus ibv ( ). the mechanism by which pseudoknots contribute to frameshifting has not been determined, but the combination of an (a+ u)-rich sequence and a pseudoknot results in a frameshift. one possible explanation is that the pseudoknot causes the ribosome to pause, allowing slippage to occur at the a+u-rich sequence. this mechanism seems to be a general one, since of sequences from a variety of viruses, known or suggested to contain frameshifting sites, have the potential to form pseudoknots ( ) . a pseudoknot of the type diagrammed in fig. a formed by a short oligonucleotide has been studied by n m r ( ) . the interproton distances, determined by nmr, were consistent with the two stem regions, one containing bp and the other containing bp, with a-form helix geometry. the distances between protons located in the two different stem regions indicate that the two stem regions stack coaxially to form one continuous helical region. the pseudoknot appears to be a normal duplex from one side, but on the other side, two loops bridge the duplex, one crossing the major groove and one crossing the minor groove. the size of the loops was varied without changing the stem sizes to show that the minimum loop size for crossing the minor groove of the -bp stem is three nucleotides, and the minimum loop size for crossing the major groove of the -bp stem is two nucleotides. the pseudoknot is only marginally more stable than either of the two potential hairpin structures that the sequence could form. the equilibrium between pseudoknot and hairpins depends on salt concentration, temperature, and nucleotide sequence ( ). there are several examples of rna molecules containing tertiary contacts between nucleotides that are in loop regions of secondary structure. phylogenetic comparison suggests that the rna component of rnase p, which catalyzes the processing of trna precursors, makes watson-crick pairs between four nucleotides in one junction loop and four nucleotides in a second junction loop ( ). these are interactions between secondary structure loop regions in the crystal structure of yeast trnaphe. two of the nucleotides in the d loop form parallel pairs with two nucleotides in the t loop: g,,*ijj, and g,,c,. two additional tertiary base-pairs are formed between unpaired nucleotides in the central four-stem junction and nucleotides in the d loop: a reverse-hoogsteen pair (fig. ) , u,.a,,, and a parallel-stranded reverse-watson-crick pair (fig. ) , g *c,, ( ) . these tertiary pairs are stabilized by hydrogen bonding and stacking interactions with adjacent nucleotides; they are partially responsible for stabilizing the l shape of trna. tertiary pairs between loop regions have also been proposed on the basis of phylogenetic comparison in the -s and rrnas ( , ). the high frequency with which known rna structures contain tertiary pairing between nucleotides that are unpaired in the secondary structure stresses the importance of learning more about such interactions. can unpaired nucleotides in all of the secondary structure loop types engage in tertiary pairing? there are several examples of pairing involving nucleotides in hairpin loops and junction loops, but it is not known whether nucleotides in bulge loops or internal loops form tertiary interactions. the fact that trna has tertiary pairing between parallel strands shows that the rules for forming tertiary pairs can be different from the rules for secondary structure. the thermodynamics of tertiary pairs is of critical importance for predicting their existence. in the trnas and pseudoknots, tertiary pairs contribute less to the free energy than secondary structure does, but it may be possible for very stable tertiary interactions to replace secondary structure. base stacking is one of the most important factors stabilizing rna structures. one of the ways in which stacking can stabilize the tertiary structure is for nucleotides that are unpaired to intercalate between base-pairs. an example of intercalation is seen in the crystal structure of yeast trnaphe, where g,,, an unpaired nucleotide in the t loop, intercalates between two nucleotides of the d loop, g,, and g,,, which form tertiary pairs with nucleotides in the t loop. to accommodate the intercalated guanine between the two tertiary pairs, the sugar-phosphate backbone is extended by a change in the g,, sugar conformation to '-endo ( ). base-triples occur when an unpaired nucleotide forms hydrogen bonds with a nucleotide that is already base-paired. the third base of a base-triple may bind to the watson-crick pair in either the major or minor groove and be stabilized by the formation of one or two hydrogen bonds as well as stacking interactions. several biological functions have been proposed for base-triples. base-triples stabilize the three-dimensional shape of trna and several other rna molecules discussed below. the formation of dna triples inhibits transcription in uitro ( ) , and it has been suggested that a small rna molecule may inhibit transcription by forming a triplex bp upstream from the transcription site of the human c-myc gene ( , ) . the self-splicing intron from tetruhymenu binds guanosines via base-triple formation during the self-splicing reaction ( ). three base-triples occur in trnaphe, all of which involve nucleotides in the junction loop binding in the major groove to watson-crick pairs of the d stem. in two of the triples, ag*a,,*u,, and g,,*g,,c,, another rna molecule that forms base-triples is the self-splicing intron from tetrahymena. the intron binds a free guanosine during cleavage at the ' exon and binds an internal guanosine during cleavage of the ' exon. it is postulated that these guanosines bind to a base-pair, g,,c,,,, in the p stem by forming a base-triple ( ). replacing the g c pair with an a.u pair abolished splicing activity, but cleavage at the ' exon was restored by adding -aminopurine instead of guanine. -aminopurine can form a basetriple with the a*u pair that is isomorphic to the wild-type g.g*c triple. this is strong evidence for the existence of the g.g.c triple in the splicing reaction of the wild-type intron. a base-triple has also been proposed to occur between a nucleotide in a junction loop and an adjacent helix in xenopus zueuis -s rrna ( ). on the basis of chemical modification and model building, it is proposed that an adenosine in the junction loop binds to a g-u pair in the minor groove by forming two hydrogen bonds, one between the adenosine n and the ' hydroxyl of the guanosine, and one between the adenosine amino and a base nitrogen of the guanosine. the formation of base-triples in junction regions where helices stack coaxially may be a recurring rna structural element. if two helical regions in a junction stack coaxially (fig. ll) , it is a consequence of a-form helix geometry that the ' strand cannot reach the major groove of the adjacent helix, and the ' strand cannot reach the minor groove of its adjacent helix. the ' strand of unpaired nucleotides can follow the minor groove of the adjacent helix, while the ' strand can follow the major groove of its adjacent helix. although oversimplified, this picture is consistent with two of the three base-triples that form in trnaphe, and with the base-triple proposed to occur in the -s rrna from x. zueuis (fig. ) . the base-triple formed during the ' splicing reaction of the intron from tetruhymenu also fits this model if the recently identified p . helix near the ' splice site of the tetruhymenu intron ( , ) is stacked on the p helix (fig. ) . to predict the existence of base-triples, the sequences that can form triples must be determined, and their structures and thermodynamic stabilities must be characterized. triple helices were originally found in polynucleotides with simple repeating sequences. fiber diffraction studies on the poly(ru).poly(ra)-poly(ru) system showed that the third strand, poly(ru), bound parallel to the purine strand of the watson-crick helix in the major groove with two hydrogen bonds between the adenine and uridine ( ) . in addition to ru.ra*ru, the following polynucleotides have been shown to form triple helices: ra-ra-ru, rc+.rg*rc (at ph . ), and rg.rg*rc ( ) (fig. ) . recent nmr studies of dna triples show that dc+ .dg.dc forms a structure isomorphic with dt*da.dt ( , ) ; the corresponding rna triples are probably isomorphic as well. structural characterization has not been done on rg-rg*rc or ra*ra.ru triple helices yet, but these base-triples can form isomorphic structures. replacing the ra.ra*ru base-triple in trnaphe with a sequence capable of forming an rg.rg*rc base-triple resulted in no loss of aminoacylation activity, whereas sequences that could not form base-triples did lose activity ( ). unpublished results from our laboratory, including uv absorption mixing curves and circular dichroism spectra, show that poly(ra)*poly(rg).poly(rc) forms a triple helix. a hydrogen-bonding scheme has not yet been determined for this structure, but the poly(ra) strand appears to bind in the minor groove of the watson-crick duplex. the evidence for minor groove binding is that a triple helix did not form when the poly(rg) strand was replaced by poly(ri), which lacks the minor groove amino group capable of hydrogen bonding to poly(ra). we have already described structures in which helical regions stack coaxially end to end. helix-helix contacts can form between the grooves of different helices when rna molecules fold into compact tertiary structures. the negatively charged sugar-phosphate backbones repel each other, but a variety of interactions found in crystal structures of nucleic acid duplexes could stabilize the structure. the importance of the ' hydroxyl groups in stabilizing helix-helix contacts is seen in the crystal structure of an rna duplex [u(ua),a],. there are intermolecular hydrogen bonds between the ' hydroxyl groups in one helix and either uracil carbonyl groups or sugars in the minor groove of another helix ( ). another type of helix-helix contact is found in the crystal structure of a dna duplex, helix ( ) . in general, helix-helix interactions could include base-phosphate, base-sugar, sugar-sugar, and sugar-phosphate hydrogen bonding. helix-helix contacts have been implicated in the function of one intriguing biological system. the tetrahymena intron is capable of binding a nicked duplex rna containing three oligonucleotides and then ligating the nick ( ). since the nicked duplex is base-paired and the reaction is independent of the duplex sequence, the intron must bind the duplex substrate through the formation of helix-helix contacts. the weakness of the contacts between the intron and the duplex substrate is evidenced by a michaelis constant greater than . mm. this system illustrates the complexity of the interactions stabilizing rna structure. determining the interactions between bases is not enough to understand the structure and function of rna; the backbone interactions must be determined as well. another system in which helical regions may interact involves sequences rich in guanosine. guanosine-rich dna sequences have been proposed to form duplex structures ( ), and there is evidence that two of these duplexes in solution dimerize to form four-stranded dna complexes ( - ). it is not known whether similar sequences in rna can form these structures, but x-ray diffraction studies have shown that poly(rg) forms a structure containing four parallel strands with the four equivalent guanosines hydrogen-bonded in a coplanar arrangement ( ). the secondary structures for a wide variety of biological rna molecules have been established by a combination of techniques such as phylogenetic comparison, chemical and enzymatic modification, and computer prediction algorithms. some biological functions of rna can be understood once the secondary structure is known, but understanding most biological functions, particularly catalytic rna activity, requires the determination of rna three-dimensional structure. the next step toward the prediction of rna three-dimensional structure is to develop methods to predict its tertiary interactions. the same two methods used to predict secondary structure (phylogenetic comparison and computer algorithms) can be used to predict tertiary interactions. as currently used, both of these methods are limited because they predict tertiary interactions only between bases. the tertiary interactions in the crystal structures of trna contain many examples of base-sugar, base-phosphate, sugar-sugar, and sugar-phosphate interactions ( , ). establishing the presence of tertiary structure from phylogeny relies on the replacement of a specific tertiary pairing by an equivalent one. phylogenetic comparison has been used to predict the existence of watson-crick tertiary pairing in the rna component of rnase p (loo), the tetruhymenu intron ( , ), and both the ( ) and -s ( , ) rrnas. phylogeny can also be used to predict the existence of base triples, as was done for trna ( ) . there are several limitations on predicting tertiary pairs using phylogenetic comparison. all but one of the tertiary pairs formed in trna is non-watson-crick; these include reverse-hoogsteen, reverse-watson-crick, and parallel-stranded pairing. if this is true for other rna molecules, phylogenetic comparison will have trouble predicting tertiary pairings. although phylogenetic evidence has been used to suggest the presence of non-watson-crick pairs ( ), in general, we do not know which non-watson-crick pairs are equivalent. since phylogenetic comparison depends on sequence variation, the structures of conserved regions cannot be predicted. this is a problem in predicting secondary structure and could be a much greater limitation in the case of tertiary structure. the nucleotides in trna that are engaged in tertiary pairing are much more highly conserved than are those involved in secondary structure. if this is true in general, it will be difficult to predict tertiary pairing by phylogeny. the use of thermodynamic stabilities to predict secondary structures has already been discussed. extending this approach to the prediction of tertiary interactions poses several problems. we discuss the problems inherent in predicting tertiary motifs and then describe an algorithm that predicts the pseudoknot tertiary structure as well as secondary structure. the prediction of tertiary interactions using computer algorithms based on thermodynamic stabilities poses three problems: ( ) evaluating free energies for all of the possible secondary and tertiary structures requires prohibitive amounts of computer time; ( ) rules governing which regions of secondary structure are sterically allowed to form tertiary interactions have not been established; and ( ) the free energies of most tertiary structures have not been determined. the distinction between secondary and tertiary structures was originally made so that an algorithm could rigorously consider all of the possible secondary structures. for an rna of n nucleotides, the number of different base-pairs possible is n(n - )/ . if tertiary pairs are allowed, the number of possible structures increases proportional to n factorial instead of n ; this renders impractical a rigorous examination of all possible secondary and tertiary structures for a biological rna. since we cannot search every possible combination of secondary and tertiary structures, we must choose criteria that limit the number of structures to be evaluated, but still find the biologically relevant structures, although the actual path by which rna molecules fold is a kinetic property, we assume that the structure that forms is the structure of lowest free energy. this means that we can fold the rna by any path we choose as long as the free energy for each step is known. one way to restrict the number of structures evaluated is first to find the low-free-energy secondary structures by standard programs ( ) . then tertiary interactions are added to a small number of calculated secondary structures to obtain the tertiary structure of lowest free energy. we cannot use the approach just outlined for predicting tertiary structures until we know how to look for secondary structure elements that can form tertiary interactions. for example, from the cloverleaf secondary structure of trna, how would the algorithm know that the junction conformation brings the d loop close to the t loop, but not to the anticodon loop? until we learn more about the conformation around the junction, internal, and bulge loops, assumptions must be made about which loop regions can interact. the simplest assumption is that any pair of loop regions can interact. prediction of tertiary interactions also requires knowledge of the free energy of forming tertiary interactions. judging from trna and pseudoknots, secondary structure is more stable than tertiary structure. if this is true, the calculation of the free energy for tertiary pairs after the calculation of the free energy for secondary structure is justified. errors in this assumption can be accounted for by predicting tertiary interactions for several secondary structures. unfortunately, free energies have not been measured for most tertiary structures. the free energy of pseudoknot formation has been determined for a very limited set of molecules ( ) , but the free energies of other tertiary interactions, such as loop-loop pairing, base-triples, or intercalation, have not been determined. despite the difficulties of predicting tertiary structure, an algorithm capable of predicting pseudoknots as well as secondary structure has been developed ( ) . the algorithm calculates the free energy of formation for all of the stem-loops that could be formed by the structure. the predicted structure starts with the stem-loop with the lowest free energy of formation; new stem-loops consistent with those already incorporated are added in order of their stability. the algorithm predicts stem-loops due to pseudoknots as well as those due to normal hairpins. the biggest limitation of this algorithm is that it only predicts a single structure. the free-energy contribution of the base-pairs in a pseudoknot were assumed to be the same as in a hairpin stem; the free-energy contribution of the two loops of the pseudoknot was empirically determined so that known pseudoknots were predicted. the program also predicted pseudoknots in sequences that had not previously been shown to contain pseudoknots. the most important information needed to improve tertiary structure prediction are the free-energy parameters for tertiary interactions such as pseudoknots, tertiary base-pairs, and base-triples. furthermore, rules must be developed indicating which tertiary contacts are sterically possible for a given structure. realistic predictions of tertiary interactions and three-dimensional structure will not be possible until the conformations around junctions, bulge loops, and internal loops are known. once the secondary structure and tertiary interactions contained in an rna molecule are known, the next step in understanding its structure is the determination of a three-dimensional structure. the concept of one threedimensional structure may be misleading, since it implies that the molecule exists in a single structure and ignores the changes the rna can undergo. ultimately, we would like to know all of the conformations an rna molecule can adopt and the dynamics of their interconversion. the first step toward this goal is determination of the three-dimensional structure of one conformation. since no three-dimensional structures of rna are known, with the exception of trna, which is discussed extensively elsewhere ( , , ) , we discuss the three-dimensional models which have been built for other rnas. three the process of model building shows the importance of secondary structure loop regions (internal loops, bulge loops, and junctions) in the threedimensional structure of rna. the ability to predict the conformation of loop regions or even the ability to rule out certain conformations would significantly improve the process of building three-dimensional structures of rna molecules. the positions of the helical regions in models depend on different types of experimental data. for example, the three-dimensional locations of the proteins bound to -s rrna ( ) were combined with protein-rna crosslinking and footprinting data to generate constraints on % of the helices in one of the models of rrna ( ). another model, based on much of the same data, is in substantial agreement with this model ( ), as is a model built from the accessibility of the -s rrna to dna oligonucleotide probes, which constrains % of the helices in the -s rrna ( ). chemical and enzymatic modification data are useful, but these data are insufficient to determine the relative positioning of helical regions. the method with the next highest level of resolution is nmr. it complements the x-ray method in that it provides distances between nearby protons (distances less than &; it can also determine backbone torsion angles. thus, nmr provides details about local conformation and can be used to determine secondary, tertiary, and, in principle, three-dimensional structures. information about the arrangement of the secondary structure elements in three dimensions can also be obtained from crosslinking and fluorescence energy transfer experiments. chemical and enzymatic modifications are used to determine the accessibility of functional groups within nucleotides, . and the effect of mutations introduced into rnas on biological activity can also determine rna structure. nmr experiments provide a powerful method for determining the structures of proteins and nucleic acids in solution. detailed explanations of nmr methodology applied to nucleic acids have been published ( , ) , so we only outline the principles of the nmr method here. then we discuss how well the information determined by nmr defines the three-dimensional structures of rna molecules. nmr methods provide three types of information that can be used in structure determination: nuclear overhauser effects (noes), scalar coupling constants, and chemical shifts. noe is the transfer of magnetization due to magnetic dipole-dipole coupling between nuclei. the effect is directly proportional to the magnetic moments of the nuclei and depends on the inverse sixth power of the distance between them. noes can be measured between protons up to a apart. both the exchangeable and nonexchangeable protons in rna are used for noe measurements. information regarding base-pairing and stacking can be obtained from the measurement of noes between imino resonances assigned to specific nucleotides. the imino protons resonate in a separate region of the spectrum from other protons, and each watson-crick base-pair contains one hydrogen-bonded imino proton. only imino protons that are hydrogen-bonded, or whose rates of solvent exchange are otherwise decreased, are seen in the nmr spectrum. noes between imino protons have been measured in molecules as large as trna ( ) . the secondary and tertiary base-pairing as well as the coaxial stacking of helical regions of trna in solution have been confirmed by this method ( , ) . noes between the nonexchangeable base and sugar proton resonances give much more detailed information about rna structure than exchangeable-proton nmr. about intranucleotide distances ( base-sugar and intra-sugar) can be measured between base and sugar protons. these distances are sufficient to define the conformation of a nucleoside. up to additional internucleotide (base-base, base-sugar, and sugar-sugar) distances can be measured, depending on the rna structure. nmr studies on the nonexchangeable protons are currently limited to oligonucleotides containing no more than or nucleotides. new nmr techniques such as isotopic labeling ( , ) and three-dimensional nmr methods ( ) are being developed, which will allow nmr studies on larger rna molecules. oligonucleotides used for nmr studies of nonexchangeable protons are designed to adopt structures found in larger rna molecules. lower-resolution studies such as chemical modification can be done to check that the structure adopted by the oligonucleotide is similar to the structure within the larger rna molecule. scalar coupling constants, also called spin-spin splittings, can be measured for two nuclei separated by two, three, or sometimes four bonds. for a three-bond splitting, for example, h-c-c-h, the value of the coupling constant depends on the torsion angle for rotation around the central bond. coupling constants are related to torsion angles by karplus-type equations ( ); these relationships have been determined for the sugar-phosphate backbone in rna by studies of model compounds ( ) . coupling constants can be measured between ribose protons as well as between protons and phosphorus atoms. these coupling constants can be used to determine four of the seven torsion angles that completely define the conformation of a nucleotide unit (fig. ). information about two of the three remaining torsion angles can be estimated from phosphorus chemical shift information as described below, and the remaining torsion angle can be determined from the intranucleotide noes. in principle, all of the torsion angles can be determined by nmr methods. the chemical shift of a resonance depends on the local magnetic field at the nucleus. the local magnetic field is extremely sensitive to the bonding, and to the proximities and types, of nearby atoms. unfortunately, no simple correlation between proton chemical shift and structure has been established so far, although chemical shifts have been calculated for protons in several molecules ( ). phosphorus chemical shifts have been correlated with the two phosphodiester torsion angles -p- ( ). normally, both phosphodiester torsion angles are in the gauche conformation. both theory ( ) and experiment ( , ) suggest that ifeither of the two angles changes to the trans conformation, the phosphorus resonance moves to the downfield region of the spectrum. the two adjacent c - torsion angles and the -p- bond angle have also been shown to effect the phosphorus chemical shift ( ). ifthe information inherent in the chemical shift could be tapped, nmr structure determination would become much more powerful. in principle, nmr experiments on rna molecules could determine their three-dimensional structures. accurate measurement of seven torsion angles per nucleotide suffices to specify an rna structure. if all of the torsion angles and a large number of intra-and internucleotide distances are determined (within experimental uncertainties), the atomic coordinates of the rna molecule can be generated by distance-geometry algorithms ( ) ( ) ( ) . in practice, not all of the torsion angles are determined, and spectral overlap can preclude the measurement of many noes. an important question not yet satisfactorily answered is: which distances and torsion angles must be measured with what accuracy to specify a three-dimensional structure for rna molecules? the first step in answering this question was made by testing the distance-geometry algorithm on a known dna duplex structure ( ). a set of distances that could be measured easily by nmr was taken from the crystal structure of a -bp dna duplex. the established base-pairing in the duplex was used to add more constraints: the distances between hydrogen-bond donors and acceptors in the base-pairs. since each nucleotide has seven degrees of freedom, independent constraints suffice to define the structure completely. although there are more than n m r constraints, the fact that they are not all independent may result in an underdefined structure. the structure generated from these constraints by the distancegeometry algorithm was compared to the starting structure. the results showed that areas of the structure where many interproton distances were used (the sugars and the bases) were well defined. however, the phosphate backbone was not very well defined. the root-mean-square deviation for the generated base hydrogen atoms was . a, whereas the deviation was . , for the phosphorus atoms. for all of the atoms in the structure, the deviation was . a, comparable to the deviation found for the atoms in a protein structure when a similar procedure is undertaken ( ). nmr data can determine structures more accurate than that of the dna duplex just described. the constraints used to generate the dna structure did not include distances to the h ', h ', and h " sugar protons because they cannot always be assigned. more importantly, no torsion angles were used to constrain the structure, although many of these angles can be determined from proton-proton and proton-phosphorus coupling constants ( , , ) . the accuracy of structures determined by nmr ultimately depends on the nucleic acid structure itself. crosslinks-covalent bonds between different bases-can be introduced into rna molecules by irradiation with uv light ( ) or by using chemical reagents such as psoralen ( ) or nitrogen mustard ( ) . the positions of such crosslinks within the structure can be determined by sequencing the rna on either side of the crosslink. the crosslinks are of two types. the first type occurs between nucleotides adjacent in the secondary structure. the more interesting crosslinks occur between nucleotides that are not adjacent in the secondary structure. the positions of these crosslinks reveal something of the three-dimensional structure of the molecule, since they show the proximity of two secondary structure elements. caution must be used when interpreting the results obtained from crosslinking studies. crosslinks generated by chemical reagents that bind to one base and then crosslink to a second base may not reflect the native structure of the rna, since the binding of the reagent to the first base may alter the rna structure.' some of the molecules studied by crosslinking include the rrna ( ), the -s rrna ( ), trna binding to the catalytic subunit of rnase p ( ), trna binding to ribosomes ( ), and protein binding to ribosomes ( ). fluorescence energy can be transferred from a donor to an acceptor group by an electric dipole-dipole mechanism. the transfer depends on the inverse sixth power of the distance between these groups. in practice, the transfer can be measured for groups separated by roughly - w ( ). measuring fluorescence energy transfer in rna requires adding acceptor and donor groups to the molecule. some of the applications of this technique include the study of trna ( ), ribosome assembly ( ), and the conformation about a four-stem dna junction, as described above ( ). chemical and enzymatic modification methods determine the accessibility of the nucleotides within an rna molecule to modification by chemical reagents or enzymes ( ). the reactivity of a nucleotide to chemical reagents is a complicated function of solvent accessibility and electrostatic environment ( , ) . the reactivity of nucleotides to chemical modification is used to confirm predicted secondary structures and to learn about tertiary interactions. the advantages of these methods are that they can be used to probe the structure of very large rnas, and that they require only picomoles of rna. chemicals that react with each of the four bases at watson-crick hydrogen-bonding positions can reveal which nucleotides are involved in basepairing. conditions are used in which each rna is modified at most only once, so that the structural information deduced is not an artifact of the modification procedure. enzymes that cleave specifically in base-paired or unpaired regions are used to determine base-pairing as well, but the large size of enzymes makes them generally less useful than chemicals, since the compact structure of a large folded rna yields very few enzymatic cleavage sites ( ). chemical modification of the nucleotides within the rna is detected by one of two methods. the simplest method induces strand scission at the site of modification; this is most useful for short rna molecules. sites of modification within large rna molecules are located by synthesis of a dna complementary to the rna using reverse transcriptase ( ). modified residues in the rna cause the reverse transcriptase to stop. separation of the synthesized dnas by gel electrophoresis determines the positions of modification. clear interpretation of the results of chemical modification is often difficult. the amount of modification ranges from essentially none through various degrees of partial modification to strong modification. strong modification of a nucleotide is good evidence that it is not involved in secondary or tertiary pairing. weak or partial modification can result from nucleotides engaged in either secondary or tertiary interactions. mutations are often introduced into rna sequences in order to determine rna structure or protein-rna interactions ( , , , - ) . the effect of these mutations is often assayed by measuring the ability of the mutated sequences to bind a protein that specifically recognizes the wildtype rna. although mutational analysis is a powerful technique for determining interactions in rna structure, caution is necessary with this approach. the results of such experiments can be unclear, since loss of protein binding can result either from a change in rna structure or from an rna sequence that maintains the same structure but is not recognized efficiently by the protein. since most cellular rna molecules are complexed by proteins, understanding protein-rna interactions is vital to understanding cellular rna functions. unfortunately, even less is known about the structure of rna binding proteins than about rna structure. the protein-rna interactions studied show that proteins recognize specific secondary structural features of rna as well as its three-dimensional shape. the interactions between the helix-turn-helix proteins and duplex dna are well-characterized ( - ) . an a-helix lies in the major groove with amino acids forming specific hydrogen bonds to the dna sequence. as shown in fig. , the major groove in typical a-form rna is much narrower than in typical b-form dna. it has been suggested that the narrower major groove in rna prevents protein structure elements from binding to the bases in the major groove ( - ) . however, the fact that a nucleotide strand is capable of binding to duplex rna in the major groove to form a triple helix, and the fact that there is considerable polymorphism in the size of the a-form major groove in x-ray studies of fibers, suggest that the bases may be accessible to protein structures through the major groove ( ). transcription factor iiia is a "zinc finger" protein that binds the dna gene for the -s rrna in x. zueuis as well as the -s rrna. there is evidence that it binds to the dna gene in the major groove ( ), and it is proposed that it binds to the -s rrna in the major groove ( ). although it could bind to the dna and rna sites by different mechanisms, the possibility that proteins bind in the major groove of rna should not be ruled out. most of the protein binding sites in rna that have been characterized are loop regions: hairpins, bulges, and internal loops. unpaired nucleotides are more conserved than base-paired nucleotides in rrna sequences, suggesting that these nucleotides are involved in either tertiary interactions or protein contacts. unpaired adenosines occur more frequently than the other nucleosides ( ). direct evidence for the role of unpaired adenosines in protein binding of the -s rrna comes from the decrease in chemical reactivity of these adenosines when the ribosomal proteins are bound to the -s rrna ( ). both bulge and internal loops bind proteins. a purine bulge is required for the coat protein to bind to the r viral rna ( ), and an adenosine bulge is part of the l protein binding site on the -s rrna ( ). an asymmetrical internal loop is required for the binding of s ribosomal protein to the -s rrna in e. coli ( , , ) . ribosomal protein l binds to the -s rrna at a large asymmetrical internal loop as well ( ). hairpin structures are commonly found to bind proteins specifically ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . hairpins with loop sequences cagugn bind to the iron-responsive element (ire)-binding protein ( ) . as proposed in other protein-rna interactions ( ) ( ) ( ) , free cysteines in the ire-binding protein are thought to form transient covalent bonds to the rna hairpin by a nucleophilic attack by the sulfhydryl group on a uracil in the rna. iron regulates this system by altering the equilibrium between reduced and oxidized sulihydryl groups. this in turn alters the amount of hairpin bound by protein and alters the translation of the iron receptor mrna ( ) . the most-characterized example of a hairpin protein binding site is the r coat protein binding site ( ). binding requires a hairpin, whose loop sequence must be anya, plus a purine bulge three nucleotides away on the ' side of the loop. it is not clear what role the purine bulge plays in protein binding, since adding substituents such as methyl groups to an adenosine bulge reduces binding iow-fold, whereas changing the bulge from adenosine to guanosine leaves binding essentially unchanged. these data are insufficient to determine whether the protein forms specific contacts with the purine bulge, or whether intercalation of the purine alters the structure of the rna. this study shows the limitation of mutagenesis experiments in which specific nucleotides are changed and the effect on protein binding is measured. without doing structural studies of the rna molecules, it is not clear whether the different binding ailhities that result from substituting specific nucleotides are due to disruption of protein contacts to that nucleotide, or whether the overall structure of the rna has changed. the first high-resolution crystal structure of a protein-rna complex to be solved was that of e. co i trnag'" and its synthetase ( ). sequencespecific contacts occur at the anticodon loop and at the end of the acceptor stem of the trna in addition to contacts along one side of the trna. three bases in the anticodon loop, which form specific contacts with the protein, are unstacked compared to the trnaphe structure. the first base-pair in the acceptor stem is unpaired, and the protein contacts the acceptor stem in the minor groove, forming hydrogen bonds with exocyclic amino protons of two guanines. overall, the crystal structure shows that the synthetase recognizes the trna shape, but distinguishes it from other trnas by forming two types of sequence-specific contacts: binding to anticodon loop nucleotides and binding to guanines in the minor groove of the acceptor stem. three-dimensional structure is not always recognized by trna syn-thetases, since a hairpin containing the major recognition feature of trnaala is efficiently aminoacylated by e. coli trnaaia synthetase ( ). the major recognition feature is a single g-u mismatch in the acceptor stem ( ). replacing the g*u mismatch by g*a, c-a, or u*u mismatches (fig. ) results in only small losses in aminoacylation activity, although replacing the mismatch by a watson-crick pair completely abolishes activity. the fact that other mismatches are almost as efficient as g*u suggests that the protein recognizes a change in the sugar-phosphate backbone caused by the mismatch rather than specific groups on the g-u pair. many of the trna synthetases probably recognize three-dimensional structure, since the nucleotides required for binding are often scattered throughout the trna ( - ) . although the structure of only one protein-rna complex has been determined, several features of protein binding sites in rna molecules have been characterized. the most frequently found protein binding sites in rna are specific nucleotides within loop regions. hairpin loops are the bestcharacterized protein binding sites, but bulge loops and internal loops have been implicated in protein binding as well. proteins recognize primary sequence [poly(a) binding protein] ( ), secondary structure (hairpin, bulge, and internal loops), and three-dimensional shape (trna synthetase binding). the specific interactions between proteins and rna include transient covalent sulihydryl-uracil interactions, positively charged amino acids binding to negatively charged phosphates, hydrogen-bond formation with the exocyclic amino group of guanine in the minor groove, and specific hydrogen bonds and stacking interactions with bases in loop regions. the rna structures that proteins recognize are better characterized than the protein structural elements that bind to rna. an -amino-acid consensus rna binding domain, which contains a sequence of eight highly conserved amino acids ( ), has been identified in several rna binding proteins, including human u a protein as well as the poly(a) binding protein ( - ) . the structure of a peptide portion of the gag polyprotein which binds hiv viral rna has been determined by nmr ( ) and is similar to the structure of a zinc finger protein that binds to dna ( ). intermolecular rna interactions occur in a wide range of biological processes, including a "spliceosome" assembly ( , ), rna "editing" ( ), and protein synthesis ( - ). intermolecular contacts include base-pairing and backbone interactions. backbone interactions occur between regions of rna already base-paired. although these interactions are not well-characterized, they probably involve the backbone of one helical region interacting with the groove of another helical region. these interactions include basesugar, base-phosphate, sugar-sugar, and sugar-phosphate hydrogen bonds. the stabilities of complexes formed between trna anticodon loops and short oligonucleotides ( ) or between complementary anticodon loops ( ) are much greater than the stabilities of short duplexes. the enthalpy changes during formation of anticodon loop complexes are similar to that of duplex formation, but the entropy of formation is significantly more favorable for the anticodons than for the duplexes ( ). the favorable entropy change for the codon-anticodon interaction is probably due to the stacked conformation of the nucleotides within the anticodon loop. often, the nucleotides in small hairpin loops are not stacked in a-form geometry, presumably making base-paired complexes with these loops much less stable than complexes with anticodon loops. the importance of backbone interactions in the formation of rna-rna complexes is demonstrated by the tetrahymena intron. as previously discussed, the intron is capable of binding an rna duplex through backbone interactions ( ) . a separate study showed the importance of the ' hydroxyl when the intron binds a single-stranded oligonucleotide ( ). a single-stranded rna oligonucleotide base-pairs to the intron, forming a complex lo times ( kal/mol) more stable than would be expected for rna duplex formation; however, the stability of a complex between the intron and a dna oligonucleotide is only as stable as the formation of short rna-dna duplexes. this suggests that either the ' hydroxyl groups of the oligonucleotide form specific interactions with the intron or that the intron forms interactions with the sugar or phosphate groups along the backbone of an a-form duplex. the complex formed between m rna, the catalytic component of rnase p, and its trna substrate is probably stabilized by the formation of backbone interactions. this suggestion is supported by a chemical crosslink that forms in the m rna-trna complex between base-paired regions of the m rna and the precursor trna ( ) . as previously noted ( ), there is striking sequence and secondary structure homology between the region of the m rna that crosslinks to the trna and the region of the rrna which was found to bind trna by chemical footprinting ( ). complexes stabilized by backbone interactions may be a general feature of catalytic rna-substrate complexes since these interactions are weak and allow dissociation of the enzyme-substrate complex after the reaction. hybrid duplexes form between rna and dna during transcription and reverse transcription. the stability of these hybrids is believed to play a role in transcription termination ( ). the combination of a stable hairpin forma-tion in the nascent rna followed by a repeating da sequence in the dna leads to termination. the explanation for this is that the stable hairpin causes the polymerase to pause and disrupts part of the hybrid duplex ( ). the polymerase complex is held together by the remaining hybrid duplex. since ru*da hybrid duplexes are much less stable than other hybrids ( , ) , the polymerase falls off and transcription terminates. the discovery that a ribonucleoprotein complex adds dna sequences to chromosomal ends ( ) presents the possibility that an rna molecule can function as a reverse transcriptase. tetrahymena telomerase adds the dna sequence ttgggg to the ' end of chromosomes; it contains a protein component and a -nucleotide rna component, including the sequence caaccccaa complementary to the synthesized dna ( ) . the role of the caacccaaa sequence as a template for dna synthesis was proven by the discovery that mutating this sequence changes the dna sequence at the ends of chromosomes in uiuo ( ) . whether the rna alone can act as a reverse transcriptase or whether it only serves as a template for dna synthesis by the protein component of telomerase has not been established. or "telomere terminal transferase witherell and . c. uhlenbeck, nares principles of nucleic acid structure nmr of proteins and nucleic acids nares . r. lavery and a. pullman witherell and . c. uhlenbeck romaniuk and we thank peter davis, john jaeger, and gabriele varani for their reading of the manuscript, and we particularly thank jacqueline wyatt for her reading of the manuscript and for many key: cord- -iuglkdcp authors: sperschneider, jana; datta, amitava; wise, michael j. title: predicting pseudoknotted structures across two rna sequences date: - - journal: bioinformatics doi: . /bioinformatics/bts sha: doc_id: cord_uid: iuglkdcp motivation: laboratory rna structure determination is demanding and costly and thus, computational structure prediction is an important task. single sequence methods for rna secondary structure prediction are limited by the accuracy of the underlying folding model, if a structure is supported by a family of evolutionarily related sequences, one can be more confident that the prediction is accurate. rna pseudoknots are functional elements, which have highly conserved structures. however, few comparative structure prediction methods can handle pseudoknots due to the computational complexity. results: a comparative pseudoknot prediction method called dotknot-pw is introduced based on structural comparison of secondary structure elements and h-type pseudoknot candidates. dotknot-pw outperforms other methods from the literature on a hand-curated test set of rna structures with experimental support. availability: dotknot-pw and the rna structure test set are available at the web site http://dotknot.csse.uwa.edu.au/pw. contact: janaspe@csse.uwa.edu.au supplementary information: supplementary data are available at bioinformatics online. macromolecules such as dna, rna and proteins have the ability to form diverse tertiary structures, which enable functionality and thus, life. for many decades, proteins were deemed the global players in the cell until rna entered the spotlight. for example, rna structures have been found to be catalytically active, which was assumed to be the privilege of proteins. furthermore, small rnas are known to regulate gene expression and rna viruses employ a plethora of structure elements to invade the host cell. to gain insight into macromolecule function, one must investigate the structure. the first step in rna folding is stable base pairing that leads to a secondary structure. as rna structure formation is of hierarchical nature, secondary structure is the basis for the tertiary fold that produces the functional structure. especially for rnas, structure determination by experimental means is an intricate and expensive task. computational rna structure prediction is therefore an invaluable tool for biologists. comparative structure prediction is considered the most reliable approach for computational rna structure prediction. single sequence structure prediction is always limited by the accuracy of the underlying folding model. three main streams have been identified for comparative rna secondary structure prediction: (i) predict a structure from a pre-computed sequence alignment; (ii) simultaneously compute an alignment and a structure and (iii) alignment-free methods (gardner and giegerich, ) . tools for multiple sequence alignments such as clustalw (thompson et al., ) are readily available and thus, structure prediction from an alignment is a tempting approach [e.g. rnaalifold (hofacker et al., ) ]. such methods heavily depend on the sequence conservation and quality of the underlying alignment. however, ncrnas are conserved rather on the structure level than on the sequence level. the gold standard of rna comparative structure prediction is the sankoff approach as it does not rely on a high-quality sequence alignment and captures the structural conservation of ncrnas. sankoff ( ) introduced a theoretical dynamic programming algorithm for simultaneous folding and aligning for a set of n sequences that takes o(n n ) time and o(n n ) space. practical variants have been derived which more or less retain the sankoff principle by sacrificing optimality. alignment-free methods aim to avoid the pragmatic restrictions made in a practical sankoff approach as well as the reliance on a high-quality alignment [e.g. carnac (perriquet et al., ) ]. note that all of these comparative structure prediction methods exclude the prediction of rna pseudoknots. rna pseudoknots are crossing structure elements with diverse functions. the principle of pseudoknot formation is that bases within a loop region pair with complementary unpaired bases outside the loop. from an algorithmic point of view, even the simplest type of pseudoknot adds considerable computational demands due to crossing base pairs. in fact, the majority of comparative rna structure prediction methods exclude pseudoknots. biologists have delivered a wealth of studies, which show that pseudoknots have an astonishing number of diverse functions and occur in most classes of rna (staple and butcher, ) . rna viruses use pseudoknots for hijacking the replication apparatus of the host (brierley et al., ) . a limited number of rna comparative structure prediction methods can handle pseudoknots due to the computational complexity. several of these methods take a sequence alignment as an input. ilm is an algorithm that takes as an input either individual sequences or a sequence alignment (ruan et al., ) . a base pair score matrix is prepared initially and helices are added to the structure in an iterative fashion. in the approach hxmatch, a maximum weighted matching algorithm with combined thermodynamic and covariance scores is used (witwer et al., ) . this program gives the option to be combined with rnaalifold. knetfold is a machine learning method, which takes a sequence alignment as an input and outputs a consensus structure allowing pseudoknots (bindewald and shapiro, ) . simulfold takes an alignment as an input and simultaneously calculates a structure including pseudoknots, a multiple-sequence alignment and an evolutionary tree by sampling from the joint posterior distributions (meyer and miklos, ) . tfold combines stem stability, covariation and conservation to search for compatible stems and subsequently for pseudoknots for a set of aligned homologous sequences (engelen and tahi, ) . several comparative structure prediction methods including pseudoknots do not rely on an initial sequence alignment. the graph-theoretical approach comrna computes stem similarity scores and uses a maximum clique finding algorithm to find pseudoknotted structures (ji et al., ) . scarna performs pairwise structural alignment of stem fragments with fixed lengths derived from the probability dot plot (tabei et al., ) . in the following, a novel comparative approach for predicting structures including h-type pseudoknots called dotknot-pw will be introduced. the input consists of two unaligned, evolutionarily related rna sequences. similarity scores between structure elements will be calculated. statistically significant pairs will be used to find the set of conserved structure elements common to two sequences, which maximize a combined thermodynamic and similarity score. using a hand-curated test set of pseudoknotted structures with experimental support, the prediction accuracy of dotknot-pw will be compared with methods from the literature. pseudoknots are functional elements in rna structures and therefore, the most promising approach for comparative prediction is a structure comparison with less focus on exact sequence matching. in fact, perfect conservation on the sequence level can be more of a curse than a blessing. especially ncrnas are known to evolve quickly and so-called consistent and compensatory base pairs in both sequences will give much more confidence for structure conservation than a sequence alignment. one strong point of the dotknot method for single sequence pseudoknot prediction (sperschneider and datta, ; sperschneider et al., ) is that the set of possible h-type pseudoknot candidates (and secondary structure elements) is explicitly computed and thus readily available for further investigation. the main steps in the pairwise pseudoknot prediction approach dotknot-pw are as follows ( fig. ) : ( ) run dotknot for two unaligned sequences seq x and seq y . this returns secondary structure element and htype pseudoknot candidate dictionaries. ( ) calculate pairwise base pair similarity scores for secondary structure elements and h-type pseudoknot candidates. keep significant pairs that have a low estimated p-value. ( ) use significant pairs to calculate the set of conserved structure elements and pseudoknots for the two sequences that maximizes a combined free energy and similarity score. the key point of the dotknot-pw approach is how to score the similarity of stems, secondary structure elements and h-type pseudoknot candidates derived from sequences seq x and seq y . related work has been done for stem finding in unaligned sequences, where stem candidates are assigned a matching score across unaligned sequences, e.g. in scarna. another point is how to assess the significance of a similarity score using pvalues. these points will be explained in detail in the following section. for two unaligned rna sequences seq x and seq y , the single sequence prediction method dotknot (sperschneider and datta, ; sperschneider et al., ) returns two stem dictionaries d s (x) and d s (y) derived from the probability dot plot. it also returns secondary structure element dictionaries d l s ðxÞ, d l s ðyÞ and d m s ðxÞ, d m s ðyÞ and h-type pseudoknot candidate dictionaries d p (x) and d p (y) (fig. ) . to detect conserved structure elements for the two sequences, a pairwise structural comparison is performed. instead of a full structure-tostructure alignment, which takes o(n ) time and o(n ) space, pairwise base pair similarity scores are calculated using the ribosum - matrix for base pair substitutions (klein and eddy, ) . for two given stems s i (x) and s j (y) with fixed lengths in sequences seq x and seq y , respectively, the base pair similarity score sim[s i (x), s j (y)] is calculated using an ungapped local structure alignment of the base pairs with the ribosum - matrix. as an example, consider the following optimal ungapped local structure alignment of the two stems with base pair similarity score of sim[s (x), s (y)] ¼ . using the ribosum - matrix. )))))--to evaluate the significance of base pair similarity scores instead of the raw score, one has to find out what the underlying probability distribution is. similar to the case of ungapped local sequence alignments (karlin and altschul, ) , it is assumed here that the base pair similarity scores follow an extreme value distribution. however, the main difference is that a comparison between fixed-length stem fragments is made. it is important to remember that parameters and k describe the extreme value distribution of optimal local alignment scores in the asymptotic limit of long sequences (altschul et al., ) . here, the parameters for the generalized extreme value distribution are pre-calculated using maximum fig. . for two unaligned rna sequences seq x and seq y , dotknot-pw produces structure element dictionaries derived from the probability dot plot. similarity scores and p-values are computed to detect conserved elements likelihood fitting of a distribution to the histogram of a large sample of random base pair similarity scores. the maximum likelihood fitting was performed using the ismev package of the r statistical language for a range of stem lengths (see supplementary material). the p-value is defined as the probability to obtain a score greater than or equal to the observed score strictly by chance. a stem s i (x) in sequence seq x and a stem s j (x) in sequence seq y are a significant pair if the score sim[s i (x), s j (y)] has an estimated p-value less than . stem pairs with a p-value larger than are not considered in the following. for two interrupted stems, the base pair similarity score is calculated by deleting bulges and internal loops and scoring stems as consecutive base pairs. base pair similarity scores for regular and interrupted stems are also calculated if the difference in number of base pairs is less than . for example, a stem with one bulge might be a conserved match with a regular stem. a stem s i (x) in sequence seq x and a stem s j (y) in sequence seq y are a significant pair if the score sim[s i (x), s j (y)] has an estimated p-value less than . calculating the base pair similarity score for two multiloop structures is complex due to the variety of inner loop elements, which may be regular or interrupted stems. a multiloop s m i ðxÞ can be decomposed into an outer stem s o i ðxÞ and a set of inner structure elements the base pair similarity score sim½s o i ðxÞ, s o j ðyÞ for the outer stems of two multi-loops can be easily obtained from the previously calculated base pair similarity scores. if the outer stem is a conserved match, a local alignment on the set of inner structure elements is used to find the base pair similarity score. here, gaps are allowed in the local alignment of inner structure elements; however, no gap penalty is used. let two sets of inner structure elements s i (x) ¼ [s (x),. . ., s n (x)] and s j (y) ¼ [s (y),. . ., s m (y)] be given. let h(i, j) be the maximum similarity score between a suffix of s i (x) and a suffix of s j (y). the optimal local alignment is calculated as follows: hði, Þ ¼ , i n hð , jÞ ¼ , j m hði, jÞ ¼ max hði À , jÞ hði À , j À Þ þ sim½s i ðxÞ, s j ðyÞ hði, j À Þ a multiloop s m i ðxÞ in sequence seq x and a multiloop s m j ðyÞ in sequence seq y are a significant pair if the similarity score sim½s m i ðxÞ, s m j ðyÞ has an estimated p-value less than . a h-type pseudoknot has two pseudoknot stems s and s . the prerequisite for a conserved pseudoknot pair is that both core h-type pseudoknot stem pairs [s (x), s (y)] and [s (x), s (y)] are significant. the base pair similarity score for two h-type pseudoknots p i (x) and p j (y) in sequences seq x and seq y , respectively, is the sum of base pair similarity scores for the core pseudoknot stems as well as the base pair similarity score from a gapped local alignment of the recursive secondary structure elements in the loops (as described for multiloops). a pseudoknot p i (x) in sequence seq x and a pseudoknot p j (y) in sequence seq y are a significant pseudoknot pair if the similarity score sim[p i (x), p j (y)] has an estimated p-value less than . the base pair similarity score calculated in the previous sections might not be powerful enough to distinguish true positive conserved structure element pairs from false-positive structure element pairs due to the finite lengths of stems and exclusion of loop sequences in the alignment. therefore, a dissimilarity score is also used to confirm whether a pair is significant. the dissimilarity for two given structure elements s i (x) and s j (y) in sequences seq x and seq y is defined as: where dissim is the difference in the stem lengths and dissim is the difference in the number of loop lengths. as an example, consider the pseudoknot pair p (x) and p (y) in sequences seq x and seq y , respectively, with stems s , s and loops l , l , l . the pseudoknot pair has dissimilarity of . [[[[[[.) )))))) [.[[[[[[[.) ))))). . a weight is assigned to a significant pair, which is a combination of the free energy, covariation and dissimilarity. the overall weight s of a significant structure element pair [s i (x), s j (y)] in sequences seq x and seq y is a combination of the free energy weights w[s i (x)] and w[s j (y)], base pair similarity score sim[s i (x), s j (y)] and dissimilarity dissim[s i (x), s j (y)]: s½s i ðxÞ, s j ðyÞ ¼  sim½s i ðxÞ, s j ðyÞ À  fw½s i ðxÞ þ w½s j ðyÞg À  dissim½s i ðxÞ, s j ðyÞ only structure element pairs with positive score s are allowed in the following dynamic programming algorithm. here, and are set to . and is set to . let p x , . . . , p x n be the number of structure elements in the first sequence seq x and p y , . . . , p y m be the number of structure elements in the second sequence seq y . each structure element has a left and right endpoint in the sequence and is a stem, interrupted stem, multiloop or h-type pseudoknot. structure elements can also be represented as nodes in a graph. in each sequence, the structure elements are ordered by their right endpoints. an edge is drawn between two structure elements in the first and the second sequence if their base pair similarity score has a p-value less than . given the set of edges between nodes p x , . . . , p x n and p y , . . . , p y m , the goal is to find the set of edges with maximum weight that are non-crossing. this relates to finding the set of non-overlapping structure elements in the two sequences that maximize the score under the requirement that the interval ordering is preserved. a set of structure elements in the first and second sequence, which preserves the interval ordering is called a feasible structure element alignment and must satisfy the following two requirements. each structure element can be aligned with at most one other structure element in the other sequence. the order of structure elements must be preserved with respect to the alignment. that is, if structure elements p x i and p x j in the first sequence are aligned with p y a and p y b in the second sequence, respectively, the pairs may never overlap: p x i p x j^p x a p x b (fig. ) . given nodes p x . . . , p x n in the first sequence seq x and p y , . . . , p y m in the second sequence seq y , let f(i, a) be the maximum sum of edge weights for nodes between and i in the first sequence and and a in the second sequence such that the edges are non-crossing (i n and a m). the nodes that maximize the sum of edge weights are called an optimal structure element alignment for the two sequences. the optimal structure element alignment is calculated using dynamic programming. for a given structure element p i with start point a i and end point b i , let pre(i) be the non-overlapping predecessor. for each structure element, its predecessor is pre-computed using the sorted list of structure elements. the recursion for calculating the optimal structure element alignment is as follows: furthermore, nested structures are taken into account for significant outer stem pairs, which have estimated p-value less than . for each significant outer hairpin loop pair, the optimal structure element alignment of inner elements is computed. for two unaligned rna sequences seq x and seq y , the single sequence prediction method dotknot returns structure element dictionaries derived from the probability dot plot. let n and m be the number of structure elements in sequences seq x and seq y , respectively. calculating the similarity scores and the optimal structure element alignment takes o(nm) time. furthermore, nested structures are taken into account for significant outer stem pairs, which have estimated p-value less than . let a be the number of significant stem pairs, where both stems are hairpin loops. for each significant outer hairpin loop pair, the optimal structure alignment of inner elements is computed. in the worst case, this increases time requirements to o(a  nm). the number of structure elements depends on the base composition of the sequence. empirically, n and m can be observed to grow linearly with the length of the sequence for uniform base distribution (see supplementary material). in practice, dotknot-pw can be expected to run in the order of minutes for sequences shorter than nt. many pseudoknot prediction programs have been evaluated using all the entries in the pseudobase database (van batenburg et al., ) . there are several caveats in this approach. first, the sequences given in pseudobase are those which exactly harbor the pseudoknot. however, in practice structure prediction algorithms will be applied to longer sequences without prior knowledge of the pseudoknot location. second, long-range pseudoknot entries appear in a truncated version in the database. third, some classes of pseudoknots have a large number of entries (such as short h-type pseudoknots in the -untranslated regions of plant viruses), whereas more complex types of pseudoknots only have one representative (such as long-range rrna pseudoknots). therefore, a hand-curated dataset of pseudoknot structures will be used here. when it comes to pseudoknots, many structures have been published based on a secondary structure predicted by free energy minimization. these predicted secondary structures are used as a working model and refined using experimental techniques such as chemical and enzymatic probing. however, the native structure remains unsolved unless tertiary structure determination methods such as x-ray crystallography are used. testing structures that are based on computer predictions with no experimental support creates a bias in the benchmark and will be avoided in this evaluation. a total of pseudoknotted reference structures from different rna types were collected, which have strong experimental support. for each reference structure, a supporting set of evolutionarily related sequences was obtained from the rfam database (gardner et al., ) . note that for the vast majority of supporting sequences, no experimentally determined structures are available. the average pairwise sequence identities vary from % to %. given a reference structure, the performance of prediction algorithms is evaluated in terms of sensitivity (s), i.e. the percentage of base pairs in the reference structure, which are predicted correctly, as well as positive predictive value (ppv), i.e. the percentage of predicted pairs, which are in the reference structure. the matthews correlation coefficient (mcc) is also reported and is in the range from À to , where corresponds to a perfect prediction and À to a prediction that is in total disagreement with the reference structure. the performance of each method for predicting the reference structure was evaluated as described in gardner and giegerich ( ) . dotknot-pw was compared with methods that are freely available and use standard input and output formats. the comparative methods are carnac, tfold and hxmatch (with the -a option using rnaalifold). all of these methods return structure predictions for only the reference structure with regards to the support set of evolutionarily related sequences. tfold and hxmatch take a sequence alignment as the input. clustalw with the default parameters was used to produce the initial sequence alignment. dotknot-pw and carnac take a set of unaligned sequences as the input. furthermore, prediction results for the reference sequence (not the supporting sequences) were obtained from the single sequence methods dotknot (sperschneider and datta, ; sperschneider et al., ), probknot (bellaousov and mathews, ) , ipknot (sato et al., ) and rnafold (hofacker et al., ) . note that all methods except carnac and rnafold allow pseudoknot prediction. the results are shown in table . dotknot-pw has the highest average mcc of . for the test sequences. for each reference structure with the support sequences from the corresponding rfam family, predictions are returned ordered by the combined free energy and similarity score. if only the fig. . a set of edges with positive scores is given between nodes p ,. . .,p in the first sequence and p ,. . .,p in the second sequence. the goal is to find the best set of non-overlapping structure elements in the two sequences such that the interval ordering is preserved. the optimal structure element alignment, which preserves the interval ordering includes structure elements p , p , p in the first sequence and p , p , p in the second sequence pairwise prediction with highest combined free energy and similarity score is taken, dotknot-pw has an improved average mcc of . . tfold and hxmatch have average mcc of . and . , respectively. carnac has average mcc of . with much higher average specificity than sensitivity. the prediction results for single sequence structure prediction for each of the reference sequences with experimentally determined structures are also shown in table . note that this does not include the prediction for the support sequences from rfam, as no experimentally determined structures are available. all single sequence pseudoknot prediction methods show improved results over using rnafold. dotknot has the highest average mcc of . , followed by ipknot and probknot. as an example, consider the s mrna pseudoknot that binds to specific proteins in the autoregulation mechanism of ribosomal protein s synthesis (philippe et al., ) . for the reference sequence s and support sequences from the corresponding rfam family, dotknot-pw returns pairwise predictions ordered by the combined free energy and similarity score. the top two pairwise predictions with the highest scores are shown in figure . we presented dotknot-pw for prediction of structures common to two rna sequences, including h-type pseudoknots. both dotknot and dotknot-pw have been designed as dedicated pseudoknot prediction tools. in the following, important aspects of pseudoknot prediction will be discussed. single sequence prediction methods are always limited by the underlying rna folding model. this may be the set of free energy parameters used by free energy minimization methods or the underlying methodological framework such as maximum expected accuracy methods. dotknot-pw shows excellent results on h-type pseudoknots with short interhelix loops. for this type of pseudoknots, dotknot-pw uses free energy pseudoknot parameters by chen ( , ) based on polymer statistical mechanics. improvements of the accuracy of free energy parameters, both for secondary structures and pseudoknots, will lead to more accurate prediction methods. however, one has to keep in mind that the algorithms themselves must be designed in such a fashion that novel parameters can be efficiently incorporated. the heuristic framework of dotknot-pw has been designed such that it can incorporate sophisticated free energy parameters for pseudoknots, secondary structures and coaxial stacking. in the future, dotknot-pw could also use contributions from basic tertiary structure elements such as base triples around the pseudoknot junction or stem-loop interactions. pseudoknot prediction algorithms come in two flavors: either they can predict a certain, restricted class of pseudoknots or they do not have a restriction on the type of pseudoknot that can be predicted. for methods using free energy parameters, the inclusion of general types of pseudoknots might be more of a each reference structure is given by its id (see supplementary material for dot-bracket notation). the following column gives the method of experimental support (nmr, nmr spectroscopy; x-ray, x-ray crystallography; sc, sequence comparison; mg, mutagenesis; sp, structure probing), length of the sequence and number of pseudoknots. for each reference structure, the corresponding rfam family id, average sequence length and average pairwise sequence identity is shown. the * symbol means that the method failed to run. the 'first' prediction for dotknot-pw is the pairwise prediction with highest combined free energy and similarity score. curse than a blessing, as no reliable free energy parameters for complex pseudoknots are available. dotknot-pw has restrictions on the type of pseudoknot that can be predicted. however, this does not always lead to poor prediction results in practice. for example, dotknot-pw shows the best result for the hdv ribozyme, which is a complex double nested pseudoknot. the results from the benchmark for structure prediction in table must be interpreted with care. first, the tested methods can be run with different parameters, possibly producing better results. however, as a typical user has no prior knowledge about the structure, the default parameters for each method are used. of course, a comprehensive benchmark should include a larger number of structures to obtain a more reliable evaluation. however, in this study, the focus has been on a test set where the structures are supported experimentally. many structures have been published, which were determined using computational tools and this will inevitably create a bias in a benchmark, and thus they were excluded here. here, an extension of the single sequence prediction method dotknot was presented based on the pairwise comparison of structure elements. this approach called dotknot-pw is designed as an algorithm for finding the structure including h-type pseudoknots common to two sequences. as shown in table , dotknot-pw can greatly improve structure predictions for rna families when compared with the single sequence prediction using dotknot. in some cases, a comparative approach might have lower sensitivity than a single sequence prediction; however, this should not generally be judged as 'inferior'. for example, ncrnas might preserve some integral base pairs throughout evolution and only these will be detected by a comparative approach, which returns the set of base pairs common to a set of evolutionarily related sequences. dotknot-pw uses a set of unaligned sequences as the input; therefore, no expert user intervention is required. in the future, dotknot-pw will be extended to include intramolecular kissing hairpins. furthermore, constrained folding will be implemented to predict a structure subject to constraints, e.g. enforce certain base pairs or regions, which must remain unpaired. dotknot-pw has been designed as a dedicated pseudoknot prediction tool and should be applied to rna sequences where pseudoknotted interactions are suspected in the structures. prediction accuracy will inevitably decrease for sequences, which are longer than say nt for any single sequence structure prediction method (reeder et al., ) . to achieve reliable results, short sequences should be folded using dotknot and predictions should be compared with results from other methods from the literature. to gain confidence in predictions, subsequent comparative prediction using dotknot-pw and other comparative methods is highly recommended. ideally, experimental verification of computationally predicted pseudoknots should be sought. fig. . pairwise prediction results for the s mrna pseudoknot (rfam family rf ) with the top two combined free energy and similarity scores. the reference structure is shown at the top and folds into two conformations in dynamic equilibrium: a h-type pseudoknot or a series of hairpins. for the pairwise prediction with highest score, the pseudoknot structure is returned. for the second-best pairwise prediction, the alternative hairpin loop structure is returned the estimation of statistical parameters for local alignment score distributions an rna pseudoknot as the molecular switch for translation of the repz gene encoding the replication initiator of inci alpha plasmid colib-p probknot: fast prediction of rna secondary structure including pseudoknots rna secondary structure prediction from sequence alignments using a network of k-nearest neighbor classifiers viral rna pseudoknots: versatile motifs in gene expression and replication predicting rna pseudoknot folding thermodynamics predicting structures and stabilities for h-type pseudoknots with interhelix loops tfold: efficient in silico prediction of non-coding rna secondary structures crystal structure of a hepatitis delta virus ribozyme a comprehensive comparison of comparative rna structure prediction approaches rfam: wikipedia, clans and the ''decimal'' release new insight into rnase p rna structure from comparative analysis of the archaeal rna fast folding and comparison of rna secondary structures secondary structure prediction for aligned rna sequences a graph theoretical approach for predicting common rna secondary structure motifs including pseudoknots in unaligned sequences methods for assessing the statistical significance of molecular sequence features by using general scoring schemes structural basis of glms ribozyme activation by glucosamine- -phosphate rsearch: finding homologs of single structured rna sequences ribosomal binding to the internal ribosomal entry site of classical swine fever virus chloroplast phosphoglycerate kinase, a gluconeogenetic enzyme, is required for efficient accumulation of bamboo mosaic virus simulfold: simultaneously inferring rna structures including pseudoknots, alignments, and trees using a bayesian mcmc framework structure of the s-adenosylmethionine riboswitch regulatory mrna element an nmr and mutational analysis of an rna pseudoknot of escherichia coli tmrna involved in trans-translation terminal rna replication elements in human parechovirus the stimulatory rna of the visna-maedi retrovirus ribosomal frameshifting signal is an unusual pseudoknot with an interstem element finding the common structure shared by two homologous rnas structural basis for ribosome recruitment and manipulation by a viral ires rna molecular dissection of the pseudoknot governing the translational regulation of escherichia coli ribosomal-protein s a three-stemmed mrna pseudoknot in the sars coronavirus frameshift signal beyond mfold: recent advances in rna bioinformatics folding of a transcriptionally acting preq riboswitch an iterated loop matching approach to the prediction of rna secondary structures with pseudoknots simultaneous solution of the rna folding, alignment and protosequence problems ipknot: fast and accurate prediction of rna secondary structures with pseudoknots using integer programming dotknot: pseudoknot prediction using the probability dot plot under a refined energy model heuristic rna pseudoknot prediction including intramolecular kissing hairpins pseudoknots: rna structures with diverse functions a fast structural multiple alignment method for long rna sequences structure and function of telomerase rna clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice pseudobase: a database with rna pseudoknots a phylogenetically conserved hairpin-type ' untranslated region pseudoknot functions in coronavirus rna replication prediction of consensus rna secondary structures including pseudoknots we thank the reviewers for their time and thoughtful comments that helped to considerably improve the quality of the manuscript.funding: international postgraduate research scholarship (iprs) from the australian government (to j.s.).conflict of interest: none declared. key: cord- -yffwd dc authors: douangamath, alice; fearon, daren; gehrtz, paul; krojer, tobias; lukacik, petra; owen, c. david; resnick, efrat; strain-damerell, claire; aimon, anthony; Ábrányi-balogh, péter; brandaõ-neto, josé; carbery, anna; davison, gemma; dias, alexandre; downes, thomas d; dunnett, louise; fairhead, michael; firth, james d.; jones, s. paul; keely, aaron; keserü, györgy m.; klein, hanna f; martin, mathew p.; noble, martin e. m.; o’brien, peter; powell, ailsa; reddi, rambabu; skyner, rachael; snee, matthew; waring, michael j.; wild, conor; london, nir; von delft, frank; walsh, martin a. title: crystallographic and electrophilic fragment screening of the sars-cov- main protease date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: yffwd dc covid- , caused by sars-cov- , lacks effective therapeutics. additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, sars-cov- or mers-cov, despite previous zoonotic outbreaks. to identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and x-ray approach against the sars-cov- main protease, one of two cysteine viral proteases essential for viral replication. our crystallographic screen identified hits that span the entire active site, as well as hits at the dimer interface. these structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments was progressed to discover improved binders. these combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against sars-cov- main protease. through ribosome frame-shifting, generates two polyproteins pp a and pp ab (bredenbeek et al., ) . these polyproteins produce most of the proteins of the replicase-transcriptase complex (thiel et al., ) . the polyproteins are processed by two viral cysteine proteases: a papain-like protease (pl pro ) which cleaves three sites, releasing non-structural proteins nsp - and a c-like protease, also referred to as the main protease (m pro ), that cleaves at sites to release non-structural proteins (nsp - ). these non-structural proteins form the replicase complex responsible for replication and transcription of the viral genome and have led to m pro and pl pro being the primary targets for antiviral drug development (hilgenfeld, ) . structural studies have played a key role in drug development and were quickly applied during the first coronavirus outbreak. early work by the hilgenfeld group facilitated targeting the m pro of coronarviruses (hilgenfeld, ) , spectrum antivirals. the most successful have been peptidomimetic α-ketoamide inhibitors (zhang et al., a) , which have been used to derive a potent α-ketoamide inhibitor that may lead to a successful antiviral (zhang et al., b) . to date, no drugs targeting sars-cov- have been verified by clinical trials and treatments are limited to those targeting disease symptoms. to contribute to future therapeutic possibilities, we approached the sars-cov- m pro as a target for high throughput drug discovery using a fragment-based approach (thomas et al., ) . we screened against over unique fragments leading to the identification of high value fragment hits, including non-covalent and covalent hits in the active site, and hits at the vital dimerization interface. here, these data are detailed along with potential ways forward for rapid follow- up design of improved, more potent, compounds. we report the apo structure of sars-cov- m pro with data to . Å. the construct we crystallised has native residues at both n-and c--terminals, without cloning truncations or appendages which could otherwise interfere with fragment binding. electron density is present for all residues, including alternate conformations, many of which were absent in previous lower resolution crystal structures. the protein crystallised with a single protein polypeptide in the asymmetric unit, and the catalytic dimer provided by a symmetry-related molecule. the structure aligns closely with the m pro structures from sars-cov- and mers (rmsd of . Å and . Å respectively). the active site is sandwiched between two β-barrel domains, i (residue - ) and ii (residue - ) ( figure a ). domain iii (residue - ), forms a bundle of alpha helices and is proposed to regulate dimerization (shi and song, ) . the c-terminal residues, cys -gln , wrap against domain ii. however, the c terminal displays a degree of flexibility and wraps around domain iii in the n inhibitor complex (shi and song, ) (pdb id lu ). his and cys comprise the catalytic dyad and dimerisation completes the active site by bringing ser of the second dimer protomer into proximity with glu ( figure b ). this aids formation of the substrate specificity pocket and the oxyanion hole (hilgenfeld, between hits. screening at more stringent conditions ( µm per electrophile; . hours; °c) resulted in . % of the library labelling above % of protein (table s a) . these hits revealed common motifs, and we focused on compounds which offer promising starting points. compounds containing n-chloroacetyl-n'-sulfonamido-piperazine or n--chloroacetylaniline motifs were frequent hitters. such compounds can be highly reactive. therefore, we chose series members with relatively low reactivity for follow up crystallization attempts. for another series of hit compounds, containing a n-chloroacetyl piperidinyl- -carboxamide motif (table s ) which displays lower reactivity and were not frequent hitters in previous screens, we attempted crystallization despite their absence of labelling in the stringent conditions. while mild electrophilic fragments are ideal for probing the binding properties around the active site cysteine, their small size prevents extensive exploration of the substrate binding pocket. we performed an additional crystallographic fragment screen to exhaustively probe the m pro active site, and to find opportunities for fragment merging or growing. the electrophile fragment hits were added to crystals along with a total of unique fragments from libraries (table s ) . non-covalent fragments were soaked (collins et al., ) , whereas electrophile fragments were both soaked and co-crystallized as previously described (resnick et al., ) , to ensure that as many of the mass spectrometry hits as possible were structurally observed. a total of soaking and co-crystallization experiments resulted in mounted crystals. while some fragments either destroyed the crystals or their diffraction, datasets with a resolution better than . Å were collected. the best crystals diffracted to better than . Å, but diffraction to . Å was more typical, and no datasets worse than . Å were included in analysis ( figure s ). we identified fragment hits using the pandda method (pearce et al., ) , all of which were deposited in the protein data bank (table s ) active-site fragments eight fragments were identified that bind in the s subsite and frequently form interactions with the side chains of the key residues his , through a pyridine ring or similar nitrogen containing heterocycle, and glu through a carbonyl group in an amide or urea moiety ( figure ). several also reach across into the s subsite. this location, which we termed the "aromatic wheel" because of a consistent motif of an aromatic ring forming hydrophobic interactions with met or π-π stacking with his , with groups variously placed in axial directions. particularly notable is the vector into the small pocket between his , met and asp , exploited by three of the fragments (z (x ), z (x ) and z (x )) with fluoro and cyano substituents ( figure ). of the four fragments exploring subsite s , three contain an aromatic ring with a sulfonamide group forming hydrogen bonds with gln and pointing out of the active site towards the solvent interface ( figure ). these hits have expansion vectors suitable for exploiting the same his /met /asp pocket mentioned above. the experiment revealed one notable conformational variation, which was exploited by one fragment only (z (x ); figure ): a change in the sidechains of the key catalytic residues his , cys alters the size and shape of subsite s ʹ and thus the link to subsite s . this allows the fragment to bind, uniquely, to both s and s ʹ. in s , the isoxazole nitrogen hydrogen-bonds to his , an interaction that features in several other hits; and in s ʹ, the cyclopropyl group occupies the region sampled by the covalent fragments. notably, the n- methyl group offers a vector to access the s and s subsites. thus, compounds that interfere with dimerization might serve as quasi-allosteric inhibitors of protease activity. in this study three compounds bound at the dimer interface. fragment z (x ; figure a ) binds in a hydrophobic pocket formed by the sidechains of met , phe , arg and val . it also mediates two hydrogen bonds to the sidechain of gln and the backbone of met . its binding site is less than Å away from ser , whose mutation to alanine in sars-cov- protease reduced both dimerization and protease activity by about % ( ser . finally, pob (x ; york d library; figure c ), binds only Å from gly at the dimer interface and is encased between lys and val of one protomer and gly , arg , phe , lys and leu of the second, including two hydrogen bonds with the backbone of phe . heterobenzyl-piperazine motif crystallized in one binding mode with respect to the piperazinyl moiety ( figure c ) (with one exception, pcm- (x )). two structures (pcm- (x ), pcm- (x )) with a -halothiophen- -ylmethylene moiety exploit lipophilic parts of s , which is also recapitulated by the thiophenyl moiety in an analogous carboxamide (pcm- (x )). the other five structures point mainly to s , offering an accessible growth vector towards the nearby s pocket. a series of compounds containing a n-chloroacetyl piperidinyl- -carboxamide motif showed promising binding modes. to follow up on these compounds we performed a rapid second- generation compound synthesis. derivatives of this chemotype were accessible in mg-scale by reaction of n-chloroacetyl piperidine- -carbonyl chloride with various in-house amines, preferably carrying a chromophore to ease purification. these new compounds were tested by intact protein mass-spectrometry to assess protein labelling ( um compound; . h incubation, rt; table s b ). amides derived from non-polar amines mostly outcompeted their polar counterparts, hinting at a targetable lipophilic sub-region in this direction. the two amides with the highest labelling pg-cov- and pg-cov- (figure g,h) highlight the potential for further synthetic derivatization by amide n-alkylation or cross-coupling, respectively. the screen revealed unexpected covalent warheads from the series of -bromoprop- -yn- two peplites, containing threonine (ncl- (x )) and asparagine (ncl (x )) bound covalently to the active site cysteine (cys ), forming a thioenolether via c- addition with loss of bromine ( figure e ,f). the covalent linkage was unexpected and evidently the result of significant non-covalent interactions, specific to these two peplites, that position the electrophile group for nucleophilic attack. we note the side-chains make hydrogen-bonding interactions with various backbone nh and o atoms of thr and thr ; in the case of threonine, it was the minor r, r diastereomer (corresponding to d- allothreonine) that bound. the only other peplite observed (tyrosine, ncl- (x )) bound non-covalently to a different subsite. the highlighted structure activity relationships is important for further optimisation. bromoalkynes have intrinsic thiol reactivity that is lower than that of established acrylamide- proteins. the data presented herein provides many clear routes to developing potent inhibitors against sars-cov- . the bound fragments comprehensively sample all subsites of the active site revealing diverse expansion vectors, and the electrophiles provide extensive, systematic as well as serendipitous, data for designing covalent compounds. it is widely accepted that new small molecule drugs cannot be developed fast enough to help against covid- . nevertheless, as the pandemic threatens to remain a long-term problem and vaccine candidates do not promise complete and lasting protection, antiviral molecules will remain an important line of defence. such compounds will also be needed to fight future pandemics (hilgenfeld, ) . our data will accelerate such efforts: therapeutically, through design of new molecules and to inform ongoing efforts at repurposing existing drugs; and for research, through development of probe molecules (arrowsmith et al., ) to understand viral biology. one example is the observation that fragment z (x ) is a close analogue of melatonin, although in this case, it is unlikely that melatonin mediates direct antiviral activity through inhibition of m pro , given its low molecular weight; nevertheless, melatonin is currently in clinical trials to assess its immune-regulatory effects on covid (clinicaltrials.gov identifier nct ). in line with the urgency, results were made available online immediately for download. additionally, since exploring d data requires specialised tools ( figure . these can be expected to result in potent m pro binders and compound synthesis is ongoing. collectively, the covalent hits provide rational routes to inhibitors of low reactivity and high selectivity. rationally designed covalent drugs are gaining traction, with many recent fda approvals (singh et al., , bauer, . their design is based on very potent reversible binding, that allows precise orientation of a low reactivity electrophile, so that formation of the covalent bond is reliant on binding site specificity, with minimal off-targets. ( protease and other impurities were removed from the cleaved target protein by reverse nickel-nta. the relevant fractions were concentrated and applied to an s / gel filtration column equilibrated in mm hepes ph . , mm nacl buffer. the protein was concentrated to mg/ml using a kda mwco centrifugal filter device. crystallisation and structure determination: protein was thawed and diluted to mg/ml using mm hepes ph . , mm nacl. the sample was centrifuged at g for minutes. initial hits were found in well f of the proplex crystallisation screen, . m licl, . m tris ph , % peg k. these crystals were used to prepare a seed stock by crushing the proteins with a pipette tip, suspending in reservoir solution and vortexing for s in the reservoir solution with approximately glass beads ( . mm diameter, biospec products). adding dmso to the protein solution to a concentration of % and performing microseed matrix screening, many new crystallisation hits were discovered in commercial crystallisation screens. following optimisation, the final crystallisation condition was % peg k, % dmso, . m mes ph . with a seed stock dilution of / . the seeds were prepared from crystals grown in the final crystallisation condition. the drop ratios were . µl protein, . µl reservoir solution, . µl seed stock. crystals were grown using the sitting drop vapor diffusion method at °c and appeared within hours. initial diffraction data was collected on beamline i at diamond light source on a crystal grown in . m mes ph . , % peg k, cryoprotected using % peg . data were processed using dials ( light source on crystals grown using the . m mes ph . , % peg k, % dmso condition. to create a high-resolution dataset, datasets from crystals were scaled and merged using aimless (evans and murshudov, ) . waters acuity uplc class h instrument, in positive ion mode using electrospray ionization. uplc separation used a c column ( Å, . μm, mm × mm). the column was held at °c and the autosampler at °c. mobile solution a was . % formic acid in water, and mobile phase b was . % formic acid in acetonitrile. the run flow was . ml/min with gradient % b for min, increasing linearly to % b for min, holding at % b for . min, changing to % b in . min, and holding at % for min. the mass data were collected on a waters sqd detector with an m/z range of − . at a range of − m/z. raw data were processed using openlynx and deconvoluted using maxent. labelling assignment was performed as previously described (resnick et al., ) . fragment screening: fragments were soaked into crystals as previously described (collins et al. libraries.html). electrophile fragments identified by mass spectrometry were soaked by the same procedure as the other libraries, but in addition, they were also co-crystallised in the same crystallisation condition as for the apo structure. the protein was incubated with to - fold excess compound (molar ratio) for approximately h prior to the addition of the seeds and reservoir solution (following resnick et al (resnick et al., ) ). data were collected at the beamline i - at k and processed to a resolution of approximately . Å using xds (kabsch, ) the corresponding amides were prepared by addition of the acid chloride ( equiv.) as a dcm solution to the pertinent amines ( equiv.) in presence of pyridine ( equiv.) in dcm. heterogeneous reaction mixtures were treated with a minimal amount of dry dmf to achieve full solubility. after stirring the reaction mixtures overnight, the solvents were removed in by rotary evaporation, re-dissolved in % aq. mecn (and a minimal amount of dmso to achieve higher solubility), followed by purification by (semi-)preparative rp-hplc in mass-directed automatic mode or manually. synthesis of peplites hatu ( . eq.), dipea ( . eq.) and the acid starting material ( . eq.) were dissolved in dmf ( - ml) and stirred together at room temperature for min. - bromoprop- -yn- -amine hydrochloride was added and the reaction mixture was stirred at °c overnight. the reaction mixture was allowed to cool to room temperature, diluted with etoac or dcm and washed with saturated aqueous sodium bicarbonate solution, brine and water. the organic layer was dried over mgso , filtered and evaporated to afford crude product. the crude product was then purified by either normal or reverse phase chromatography. ( -bromoprop- -yn- -yl) n-( -bromoprop- -yn- -yl)- -(tert-butoxy) butanamide ( s, s)- -acetamido-n-( -bromoprop- -yn- -yl)- -(tert-butoxy)butanamide the reaction mixture was allowed to warm to room temperature and was stirred at room temperature for h then evaporated to dryness to afford crude product. the crude product was purified by flash silica chromatography, elution gradient - % meoh in dcm. pure fractions were evaporated to dryness to afford ( s, s)- -acetamido-n-( -bromoprop- -yn- (s)- -acetamido-n -( -bromoprop- -yn- -yl)succinimide (asparagine peplite) (s)- -acetamido-n -( -bromoprop- -yn- -yl)succinamide was synthesized according to general procedure a using (s)- -acetamido- -amino- -oxobutanoic acid ( mg, . mmol) and evaporating the reaction mixture to afford the crude product without aqueous work-up. the crude product was purified by flash silica chromatography, elution gradients - % meoh in dcm. pure fractions were evaporated to dryness to afford (s)- -acetamido-n - ( - bromoprop- -yn- -yl)succinamide ( mg, %) as a white solid. coronavirus main proteinase ( cl(pro)) structure: basis for design of anti-sars drugs the promise and peril of chemical probes new substructure filters for removal of pan assay interference compounds (pains) from screening libraries and for their exclusion in bioassays seven year itch: pan-assay interference compounds (pains) in -utility and limitations targeted covalent inhibitors for drug design long-range cooperative interactions modulate dimerization in sars cl(pro) covalent inhibitors in drug discovery: from accidental discoveries to avoided liabilities and designed therapies rapid experimental sad phasing and hot-spot identification with halogenated fragments severe respiratory illness caused by a novel coronavirus the primary structure and expression of the nd open reading frame of the polymerase gene of the coronavirus mhv polymerase is expressed by an efficient ribosomal frameshifting mechanism residues on the dimer interface of sars coronavirus c-like protease: dimer stability characterization and enzyme catalytic activity analysis quaternary structure of the severe acute respiratory syndrome (sars) coronavirus main protease gentle, fast and effective crystal soaking by acoustic dispensing a poised fragment library enables rapid synthetic expansion yielding the first reported inhibitors of phip( ), an atypical bromodomain covalent inhibitors design and discovery an interactive web-based dashboard to track covid- in real time design and synthesis of shape diverse -d fragments how good are my data and what is the resolution michelanglo: sculpting protein views on web pages without coding an improved model for fragment- based lead generation at astrazeneca structure-based design,synthesis, and biological evaluation of peptidomimetic sars-cov clpro inhibitors conservation of substrate specificities among coronavirus main proteases from sars to mers: crystallographic studies on coronaviral proteases enable antiviral drug design critical assessment of important regions in the subunit association and catalytic action of the severe acute respiratory syndrome coronavirus main protease two adjacent mutations on the dimer interface of sars coronavirus c-like protease cause different conformational changes in crystal structure structure of m(pro) from sars-cov- and discovery of its inhibitors integration, scaling, space-group assignment and post-refinement. acta crystallographica section d-biological crystallography dimple: a difference map pipeline for the rapid screening of crystals on the beamline heterocyclic electrophiles as new mura inhibitors design and characterization of a heterocyclic electrophilic fragment library for the discovery of cysteine-targeted covalent inhibitors the xchemexplorer graphical workflow tool for routine or large-scale protein-ligand structure determination early dynamics of transmission and control of covid- : a mathematical modelling study how significant are unusual protein-ligand interactions? insights from database mining newly discovered coronavirus as the primary cause of severe acute respiratory syndrome interactive jimd articles using the isee concept: turning a new page on structural biology data acedrg: a stereochemical description generator for ligands the alkyne moiety as a latent electrophile in irreversible covalent small molecule inhibitors of cathepsin k refmac for the refinement of macromolecular crystal structures crystallographic screening using ultra-low-molecular-weight ligands to guide drug design a multi-crystal method for extracting obscured crystallographic states from conventionally uninterpretable electron density electrophile-fragment screening the catalysis of the sars c-like protease is under extensive regulation by its extra domain the resurgence of covalent drugs assessment of chemical libraries for their druggability sars -beginning to understand a new virus mechanisms and enzymes involved in sars coronavirus genome expression structure-guided fragment-based drug discovery at the synchrotron: screening binding sites and correlations with hotspot mapping a structural view of the inactivation of the sars coronavirus main proteinase by benzotriazole esters data processing and analysis with the autoproc toolbox decision making in xia dials: implementation and evaluation of a new integration package fraglites-minimal, halogenated fragments displaying an efficient approach to druggability assessment and hit generation a new coronavirus associated with human respiratory disease in china structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design design of wide-spectrum inhibitors targeting coronavirus main proteases the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor isolation of a novel coronavirus from a man with pneumonia in saudi alpha-ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication: structure-based design, synthesis, and activity assessment crystal structure of sars-cov- main protease provides a basis for design of improved alpha-ketoamide inhibitors recent advances in selective and irreversible covalent ligand development and validation a novel coronavirus from patients with pneumonia in china key: cord- -be ith z authors: wang, qi; pang, yuan-ping title: accurate reproduction of small-molecule complex crystal structures using the eudoc program: expanding the use of eudoc to supramolecular chemistry date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: be ith z eudoc is a docking program that has successfully predicted small-molecule-bound protein complexes and identified drug leads from chemical databases. to expand the application of the eudoc program to supramolecular chemistry, we tested its ability to reproduce crystal structures of small-molecule complexes. of selected crystal structures of small-molecule guest-host complexes, eudoc reproduced all these crystal structures with guest structure mass-weighted root mean square deviations (mwrmsds) of < . Å relative to the corresponding crystal structures. in addition, the average interaction energy of these guest-host complexes (− . kcal/mol) was found to be nearly half of that of previously tested small-molecule-bound protein complexes (− . kcal/mol), according to the interaction energies calculated by eudoc. of the complexes could not be reproduced with mwrmsds of < . Å if neighboring hosts in the crystal structure of a guest-host complex were not included as part of the multimeric host system, whereas two of the complexes could not be reproduced with mwrmsds of < . Å if water molecules were excluded from the host system. these results demonstrate the significant influence of crystal packing on small molecule complexation and suggest that eudoc is able to predict small-molecule complexes and that it is useful for the design of new materials, molecular sensors, and multimeric inhibitors of protein-protein interactions. in , a computer was used to screen , chemicals in the cambridge structural database (csd) [ ] , leading to the identification of a haloperidol analog capable of inhibiting hiv- and hiv- proteases with a k i of < mm [ ] . the screening was accomplished using a computer docking program, dock, that docked each chemical of the database into the active sites of the enzymes and evaluated the shape complementarity of the docked compound relative to the active sites. inspired by this seminal work, the eudoc program was devised to search for the specific conformations, positions, and orientations of two threedimensional ( d) structures that permit the strongest nonbonded intermolecular interactions between the two. the eudoc program uses docking algorithms that differ from those of dock [ ] . it addresses molecular flexibility by using conformation selection and conformation substitution mechanisms that enable massively parallel computing [ ] . eudoc was devised to perform on a cluster of more than loosely connected processors [ ] and has recently been ported to the ibm blue gene/l supercomputer [ , ] . this program has successfully predicted small-moleculebound protein complexes and identified drug leads from chemical databases [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the eudoc program is also efficient. in a computational screen of , chemicals (at a resolution of . Å translation and u of arc rotation) for inhibitors of a chymotrypsin-like cysteine protease of the severe acute respiratory syndrome-associated coronavirus, the eudoc program is able to reduce the wall-clock time of the screen from minutes using xeon processors ( . ghz) on a beowulf cluster to and minutes using and powerpc- processors ( mhz) on blue gene/l, respectively [ , ] . because a large database can be divided into subsets, a sustained petaflops capability would be able to screen million chemicals in about minutes or to screen billion chemicals for one drug target in a year [ ] . this capability offers the possibility of identifying inhibitors that are effective enough for in vivo testing, eliminating the need of medicinal chemistry to improve the efficiency of inhibitor leads identified by terascale computers [ ] . in the context of this promise, we seek to extend the application of the eudoc program to supramolecular chemistry. supramolecular chemistry deals with creation of a large molecule assembled with noncovalent bonding among small molecular units, in contrast to organic synthesis that involves breaking and making covalent bonds to create a new molecule [ ] . such noncovalent bonding is reversible and comprises hydrogen bonding, metal coordination, hydrophobic force, van der waals force, p-p interaction, cation-p interaction, and/or long-range electrostatic interaction to assemble small molecules into a multimolecular complex. supramolecular chemistry principles have been used to develop new materials, molecular sensors, and multimolecular complexes designed to disrupt protein-protein interactions. to expand the application of the eudoc program to supramolecular chemistry, we tested its ability to reproduce the crystal structures of small-molecule guest-host complexes. previously we had tested the ability of the program to reproduce crystal structures of proteins in complex with small molecules and found that eudoc reproduced % of crystal structures using the bound conformations of both proteins and their smallmolecule partners [ ] . this success may not transfer to with smallmolecule guest-host complexes such as a crown ether in complex with -nitrobenzene- , -diamine, however, because the binding pocket or cavity in a small-molecule host is not as well formed as that in a protein. herein we report the results of our docking studies with selected crystal structures of small-molecule guest-host complexes using the eudoc program. these results show that the program is able to reproduce all crystal structures and that the average interaction energy of these small-molecule complexes ( . kcal/mol) is nearly half of that of the small molecule-bound protein complexes we studied in previous tests ( . kcal/mol). the results also demonstrate the significant influence of crystal packing on small-molecule complex crystal structures and suggest that the eudoc program is able to predict d structures of small-molecule guest-host complexes with reasonable reliability. the ability of eudoc to reproduce crystal structures of smallmolecule guest-host complexes was evaluated with the following procedure. the guest and host molecules in the complex crystal structure were separated, and the guest structure was then docked back into the host structure by the eudoc program. this docking process used translational and rotational increments of . Å and u of arc, respectively, and a docking box that was defined to enclose the guest structure in the guest-host complex crystal structure. of many eudoc-generated guest-host complexes, only the complex with the strongest interaction energy was compared to the corresponding crystal structure of the complex. in this comparison, the host portion of the eudoc-generated complex was superimposed onto the host portion of the crystal structure, and the mass-weighted root mean square deviation (mwrmsd) of the guest portion between the two superimposed complexes was calculated. if the mwrmsd was , . or . Å , the crystal structure of the complex was reproduced or accurately reproduced, respectively, by the eudoc program [ ] . because the uncertainty in calculating the interaction energy using the eudoc program was estimated to be . kcal/mol [ ] , occasionally, a few eudoc-generated complexes were considered to have the strongest interaction energy and compared to the crystal structure thus resulting in multiple mwrmsds, if their interaction energies differed from the strongest interaction energy by # . kcal/mol. in that case, as long as one of the mwrmsds was , . or . Å , the crystal structure of the binary complex was reproduced or accurately reproduced, respectively, by the eudoc program. a total of crystal structures of small-molecule guest-host complexes were obtained from csd for this study [ ] . the selection criteria included the followings: ( ) no covalent bond between a host and a guest; ( ) the r factor of , to ensure good crystallographic quality; ( ) a guest to host ratio of in a unit cell; ( ) no structures containing ni + , ag + , pd + , pt + , au + or ru + because force field parameters for these ions were unavailable in the eudoc program. the results of the docking studies with the guest-host complexes using the procedure described above are listed in table . as apparent from the mwrmsd distribution listed in table , the eudoc program reproduced % and accurately reproduced % of the complexes. the deviations between the eudoc-generated and crystal complexes at different mwrmsd values are depicted in figure . docking with consideration of the influence of crystal packing figure shows the difference (mwrmsd of . Å ) between the eudoc-generated and crystal structures (csd code: xag-mat). complex xagmat is one of the complexes that the eudoc program failed to reproduce. despite a favorable p-p interaction between the guest and host structures predicted by the eudoc program, in the corresponding crystal structure the guest structure surprisingly docks at a region at which it partly interacts with the host via a p-p interaction (see fig. ). this discrepancy suggests that the guest might partly interact with host(s) and/or guest(s) in neighboring unit cells of the crystal structure. to confirm this, the docking study with complex xagmat was repeated with consideration of the influence of crystal packingnamely, the guest was docked into a multimeric host system that included neighboring host(s) and/or guest(s). these neighboring structures were generated by applying the symmetry of the space group of the crystal structure. the host(s) and/or guest(s) in neighboring unit cells were excluded if these structures were . . Å away from the guest to be docked. interestingly, when the influence of crystal packing was taken into account, the eudoc program accurately reproduced complex xagmat with an mwrmsd of . Å , instead of the . Å obtained without consideration of crystal packing. this result prompted a new docking study that considered the influence of crystal packing. the results of the docking studies with consideration of the influence of crystal packing are listed in table . the complexes (csd codes: ajuxuy, atukef, baxzab, bif-kik, cramcc , fanjag, gugguk, kolmaz, lay-maz, nebqoa, qajkan, and ralqaw ) that were not reproduced previously by the eudoc program were accurately reproduced after the influence of crystal packing was taken into account. with consideration of the influence of crystal packing, the eudoc program reproduced all complexes and accurately reproduced % of them (see table ). two crystal structures (csd codes: xaqjaa and xaqjee) could not be accurately reproduced by the eudoc program even after consideration of the influence of crystal packing (xaqjaa: mwrmsd = . Å ; xaqjee: mwrmsd = . Å ). visual inspection of these structures revealed that the binding between the guest and host structures was mediated by crystallographically determined water molecules. this mediation suggested that, similar to the crystal packing, water molecules might also play an important role in guest-host complexation, and it might be necessary to include them in the multimeric host system for docking. accordingly, the docking studies with the complexes were repeated with consideration of the influences of both crystal packing and structural waters. the results are listed in table . indeed, the eudoc program accurately reproduced complexes xaqjaa and xaqjee with mwrmsds of . and . Å , respectively. taking into account the influences of both crystal packing and structural waters, the eudoc program accurately reproduced all complexes (see table ). the influences of crystal packing and structural water on docking this study shows that ( %) of the complexes could not be accurately reproduced with mwrmsds of , . Å by the eudoc program if neighboring host(s) and/or guest(s) in the crystal structure were not included as part of the multimeric host system, whereas only ( %) of these complexes could not be accurately reproduced with mwrmsds of , . Å if neighboring structures were included but water molecules were excluded from the host system. these results show that the influence of crystal packing or crystal environment on crystal structures of guest-host complexes is significant, which is consistent with the reported influence of crystal packing on protein structures [ ] . these results also show that the influence of structural waters on guesthost complex crystal structures is insignificant, which is consistent with our reported finding that complexation between a small molecule and a protein is not commonly mediated by water molecules [ ] . this study therefore suggests that crystal packing should be taken into account when reproducing crystal structures of small-molecule guest-host complexes through docking studies, whereas water molecules, counterions or other companying molecules such as ethanol can be excluded from the host system. this study also suggests that, ideally, to perform prospective and accurate docking of a small molecule into another small molecule, repetitive docking of a guest or host into a guest-host complex that is generated by the previous docking is preferred, because a host is sometimes too small to prevent a guest from interacting with nearby guest(s) and/or host(s). it is worth noting, however, that the success rate of docking a small molecule into another small molecule is about % if the influence of crystal packing is ignored. in the crystal structure of rebek's acridine diacid in complex with quinoxaline (csd code: yawjip) there are two crystallographically independent forms of the complex in the asymmetric unit [ ] . this structure was used to benchmark the all-atom amber/opls force field [ ] . in this study, complex yawjip was accurately reproduced by the eudoc program using the nonbonded force field parameters of the second-generation [ , ] ; the mwrmsds of the guest position between the eudoc-generated and crystal complexes for forms a and b are both . Å . the accurate reproduction of the remaining crystal structures (see table ) further demonstrates the accuracy of the nonbonded force field parameters of the secondgeneration amber force field (parm .dat) for reproducing crystal structures [ , ] . the above results suggest that the eudoc program can predict small-molecule guest-host complexes with a reasonable success rate ( %), without consideration to the mechanism of small-molecule complex aggregation-namely, without being given the multimeric host system. to demonstrate this ability herein, the crystal structure of complex yawjip is used as a model system. based on the nmr spectroscopic data of complex yawjip, the guest structure quinoxaline was proposed to have face-to-face pstacking with the acridine portion of rebek's acridine diacids in an early report of the complex [ ] . however, the face-to-face pstacking was found in neither the crystal structure of complex yawjip [ ] nor the monte carlo statistical mechanics calculations of the complex [ ] . to perform a prospective docking study, the two-dimensional structures of quinoxaline and rebek's acridine diacid were the van der waals component of the intermolecular interaction energy; the electrostatic component of the intermolecular interaction energy. eudoc identified one alternative binding mode that is energetically indistinguishable from the binding mode of the crystal structure. , in) , respectively. both d structures were refined with energy minimization monitored with a normalmode (nmode) analysis to ensure that the energy minimization stopped when the minimized conformation reached a local potential energy minimum. the energy minimization was performed by using the sander module of the amber program [ ] with the second-generation amber force field [ ] , and the nmode analysis was carried out using the nmode module of the amber program [ ] . given the refined d structures of quinoxaline and rebek's acridine diacid, the eudoc program generated a complex nearly identical to the crystal structure of form a (mwrmsd: . Å ) but not the proposed complex with nearly face-to-face p-stacking. this perspective docking result suggests that the eudoc program is a useful tool for predicting d models of guest-host complexes to aid the design of new molecular entities according to the principles of supramolecular chemistry. visual inspection of reported crystal structures of proteins in complex with small molecules [ ] and the crystal structures of guest-host complexes reported herein suggested that the noncovalent interactions of the guest-host complexes are in general weaker than those of the protein complexes. the average of the interaction energies of the guest-host complexes listed in the van der waals component of the intermolecular interaction energy; the electrostatic component of the intermolecular interaction energy; structural water molecules were present in the multimeric host system. eudoc identified one alternative binding mode that is energetically indistinguishable from the binding mode of the crystal structure. table vi (excluding pha) of reference ( . kcal/mol) quantitatively confirm the relatively weak noncovalent interactions of the guest-host complex. this confirmation suggests that to design high-affinity guest-host complexes it is of advantage to incorporate the entropic energy into the binding, because the number of functional groups that can be introduced onto the guest and host structures to confer the nonbonded interactions is limited by the size of the two partners. this ''saturation'' problem is more apparent for small-molecule complexes than for protein complexes. it is therefore conceivable that the eudoc program is also a useful tool for estimating the interaction energies of guest-host complexes to aid the design of new molecular entities according to the principles of supramolecular chemistry. the guest and host structures were taken from the crystal structures of their corresponding complexes obtained from csd [ ] . water molecules, counterions, and solvent molecules such as ethanol were removed from the guest or host structure. hydrogen atoms were added by using the quanta program (accelrys software, inc, san diego, california) followed by energy minimization of the hydrogen atoms using the sander module of the amber program [ ] with the second-generation amber force field (parm .dat) [ ] and a positional constraint on all non-hydrogen atoms. the protonation states of the guest and host structures shown in figures s , s , s , s , s , s , s , s , s , s , s , s , s , s , s and s of supporting information were determined according to pka values of functional groups at ph of . . the atomic charges of the guest and host structures listed in table s of supporting information were generated according to the resp procedure [ ] with ab initio calculations at the hf/ - g* level using the gaussian program [ ] . the amber atom types of the guest and host structures listed in table s of supporting information were assigned by the antechamber module of amber [ ] . the algorithm of the eudoc program has been reported elsewhere [ ] . briefly, it uses a systematic search protocol, translating and rotating a guest in a putative binding pocket of a host to search for energetically favorable orientations and positions of the guest relative to the host. a docking box is defined within the binding pocket to confine the translation of the ligand. the intermolecular interaction energy is the potential energy of the guest-host complex relative to the potential energies of the two partners in their free state. this energy was calculated according to equations and using the second-generation amber force field [ ] . in calculating the intermolecular interaction energy, the dielectric constant was set to . , and the distance cutoffs for steric and electrostatic interactions were set to Å . a docking box was defined to enclose the guest structure in the crystal structure of the guest-host complex. the size of the docking box and the cutoff for the interaction energy used by the eudoc program are listed in table s of supporting information. the complex-prediction module of the eudoc program (version , executable available from ypp) was used to translate and rotate the guest around the host at increments of . Å and u of arc, respectively, unless noted otherwise in table . to consider the influence of crystal packing, the pymol program (delano scientific llc, south san francisco, california) was used to generate a multimeric host system by applying the symmetry of the space group of the crystal structure. the host or guest structure was excluded from the multimeric host system if the shortest distance between a heavy atom of the guest structure to be docked and the heavy atom of the host/guest structure in neighboring unit cells was . . Å . energy minimization was performed with the sander module of amber [ ] using ( ) maxcyc = , ( ) dielc = , ( ) scnb = . , ( ) scee = . , ( ) ntr = , ( ) ntmin = or , ( ) ncyc = , ( ) cut = , and ( ) drms = . nmode analysis was performed with the nmode module of amber [ ] using ( ) ilevel = or , ( ) cut = , ( ) scnb = . , ( ) scee = . , ( ) dielc = , and ( ) idiel = . the mwrmsds were calculated by superimposing the host portion of the eudoc-generated complex over the corresponding host portion of the crystal structure followed by a calculation for the mwrmsd of all atoms of the guest portion in the two superimposed complexes using the ptraj module of amber [ ] . the cambridge structural database: a quarter of a million crystal structures and rising structure-based design of nonpeptide inhibitors specific for the human immunodeficiency virus protease eudoc: a computer program for identification of drug interaction sites in macromolecules and drug leads from chemical databases in silico drug discovery: solving the ''target-rich and lead-poor'' imbalance using the genome-to-drug-lead paradigm eudoc on blue gene: accelerating the transfer of drug discoveries from laboratory to patient successful virtual screening of a chemical database for farnesyltransferase inhibitor leads chemical database techniques in drug discovery structure of acetylcholinesterase complexed with the nootropic alkaloid discovery of a new inhibitor lead of adenovirus proteinase: steps toward selective, irreversible inhibitors of cysteine proteinases re-engineering butyrylcholinesterase as a cocaine hydrolase from genome to drug lead: identification of a small-molecule inhibitor of the sars virus serotype-selective, small-molecule inhibitors of the zinc endopeptidase of botulinum neurotoxin serotype a the limit of accuracy of protein modeling: influence of crystal packing on protein structure molecular-sructure of rebek diacid quinoxalineconfirmation of -point binding structure and binding for complexes of rebek's acridine diacid with pyrazine, quinoxaline, and pyridine from monte carlo simulations with an all-atom force field a second generation force field for the simulation of proteins, nucleic acids, and organic molecules molecular recognition: ionic and aromatic stacking interactions bind complementary functional groups in a molecular cleft amber, a package of computer programs for applying molecular mechanics, normal mode analysis, molecular dynamics and free energy calculations to simulate the structural and energetic properties of molecules application of the multimolecule and multiconformational resp methodology to biopolymers: charge derivation for dna, rna, and proteins gaussian , revision c. the authors acknowledge computing support from the university of minnesota supercomputing institute. key: cord- -ob euspo authors: durdagi, serdar; dag, cagdas; dogan, berna; yigin, merve; avsar, timucin; buyukdag, cengizhan; erol, ismail; ertem, betul; calis, seyma; yildirim, gunseli; orhan, muge d.; guven, omur; aksoydan, busecan; destan, ebru; sahin, kader; besler, sabri o.; oktay, lalehan; shafiei, alaleh; tolu, ilayda; ayan, esra; yuksel, busra; peksen, ayse b.; gocenler, oktay; yucel, ali d.; can, ozgur; ozabrahamyan, serena; olkan, alpsu; erdemoglu, ece; aksit, fulya; tanisali, gokhan; yefanov, oleksandr m.; barty, anton; tolstikova, alexandra; ketawala, gihan k.; botha, sabine; dao, e. han; hayes, brandon; liang, mengning; seaberg, matthew h.; hunter, mark s.; batyuk, alex; mariani, valerio; su, zhen; poitevin, frederic; yoon, chun hong; kupitz, christopher; sierra, raymond g.; snell, edward; demirci, hasan title: near-physiological-temperature serial femtosecond x-ray crystallography reveals novel conformations of sars-cov- main protease active site for improved drug repurposing date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: ob euspo the covid pandemic has resulted in + million reported infections and nearly . deaths. research to identify effective therapies for covid includes: i) designing a vaccine as future protection; ii) structure-based drug design; and iii) identifying existing drugs to repurpose them as effective and immediate treatments. to assist in drug repurposing and design, we determined two apo structures of severe acute respiratory syndrome coronavirus- main protease at ambienttemperature by serial femtosecond x-ray crystallography. we employed detailed molecular simulations of selected known main protease inhibitors with the structures and compared binding modes and energies. the combined structural biology and molecular modeling studies not only reveal the dynamics of small molecules targeting main protease but will also provide invaluable opportunities for drug repurposing and structure-based drug design studies against sars-cov- . one sentence summary radiation-damage-free high-resolution sars-cov- main protease sfx structures obtained at near-physiological-temperature offer invaluable information for immediate drug-repurposing studies for the treatment of covid . in late , after the first patient was diagnosed with pneumonia of unknown etiology reported to the world health organization (who) from china, millions of cases followed in a short span of four months (who). on th of march, ; who declared covid outbreak as a pandemic, which originated from severe acute respiratory syndrome corona virus- (sars-cov- ) infection. sars-cov- has a high spread rate (rₒ) value, deeming the pandemic difficult to control (petersen et al., ) . moreover, the absence of a vaccine to provide immunity and the lack of effective treatments to control the infection in high comorbidity groups make this pandemic a major threat to global health (ahn et al., ) . the first human coronavirus to cause a variety of human diseases, such as common cold, gastroenteritis, and respiratory tract diseases was identified in the s (tyrrell et al., ) . in , a deadly version of coronavirus responsible for sars-cov was identified in china (heymann et al., ) . sars-cov- , the most recent member of the coronavirus family to be encountered, is a close relative of sars-cov and causes many systemic diseases (andersen et al., ; dutta and sengupta., ; braun et al., ) . covid patients exhibit (i) high c-reactive protein (crp) and pro-inflammatory cytokine levels; (ii) macrophage and monocyte infiltration to the lung tissue; (iii) atrophy of spleen and lymph nodes which weakens the immune system; (iv) lymphopenia; and (v) vasculitis . release of a large amount of cytokines results in acute respiratory distress syndrome (ards) aggravation and widespread tissue injury leading to multi-organ failure and death. therefore, mortality in many severe cases of covid patients has been linked to the presence of the cytokine storm evoked by the virus (ragab et al., ) . the sars-cov- genome encodes structural proteins including surface/spike glycoprotein (s), envelope (e), membrane (m), and nucleocapsid (n) proteins; and the main reading frames named orf a and orf b that contain non-structural proteins (nsp) (gordon et al., ; chen, liu & guo., ) . among these, orf a/b encodes papain-like protease (plpro), main protease (mpro), a chymotrypsin-like cysteine protease, along with polyproteins named polyprotein a (pp a) and polyprotein b (pp b) (astel et al., ) . encoded polyproteins are then proteolyzed to nsps by precise mpro and plpro cleavages of the internal scissile bonds. nsps are vital for viral replication, such as rna-dependent rna polymerase (rdrp) and nsp , which are used for the expression of structural proteins of the virus thiel et al., ; ullrich & nitsche., ; ziebuhr et al., ) . sars-cov- mpro has no homologous human protease that recognizes the same cleavage site (pillaiyar et al., ) . therefore, drugs that target its active site are predicted to be less toxic and harmful to humans (l. . high sequence conservation of mpro provides minimized mutation-caused drug resistance . given its essential role in the viral life cycle, the sars-cov- mpro presents a major drug target requiring a detailed structural study. drug repurposing is a rapid method of identifying potential therapies that could be effective against covid compared to continuous investigative efforts (e.g identification of new drugs and development of preventive vaccine therapies). the well-studied properties of food and drug administration (fda) approved drugs means such molecules are better understood compared to their counterparts designed de novo. a putative drug candidate identified by drug-repurposing studies could make use of existing pharmaceutical supply chains for formulation and distribution, an advantage over developing new therapies pushpakom et al., ; jarada et al., ) . in typical drug repurposing studies, approved drug libraries are screened against the active site or an allosteric site of target protein structures obtained by methods that have limitations in revealing the enzyme structure, such as cryogenic temperature or radiation damage and investigations that only involve screening of drugs currently on the market (zhou et al., ; choudhary et al., ; beck et al., ; wang, ) . current structural biology-oriented studies that display a repurposing approach to sars-cov- research focused on target-driven drug design, virtual screening or a wide spectrum of inhibitor recommendations (rathnayake et al., ; chauhan and kalra, ; muralidharan et al., ; khan et al., ; kumar et al., ; joshi et al., ) . it is critical to interfere with the ongoing pandemic, instead of time and resource-consuming drug design studies, drug repurposing studies with the existing drugs are crucial in short terms should be a priority. the main purpose of our study is to reveal the conformational dynamics of mpro, which plays a central role in the viral life-cycle of sars-cov- . linac coherent light source (lcls) with its ultrafast and ultrabright pulses enables outrunning secondary radiation damage. the updated highthroughput macromolecular femtosecond crystallography (mfx) instrument of lcls that is equipped with the new autoranging epix k detector that provides a dynamic range of eleven thousand kev photons in fixed low gain mode to collect secondary radiation-damage-free structural data from mpro microcrystals remotely at ambient-temperature (sierra et al., ; blaj et al., ; . here we present two sfx structures of sars-cov- mpro which provide structural dynamics information of its active site and their deep in silico analysis. our results emphasize the importance of structure-based drug design and drug repurposing by using the xfel structures. we obtained more relevant results of flexible areas over two different structures. radiation-damage-free sfx method which enables obtaining the novel high-resolution ambient-temperature structures of the binding pocket of mpro provides an unprecedented opportunity for identification of highly effective inhibitors for drug repurposing by using a hybrid approach that combines structural and in silico methods. besides, structure-based drug design studies will be more accurate based on these novel atomic details on the enzyme's active site. conformations of the drug-binding pocket. we determined two radiation-damage-free sfx crystal structures of sars-cov- mpro in two crystal forms at . Å and . Å resolutions with the following pdb ids: cwb and cwc, respectively (fig. a, b) (supplementary table & the diffraction data collected remotely at the mfx instrument of the lcls at slac national laboratory, menlo park, ca (sierra et al., analysis for sfx studies at lcls). we used an mpro structure determined at ambient-temperature using a rotating anode home x-ray source (pdb id: wqf; as our initial molecular replacement search model for structure determination. two high-resolution sfx structures obtained in different space groups were superposed with an overall rmsd of . Å (fig. s ). they reveal novel active site residue conformations and dynamics at atomic level, revealing several differences compared to the prior ambient-temperature structure of sars-cov- mpro that was obtained at a home x-ray source (fig. a, b ). mpro has a unique n-terminal sequence that affects enzyme's catalytic activity (chang, ; l. zhang et al., ) . besides the native monoclinic form of mpro at . Å (fig. a , pdb id: cwb) we also determined the structure of the mpro with additional extra n-terminal amino acids (generated by thrombin specific n-terminal cleavage) at . Å resolution (fig. b , pdb id: cwc). the structure obtained from this modified version of mpro reveals how the minor changes introduced at the n-terminus affects both the three-dimensional structure of the mpro and promotes the formation of a new orthorhombic crystal form. biologically relevant dimeric structure of native monomeric mpro can be generated by adding the symmetry-related chain b ( fig. a) . each protomer of sars-cov- mpro is formed by three major domains s ). domain i starts from the n-terminal of protein and includes anti-parallel beta-sheet structure. this beta-sheet forms a beta-barrel fold which ends at residue . the domain ii of mpro resides between residues through and mostly consists of anti-parallel beta-sheets. the third domain of the mpro is located between residues through and consists of mostly alpha helices and has a more . previous biochemical studies of mpro suggested there is a competition for dimerization surface between domains i and iii. in the absence of domain i, mpro undergoes a new type of dimerization through the domain iii (zhong et al., ) . during our purifications, we repeatedly observed a combination of monomeric and dimeric forms of mpro on the size exclusion chromatography steps which may be caused by this dynamic compositional and conformational equilibrium. the two sars-cov- mpro sfx crystal structures reveal a non-flexible core active site and the catalytic amino acid cys fig. s . temperature factor analysis revealed that the active site is surrounded by mobile regions (fig. s ). the presence of these mobile regions was observed in both sfx crystal structures, suggesting an intrinsic plasticity rather than an artifactual finding that could have arisen based on the crystal lattice contacts ( fig. s ; indicated with red circles). further, this plasticity suggests that molecules that interact via non-covalent bonds to the mpro binding pocket would do so weakly. our investigation of pdb structures with available electron densities identified that the majority of the mpro inhibitors formed covalent bonds with the active site residue cys . additionally, few non-covalent inhibitors were identified and exhibited weak electron densities (fig. we compared our radiation-damage-free ambient-temperature sfx structure (pdb id: cwb) with ambient-temperature x-ray structure (pdb id: wqf). structures were similar with an rmsd value of . Å (fig. a) . however, we observed significant conformational differences especially in the side chains of thr , ser , glu , leu , asn , cys , met , and gln residues (fig. b & fig. s ). the calculated bias-free composite omit map that covers the active region has been shown ( fig. b & fig. s ). this structure offers previously unobserved new insights on the active site of mpro in addition to wqf structure which is important for the future in silico modeling studies. all three domains contribute to the formation of the active site of the protein. the intersection part of the domain i residue his , domain ii residues cys and his interact via a coordinated water molecule with asp located at the n-terminal loop region of domain iii to form the active site, that is an important drug target area (fig. c & fig. s ). the n-terminal loop of domain iii is suggested to be involved in enzyme activity (ma et al., ) . the distance between cys s and his nε is . Å, very similar to the ambient-temperature structure ( wqf) . oδ -oδ atoms of asp and nh -nε atoms of arg contribute to a salt bridge between these two residues and stabilize the positions of each other. the w water molecule (indicated with a red sphere at the figure) in the active site plays crucial roles for catalysis. w forms triple h-bonds with his , his , and asp side chains with the distances of . Å, . Å, and . Å, respectively (fig. c & fig. s ). when compared to the room temperature structure of mpro ( wqf) our structure displays additional active site residue dynamics while it has an overall high similarity (fig. d) . canonical chymotrypsin-like proteases contain a catalytic triad composed of ser(cys)-his-asp(glu) in their catalytic region, however, sars-cov- mpro possesses a cys and his catalytic dyad which distinguishes the sars-cov- mpro from canonical chymotrypsin-like enzymes (gorbalenya & snijder., ; . during the catalysis, the thiol group of cys is deprotonated by the imidazole of his and the resulting anionic sulfur nucleophilically attacks the carbonyl carbon of the substrate. after this initial attack, an n-terminal peptide product is released by abstracting the proton from the his , resulting in the his to become deprotonated again and a thioester is formed as a result. in the final step, the thioester is hydrolyzed which results in a release of a carboxylic acid and the free enzyme; therefore, restoring the catalytic dyad (pillaiyar et al., ; ullrich & nitsche., ) . catalytic residue conformations of sfx structures are consistent with wqf and support the proposed mpro catalytic mechanism (fig. c, d) . the crystal contact of the symmetry-related molecule with the n-terminal region of the mpro is essential for the formation of the crystal lattice in the c space group ( cwb) (fig. a& fig. s ). & s for cwb and cwc, respectively) to determine the flexible regions. in simulations for both cwb and cwc, the protomers exhibit non-identical behavior as also observed by others in apo form of mpro (suarez and diaz., ; amamuddy et al., ) though as expected, higher rmsf values were observed for the loop regions of protomers. additionally, the protomers of cwc display higher fluctuations compared to cwb around the loops covering the active site (such as loops containing residues - and - ) which could affect the accessibility of the active site by inhibitor compounds. on the drug-binding pocket dynamics. the trajectory frames obtained from md simulations were used to perform pca to determine the variations of conformers of protein structures, i.e. to observe the slowest motions during md simulations. as pca and pca-based methods are useful to reveal intrinsically accessible movements such as domain motions (bahar et al., ) , we have performed pca for backbone atoms of dimeric units for the structures belonging to different space groups. we focused on the first three pcs, that show around % of the total variance in md trajectories to determine the regions of protein structures that display the highest variation (fig. s & s for cwb and cwc, respectively). the first three pcs were projected onto the protein structures to determine the contributions of each residue to specified pcs which displays the motions of specific regions with blue regions with higher thickness representing to more mobile structural parts of the protein along the specified pcs. it can be seen that both in c and p space group structures, again protomers a and b display asymmetric behavior and domain movements along all considered pcs ( fig. a-c for cwb and fig. d -f for cwc) which was also observed in residue dynamics for the same domain between alternate chains (amamuddy et al., ) . when the motions displayed by two dimeric forms are compared to each other cwb (supplementary movies m - for cwb and m -m for cwc), we observe that domain iii is more mobile in cwb (the crystals in the c space group) compared to cwb (the crystals in the p space group). however, the loop region containing residues - around the catalytic site is more mobile for both protomers in cwc while it is only mobile for the chain b of cwb. interestingly, protomer a of cwc is more mobile compared to protomer a of cwb which could reflect the differences in dimeric interface due to the additional n-terminal amino acids and missing interactions with chain b n-terminus residues in the dimer interface. however, the loop regions surrounding the binding pockets with residues - and - display higher flexibility in chain a of cwb while its mobility is somewhat restricted in cwc. we have also performed cross correlation analysis of residues along the pc space to understand the correlation between motions especially for different domains. the correlation between the motions along the first three pcs were plotted as dynamical cross correlation maps displayed in in which motions of some residues are along the same directions, others are in opposite directions within both protomers a and b of cwb while domain ii has limited mobility as being more buried than other domains (suarez and diaz., ) except for the β-strand segment of residues - of protomer a. surprisingly, this segment also has some mobility albeit limited in protomer b of cwc instead of protomer a (supplementary movies m , fig. h ). glu of this segment is actually an important residue that plays a role in stabilizing the substrate binding site s by interacting with ser of the alternate protomer along with phe (ghahremanpour et al., ; and we observed that along pc , for protomer a of cwb and protomer b of cwc, the motions of these residues are correlated. this could have implications about information transfer from one protomer to the other though other mpro structures of sars-cov- needs to be studied in atomistic details. in addition, we observe that correlations between domains of protomers a and b for cwb and cwc are dissimilar. for instance, domains i of cwb have defined anti-correlated motions as can be seen from the purple areas in . space groups of these structures are c , p , p and i respectively. the completeness of the structures was also evaluated, wqf, and y e crystallized in full-length sequence ( - ), however, w only lacks gln at both c-terminal ends of its chains. among compared structures, c y, which has the same space group as cwc has, and lacks amino acids (chain a, - , - , - , and ; chain b, - , - , - , and - ) . in our structures cwb has a full-length sequence, however, cwc lacks its last amino acid residues from c-terminal in chain a, and starts with phe (lacks ser and gly ), and ends at ser , lacks the last residues. when six structures ( w , y e, wqf, c y, cwb, and cwc) were compared based on hydrogen bonding interactions at the dimerization interfaces, more similarities were observed for the latter three ( c y, cwb, and cwc). the only difference between our structures and the other four is the hydrogen bond between ser (chain a) and gln (chain b) (fig. s ). although this hydrogen bond is not observed in other structures, the corresponding residues are close to each other, however they are not within hydrogen bonding distance we also monitored all interface interactions throughout the md simulations and compared cwb and cwc. interestingly, the hydrogen bond between ser (chain a) and gln (chain b) was lost and the interaction was turned into a van der waals interaction in cwb, and conserved as by % of the simulation time. in cwc structure, ser (chain a) was not in the vicinity of gln (chain b) (fig. s ). more interestingly, the same interaction but this time between gln (chain a) and ser (chain b), that is present at the cwb crystal structure, was only observed % of the simulation time ( fig. s ). in the cwc crystal structure, there was no hydrogen bonding interaction between gln (chain a) and ser (chain b). however during simulation, this bond was formed and retained in % of the simulation time (fig. s ). when static structures were compared based on hydrogen bond formation analysis, c y, cwb, and cwc clustered together, having the same hydrogen bonding network at the dimerization interface. the explanation for the hydrogen bond differences observed at the interface is the lack of amino acids at the n-terminal and c-terminal ends of the c y and cwc structures, compared to the cwb. simulation trajectories of cwb and cwc were compared based on hydrogen bond occupancies. the major differences were lys (chain a) and arg (chain b), asn (chain a) and ser (chain b), and arg (chain a) and tyr (chain b). the first interaction was not observed in cwc, however the latter two interactions were not presented in cwb. lys (chain a) and arg (chain b) was conserved % of the simulation time; asn (chain a) and ser (chain b); and arg (chain a) and tyr (chain b) retained % and % of the obtained trajectory frames, respectively (figs. s and s ). we also observed that during md simulations, in the case of cwc, gln (chain a) and phe (chain b) come closer to each other, and this van der waals contact was conserved % of the simulation time and not observed in the case of cwb (fig. s ). mpro. we used three well-known sars-cov- mpro inhibitors (i.e., ebselen, tideglusib, and carmofur) at the investigation of ligand-target interactions. these compounds were docked to the cwb and cwc structures and all-atom md simulations were performed for the top-docking poses. these three compounds were also docked to structures with the following pdb ids: w and y e, for comparison. md simulations were performed using the same md protocol. results showed that especially for apo form dimer targets, ebselen and carmofur are quite flexible at the binding pocket throughout the simulations and in most of the cases, they do not form a stable complex structure ( fig. s ). tideglusib has a more stable structure at the binding pocket of mpro, however, its binding modes are different (fig. ) . in the following section, details of md simulations results of tideglusib at the binding site of the mpro will be discussed. comparison of binding pocket volumes of cwc and ye shows that latter has a bigger average binding pocket volume. average binding pocket volumes were and Å , for the tideglusib bound ye and cwc structures, respectively. corresponding solvent accessible surface area (sasa) values throughout the md simulations also support this result. the sasa, which is the surface area of a molecule accessible by a water molecule, of cwc is smaller than y e ( fig. s ). tideglusib. tideglusib initially docked to the binding pockets of y e and cwc structures using induced-fit docking (ifd) approach. top-docking poses of these compounds were then used in allatom md simulations using the same md protocols. while tideglusib was structurally very stable at the binding pocket of the cwc during the simulations, it was not so stable at the y e the binding site (fig. a ). representative trajectory frames (i.e., the frame that has the lowest rmsd to the average structures) were used in the comparison of binding modes (fig. b) . results showed that while the binding mode of tideglusib forms hydrogen bonds and pi-pi stacking interactions with glu , gln , and his , respectively at the cwc; its corresponding binding mode at the y e only forms van der waals type interactions with hydrophobic moieties. a timeline protein-ligand contacts were visualized throughout the simulations (fig. c ). results showed that thr , leu , met , glu , and gln form stable interactions with the ligand. d ligand atom interactions with protein residues are also represented (fig. d ). interactions that occur more than % of the simulation time are shown. however, corresponding interactions of the ligand at the y e were not stable ( fig. s ). forms of mpro, we applied ifd protocol to predict the binding mode of tideglusib at the binding pocket of wqf and cwb. carbonyl oxygens of the thiadiazolidine ring of the tideglusib formed hydrogen bonds between asn , gly , and glu from their backbone atoms. the naphthalene ring of the ligand formed a pi-pi stacking interaction with the his in the cwb structure. a similar binding mode of tideglusib was observed when wqf was used. however, in this binding mode, his and gly are found to be important. a backbone hydrogen bond was observed between one of the carbonyl oxygen of the thiadiazolidine ring and gly . his formed two pi-pi interactions between the benzyl and the naphthalene rings of the tideglusib (fig. s a, b) . these two binding modes predicted by ifd were used in ns classical all-atom md simulations. each mpro-tideglusib system is evaluated based on rmsd changes from the average structure and we obtained representative structures from corresponding trajectories. in the representative structure of cwb-tideglusib complex, we observed van der waals interactions between surrounding residues, thr , his , cys , thr , ser , met , asn , cys , his , his , met , glu , and gln . in the representative structure of wqf-tideglusib complex, in addition to the similar van der waals sfx utilizes micro-focused, ultrabright and ultrafast x-ray pulses to probe small crystals in a serial fashion. structural information is obtained from individual snapshots; capturing bragg diffraction of single crystals in random orientations (martin-garcia et al., ) . the main advantages of sfx over its counterparts are the capability of working with micron to nanometer sized crystals which does not necessitate the lengthy and laborious optimization steps and enables working with multiple crystal forms and space groups. it enables obtaining high resolution structures at physiologically meaningful temperature and confirms the dynamic regions of the active site without secondary radiation damage. sfx offers great potential and provides much needed critical information for future high-throughput structural drug screening and computational modeling studies with sensitivity to dynamics, insensitivity to potential radiation-induced structural artifacts resulting in production of detailed structural information. sars-cov- mpro catalyzes the precise cleavage events responsible for activation of viral replication and structural protein expression . it has been the focus of several structural and biochemical studies, many of which have been performed at cryogenic temperatures, aiming to provide better understanding of the active site dynamics and reveal an inhibitor that affects the enzyme based on structural information. two crystal forms of mpro, native and modified, were determined at ambient-temperature with resolutions of . Å and . Å respectively. the two forms produced are optimal for co-crystallization and soaking respectively. co-crystallization experiments provide efficient interaction in the binding pocket as both drug and protein are stabilized before the formation of crystals. due to the close crystal lattice contacts, co-crystallization seems to be the preferred method for the native form of mpro (c ). the n-terminal of mpro which plays a critical role in crystal packing. elimination of the h-bonding network produced orthorhombic crystals in the p space group yielding a wider binding pocket, increasing the probability of capturing an expanded number of protein-drug complexes by soaking. an added advantage of two different crystal forms was the elimination of the artifacts introduced by specific lattice packing restraints of each crystal form in the dynamics analysis. the high-resolution mpro sfx structures presented here in two different crystal forms collectively revealed the intrinsic plasticity and dynamics around the enzyme's active site. due to the anionic nature of cys , it seems challenging to design molecules that interact with the active site only through non-covalent bonds as it has a very flexible environment. these findings provide a structural basis for and is consistent with studies claiming that the majority of inhibitors form covalent bonds with the active site of mpro , mainly through cys (figs. especially, unlike some studies, ebselen does not form a stable complex structure (sies et al., ; menendez et al., ; zmudzinski et al., ; węglarz-tomczak et al., ) . in addition to the importance of cys residue in our two different structures, a coordinated w molecule, regulating the catalytic reaction via triple hydrogen-bonding interactions with the his , and the asp that stabilizing the positive charge of his residue . this active site residue conformations of sfx structures are consistent with previous ambienttemperature structure ( wqf) (fig. c, d) . gln contributes to the stability of the ligands , along with asn and ser , are the active site residues forming the flank of the cavity (fig. ) . in a recent study, asn and gln have been indicated to interact with a and b inhibitors that have proven in vitro effectiveness . along with these amino acids, thr , which makes van der waals interaction with the n inhibitor, and ser which undergoes conformational changes in the presence of this inhibitor (pdb id: lu , and leu binding to n , gave different side-chain conformations with sfx, according to wqf structure (fig. b ). there were differences in the crystallization conditions between the sfx and wqf studies, the former making use of charge effects and the latter molecular crowding. the sfx study is also secondary radiation damage-free, eliminating potential artifacts to examine crucial amino acids at the atomic level, especially in terms of catalytic and inhibitor binding sites (figs. s -s ). these two highresolution sfx structures in different space groups reveal new active site residue conformations and intra-and inter-domain network and their dynamics at the atomic level, which helps us to better understand any related structural allosteric transitions of mpro structure interacting with the inhibitors (fig. & figs. s - ) . therefore, considering the importance of the required sensitivity in drug design or the use of natural compounds studies, these active site residue conformations reveal the critical importance of our study more clearly. there are many in silico docking studies performed based on cryogenic mpro protein structures of sars-cov- ton et al., ; pillaiyar et al., ; durdagi et al., ) . although potent antiviral drug candidates are identified and a vaccine research is still ongoing, they have not yielded a desirable final treatment/cure yet. in pursuit of effective drugs against covid , the key role of mpro in viral replication of sars-cov- , highly conserved structure, and the low toxicity of the antiviral molecules targeting this protein due to the absence of homolog of this protease in humans made mpro the target of our study. having access to the alternative ambienttemperature structures of mpro and observed conformational changes on active site residues will be a significant boon for the development of therapeutics and provide better understanding on ligand and inhibitor binding. at this point, our work has two original aspects. firstly, we used a comprehensive platform, sfx, which helps to deeply understand the complexity of sars-cov- to gain access to the high-resolution and radiation damage-free structure and the structural dynamics of the target protein mpro at an unprecedented level at near-physiological-temperatures. secondly, we determined two high-resolution sfx structures of sars-cov- mpro in two different space groups due to the new high-throughput data collection setup offered by the mfx instrument of the lcls-ii. drug repurposing has been the preferred area of research in the insufficiency of time and resources in emergency cases such as a novel pandemic. the most important advantage of the repurposing research is the bypass of several lengthy early stages in drug development. also, knowing the beneficial and detrimental effects of targeted drugs, along with well-established precautions will help with time limitations. this procedure has emerged as a fundamental and very strategic approach not only for prospective cohort design but also for many types of clinical trials, particularly crosssectional studies because as the molecules considered in repurposing studies passed through several stages, have well-defined profiles, they would not require prolonged pre-clinical studies, and hence they are important candidates to consider in case of disease emergencies or outbreaks (su et al., ; cavalla et al., ; choo et al., ; aguila et al., ) . therefore, enormous contribution to clinical studies, repurposing is a rapid step towards the conclusion of not only randomized controlled trials but also critical structural biology investigations (bumb et al., ; pihan et al., ; choudhary et al., ) . considering all this, adopting the drug repurposing approach and using known inhibitors ebselen, tideglusib, and carmofur to carry out mpro-based in silico molecular docking, md simulations and post-md analyses make our combined study more specific. a grave issue with drug research is the variability of the target proteins as the mutations and modifications may render the found drugs ineffective (dinesh et al., ) . for this reason, it is only sensible to work on a protein, whose biochemical properties are conserved over time and among different strains. evolutionarily, viruses try to hide by mimicking the proteins involved in the functioning of the host organism. cytomegalovirus (hcmv) can mimic a common host protein to hijack normal cell growth machinery or human immunodeficiency virus (hiv) can mimic a high percentage of human t cell receptors (bernstein, ; robertson, ) . sars-cov- virus contains the plpro enzyme, which is highly similar to the deubiquitinating enzymes (usp , usp ) in human metabolism (ratia et al., ) . that's why targeting this enzyme carries a high risk. in addition, spike protein, one of the other targeted proteins, also has a high mutation rate . also, spike protein includes a similar restriction site with epithelial channel protein (anand p et al., ) . besides that, inhibitors which target spike protein may act only for preventive aims (i.e., before the virus infection), if the virus already infected the host cell, targeting this region may not be useful. there is no mpro homolog in the human genome, targeting this protease is therefore safer and harmless to humans with reduced cross reactivity and side effects making it an ideal candidate for drug therapy. contributions of residues to the first three pcs for a-c) cwb and d-f) cwc with chain a displayed on the right and chain b on the left side. the aligned trajectory frames were generated to interpolate between the most dissimilar structures in the distribution along specified pcs. color scale from red to blue represents low to high displacements along specified pcs with broadening of the tubes depict the trajectory movements. the cα atoms of catalytic residues his and cys are displayed as spheres with cyan and lime colors, respectively. g-h) dynamic cross correlation matrix generated from the motions observed in pc space with values ranging from - (complete anticorrelation) to + (complete correlation) g) for cwb and h) for cwc. the boundary between chain a and b is denoted by dashed lines. bacterial vector by using ndei and bamhi restriction cleavage sites at ' and ' ends respectively. nterminal canonical sars-cov- mpro autocleavage cut site is indicated by green and purple which generates the native n-terminus. c-terminus has the prescision tm restriction site shown in red which is used to generate the native c-terminus after ni-nta hexa-histidine affinity purification chromatography. in-frame hexa-histidine tag and stop codon is shown in blue color. standard chromatography purification methods were applied to both constructs with slight modifications as described below. soluble mpro proteins were purified by first dissolving the bacterial cells in the lysis buffer containing mm tris ph . , mm nacl, % v/v glycerol supplemented with . % triton x- followed by sonication (branson w sonifier, usa). after sonication step, cell lysate was centrifuged by using the beckman optima™ l- xp ultracentrifuge at rpm for minutes at °c by using ti rotor (beckman, usa). after ultracentrifugation the pellet which contains membranes and insoluble debris was discarded and clear supernatant applied to nickel affinity chromatography by using a ni-nta agarose resin (qiagen, usa). to purify the mpro protein, first the chromatography column was equilibrated by flowing column volume of the loading buffer containing mm tris-hac ph . , mm imidazole, mm nacl. after equilibration, the supernatant containing the overexpressed mpro protein was loaded into the ni-nta agarose column at ml/minute flow rate. unbound proteins were removed by washing with column volumes of the loading buffer to clear the non-specific binding. after washing, hexa-histidine tagged mpro proteins were eluted from the column with the elution buffer containing mm tris-hac ph . , mm nacl, mm imidazole in ml of total volume. after elution, purified protein was placed in a kda cut off dialysis membrane and dialyzed against the buffer containing mm tris-hac ph . , mm nacl overnight to get rid of the excess imidazole. after the dialysis step we applied : stoichiometric molar ratio c protease (prescission protease, genscript, usa) to cleave the c- for initial crystallization screening, we employed sitting-drop microbatch under oil screening method by using well terasaki crystallization plates (greiner-bio, germany). purified mpro protein at mg/ml mixed with : volumetric ratio with ~ commercially available sparse matrix crystallization screening conditions. the sitting drop solutions were then covered with μl of % paraffin oil (tekkim kimya, turkey). all the crystallization experiments were performed at ambienttemperature. for our native construct- we were able to obtain multiple hit conditions and among them the best crystals were obtained at pact premier tm crystallization screen condition # from molecular dimensions, uk. the best crystallization condition has contained mm mmt buffer ph . and % w/v peg [mmt buffer; dl-malic acid, -morpholine ethane sulfonicacid (mes) monohydrate, -amino- -(hydroxymethyl)- , -propanediol (tris)-hcl]. for the modified construct only one crystallization condition yield the macrocrystals. after multiple optimization of the seeding protocol by using crystals obtained by microbatch under oil, we scaled up the batch crystallization volume to total of ml for native construct- and total volume of ml for modified construct- . microcrystals - × - × - μm in size were passed through micron plastic mesh filters (millipore, usa) in the same mother liquor composition to eliminate the large single crystals and other impurities before the data collection. crystal concentration was approximated to be - particles per ml based on light microscopy. due to covid travel restrictions none of the initial crystals or the batched crystalline slurry were able to be pretested for their diffraction quality before the scheduled xfel beamtime. . ml total volume of crystal slurry was transferred to ml screw top cryovial (wuxi nest biotechnology, china cat# ). to absorb the mechanical shocks during transport from istanbul to menlo park, ca these vials were wrapped loosely by kimwipes (kimberly-clark, usa) and placed in ml screw top glass vials and tightly closed to provide insulation during transport via air. the vials were wrapped with excess amounts of cotton (ipek, turkey) and placed in a ziploc tm bag (sc johnson, usa) to provide both added layer of insulation and mechanical shock absorption. the ziploc tm bags were placed in a styrofoam box that was padded with ~ kg of cotton to provide more insulation and mechanical shock absorption during the transport. the styrofoam box was sealed and wrapped with an additional layer of cm thick loose cotton layer and duck taped all around to further insulate the delicate mpro crystals during ambient-temperature transport. all these packing materials and techniques provided us with crystals diffracting to . Å - . Å resolution as described below. the . ml sample reservoir was loaded with mpro crystal slurry in their unaltered mother liquor as described above. we used standard microfluidic electrokinetic sample holder (mesh) (sierra et al., ; sierra et al., ) injector for our sample injection. the sample capillary was a μm id × μm od × . m long fused silica capillary. the applied voltage on the sample liquid was typically - v, and the counter electrode was grounded. the sample ran typically between . and μl/min. the sfx experiments with native mpro microcrystals were carried out at the lcls beamtime id: mfx at the slac national accelerator laboratory (menlo park, ca). the lcls x-ray beam with a vertically polarized pulse with duration of fs was focused using compound refractive beryllium lenses to a beam size of ~ × μm full width at half maximum (fwhm) at a pulse energy of . mj, a photon energy of . kev ( . Å) and a repetition rate of hz. om monitor and psocake (damiani et al., , thayer et al., were used to monitor crystal hit rates, analyze the gain switching modes and determine the initial diffraction geometry of the new epix k m detector . a total of , , detector frames were collected in h m s continuously with the new from native (construct- ) mpro microcrystals. a total of , detector frames were collected in h m s continuously with the new epix k m pixel array detector from modified (construct- ) mpro microcrystals. the total beamtime needed for native (construct- ) and modified (construct- ) datasets were h m s and h m s respectively, which shows the efficiency of the mfx beamline installed with the new epix k m detector and robust injector system, as due to lack of blockages no dead time was accumulated. individual diffraction pattern hits were defined as frames containing more than bragg peaks with a minimum signal-tonoise ratio larger than . , which were a total of , and , images for native and modified respectively. the detector distance was set to mm, with an achievable resolution of . Å at the edge of the detector ( . Å in the corner). an example diffraction pattern is shown in fig. s . the diffraction patterns were collected at the mfx instrument at the lcls using the epix k m detector . the raw data images were subjected to detector corrections with cheetah (barty et al., ) , as well as for hit finding based on bragg reflections. the hitfinding parameters for all datasets classifying a hit were as follows (using peakfinder ): a minimum pixel count of above an adc-threshold of with a minimum signal to noise ratio of was considered a peak, and an image containing at least peaks was classified as a crystal hit. the crystal hits were then indexed using the software package crystfel version . (white et al., ) using the peaks found by cheetah. indexing was attempted using the indexing algorithms from xgandalf (gevorkov et al., ) , dirax (duisenberg et al., ) , mosflm (powell et al., ) and xds (kabsch et al., ) , in this order. after an approximate cell was found, the data was indexed using cell axis tolerances of Å and angle tolerances of º (--tolerance option in crystfel). the integration radii were set to , , and the "multi" option was switched on to enable indexing of multiple crystal lattices in a single image. the indexed reflections were subsequently integrated and merged using partialator ) applying the unity model over iterations and the max-adu set to . the complete reflection intensity list from crystfel was then scaled and cut using the truncate program from the ccp suite (winn et al., ) prior to further processing. for the native mpro protein crystals the final set of indexed patterns, containing , frames ( . % indexing rate), was merged into a final dataset (overall cc* = . ; . Å cutoff) for further analysis (c , unit cell: a = . Å, b = . Å, c = . Å; α = °, β = °, γ = °). the final resolution cutoff was estimated to be . Å using a combination of cc* (karplus & diederichs, ) and other refinement parameters. the final dataset had overall rsplit = . %, and cc* = . in the highest resolution shell. for the n-terminally modified mpro protein crystals the final set of indexed patterns, containing , frames ( . % indexing rate), was merged into a final dataset (overall cc* = . ; . Å cutoff) for further analysis (p , unit cell: a = . Å, b = . Å, c = . Å; α = β = γ = °). the final resolution cutoff was estimated to be . Å using a combination of cc* and other refinement parameters. the final dataset had overall rsplit = . %, and cc* = . in the highest resolution shell. we determined two ambient-temperature mpro structures by using two crystal forms in space group c and p structures using the automated molecular replacement program phaser (mccoy et al., ) implemented in phenix (adams et al., ) with the previously published ambienttemperature structure as a search model (pdb id: wqf) . this choice of starting search model minimized experimental temperature variations between the two structures. coordinates of the wqf were used for initial rigid body refinement with the phenix software package. after simulated-annealing refinement, individual coordinates and tls parameters were refined. we also performed composite omit map refinement implemented in phenix to identify potential positions of altered side chains and water molecules were checked in program coot (emsley & cowtan, ) , and positions with strong difference density were retained. water molecules located outside of significant electron density were manually removed. the ramachandran statistics for native monoclinic mpro structure (pdb id: cwb) (most favored / additionally allowed / disallowed) are . / . / . % respectively. ramachandran statistics for orthorhombic mpro structure (pdb id: cwc) (most favored / additionally allowed / disallowed) are . / . / . % respectively. the structure refinement statistics are summarized in supplementary table s . structure alignments were performed using the alignment algorithm of pymol (www.schrodinger.com/pymol) with the default σ rejection criterion and five iterative alignment cycles. all x-ray crystal structure figures were generated with pymol. the two ambient-temperature mpro in space group c and p were examined to generate ellipsoid structures based on b-factor with pymol and these two structures were compared with the mpro structures at k (pdb id: xkh) to provide better understanding on the flexibility of atoms, side chains and domains. the all ellipsoid structures were colored with rainbow selection on pymol. we have used different crystal structures of mpro available in literature as well as the obtained crystal structures in this study (pdb ids: cwb and cwc) as target structures for molecular docking and md simulations. the biologically-relevant dimeric form of cwb is generated by application of a symmetry operator. as the crystal structures in this study were obtained at ambient-temperature, for comparison another ambient-temperature structure of mpro (pdb id: wqf) in apo form was also selected as target structure. another apo form structure of mpro (pdb id: y e) in dimeric form was also chosen for comparison. additionally, mpro structure bound to a non-covalent inhibitor (pdb id: w ) in both monomeric and dimeric forms was utilized as target structure. for ligands, we have considered three compounds that have shown promising inhibitory activity based on the highthroughput screening of over , compounds by jin et al., namely; ebselen (ic = . ± . μm), tideglusib (ic = . ± . μm) and carmofur (ic = . ± . μm) . all the target structures considered in this study were firstly prepared using protein preparation module of maestro modeling program in which missing atoms were added, water molecules not in the vicinity of co-crystallized ligands were removed and bond orders were assigned (madhavi sastry et al., ) . the protonation states of amino acids at physiological ph were adjusted using propka (bas et al., ) to optimize the hydrogen binding and charge interactions. as a final step of preparation, a restrained minimization was performed with opls e force field parameters (harder et al., ) . the structures of three compounds were taken from pubchem; ebselen (pubchem id, ), tideglusib (pubchem id: ) and carmofur (pubchem id: ). the compounds also needed preparation hence, ligprep module (schrödinger release - , ) of maestro modeling program was employed with opls e force field parameters (harder et al., ) . the ionization states of the molecules were predicted by epik module (shelley et al., ) at physiological ph of . . the prepared target protein and ligand structures were used for molecular docking studies. we have employed a grid-based docking method, induced fit docking (ifd) protocol of maestro (sherman et al., a,b) which uses glide halgren et al., ; friesner et al., ) and prime (jacobson et al., ) the selected docking poses at each considered target structure of mpro with the three compounds were subjected to md studies. the apo form structures obtained in this study were also subjected to md simulations. for comparison reasons, md simulations were also performed for the holo form structure (pdb id: w ) with its co-crystallized ligand, x . the target protein-ligand complexes were placed in simulation boxes with orthorhombic shape in which box sizes were calculated based on buffer distance of . Å along all three dimensions and solvated with explicit water molecules of spc (berendsen et al., ) model. the simulation systems were neutralized by the addition of counter ions (na+ or cl− depending on the charge of the systems) and . m nacl solution was added to adjust concentration of the solvent systems. all atom md simulations package desmond (bowers et al., ) was employed. proceeding the production md simulations, the systems were equilibrated using relaxation protocols of desmond package in which a series of minimizations and short md simulations which are performed with small time-steps at lower temperature and restrains on the nonhydrogen solute atoms in the initial stages and slowly time-steps are increased as well as simulation temperature and restrains on solute atoms are released. the production simulations were performed under constant pressure and temperature conditions, i.e. npt ensemble. temperature was set as k while being controlled by nose-hoover thermostat (nosé, ; hoover, ) . the pressure was set as atmospheric pressure of . bar with isotropic pressure coupling and controlled by martyna-tobias-klein barostat (martyna et al., ) . smooth particle mesh ewald method (essmann et al., ) was utilized to calculate long range electrostatic interactions with periodic boundary conditions (pbc). for short range electrostatics and lennard-jones interactions, the cut-off distance was set as . Å. the multi-step integrator respa was employed in which the time steps were varied for interaction types as followed in fs for: bonded, . ; near . and far . . principal components analysis (pca), a statistical data processing method, were performed to reduce the large-dimensional data by extracting large amplitude motions onto collective sets. a covariance matrix were generated from md trajectory data for backbone atoms of protein structures as follow here, i and j represent the backbone atom number, i.e. residue numbers of proteins while n is the number of backbone atoms considered in analysis. the cartesian coordinates of atoms are denoted by and for ith and jth atom, respectively with and representing the time-averaged values over md simulations. by diagonalization of covariance matrix, a collection of eigenvectors and corresponding eigenvalues were obtained. the eigenvectors of the diagonalized matrix are referred as principal components (pcs) and constitute a linear basis set that matches the distribution of observed structures. the corresponding eigenvalues of the diagonalized matrix display the variance of the distribution along each pcs. in this study, we have utilized the bio d package (grant et al., ; yao et al., ) , a platform independent r package to perform pca for considered simulation systems. the trajectories obtained from independent md simulations were concatenated and frames were aligned with respect to the initial (reference) frame before pca. the correlation of atomic displacements is evaluated by cross-correlation analysis to appreciate the coupling of motions. the magnitudes of all pairwise cross-correlation coefficients were investigated to assess the extent of atomic displacement correlations for each simulation system in principal component space. the normalized covariance matrix of atomic fluctuations was calculated as where and are the displacements of residues i and j, i.e., mean square atomic fluctuations. the values of varies between - to with representing completely correlated motions (same period and same phase), representing completely anticorrelated motions (same period and opposite phase) while value indicates motions are uncorrelated. (ichiye et al., ; mccammon et al., ) . bio d package (grant et al., ; yao et al., ) in r environment was employed to generate atom-wise crosscorrelations of motions observed in pcs to and dynamical cross-correlation map, or dccm were generated and displayed as a graphical representation of cross-correlation coefficients. interface analysis of crystal structures and md trajectories were carried out with the getcontacts python scripts (https://getcontacts.github.io/). two different approaches were followed, in the first one only hydrogen bonds at the dimerization interface were taken into account, and in the second approach all possible interactions, namely; salt bridges, pi-cation, pi-pi stacking, t-stacking, van der waals (vdw), and hydrogen bonds were calculated. if the distance between the acceptor and the donor atoms is < . Å and the angle < ° hydrogen bond is defined between atom groups. salt bridges were defined between atoms of negatively charged [asp (od , od ) and glu (oe , oe )] and positively charged [lys (nz) and arg (nh , nh )], where distances were < . Å. t-stacking, pi-cation, and pi-stacking distance criteria were . , . , and . Å, respectively. hydrophobic and vdw interactions were calculated based on atom r (radii), if the distance between atoms is less than the sum of r of atom a, r of atom b, and . Å. one frame and trajectory-based calculations were performed. one frame calculations were applied for the crystal structures, and means the corresponding residues are in contact, and means no interaction. however, in trajectory-based calculations, we used all available frames from our md trajectories. for the interaction frequencies, we applied a . threshold to only take into account the contacts that are occurred at least % of the simulations. fig. s . superposition of two crystal forms. native mpro in space group c colored in darksalmon and its symmetry mate in green. modified mpro in space group p is colored in palecyan and light blue. two crystal structures align with an overall rmsd of . Å. domain i is colored in light blue, domain ii is colored in pale cyan and domain iii is colored in dark salmon. active site formed by residues from all three subdomains such as critical cys is shown as spheres, his , his and asp shown in sticks. red sphere labeled as w represents the water molecule in the binding pocket. s . projection of md trajectory frames onto subspaces defined by the first three largest pcs as well as distribution of variance observed for eigenvalues for cwc. instantaneous conformations (i.e. trajectory frames) colored from blue to red in order of trajectory time ( ns). the black triangle represents the initial crystal structure conformation projected onto specified pcs. domain i residues depicted in light blue, domain ii and domain iii depicted in pale cyan and dark salmon, respectively. blue color shows hydrogen bonding, lines with red ones lack hydrogen bonding. tables supplementary table s . data collection and refinement statistics for x-ray crystallography 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simulation based drug-repurposing cheetah: software for high-throughput reduction and analysis of serial femtosecond x-ray diffraction data phaser crystallographic software very fast prediction and rationalization of pka values for protein-ligand complexes linac coherent light source data analysis using psana structural plasticity of sars-cov- cl mpro active site cavity revealed by room temperature x-ray crystallography opls : a force field providing broad coverage of drug-like small molecules and proteins constant pressure molecular dynamics algorithms protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments the missing term in effective pair potentials autoindexing diffraction images with imosflm dynamics of proteins and nucleic acids epik: a software program for pka prediction and protonation state generation for drug-like molecules bio d: an r package for the comparative analysis of protein structures indexing in single-crystal diffractometry with an obstinate list of reflections data systems for the linac coherent light source proceedings of the acm/ieee conference on supercomputing overview of the ccp suite and current developments a hierarchical approach to all-atom protein loop prediction linking crystallographic model and data quality phenix: a comprehensive python-based system for macromolecular structure solution coot: model-building tools for molecular graphics glide: a new approach for rapid, accurate docking and scoring. . method and assessment of docking accuracy extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem ii nanoflow electrospinning serial femtosecond crystallography a unified formulation of the constant temperature molecular dynamics methods glide: a new approach for rapid, accurate docking and scoring. . enrichment factors in database screening processing serial crystallography data with crystfel: a step-by-step guide crystfel: a software suite for snapshot serial crystallography recent developments in crystfel collective motions in proteins: a covariance analysis of atomic fluctuations in molecular dynamics and normal mode simulations the epix k -megapixel hard x-ray detector at a smooth particle mesh ewald method onda: online data analysis and feedback for serial x-ray imaging canonical dynamics: equilibrium phase-space distributions domain-opening and dynamic coupling in the α-subunit of heterotrimeric g proteins structure of mpro from sars-cov- and discovery of its inhibitors hd acknowledges support from national science foundation (nsf) science and technology centers grant nsf- (biology with x-ray lasers, bioxfel) and the scientific and technological research council of turkey (tubitak) grant ( c ). hd would like to thank michelle young, ritu khurana, lori anne love and tracy chou for their invaluable support and discussions. use of key: cord- -kw psrmz authors: beniac, daniel r.; booth, timothy f. title: structure of the ebola virus glycoprotein spike within the virion envelope at Å resolution date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: kw psrmz we present the structure of the surface ebola virus (ebov) trimeric glycoprotein (gp) spike at Å resolution, in situ within the viral plasma membrane of purified virus particles. gp functions in cellular attachment, endosomal entry, and membrane fusion to initiate infection, and is a key therapeutic target. nevertheless, only about half of the gp molecule has yet been solved to atomic resolution, excluding the mucin-like and transmembrane domains, and some of the glycans. fitting of the atomic resolution x-ray data from expressed, truncated deletion constructs within our Å structure of the entire molecule demonstrates the relationship between the gp -gp domains, the mucin-like and transmembrane domains, and the bilaminar lipid envelope. we show that the mucin-like domain covers the glycan cap and partially occludes the receptor binding sites prior to proteolytic cleavage. our structure is also consistent with key antibody neutralisation sites on gp being accessible prior to proteolysis. based on the findings of us and others, gp-mediated binding may create an angle of degrees between the planes of viral and endosomal membranes. truncated mutants without a transmembrane domain, or as smaller sub-domains of the gp molecule, or as artificial virus-like particles (vlps) , , , , [ ] [ ] [ ] [ ] . the structure presented in the current investigation is based solely on data from the entire glycosylated gp on the surface of ebov, using virions purified from ebov infection in cell culture, and not recombinant expressed versions of the gp spike. to establish a more definitive structure for the native spike within the ebov particle, we analysed purified bona-fide ebov in order to image the gp spike within the virion envelope (fig. a,c) . these gp spike images were analysed using the single particle method only (fig. s ,s ) , as a comparison to structures previously obtained by us and others using tomographic methods. discrepancies had been observed between the structures of the entire, untruncated ebov gp determined using material produced with differing heterologous expression systems, and between structures obtained using alternative tomographic or single-particle three-dimensional image processing methods , . due to safety concerns, the virus preparation was treated using paraformaldehyde crosslinking (after centrifugation) in a protocol that has previously been shown to preserve protein and lipid structures , . ebola virions are flexible, and viral filaments are frequently curved when prepared in the frozen-hydrated state for cryo-electron microscopy (fig. b) . therefore, regions of virions that were as straight as possible were selected for image processing (fig. c) . our data included , individual spike images for single particle analysis. in addition, , images were selected for reference-free analysis of the half-diameter of ebov to investigate the spatial distribution of the gp spikes, as well as the periodicity and symmetrical relationships between gp and the matrix protein vp in the envelope, and the underlying nucleocapsid layer (figs c and s ) . a -d structure for the gp spike trimer in situ, within the viral envelope, with a resolution of Å was calculated (figs and , supplementary fig. s ) using the projection-matching procedure on masked images , . we were able to dock the atomic structure of the ebov gp ( jq ) containing the full-length gp and gp domains, but with the mucin-like domains truncated (fig. b) . this clearly demonstrates a number of features of the viral-derived spike, including the structure and arrangement of the mucin-like domains with respect to the gp -gp structure. in particular, the base of the spike, consisting of the gp fusion domains with the heptad repeat motifs, fits extremely well: the atomic resolution structure jq fills the volume of our -d cryo-em structure (fig. a) . the closeness of fit, especially in the stalk or "neck" area of the gp trimer, is apparent when the spike structure is displayed at a density where the nm thick bilayer nature of the virion envelope is clearly visible (figs a,c and a) and also when the surface is truncated to a level where the volume approximates to a molecular weight of kda (figs and b) . the alpha-helices of gp are visible at the base of the spike, (fig. a) and when the reconstruction is displayed at a slightly higher contour level, densities that cross the interior of the membrane are visible (fig. c) . since the n-terminal ends of the docked alpha- gp domains ( ebo ) appear to line up with these transmembrane densities, the latter may be an indication of the putative hydrophobic alpha-helical transmembrane regions of gp (fig. c ). using the predicted mass of the gp (as measured by gel electrophoresis of virion-derived gp ) we adjusted the volume of the -d structure in fig. , using a value of . da/Å as an approximate density for protein . since the structure jq is kda , the truncated mucin-like and transmembrane domains, including glycans, represent ~ - kda, approximately half the mass of the spike. when viewed from above, the gp spike looks like a three-bladed propeller (figs a and b) . the length of the three "blades" encompasses a circle that is nm in diameter. when viewed from the side, the gp spike has a stalk region adjacent to the viral envelope, which then spreads out to the upper part of the gp spike. the toremifene-binding "pocket" or "tunnel" identified in jq is near the surface of the -d em structure, on the side of the stalk at the base of the propellers (figs a and b) . at the base of the stalk in fig. a , the three heptad repeat helices at the base of gp fit neatly into three strands visible in our em -d structure, that appear to penetrate the virion membrane (figs a and a) . also, when viewed from the top, each blade has a smaller nub closer to the -fold axis that protrudes distally (figs a and b ). using the kda contour level, these nubs correspond closely with the receptor binding site, covering most of the residues known to be involved in binding, as well as protruding adjacent to the glycan cap regions. each propeller blade, which is known to contain the mucin-like domain , completely covers the site known to bind to the npc- receptor (fig. b) . thus approximately half of the mass in our structure, external to the envelope, are the mucin-like domains. when the density map is contoured to a level that removes the lipid bilayer from display, the stalk still conforms closely to the surface of the atomic resolution structure, whereas there distal ends of the blades and the glycan cap "nubs" are slightly truncated. this indicates that these regions of the structure probably have a lower density, consistent with their being highly glycosylated, as predicted by the amino acid sequence of the mucin-like domain.we also docked the jnx structure , which includes part of the cleaved glycoprotein (gpcl) in complex with full-length human npc , and and jnx (npc -gp, magenta) were docked within the ebola gp using chimera (gp trimer subunits coloured in green, blue and yellow; npc -gp in magenta). the cryo-em reconstruction is presented at a threshold to show the viral envelope, and using the same colour code as in fig. . (b) the gp spike reconstruction is presented at a threshold equivalent to its molecular mass to illustrate the tight fit of jq . the inset shows the location of toremifene (red), and the residues of the receptor-binding site are coloured red. (c) the atomic resolution structures of the neutralizing antibodies kz fab ( csy, beige), mr fab, ( x d, pink) were docked within the ebola gp using chimera. overlaid the atomic resolution structure onto one monomer using the program module "fit in map" in ucsf chimera (fig. a) . this indicates that the mucin-like domain, (all of the unoccupied density remaining in the -d em structure when gp -gp are fitted) completely covers the glycan cap, with the nub at the side of the propeller covering most, if not all, of the receptor binding site (fig. a) , indicating that removal or cleavage of the mucin-like domain (probably including the nub), as well as the glycan cap, may be prerequisites for receptor binding to be achieved. the docked jnx structure also includes the transmembrane region of npc , and thus we aligned the approximate plane of the plasma membrane when the gp spike docks with the receptor npc , shown in transparent blue in fig. a . in the absence of any bending of endosomal membrane to accommodate the contour of the gp trimer and npc complex, the plane of the plasma membrane would make an angle of about degrees from the viral membrane when the spike is perpendicular to the viral envelope (fig. a) . this is consistent with the suggestion by gong et al. that perhaps only one npc -receptor binding site out of three on each trimer can be occupied at once, and that binding to more than one receptor might be precluded due to stearic interference. the possible line of cleavage delineating the mucin-like domain and glycan cap from the rest of the trimer is shown in fig. a and b. our structure suggests that the mucin-like domain and the glycan cap might be cleaved at the same time, since the latter is entirely within the density of the putative mucin-like domain, and is consistent with a molecular weight for the mucin-like domain of about kda, which is similar to that predicted by the sequence as well as gel electrophoresis, although the length of the sugar side-chains is still unknown. in addition, we fitted the atomic-resolution fab antibody-gp structures that bind to each of the two major ebov gp neutralising epitopes that have been previously investigated, mr ( x d ) and kz ( csy ): (fig. c) . this indicates that, within the limitations of the resolution achieved in our -d structure of the spike in situ in the envelope, the putative mucin-like domain (likely to consist of the propeller blade and possibly the nub features described here) is unlikely to interfere or stearically hinder either of these antibody neutralisation sites, consistent with neutralisation being possible prior to cleavage of the mucin-like domain in the endosome. it is clear that our structure differs from the -d em structures of the heterologously-expressed ebola gp spike observed in situ in the plasma membrane previously published by us and others (fig. ) . both of these structures included engineered expressed material using mutated or partially sequence-truncated proteins made in virus-like particles. the spike structure of beniac et al imaged within the virus-like particles was somehow clipped, since this should have included the mucin-like domains: in addition, tomography was combined with single-particle analysis, which may have distorted the results . the structure that we report here is based entirely on the well-accepted method of single-particle analysis using projection matching, and is broadly similar to that published of expressed gp in virus-like particles using tomography , : there are noticeable differences in shape and size of the spike, as well as in resolution (fig. ) . the previously determined atomic resolution structures of gp appear to fit well within the cryo-em structure we report here (figs and ) and the resolution, although modest ( Å), is an improvement compared to ~ Å for the structures generated from tomography alone that appear to have a slightly different shape (fig. ). while our structure shows a spike length of nm and a stalk length of nm and width of . nm, the tomographic structures show a spike length of nm, with a more pinched, shorter and more narrow stalk region of . nm in length and nm width, while the virion envelope at the base also appears to be sloped away from the gp stalk (fig. ) . our structure clearly delineates the nm bilayer of the virion envelope (that has an internal spacing of about Å, fig. a ) as well as the alpha helices of the heptad repeat domains (that have a diameter of about Å) and the nub feature at the base of the propellers, whereas these features were not discernable in the previous structures , (fig. b) . these inconsistencies could be due to the differences between the expressed and viral materials, the latter of which was used in the present report. differences in the methods used could also be significant, and a factor in the improved resolution was the use of an optimally sized t-shaped mask, and the selection of straight regions of the virion membrane for analysis (supplementary figure s ) . this allowed accurate alignment and selection of images taken at right-angles to the three-fold axis of the spikes, while getting an excellent coverage of side views, since the spikes are randomly distributed on their -fold axes, using euler angles of - degrees of rotation around the z-axis (since it is a trimer). thus, we avoided the "missing wedge" of information associated with tomography, where the constraint of tilt angle limits the angular distribution of views. our analyses did not reveal any longitudinal symmetry or well-ordered periodicity of the gp spikes along the axis of the virion filament, showing that the spike arrangement of virions may not follow a rigid symmetry. nor was any longitudinal symmetry apparent in the virion vp matrix layer (although a nm lattice spacing in the vp layer has been observed in images showing a perpendicular view of the vlp membrane ). we have shown that the gp spikes of ebov virions likely have an inconsistent or variable spacing, similar to that previously shown for ebov vlps (supplementary fig s ) . we also confirmed previous results showing that the nucleocapsid layer of virions maintains a consistent ordered longitudinal spacing of about nm (supplementary fig s ) , despite the curves and bends of the filament. thus any contacts between gp and vp and/or the nucleocapsid proteins may be variable and non-equivalent: or if a preferred alignment and stoichiometry exists, it was not detectable by cryo-em of whole virions with our current data. in conclusion, the current structure is consistent with the mucin-like domain occluding the glycan cap and receptor-binding region, such that cleavage is required for the functioning of the latter in the endosome to reveal the receptor binding site on gp , , [ ] [ ] [ ] . it is likely that the cleavage of the glycan cap also includes removal of both the "propeller" and "nub" structures described here. it is clear that the density in our structure partially covers the receptor binding domain, and would likely inhibit npc- binding until cleavage of the mucin-like domain occurs. further high resolution studies of the viral-derived gp structure and virion particles are needed to answer these questions, and for progressing knowledge of ebov morphogenesis. in future studies, analyses of the spatial arrangement of the spikes in the membrane, the structural arrangement of the transmembrane and cytoplasmic domains, and analysis of flexible, quasi-equivalent connections between the envelope matrix protein vp and the nucleocapsid, will all be important to further our understanding of how these viral components function in the replication cycle of ebov. scientific reports | : | doi: . /srep methods cells and viruses. zaire ebola virus (mayinga strain) was produced in vero (e ) culture and purified and concentrated by centrifugation . viral preparations were characterised using polyacryalamide gel electropheresis and western blotting. samples were inactivated by using paraformaldehyde fixation ( %) followed by dialysis against pbs to remove the excess. dialysis was carried out using a . ml slide-a-lyzer g cassette ( , mwco: thermo scientific, usa). all virus culture and purification, and handling of infectious materials was carried out at the national microbiology laboratory within the biosafety level laboratories in winnipeg, canada. virus specimens ( ul) were plunge-frozen on glow-discharged quantifoil grids ( um holes, at um intervals: quantifoil, germany). as a focussing aid, a bsa-colloidal gold suspension ( nm particles: aurion, the netherlands) was added to virus preparations at a rate of per cent by volume. freezing was carried out using liquid ethane as a cryogen with a vitrobot (mark iv: fei company, usa). images were taken at kv with a tecnai g electron microscope (fei) using a gatan ct tr single tilt rotation specimen holder at − °c. data was recorded with an eagle k ccd camera (fei company, usa) at , x magnification at - um defocus, with a dose of electrons/Å . this gave a pixel size of . Å at the ccd chip. image recording used the automated low-dose tia software (fei company, usa). xplore d data acquisition software (fei company, usa) was used to automate eucentric height and focus. image processing. particle selection was carried out using eman, and correction for contrast transfer function (ctf) was made using eman . image processing and -d structures used the spider and web programs . analyses were carried out on a mac pro -core computer (apple inc, with . ghzintel xeon nehalem processors, gb ram, running os x . . ) and on a dell -core power edge (r , -bit xeon x . ghz cpus, gb ram, running on linux os . ). correction of images for the ctf was carried out with the "e ctf.py" module in eman this estimated defocus, and used phase-flipping to correct for the ctf. spike images (n = , ), and half-width images of the virus (n = , ) were selected for image analysis. in all subsequent sections, spider software was used for image processing except where stated otherwise. methods are described and illustrated in supplementary figs s -s . resolution of the final cryo-em structure was estimated using the fourier shell correlation . criterion (supplementary fig. s ). single particle image analysis: half width of virus images. specific image regions were analysed for single particle image processing. two-dimensional masks were generated in the canvas x software package (acd systems, seattle, washington, usa). masks were imported as tiff files for image processing in spider. reference-free classification, to bring all images into register, was carried out with the eman software package. the class average was then used as reference to bring all the images into rotational and translational register. these pre-aligned images were then used with a series of masks for various regions of interest, both including the lipid bilayer, and with this region masked. eman was again used to perform a reference free classification, and the averages generated were processed in spider to perform a multi-reference alignment. the results of these analyses were then applied to both masked and unmasked images to investigate effect of the masking procedure and periodicity of the different layers including the spikes, viral envelope and nucleocapsid. single particle image analysis: gp spike analysis. the masking described for -d image analysis was adapted to -d projection matching of ebov spike images ( supplementary figs s -s ) . for -d processing, images were pre-aligned to a global average by reference-free classification in eman, followed by data alignment to this average in spider. pre-aligned images were then masked with an upside down "t"-shaped mask, thus selecting image regions containing both the lipid bilayer and a single spike (supplementary fig s ) . a dual set images was then sub-filed, one masked with the "t"-shaped mask, and the other unmasked. the d reconstruction of the gp spike was then generated by projection matching, using methods similar to those previously used with other viral spikes within their envelopes , . the main difference was the use of "t"-masked images for two-dimensional analysis and alignment, and unmasked images to apply the alignment parameters to generate new image averages. in addition a cylindrical mask was used on the -d reference volume. in both cases, the -d and -d masks selected data from the envelope and spike, and suppressed noise from adjacent spikes, as illustrated in supplementary figs s and s . structure visualisation and docking atomic resolution data. the d cryo-em data, including docked atomic resolution structures csy, x d, and ebo were visualised with the chimera software package (computer graphics laboratory, university of california, san francisco, supported by nih p rr- ). data and materials availability. the -d electron microscopy structure of the gp spike has been deposited into the electron microscopy data bank, www.emdatabank.org (accession number emd- ). marburg and ebola viruses. fields virology ebola virus disease in west africa-the first months of the epidemic and forward projections structural rearrangement of ebola virus vp begets multiple functions in the virus life cycle the organisation of ebola virus reveals a capacity for extensive, modular polyploidy how do filovirus filaments bend without breaking ? cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes a system for functional analysis of ebola virus glycoprotein ebola virus enters host cells by macropinocytosis and clathrin-mediated endocytosis inhibition of ebola virus glycoprotein-mediated cytotoxicity by targeting its transmembrane domain and cholesterol ebola virus entry requires the cholesterol transporter niemann-pick c small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner cross-reactive and potent neutralizing antibody responses in human survivors of natural ebolavirus infection toremifene interacts with and destabilizes the ebola virus glycoprotein structural basis for marburg virus neutralization by a cross-reactive human antibody structures of protective antibodies reveal sites of vulnerability on ebola virus biochemical and structural characterization of cathepsin l-processed ebola virus glycoprotein: implications for viral entry and immunogenicity structure of the ebola virus glycoprotein bound to an antibody from a human survivor ebola virus entry requires the host-programmed recognition of an intracellular receptor role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein endosomal proteolysis of the ebola virus glycoprotein is necessary for infection the primed ebolavirus glycoprotein ( -kilodalton gp , ): sequence and residues critical for host cell binding role of ext and glycosaminoglycans in the early stage of filovirus entry core structure of the envelope glycoprotein gp from ebola virus at . -a resolution structure and orientation study of ebola fusion peptide inserted in lipid membrane models crystal structure of the ebola virus membrane fusion subunit, gp , from the envelope glycoprotein ectodomain the creation of stable cell lines expressing ebola virus glycoproteins and the matrix protein vp and generating ebola virus-like particles utilizing an ecdysone inducible mammalian expression system spatial localization of the ebola virus glycoprotein mucin-like domain determined by cryo-electron tomography mapping of ebolavirus neutralization by monoclonal antibodies in the zmapp cocktail using cryo-electron tomography and studies of cellular entry grafix: sample preparation for single-particle electron cryomicroscopy the ribosome at improved resolution: new techniques for merging and orientation refinement in d cryo-electron microscopy of biological particles optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy gp mrna of ebola virus is edited by the ebola virus polymerase and by t and vaccinia virus polymerases the potential and limitations of neutrons, electrons and x-rays for atomic resolution microscopy of unstained biological molecules structure of glycosylated npc luminal domain c reveals insights into npc and ebola virus interactions structural insights into the niemann-pick c (npc )-mediated cholesterol transfer and ebola infection replication-deficient ebolavirus as a vaccine candidate eman: semiautomated software for high-resolution single-particle reconstructions spider and web: processing and visualization of images in d electron microscopy and related fields architecture of the sars coronavirus prefusion spike ucsf chimera-a visualization system for exploratory research and analysis we would like to thank the canadian government genomics research and development initiative for funding, dr. steven jones (national microbiology laboratory, canada) for providing us with ebov and shannon hiebert for excellent technical assistance. key: cord- - lia db authors: xu, ying; liu, zhijie; cai, liming; xu, dong title: protein structure prediction by protein threading date: - - journal: computational methods for protein structure prediction and modeling doi: . / - - - - _ sha: doc_id: cord_uid: lia db the seminal work of bowie, lüthy, and eisenberg (bowie et al., ) on “the inverse protein folding problem” laid the foundation of protein structure prediction by protein threading. by using simple measures for fitness of different amino acid types to local structural environments defined in terms of solvent accessibility and protein secondary structure, the authors derived a simple and yet profoundly novel approach to assessing if a protein sequence fits well with a given protein structural fold. their follow-up work (elofsson et al., ; fischer and eisenberg, ; fischer et al., a,b) and the work by jones, taylor, and thornton (jones et al., ) on protein fold recognition led to the development of a new brand of powerful tools for protein structure prediction, which we now term “protein threading.” these computational tools have played a key role in extending the utility of all the experimentally solved structures by x-ray crystallography and nuclear magnetic resonance (nmr), providing structural models and functional predictions for many of the proteins encoded in the hundreds of genomes that have been sequenced up to now. the seminal work of bowie, liithy, and eisenberg (bowie et ai., ) on "the inverse protein folding problem" laid the foundation ofprotein structure prediction by protein threading. by using simple measures for fitness ofdifferent amino acid types to local structural environments defined in terms of solvent accessibility and protein secondary structure, the authors derived a simple and yet profoundly novel approach to assessing if a protein sequence fits well with a given protein structural fold. their follow-up work (elofsson et ai., ; fischer and eisenberg, ; fischer et ai., a,b) and the work by jones, taylor, and thornton (jones et ai., ) on protein fold recognition led to the development of a new brand ofpowerful tools for protein structure prediction, which we now term "protein threading." these computational tools have played a key role in extending the utility of all the experimentally solved structures by x-ray crystallography and nuclear magnetic resonance (nmr), providing structural models and functional predictions for many ofthe proteins encoded in the hundreds of genomes that have been sequenced up to now. what has made protein threading particularly attractive as a protein structure prediction tool is the observation that the number ofunique structural folds in nature is a few orders ofmagnitude smaller than the number ofproteins in nature (finkelstein andptitsyn, ; bairochetai., ) . although this is still not a fully resolved issue, both theoretical and statistical studies (murzin et ai., ; brenner et ai., ; li et ai., li et ai., , li et ai., , wang, ; orengo et ai., ; holm and sander, a; zhang and delisi, ) suggest that the number ofunique structural folds in nature ranges from a few hundred to a few thousand. clearly this is a significantly smaller number than the number ofproteins in nature -as we understand now, the number of different living organisms on earth could range from millions to hundreds ofmillions (may, ) . since each organism often has at least thousands of different proteins encoded in its genome, the total number of different proteins in nature is at least in the tens ofbillions or possibly significantly higher, even without considering protein variants such as alternatively spliced proteins. this disparity suggests an effective paradigm for possibly solving all protein structures through combining experimental and computational approaches, that is, to solve structures of proteins with unique structural folds using the expensive and time-consuming experimental techniques and computationally model the rest ofthe proteins using the experimental structures as templates. this is the key strategy currently being employed by the worldwide structural genomics efforts (gaasterland, ; skolnick et ai., ; baker and sali, ) . the basic idea of protein threading is to place (align or thread) the amino acids of a query protein sequence, following their sequential order and allowing gaps, into structural positions of a template structure in an optimal way measured by fitness scores outlined above. this procedure will be repeated against a collection of previously solved protein structures for a given query protein. these sequencestructure alignments, i.e., the query sequence against different template structures, will be assessed using statistical or energetic measures for the overall likelihood of the query protein adopting each ofthe structural folds. the "best" sequence-structure alignment provides a prediction of the backbone atoms of the query protein, based on their placements in the template structure. currently, protein threading is being widely used in molecular biology and biochemistry labs, often for initial studies of target proteins, as it may quickly provide structural and functional information, which could be used to guide further experimental design and investigation. as a prediction technique, protein threading has a number of highly challenging computational and modeling problems. these include (a) how to effectively and accurately measure the fitness of a sequence placed in a template structure, (b) how to accurately and efficiently find the best alignment between a query sequence and a template structure based on a given set of fitness measures, (c) how to assess which sequence-structure alignment among the ones against different template structures represents a correct fold recognition and an accurate (backbone) structure prediction, and (d) how to identify which parts of a predicted structure are accurate and which parts are not. as researchers find more effective solutions to these and other challenging problems, we expect that protein threading will play an increasingly significant role in structural and functional studies of proteins. as of now, over a million protein sequences have been determined (bairoch et ai., ) , among which~ , have had their tertiary structures experimentally solved (dutta and berman, ) . given that there could be at least tens of billions of different proteins in nature as discussed above, one interesting question, particularly relevant to the idea of protein threading, is how many unique protein structures or structural folds these proteins might have adopted. to answer this question, we need to first look at the basic structural units of proteins, called protein domains (wetlaufer, ; richardson, ) . protein domain is extensively discussed in chapter of this book. here we describe briefly the concept of a domain from the perspective of threading. a structural domain is a distinct and compact structural unit that could fold independently of other domains. while many proteins are single-domain proteins, there are proteins with two, three, or even more structural domains (ekman et ai., ) . our study shows that in the fssp (fold classification based on structure-structure alignment of proteins) nonredundant set (holm and sander, b) of the pdb, % of the proteins have single domains, % have two domains, % have three domains, and the remaining % have four or more domains (xu et ai., a) . this distribution may not necessarily reflect the actual domain distribution for all the proteins in nature as the set of known protein structures in pdb might have overrepresented small proteins due to the relative ease in solving these structures. for eukaryotic organisms, it was estimated that at least two-thirds oftheir proteins are multidomain proteins (gerstein and hegyi, ; gerstein, gerstein, , doolittle, ; apic et ai., a,b) . this number is somewhat smaller for bacterial and archaeal organisms but still represents a significant percentage of all their proteins. previously domain partition of multidomain proteins was typically done manually. to keep up with the rate at which protein structures are being solved, there is a clear need for more automated domain-partitioning methods to process the newly solved structures. currently computer programs are being used for partitioning a protein structure into individual domains. popular programs for this purpose include dali (dietmann and holm, ) , domainparser (xu et ai., a) , and pdp (alexandrov and shindyalov, ) . a protein domain could be part of different protein structures, through combining with other domains. figure . shows an example of a domain in different proteins. both proteins, rna '-terminal phosphate cyclase and glutathione s-transferase, have the thioredoxin fold domain, which has two layers, one with two a-helices and one with four antiparallel r -strands, although some details differ in the two proteins. the parts other than the thioredoxin fold domain in the two proteins have no structural relationship. since domains are the basic structural units of proteins, current studies on the number of unique structural folds in nature have been carried out on protein domains rather than whole proteins (hereafter, the term "proteins" will refer to single-domain proteins for simplicity of discussion). to estimate the number of unique folds of proteins, one popular approach is through examining all protein families and the relationships between protein families and unique structural folds. using the definition of scop classification scheme (murzin et ai., ; brenner et al., ) , a proteinfamily represents a group of orthologous proteins (makarova et ai. ; gerlt and babbitt, ; tatusov et ai., ; gelfand et al., ) . the number ofprotein families in nature could possibly be estimated through finding orthologous gene groups covering all the genes in the genomes that have been sequenced. one such estimate suggests that there are , such protein families (orengo et al., ) . this number has been used in later studies on estimating the number of unique structural folds in nature. other studies estimate this number to be in the tens of thousands (koonin et al., ) . whether it is , or some other number ranging from , to , , the idea is that all proteins in nature fall into one of these families. the estimation on the number of unique structural folds is obtained through estimating the number of families that each structural fold covers and studying the coverage distribution by all the known structural folds. one of the estimates by zhang and delisi ( ) suggests that the number n of unique structural folds is probably around . this estimation is based on the observation that the number of structural folds covering x number of protein families follows a power-law distribution, withxbeing a variable. in essence it says that a few structural folds each cover many families (e.g., tim barrel fold covers protein families) while many structural folds each cover only a small number of families ; more generally, the number of structural folds decreases as their coverage of protein families increases . specifically, zhang and delisi proposed a formula which matches well with the known protein families and structural folds. let m and n represent the number of families and the number of unique folds in nature, respectively. the probability that a fold covers exactly x families is given by let m, and n, be the numbers of protein families and unique structural folds currently having solved structures, respectively. through a simple algebraic transformation, zhang and delisi showed that which is used to estimate the number of unique structural folds. using the known numbers of m, = and n, = at the time of the estimation, they estimated that n is roughly about , which many researchers will argue to be too small (see following for more discussion). similar estimations have been made by other researchers, estimating the size of n ranging from a few hundred to a few thousand (orengo et ai., ; wang, ) , based on somewhat different assumptions. coulson and moult ( ) recently developed a new model for estimating n, based on the work of zhang and delisi. using the more recent data on the numbers of genes, gene families, and structural folds, they argued that there are two "special" cases that have not been treated well by previous estimation models. based on their argument, they consider that there are three classes of structural folds, which are termed unifolds, mesofolds, and superfolds. unifolds represent structural folds that each covers only one family of proteins, superfolds represent structural folds, each ofwhich covers many structural folds, and mesofolds represent structural folds in between. for example, tim barrel covers families, while many unifolds exist in sco~based on their observation, they argued that previous models such as zhang and delisi did not fit well with the data of unifolds and superfolds. so a new piecewise model was then developed which treats unifolds, mesofolds, and superfolds, separately. using this new model, coulson and moult ( ) estimated that less than % of the protein families belong to unifolds, while % of the families belong to a few dozen superfolds and the rest of the protein families belong to mesofolds. considering that the estimated number of protein families ranges from , to , (or , as one of the popular estimates suggests), we can infer that the number of unifolds ranges from , to , . the number of mesofolds could be estimated using the zhang and delisi model, based on the sizes of m, and n s , after excluding the unifolds and superfolds. hence, coulson and moult concluded that the most probable size for the number ofmesofolds is about . the number of superfolds is believed to be very small, possibly in the range of low dozens. overall this model suggests that over % ofthe protein families fold into a little over structuralfolds, the majorityofwhichare alreadyknown, whilethe restoftheprotein families each belongs to a unique unifold. the implication ofthis estimation is that about % ofthe protein families are amenable for structural modeling using protein threading techniques, assuming that at least one protein in each of the meso-and superfolds has its structure solved. if experimental facilities for structure solution will strategically select their solution targets to maximally cover all the meso-and superfolds, we could expect that at least % of the protein families will be structurally modelable in the near future. this is exactly the strategy that the national institute of health (nih) is using in its protein structure initiative (http: //www.nigms.nih.gov/psi/) . for the remaining % of protein families, it might take some time to have at least one solved structure in each of the unifolds. hence, the threading technique will be less applicable for this class of proteins, at least in the near future. there are a number of popular schemes and associated databases for classification of proteins into structural folds, including scop (murzin et ai., ) , cath (orengo et ai., ) , and fssp (holm and sander, b) . these classification schemes classify all solved protein structures into different structural folds and subclasses of structural folds. the classification of protein structures is essentially achieved through grouping protein structures into clusters ofsimilar structures, which can be done computationally through structure-structure alignments (holm and sander, a) . scop (murzin et ai., ; brenner et ai., ; andreeva et ai., ) groups all protein structures essentially into a three-level classification tree. at the top level, scop (scop . ) currently consists of about structural folds, each ofwhich is further divided into superfamilies and then into families. while a family represents a group of orthologous proteins, a superfamily represents a group of homologous proteins, possibly made ofmultiple families. currently scop consists ofabout superfamilies and about families. among the structural folds, have only one family each, which might represent unifolds; and cover a large number offamilies each, which are considered as superfolds by coulson and moult. one thing worth noting is that among the scop folds, only represent membrane proteins. this is a reflection of the fact that only % of all the solved protein structures are membrane proteins (http: //blanco.biomol.uci.edu/membraneyroteins...xtal.html) . this suggests that threading is generally not applicable to structure prediction of membrane proteins, at the present time. scop's hierarchical classification ofstructural folds provides a convenient tool for applications ofthreading methods, as query proteins falling into a scop protein family are generally expected to have accurate structure predictions, while proteins with structural homologues in a scop superfamily will have a good chance to have the correct structural folds identified and some portions oftheir backbone structures predicted accurately. in general, it still represents a challenge to correctly identify the structural fold by a threading method if a query protein only has a structural analogue (i.e., similar structure but not homologous) in sco~ the realization that protein structures are clustered into structural folds in the structure space and the number ofsuch clusters is possibly quite small has led to a new way ofpredicting protein structures in a more efficient and effective manner. the general belief is that different proteins fold into similar d shapes because at some level, they share similar interaction patterns among their residues and between the residues and the environments. it has been shown that these interaction patterns could possibly be captured using simple statistics-based energy models as exemplified by the earlier work ofeisenberg and colleagues (bowie et al., ; fischer et al., a,b; fischer and eisenberg, ) , the work of sippl and colleagues (sippl, ) , and others (jones et al., ; rost et al., ) . these simple statistics-based energy functions have been used, for many cases, to distinguish the correct structural folds from the incorrect ones and to distinguish the correct placements of the residues in a query protein into the structural positions of a correct structural template from the incorrect ones. placing the (backbone atoms of) residues of a query protein into the correct structural positions in a correct structural fold gives a prediction of the backbone structure of the query protein. to accomplish this, one would need two capabilities: (a) an energy function whose global minimum will correspond to the correct placement of residues into the correct structural template, and (b) a computational algorithm that can find the global minimum of the given energy function. we explain the basic idea of developing such energy functions in this section and leave the algorithmic issues to the next one. in their earlier work (bowie et al., ; fischer et al., a,b, fischer and eisenberg, ) , eisenberg and colleagues demonstrated that simple residue-based, instead of atom-based, energy functions could provide substantial discriminating power in separating good from poor placements of individual residue types into different structural environments, justifying the usage ofresidue-based energy functions. in their work, structural environments are simply defined in terms oftwo parameters, solvent accessibility sol and secondary structure ss. specifically the quantity sol of solvent accessibility is discretized into a number of intervals, say -- % exposed to the solvent. a secondary structure could be a helix, a beta-strand, or a loop, or it could be defined in terms of more refined categories of secondary structures, say including different types oftums. then a structural environment for each residue in a template structure could be defined using (sol, s), say ( - % exposed, alpha-helix). statistics could be collected from a collection of solved protein structures about how frequent a particular type of amino acid appears in a particular structural environment as we just defined. this can be done by going through all protein structures under consideration to count the number of occurrences of each amino acid type in each encountered structural environment. if we consider, say, three levels of solvent accessibility, {exposed, intermediately exposed, buried}, and three types of secondary structures, we will have nine types of structural environment. under this assumption, the result of counting the numbers of occurrences above will be a by table, with each of the rows representing an amino acid type and each of the columnsrepresenting a structuralenvironment. based on the collected statistics, we can build a simplepreference model to measure how preferred a particular amino acid type is to a particular structural environment. this can be done using the following measure: where oi,} represents the observed frequency of amino acid type i in structural environment j and ei,l represents the expected frequency of amino acid type i in structuralenvironment j. in the work of eisenberg and colleagues, ei,l is estimated using the frequency of amino acid type i in all proteinsunder consideration. hence, if an aminoacidtype i has a higherfrequency in a particularstructuralenvironment j than its frequency overall, it willbe assigneda negative score-in(oi,}i ei,l); otherwise it will get a positivescore or zero (when oi,l = ei,j). the higher oi,l is comparedto ei.j» the more negative the corresponding energy is. a popular name for this type of energyfunction is singletonenergy.by performingstatistical analysis on a database, one can get the scoring functionusing the above formulation for the amino acid types appearing in the nine structuralenvironments. when building such statistics-based energy functions, one needs to be careful in selectingthe data set for statistics collection. forexample, someproteinsin scop (or in pdb) havemore homologous structuresthan the others,which could possibly leadto biased statistics. toremove this type of statisticbias in our data set, one needs to remove homologous structuresin the data set for statisticscollection. there are a numberof databases forthis purpose,suchas the nonredundant sequencerepresentativesin fssp, pdb-select (hobohmet al., ) , and pisces (wang and dunbrack, ) ,whereno two proteinshavehigher than a certainlevelof sequencesimilarity. another statistics-based energy functionwidelyused in threadingprogramsis often calledpairwise interaction energy. it measures the preference of having two particulartypes of amino acids that are spatiallyclose. one particular form of such an energy function was developed by sippl ( ) . the basic idea of this energy function is to compare the observed frequency of a pair of amino acids within a certain distancein solvedprotein structureswith the expectedfrequency of this pair of aminoacid types in a protein structure. the basic idea of such an energyfunction comes from statistical mechanics where the probability pi} of having a pairwise interaction betweenresidues i and j has the boltzmanncorrelationto its energygij (gibbs free energy), definedas wherek is the boltzmannconstant, t is the temperature, andz is a partitionfunction. when using a residue-averaged state as a reference state g(p), a knowledge-based potential can be derivedusing . specifically, if n o(i, j) and ne(i, j) represent, the observedand expectednumbers of amino acid types i andj within a certain distance, respectively, we can use the following to measurethe preferenceof havingthese two types of amino acids close to each other: while no(i, j) canbe collectedby goingthroughtheproteinstructuresin the sample set, an accurate estimation of ne(i, j) represents a challenge. there have been a number of proposed models for estimatingthis quantity. among these models, the simplestone is the "independentreference state" model (xu et al., ) ,in which ne(i, j) is estimatedas follows: table . shows a scoring function for the preference between types of amino acids using the above formulation (xu et ai., ) . there are more sophisticated models for defining the reference state, one of which is the uniform distribution model (sippl, ; dewitte and shakhnovich, ; lu and skolnick, ; samudrala and moult, ) , as discussed later. these more complex models take more factors into consideration in building the reference state, hence making the energy models more likely to be accurate. in addition to using physics-or statistics-based energy functions, researchers have incorporated evolutionary information into energy model building. one of the earlier major improvements in energy function modeling is the incorporation of sequence profile information (panchenko et ai., ; zhou and zhou, ) derived from homologous proteins into the energy models outlined above. it was noticed that when using a sequence profile ofa protein family (or superfamily) rather than a single (query) protein sequence, threading accuracy could be significantly improved (panchenko et ai., ; zhou and zhou, ) . the very basic idea ofthis generalized approach is that rather than asking the question "will protein sequence a fit well with structural fold b?" we ask the more general question "will the whole family of protein a fit well with structural fold b?" clearly if done properly, this approach could iron out some ofthe spurious predictions, caused by the appearance ofspecific individual sequences. now a threading problem becomes a fitting problem between a sequence profile and a structural fold. a sequence profile is defined in terms ofa multiple sequence alignment ofthe members ofthe query protein's family (or superfamily), with each element being the frequency distribution ofthe amino acid types in this aligned position rather than a specific amino acid. to generalize the aforementioned energy functions to take into account the profile information, we can simply use the relative frequency of each amino acid in the position-dependent distribution as a weight factor when calculating the energy values for each amino acid or amino acid pairs, and then sum over all possible amino acids or amino acid pairs. specifically,let pibe the relative frequency ofamino acid type i in a particular aligned position with l pi = . . then the eisenberg type of energy can be calculated as similarly, pairwise interaction energy could be generalized as follows: other types of energy functions have also been used in existing threading programs, including fitness scores between specific amino acids and the secondary structures in the template structure and threading alignment gap penalties. typically these energy scores are combined using a simple weighted sum while often the scaling factors are empirically determined, based on some training data. it has been observed that distance-dependent pairwise interaction energy could provide more accurate threading results than distance-independent models as outlined above. a distance-dependent energy could be estimated as follows: where r is the distance between residues i and j (possibly measured between their c-beta atoms), no(i, j, r) is the observed number of pairs of residues (i, j) within a distance bin from r -~r to r +~r/ in a database of folded structures for some bin width sr, and ne(i, j, r) is the expected number of pairs (i, j) within the same distance bin. the challenging issue in accurately estimating the interacting energy u(i,j,r) is how to estimate ne (i, j, r) . under the assumption that we are dealing with an ideal infinite liquid-state system within a volume v and residues are distributed uniformly (sippl, ; dewitte and shakhnovich, ; lu and skolnick, ; samudrala and moult, ), ne(i, j, r) can be estimated using where n, and n, are the numbers of amino acid types iand j in the protein database, respectively. researchers have realized that this model needs to be corrected when dealing with finite systems like a protein structure, to make the model more accurate when used in threading programs. twoparticular corrections are made in the dfire (distance-scaled ideal gas reference state) energy model (zhou and zhou, ) , a popular energy function for threadirtg. in the first correction, dfire used r" instead of r , considering that the number of interaction pairs in a finite system could not actually reach the level of r as in an infinite system, where a < is determined throughminimizing the distribution fluctuation of interaction distanceon a set of trainingdata. in the secondcorrection, dfireassumes that only short-range interactions need to be considered. that is, interaction energy becomes zero when the distance betweenthe interacting pairs is beyonda cutoffdistancercut. afterthese corrections, the interactionenergy could be estimatedusing the following formula: where constant is related to the systemtemperature and can be determinedempirically. these simple energy models have played key roles in making threading programs as popular and as useful as we have seen today. while they have been used to help to solve many structurepredictionproblems, the limitations of these simple models have also become quite clear as we have seen from the recent casp prediction results-the improvement in predictionaccuracyhas been only incremental in the past few casps (kinch et ai., ) . one of the key reasons for the incremental improvement comes from the crudeness of the threading energy functions. currently, the algorithmic techniques for protein threadinghaveadvanced to a stage that should be able to handle more sophisticated energy models in the threading framework, which could lead to more accuratepredicted structures. we can expect that more detailedand more physics-based energyfunctions will emergein the near future as the field is clearly in need of more accurate energy models for protein threading. the general form of threadingenergy function could be written as follows: wherees measuresthe overall fitness of puttingindividual residuetypesinto specific structuralenvironments, e p measuresthetotalinteraction energyamongpairswithin the cutoffdistance, and egap represents the total penalty for the gaps in a sequencestructurealignment. the scalingfactorsfi andj are typicallydeterminedempirically through optimizingthe performance of a threadingprogram on a representative set of proteinpairs. withthe optimizedfi andj values, the goal of threadingis to findan alignment(or placement) betweena queryprotein sequence and a templatestructure that optimizesthe energy function. in a sense,proteinthreadingis like sequence-sequence alignmentas it finds an alignmentbetweena sequence of aminoacids and a sequence of structuralpositions in a d structure. what makes threading more difficult than a sequence alignment problem is the pairwise interaction energy term ep. for a sequence alignment problem, a simple dynamic programming can guarantee to find the global optimal alignment between two sequences as the problem formulation follows the principle of optimality (brassard and bratley, ) . this type of simple dynamic programming algorithm does not work for a threading problem as the global optimal threading alignment could not be easily reduced to a small number of optimal threading alignments for the partial problems as in a sequence alignment problem. intuitively, a simple dynamic programming approach, like the one used for sequence alignment that goes from the starts of the sequences to their ends and extends partial optimal alignments to include more elements at each step, will not work for protein threading as at each current point, we do not know what residues will be available to be assigned to future structural positions, which might have interactions with the previously aligned positions. such interactions will have a global impact on a sequence-structure alignment. it is such a global nature of the problem that makes the threading problem more challenging from the algorithmic point of view. there have been a number of studies attempting to understand the "intrinsic" difficulty, or computational complexity, ofthe threading problem. under the assumption that all pairwise interactions need to be considered, the threading problem was proved to be an np-hard problem by a number of authors (lathrop, ; calland, ) . while these mathematical proofs provide some evidence that the problem is computationally difficult, they might not be particularly relevant to the true difficulty of a threading problem as previous studies have shown that pairwise interactions beyond certain cutoffdistances (e.g., - a between c-beta atoms) do not contribute to fold recognition and threading alignment (lund et al., ; melo and feytmans, ; xu et al., ; zhang and skolnick, ) , and hence need not be considered. as of now, it remains an open problem regarding whether the threading problem, using a distance cutoff for pairwise interactions, is polynomial-time solvable. the challenging issue in studying the "intrinsic" computational complexity of a threading problem with a cutoff for pairwise interactions is that we do not have a good and realistic characterization for the overall interaction patterns for such a threading problem. because of the algorithmic challenges, earlier threading programs have employed various heuristic strategies for solving the optimal sequence-structure alignment problem. one particular strategy is called "frozen approximation" (westhead et al., ) . the basic idea is that it uses a dynamic programming approach to find a sequence-structure alignment and uses an approximation scheme to calculate the interaction energy. when the algorithm assigns an amino acid to a specific structural position from the beginning to the end of the query sequence during the dynamic programming procedure, it calculates the relevant interaction energy using the amino acids in the template structure rather than assigned amino acids from the query protein, for all the unassigned structural positions up to the current point of the dynamic programming procedure, within a certain cutoffdistance. intuitively the algorithm should work to some degree in capturing some ofthe interaction "patterns" encoded in the query protein sequence as some of the position-equivalent residues between the native structure and the native-like template structure should have similar physicochemical properties, suggesting the validity ofthe frozen approximation scheme. practical applications have also confirmed that the frozen approximation, while not guaranteeing global optimal threading alignment, does have an advantage over threading programs that do not consider pairwise interactions (westhead et al., ; skolnick and kihara, ; zhang et al., ) . a number ofrigorous threading algorithms have been developed that guarantee to find the global optimal threading alignments, measured in terms of energy functions that consider pairwise interactions. these include a divide-and-conquer algorithm employed in the prospect threading program and an integer programming algorithm used in the raptor program (xu et ai., a,b) . it was convincingly demonstrated, through applications ofthese programs at the casp contests (xu et ai., ; xu and li, ) , that threading programs with guaranteed global optimality do have an advantage over programs without this property. one particular advantage of such programs is that they can be used to rigorously benchmark a proposed energy function. when using programs without such a guarantee to assess an energy function, it will be difficult to decide whether it is the energy function or the lack of rigor in the threading algorithm that has resulted in a subpar performance by a particular energy function. the following provides some detailed information about three types of threading algorithms. the basic idea of the divide-and-conquer algorithm in prospect can be outlined as follows. the algorithm first divides the query protein sequence into two subsequences and also divides the template structure into two substructures by cutting at one of its loop regions. then it tries to find the globally optimal threading alignments between the first subsequence/substructure pair and between the second subsequence/substructure pair, respectively. when calculating pairwise interaction energy for each "half" of the sequence-structure alignment problem, we might need information about which amino acids are assigned to which structural positions in the other "half" of the problem. to facilitate this calculation, the algorithm uses a simple data structure for each structural position l in the current substructure, that keeps a list of structural positions in the other substructure that are close enough to l to have interactions with the amino acid to be assigned to it. figure . depicts schematically the situation where each of the two substructures has a number ofstructural positions, which are close enough to structural positions in the other substructure so that their alignments with amino acids need to be considered when calculating the interaction energy in the other substructure. these structural positions can be considered as extended parts of the other substructure (shown as extended arms for each substructure in fig. . ) . the difference between these extended parts and the original positions ofa substructure is that when doing alignment between the substructure and the corresponding subsequence, we do not have any knowledge about which amino acids are assigned to these positions in the other substructure. hence, the optimal threading alignment for each substructure/subsequence pair depends on the optimal threading alignment for other substructure/subsequence pair. this codependence relationship makes the problem challenging. in prospect, this problem is overcome using the following strategy : consider all possible combinations of amino acids possibly assigned to these extended positions (in the other "half" of the problem); and then solve an optimal threading alignment problem for each substructure/subsequence pair under each possible combination of amino acid assignments to these extended structural positions. assume that we can solve the optimal threading problem for each pair of (extended) substructure/subsequence and for each combination of such an assignment. then it can be checked that the global optimal threading alignment for the whole structure and sequence must be the union of two optimal threading alignments for the two extended subproblems, under one specific combination of the amino acid assignment to the extended part of each subproblem. this realization lays the foundation for the divide-and-conquer algorithm of prospect as it allows reducing a whole threading problem to two smaller threading problems. if we can solve the smaller threading problems, the whole threading problem can be solved by simply going through all combinations of the amino acid assignments to the extended parts for each subproblem to find the one that gives the overall best combined score. during the "conquer" step, it needs to make sure that the two optimal solutions, to be combined, to the subproblems is consistent. to solve a smaller threading problem, we can apply the same divide-and-conquer strategy to reduce it to even smaller problems. this procedure can continue until the size ofthe problem is small enough that it can be solved using a brute force exhaustive search strategy. the trick is how to make this algorithm run efficiently.note that ifnot done carefully, the number of possible combinations of assignments needed to be considered could be very large. in prospect, (sub)structures are divided in such a way which minimizes such a number ofcombinations, through cutting at the "weakest" link with the least interactions between two substructures (xu et ai., ) . the overall computational complexity of the divide-and-conquer algorithm is dominantly determined by the thickest link among all weakest links throughout the bipartitioning of a protein structure into a series of small structures during the whole divide-and-conquer scheme. intriguingly, we found that this thickest link is generally a small number for the vast majority ofthe solved protein structures (xu et ai., ) , making the actual computing time of prospect threading practically acceptable. this observation has also raised an interesting question: "is there something special about the topology of protein structures, which could be further and more rigorously exploited for efficient threading algorithm development?" . . raptor raptor uses a more general framework to rigorously solve the threading problem (xu et ai., a,b) than prospect. it formulates a threading problem as a linear integer programming (lip) problem. it uses an integer variable (" " or " ") to represent if a particular residue in the query protein sequence is assigned to a particular structural position. then the set ofall feasible solutions to a threading problem could be defined in terms of a set of equalities or inequalities (called constraints), each of which is defined in terms of the above and other integer variables. the global optimal threading problem is then defined to find a feasible threading alignment that optimizes a given energy function. generally a linear integer programming problem requires an exponential computing time to find an optimal solution, and hence intractable for large-size problems. branch-and-bound represents a popular technique for solving linear integer programming problems. typically, an integer programming problem is first relaxed to a linear programming problem, i.e., variables could take real values as possible solutions. there are efficient algorithms for solving linear programming problems as they are polynomial-time solvable (papadimitriou and christos, ) . if by chance the solution to the relaxed linear programming problem is all integral, a solution to the original linear integer programming is found. otherwise the linear solution will be used to constrain the search space, through fixing one variable to " " or" " and then the algorithm iterates this process until all solutions have integral values. an interesting observation made is that for the vast majority of threading problems, this relaxation procedure stops after a few iterations, solving the threading problem efficiently and also indicating that threading problems seem to have a special structure in terms of the integer programming formulation. such special characteristics could possibly be utilized for developing more efficient threading algorithms, using more specialized algorithmic techniques. one particularly interesting technique which is being actively investigated by a number of researchers is based on the idea of tree decomposition of an interaction graph representing possible alignments between a query sequence and a template structure (song et al., ; xu et al., ) . in a sense this type of technique is a generalization of the divide-and-conquer outlined above. tree decomposition technique has been widely used for various graph-related optimization problems, for example finding the maximum independent set and dominating set (arnborg and proskurowski, ) . we now provide a detailed description of one such algorithm for solving the protein threading problem. using a tree decomposition algorithm, both the template structure and the query sequence are represented as graphs; vertices denote core secondary structures (or simply cores) and edges represent interactions between cores (two cores are considered to be in interaction if their shortest distance is within a predefined cutoff distance). a sequence-structure alignment problem essentially corresponds to finding an isomorphic mapping from the structure graph to a subgraph of the sequence graph. the efficiency of the alignment hinges on the tree width of the structure graph. intuitively, the tree width of a graph measures how much the graph is "treelike." a graph can be represented as a "tree having thick trunks," where the "trunk thickness" is quantified by the tree width of the graph. this technique of "treelike" representation for graphs is called tree decomposition. in a tree decomposition of a graph, vertices of the graph are grouped into possibly overlapping subsets, each of which is associated with a node in the tree. the maximum size of such a subset corresponds to the tree width ofthe tree decomposition. given a tree decomposition of a structure graph with tree width t, a dynamic programming algorithm can be employed to find the optimal sequence-structure alignment in time oik: n ) , for some small integer parameter k and n being the number of amino acids in the template structure. the alignment algorithm is very efficient since the tree width for such structure graphs is small in general (by the nature of protein structures). for example, among protein tertiary structure templates compiled using pisces (wang and dunbrack, ) only . % of them have tree width t > and % have t < , when using a . -a c~-c~distance cutoff for defining pairwise interactions [see fig. . (a)]. we now provide the details of a tree-decompositionbased threading algorithm. a sequence-structure alignment can be formulated as a generalized subgraph isomorphism optimization problem, for which both the template structure and the query sequence are represented as mixed graphs that contain both directed and undirected edges. we use v(g), e(g), and a(g) to denote the vertex set, the undirected edge set, and the directed edge (arc) set of a mixed graph g, respectively. the graph h for the template structure is constructed as follows: each vertex in v(h) represents a core, each undirected edge in e(h) represents the interaction between two cores, and each directed edge (arc) in a(h) represents the loop between two consecutive cores (from the n-terminal to the c-terminal). for technical convenience, both n-and c-terminals are presented as vertices. figure . gives a protein tertiary structure and its corresponding structure graph representation. a query sequence is preprocessed so that for each core in the template, all substrings (called candidates) of the query sequence that align well with the core are identified (xu et ai., ) . by representing each candidate as a vertex, a query sequence can also be represented as a mixed graph. that is, each edge in e(g) connects a pair of candidates that may possibly interact but do not overlap in the sequence, and each arc in a( g) connects two candidates (from the n-terminal to the c-terminal) that do not overlap. as in a structure graph, both n-and c-terminals are represented as vertices in the sequence graph . figure . illustrates the sequence graph with a simple example. the relationship between a core v in the template and its candidates in the query sequence can be constrained using a mapping function m such that m(v) contains all possible candidates of v. the less restricted m is, the more accurate the alignment is expected to be and the more time it may take to compute. xu et a . ( ) used a similar approach in their divide-and-conquer threading algorithm, which can find all suitable candidates for each core. the maximum size k = m(v) lover all cores v is called the map width ofm, an important parameter for the alignment algorithm. now a sequence-structure alignment problem can be formulated as a problem offinding an isomorphism mapping f between the structure graph and a subgraph of the sequence graph g such that the following sum ofthe alignment energy functions l ecore (u, f(u) achieves the minimum, where e eore is the alignment energy score (singleton type of energy) between a core u in the template and its candidate feu) in the sequence, e pair represents interaction energy (pairwise interaction energy) between residues (f(u),f(v)) assigned to cores (u, v) , and el oop is the alignment score between the loop < u, v > in the template and the corresponding <feu), i(v» in the sequence. for a structure graph h, its tree decomposition (t, x) is defined by a tree topology t, which is a binary tree, and a collection x = {xl, x , ... , x m } , where each xi is a subset of v(h),called a bag for tree node i in t. any tree decomposition (t, x) of a graph h should satisfy the following conditions: (a) uxi = v(h); (b) for any u, v forming an (un) directed edge in the graph, u and~are contained in the same bag; and (c) the tree nodes whose bags contain the same vertex should form a connected subtree of t. intuitively, a tree decomposition groups locally connected vertices together into bags, and the set of vertices in the bag for each tree node can actually separate the graph into two disconnected components. the tree width of a tree decomposition is the maximum size ofthe bag associated with a tree node minus one. the tree width of the graph is the minimum tree width over all possible tree decompositions for the graph (robertson and seymour, ; bodlaender, ) . there are algorithms oftime (c' n) that can find an optimal tree decomposition for a graph of n vertices, provided that the graph has a tree width t. however, the constant c may be too large for the algorithm to be practically useful. for structure graphsformulated in ourcurrentcontext, wefoundthat fastheuristic algorithms may producetree decompositions with a tree widthreasonably closeto the minimum tree width. the following describes one such heuristic algorithm for tree decomposition. given a structure graph, two undirected edges (representing two interactions between cores)crosseachotherifthefourcoresinvolved interleave intheirsequential positions. tofind a tree decomposition of the graph, an edgethat crosses the greatest number of other edges is selected and removed from the graph (but the endpoints of the edge are kept). this process is repeated until the remaining graph does not contain any crossing edges. the remaining graph is called an outerplanar graph (frederickson, ) that has tree width , whose optimal tree decomposition can based on the tree decomposition for the derived outerplanar graph, a tree decomposition for the original graph can be constructed by adding u, for every removed edge (u, v) , to everybag on the shortestpath from the bag containing u to the bag containing v in the tree. forexample, fig. tree width (b) fig. . (a) tree width distribution of the structure graphs for protein tertiary structure templates compiled using pisces (wang and dunbrack, ) . (b) a tree decomposition for the structure graph given in fig. l . (b) , constructed by first removing edge ( , ), building a tree decomposition for the remaining graph, and then putting vertex (in the bold font) in node {i , , } on the path from node { , , } to { , , } . of the structure graph in fig. . (b) . in fig. . (b) the crossing edge ( , ) is first removed and a tree decomposition is constructed for the derived outerplanar graph, which is then extended to a tree decomposition for the original graph by placing vertex (in the bold font) in tree node bag {i , , } on the path from node {i , , } to node { , , }. this heuristic strategy produces a tree decomposition of size at most + c if there are c crossing edges removed. in actual applications, the obtained tree decomposition in general has a much smaller tree width than + c. once a tree decomposition (t, x) and a mapping constraint m are given, a dynamic programming can be designed to traverse the tree to find an optimal alignment between the template structure graph and a subgraph of the sequence graph g, as follows. in a bottom-up fashion, the algorithm establishes one table for each tree node i, such a table consists of t + columns, where t is number of vertices (i.e., cores) contained in the bag for the tree node. each row in the table is a combination f of candidates for the t cores constrained by m. the additional three columns store the validity, the score, and the optimality for each such combination. the computation ofthese three columns is performed by considering the cores in the present tree node bag that also occurs in its child node bags and in its parent node bag. in particular, the validity asserts that the combination f ofthe cores' candidates is legal not only for all cores in the present tree node bag xi, but also consistent with at least one valid combination in the tables of its two child nodes (if it has children). the score for each combination f in the present table mk is calculated as the sum of the following three scores: . the alignment score of the core-candidate correspondences defined by the combination f for the cores in xi only; . the maximum score over all combinations in the table ni; for the left child node k which are consistent with combination f; and . the maximum score over all combinations in the table m j for the right child node j which are consistent with combination f. the optimality column indicates if the score for each combination f is optimal over all combinations with the same candidate choices as f for the subset of cores of xi that also occur in its parent node bag. figure . illustrates the computation for a row in the table of an internal node that has two child nodes. the optimal score over all combinations computed in the table for the root of the tree t is the best alignment score. a recursive routine can be used to trace back the corresponding optimal alignment. there are three steps to accomplish the structure template-target sequence alignment. the time complexity for each step is analyzed in the following. the running time for the tree-decomposition-based structure-sequence alignment is otk' t n), where k is the maximum number of candidates of a core, t is the width of the tree decomposition, and n = iv(h)i. this is because the tree decomposition contains at most n nodes, each maintaining a table of at most o(k t ) rows. for each row in the table, the combination of core-eandidate correspondences needs to be validated and a score is computed according to the objective function given in section . . . (by looking up precomputed values of the three alignment energy functions e eor e , epair, and e loop ) ' the former step needs (t ) and the latter (t + t log~k) (note that the table for a child node can be ordered so as to facilitate binary search by the computation for its parent node). it takes o(nn) time to preprocess the target sequence of length n to construct the sequence graph. simultaneously, this step precomputes the values of alignment functions e eor e and epair' the values of function e loop can then be precomputed, where ii is the length of the segment (between two candidates of cores) to be aligned to the ith loop in the structure, and i is the total number of loops in the structure. putting together the time needed by the preprocessing, tree decomposition, and the alignment gives us a loose upper bound oik' nn), or oik' n ) , for the total time needed by the whole algorithm for the structure-sequence alignment part of the threading. based on preliminary computational results, the tree-decompositionbased threading algorithm seems to run faster than both the divide-and-conquer and the integer programming-based threading programs. protein threading is used mainly for two purposes: (a) identification of the correct structural fold or folds from a collection ofprotein structures for a query protein and (b) prediction of the backbone structure through identifying the correct assignment (or alignment) of the amino acids of a query sequence to the structural positions in the correct structural fold. in a way, protein structure prediction through protein threading could be considered as a problem to find a global optimal threading result among many sequence-structure alignments. the threading algorithms outlined in the previous section could only provide the best alignment between a query sequence and a particular template structure, i.e., a local optimum. the problem of finding a global optimum from the many local optima for the threading problem represents a highly challenging problem. the key reason is that the alignment scores between a query sequence and different template structures are in general not comparable directly with each other. some structures might tend to have higher baseline threading scores than the others, no matter what query sequences are used. we can think of this problem using the following analogy from local sequence-sequence alignment problems. suppose we have two "template" dna sequences (consisting of four different letters). we assume that one sequence is significantly longer than the other one. for simplicity of discussion, we can regard the length of the longer one to be infinite. it is not hard to convince ourselves that for a randomly selected dna sequence, its best (local) alignment with the longer sequence will have a higher probability to have a better score than with the shorter sequence. the reason is that the longer sequence, by chance, should have a higher probability to have portions of its sequence similar to the query sequence, compared to the shorter sequence. this indicates that the background alignment-score distributions for the two "template" sequences are different. hence, alignment scores against these two sequences could not be compared with each other directly in a meaningful way. similar can be said about structural templates for protein threading. to compare threading alignment scores, we need to first "normalize" the scores so that the quality ofthreading alignments against different templates could be compared with each other. for sequence-sequence alignments, there have been a number of methods developed for assessing the quality of an alignment through assessing the statistical significance of its alignment score. the p-value calculation for blast represents a popular method for such a purpose (altschul and gish, ) . developing rigorous and effective statistical models for threading has proven to be more challenging than sequence alignment problems. hence as of now, there has not been a dominating method, like the p-value calculation for blast, in estimating the statistical significance of a threading alignment score. early studies on statistical significance analysis ofthreading results were mostly empirical in nature. a representative of these studies was by bryant and co-workers (bryant and altschul, ; panchenko et ai., ) . in their model, they assume that threading scores between a template structure and a large set of sequences of the same length and with the same amino acid composition follow a gaussian distribution, and have estimated the parameters of the gaussian distribution through .calculating these threading scores. specifically, for a query sequence and a template structure, they have generated a large number of sequences with the same length and the same amino acid composition as those of the query sequence by randomly reshuffling the query sequence and have used these sequences to estimate the parameters ofthe gaussian distribution. with the estimated gaussian distribution, they then calculate the probability (p-value) that a random sequence would have a better threading score than the score between the template and the query sequence. the effectiveness of this p-value calculation was demonstrated through their successful performance at the casp contest (panchenko et ai., ) . while effective, the approach has a number of limitations which have limited its wider range of applications. these limitations include (a) its high demand for computing time as it requires solving a large number of threading problems involving the reshuffled sequences and (b) its not well justified assumption that the threading scores follow a gaussian distribution. it has been shown that the scores ofoptimal sequence-sequence and structurestructure alignments generally follow extreme-value distributions (levitt and gerstein, ) . based on the assumption that sequence-structure alignment scores also follow an extreme-value distribution, sommer et al. ( ) have recently developed another scheme for p-value estimation, in which parameters of the model are estimated by fitting the threading scores against an extreme value distribution. a key characteristic of the work is that it attempts to have a unified model for threading problems involving different lengths and compositions of the proteins involved. based on the random energy model (rem), shakhnovich and co-workers systematically studied the distribution ofthreading alignment scores (chen et ai., ; mimy et ai., ) . rem provides an adequate description ofequilibrium properties of random heteropolymers, which assumes that for a query sequence, the energy of a set ofalignments, including the "native" alignment with energy en and m decoys, followed a gaussian distribution with an average energy e av and standard devia-tion~. assisted by a null hypothesis where the optimal native sequence-template alignment should be the highest ranked, they proved that the threading scores for random sequence-structure pairs follow an extreme-value distribution. a critical energy e; was derived, which was a quantity in the rem, for characterizing the probable lower boundary ofthe density of states and it represents the most probable energy value of an optimal random sequence-structure alignment, which is determined by eq. ( . ) . they used another parameter e for the statistical significance [eq. ( . )] based on e e , corresponding to the relative energy gap for an alignment. this e score has been tested for both gapless threading and limited-gap threading results. they found that when e > , especially when e > . , there was a significant energy gap between the optimal alignment and the decoy alignments. hence, this quality could be used as a measure for assessing the significance of a threading alignment. a good method for assessing the statistical significance of a threading score should not only allow comparing threading results on the same footing but also provide a way to indicate if a particular fold is possibly the correct fold for a query sequence, without using other reference information. a typical threading program consists of four key components from the implementation perspective: (a) a database oftemplate protein structures, (b) an energy function, often residue-based, (c) a threading algorithm that can find the optimal threading alignment between a query sequence and a template structure, and (d) a method for calculating "significance" scores of threading alignments. from the prediction accuracy point of view, the larger a template structure database is, the more accurate we can expect the threading prediction will be. however, it might not always be realistic to use the whole pdb database as the template database due to the amount of time required to thread a query sequence against each pdb structure. often a template structure database consists of a representative subset of all the structures in pdb, say pdb-select, which consists of pdb structures with the "redundant" structures removed. here "redundant" refers to structures that have high sequence similarities to other structures in the database. to make prediction more accurate, some threading programs employ a two-stage strategy: (a) thread a query sequence against a representative structure database and to identify a few possible native-like structures, and (b) thread the query sequence against all family/superfamily members ofthe identified structures in (a). certain preprocessing of the template database might be needed for some threading programs. for example, as we discussed in section . , protein structure needs to be represented as structure graphs as required by the threading algorithm. the majority ofthe current threading programs employ the types ofenergy functions outlined in section . or their variations. these energy functions are statistics, rather than physics, based. they are used to distinguish correct structural folds from the incorrect ones and to distinguish accurate alignments against the correct structural folds from inaccurate alignments. threading programs have been using these simple energy forms, mainly because of the consideration of computational efficiency and also partly due to the constraints of limited available structural data for more sophisticated energy forms when such statistics-based energy functions were first developed. as those simple energies began to reach their limits, we began to see more physics-based energy functions developed, as we discussed in section . . we expect that as the threading algorithms become more efficient, we will see more physics-based energy functions. we expect that one particular type ofextension to the existing energy functions is to consider multibody interactions, which have been mostly ignored by existing threading programs. recent studies have shown that multibody interactions could help to improve the performance ofthreading programs (munson and singh, ; li and liang, ) , and hence should be considered. existing threading programs employ various algorithmic techniques for solving the sequence-structure alignment problem, including dynamic programming with enhanced heuristics (westhead et ai., ; skolnick and kihara, ; zhang et ai., ) , divide-and-conquer algorithm , and integer programming (xu and li, ; xu et ai., a,b) . in this chapter, we presented a new class of threading algorithm based on a tree decomposition of sequence and structure graphs. while integer programming might represent the most general framework for handling sequence-structure alignments, particularly so for threading problems considering multibody interactions, tree-decomposition-based algorithm could prove to be more popular down the road because of its conceptual simplicity and computational efficiency. we expect that a class of more general threading algorithms will begin to emerge to deal with more complex threading problems as the existing threading algorithms become faster and faster. this general class of threading algorithms should be able to handle simultaneous backbone threading and side-chain packing problems, leading to significantly more accurate capabilities in fold recognition and protein structure prediction. existing threading programs use various ideas and techniques to assess the "significance" of threading results. these methods include z-score calculation (sommer et ai., ) , normalized threading scores using techniques such as support vector machines or neural network (xu et ai., ; ding and dubchak, ) , and (panchenko et al., ; bryant and altschul, ) . while useful to some degree, none ofthese methods have reached the level of performance comparable to p-value calculations for blast sequence alignments (altschul and gish, ) . this is possibly due to a combination of the inadequacy of the existing threading energy functions for accurate threading prediction and the lack of general understanding about distinguishing characteristics between correct and incorrect native folds and between correct and incorrect placements of amino acids into structural positions. overall, compared to other areas of protein threading, this is a somewhat underdeveloped area. new ideas and techniques are clearly needed to fill the holes in this area. because of the importance of solving protein structures for functional studies and the power of threading techniques, many protein threading programs have been developed. using these programs, a large number of protein structures have been predicted prior to the solution of their experimental structures, providing highly useful information for guiding experimental design in investigation ofthese proteins. examples ofsuch predictions include an obese gene (madej et al., ), vitronectin (xuet al., , and a sars protein (von grotthuss et al., ) . table . provides a list of popular threading programs and urls for accessing these prediction tools. we now summarize the highlights of some of these threading programs which use different energy functions and different computational techniques, each ofwhich has its strengths and limitations. prospect kim et al., ) : the prospect program employs a divide-and-conquer algorithm for rigorously solving the global optimal threading problem, which employs a somewhat standard threading energy function, including a singleton energy term and a pairwise interaction energy term plus a secondary structure fitness score and a gap penalty score. for a typical threading problem, it can find the best alignments against a template structure database of + within a couple of days on a single cpu while it can virtually get a linear speed-up using multiple cpus when the number ofcpus is smaller than the number of structures in the template structure database. it achieves its computational efficiency by taking advantage of the fact that protein structures generally have small topological complexities (xu et ai., ) and through using a filtering procedure to filter out "improbable" alignment positions for each core secondary structure in the template structure. while this heuristic filtering works well for the vast majority of the threading cases, it might filter out the correct alignment positions for some cases. prospect normalizes the threading scores, along with various parameters of the template structure and the query sequence, using a support vector machine. then a z-score is calculated based on the "normalized" threading scores. raptor (xu and li, ; xu et ai., a,b) : the raptor program formulates a threading problem as a linear integer programming problem, and solves the problem using a branch-and-bound method plus a standard integer programming solver. raptor employs the same energy functions ofprospect and uses a similar approach for assessing the "statistical significance" of threading results to that of prospect. a unique feature of the program is that its threading algorithm is more rigorous than that of prospect as it does not use a heuristic filter to filter out "improbable" alignment positions. for a typical threading problem, it takes minutes to hours to thread the query sequence against structures in its structure database. since the program is data-parallelizable, its speed-up is virtually linear when running on multiprocessor computers. genthreader (jones, b) and an improved version mgenthreader (mcguffin and jones, ) use psi-blast profile (altschul et ai., ) and predicted secondary structures by psipred (jones, a) for threading. it employs a double dynamic programming strategy (jones et ai., ) in its threading program. the algorithm does not treat pairwise interactions rigorously but its performance has been among the top threading programs, indicating the effectiveness ofthis strategy. a web server for this program has been set up at http: //bioinfcs.ucl.ac.uklpsipred/. a user in general can expect the return of a threading prediction in minutes. the prediction program runs fast enough that it can be used for genome-scale protein structure predictions. prospector (skolnick and kihara, ) recognizes native-like structural folds using a hierarchical strategy for obtaining sequence profiles. it uses two types of sequence profiles, one type derived using close homologous sequences whose sequence identity lies between % and %, and another type constructed using more remote homologous sequences with a fasta e-value less than . both types of sequence profiles are incorporated into a typical threading energy as described in section . to screen a structural database. the program uses a dynamic programming algorithm to find the best threading alignment, and employs z-scores for assessing the significance of each threading alignment. fugue (shi et ai., ) uses a typical threading energy function as described in section . , with some unique features: ( ) its structural environment singleton term includes a term for hydrogen bonding status, and the singleton term was derived from structural alignments in the homstrad database (de bakker et ai., ; http://www-cryst.bioc.cam.ac.uk/homstrad/); ( ) its gap penalties are structure-dependent based on solvent accessibility, its position relative to the secondary structure elements, and the conservation ofthe secondary structure elements; and ( ) its alignment is based on multiple sequences against multiple structures to enrich the conservation/variation information. fugue uses dynamic programming as its threading algorithm and employs z-scores to assess the statistical significance of a threading result. since each of these threading programs has its own strengths and limitations, a popular strategy for predicting a protein structure is to use multiple prediction programs and combine their prediction results. further discussion on this topic is given in chapter . we now use prospect as an example to illustrate how to use a threading program for predicting structures of sars-co v proteins (wan et ai., ) , which playa role in the development ofthe sars disease. we used the prospect pipeline to survey all ofthe open reading frames (orfs) in sars-coy strain urbani (genbank id: ), one of the first sars-cov genomes. among the orfs, the sand m proteins playa key role in the virus infection process. interestingly, both the m protein and the s domain in the s protein are predicted to adopt the fold ofig-like beta sandwich. the structural similarity suggests that the s domain and the m protein may be evolutionarily related through gene fusion and duplication, although their sequences do not have significant similarity after a long period of evolution. the threading results might explain how the m protein interacts with the s domain, for the virus assembly: since the s domain with the fold ofig-like beta sandwich can interact with the s domain, the m protein with the same fold could possibly interact with the s domain. this suggests that the si domain may act as an on/off switch between the s domain and the m protein. such a mechanism may suggest that the m protein could also be involved in the virus-host cell interaction. this hypothesis was supported by a recent study in the murine hepatitis coronavirus study, which showed that glycosylation ofthe m protein affected the interferogenic capacity of the virus (de haan et al., ) . threading programs have been used for genome-scale applications. a recent study (guo et ai., ) performed structure prediction for all ofthe orfs ofpyrococcusfuriosus, which is found in the marine sand surrounding sulfurous volcanoes and can grow at temperatures above a e.the microbe utilizes peptides, proteins, and some carbohydrates as carbon sources. its entire genome is about mb in length with annotated orfs. out of a total of orfs, are predicted to be membrane proteins, and proteins can be predicted with structures in high confidence, among which orfs cannot be detected using psi-blast. recent prediction results in casps indicate that even when the correct structural folds are identified, the threading alignments could often be off. from the same statistics, we found that the overall alignment accuracy has not been improved over the past few casps. in addition, the alignment accuracy of the best casp models using templates with < % sequence identity ranges from % to % (venclovas et al., ) . all these statistics suggest that there is significant room for improvement in threading alignments. the prediction accuracy of the current threading programs is mainly limited by the inaccuracy of statistics-based and residue-based threading energy functions. while improving the threading energy functions represents one direction to take for improving threading performance, other approaches may also help, which include (a) application ofpartial experimental data as constraints in the threading process and (b) refinement of threaded structures using molecular dynamics and energy minimization. we refer the reader to chapter for related discussions. often partial structural data is available for specific proteins before the determination ofthe detailed structure ofa protein. these partial structural data might be in the form of (a) residue-residue distances such as disulfides between specific cysteines, (b) specific residues involved in particular active sites, binding sites, or other functionally important sites, (c) specific residues known to be on the surface ofa protein structure, or any other information providing geometric information about specific residues in a protein structure. in addition to such information about specific residues, there are experimental techniques that can be used to generate geometric information in a systematic manner. nmr represents one such technique. nmr methods solve a protein structure through generating either distance restraints [called noe, or nuclear overhauser effect, distances (prestegard, ) ] between different residues (or more specifically different atoms) or orientations of certain chemical bonds in a protein, called residual dipolar coupling (tolman et ai., ) . then a protein structure is solved through finding structural models that are consistent with the collected geometric constraints and have their energy minimized. to accurately solve a protein structure using nmr technique, it typically requires - distance restraints per residue (clore et ai., ) , which will require multiple nmr experiments. partial distance restraints could possibly be collected using fewer nmr experiments, possibly involving labeling of specific amino acid types. similar can be said about orientation information collected through residual dipolar coupling experiments. while these partial nmr data might not be sufficient for solving a protein structure accurately, they provide highly useful constraints for protein fold recognition and backbone structure prediction by a threading method. chemical cross-linking experiments provide another systematic approach to generating partial structural information for a protein. in such experiments, chemical cross-linkers, with customized arm lengths, are designed to link specific types of amino acids within a certain distance range (cohen and sternberg, ) . such experiments followed by tandem mass spectrometry experiments and data interpretation could provide distance information between certain amino acids. such an approach has been used for structural data collection for both soluble and membrane proteins (yan et ai., ) , which have then been used for protein structure prediction (young et ai., ) . one way to use such structural information, such as distances or structural locations, in a threading program is to add an energy term in the threading energy function, which measures the consistency between the collected structural data and a threading alignment. for example, if residues a and b are known to be within a certain distance, then an energy term could be specifically designed to penalize threading alignments which violate this particular geometric constraint. the energy term could be designed so that the bigger the violation, the larger the penalty. similarly, if a residue x is known to be on the surface of a protein structure, an energy term could be specifically designed for this knowledge so that it penalizes threading alignments that do not put x into a surface position in the template structure, and the amount of penalty could be designed to reflect the degree of violation of this particular knowledge. to deal with all geometric constraints, we can design a new energy term eg ,which is the sum of the individual penalty functions for all the specific geometric constraints. the overall scaling factor for this new energy term in a threading energy function could be empirically determined based on a training data set, for which actual partial experimental data is available. the effectiveness of applying such partial structural data in a threading program has been documented in a number of studies (xu et ai., b,c; young et ai., ; qu et ai., a,b) . table . shows a systematic study on improving the "query" and "temp" represent the pdb codes of the query and template proteins, respectively. "rmsd/rank vs. percentage of assigned ss" are the ca-rmsd between the experimental structure and the predicted structure using mod eller based on threading alignments for the alignable portions in the structure-structure alignment between the query protein and the template, and the rank of the correct template structure among templates. %, %, %, ... , % represent the percentage of residues with secondary structure assignment, respectively. the highlighted numbers show improvement between using no secondary structure information and using full secondary structure assignments. threading performance by incrementally increasing the number ofnmrlnoe distance constraints used in a threading process. it is seen that distance constraints help both fold recognition and threading alignment accuracy. in the best scenario, a threading program can provide an accurate prediction of the backbone atoms of a protein structure, which is still a long way from having a detailed all-atom structure. in the most general situation, a threading program could provide a somewhat accurate structure for the backbone atoms in the core secondary structures while predictions for the loop regions are often not accurate. the reason is that threading predicts a structure based on a known template structure. while the core secondary structures among homologous proteins are generally "well" conserved, loops are often not. hence, template-based loop predictions are generally not accurate. fortunately, existing methods for short loop prediction « residues) have reached a level that the predicted loops could be as accurate as the predicted core structure. for example, tile recent work by jacobson et al, ( ) has achieved prediction, accuracy of . a for -residue loops, . a for -residue loops, and . a for i l-residue loops, using an accurate all-atom energy function and hierarchical refinement protocol. potentially the predicted full backbone structure, after adding loop structures, could be refined using an energy-based approach. to do this, one needs to put all the atoms, backbone and side chains, into a structural model. one can use alignments with the selected templates in fold recognition to produce a d atomic model through homology modeling tools, such as modeller (sali and blundell, ) , which runs a protocol ofenergy minimization and molecular dynamics simulation to refine a structural model. after a structure model is generated, one can apply structure assessment tools such as whatif (vriend, ) and procheck (laskowski et ai., ) to evaluate the packing and backbone conformations, the inside/outside occupancies ofhydrophobic and hydrophilic residues, and stereochemical quality of a predicted structure. based on this assessment, a user can pick the best among the multiple structures derived from an alignment. while widely used, the potential of protein threading as a protein structure and function prediction technique is far from being fully realized. there are a number of factors that have limited its wider range of applications. first, fold recognition for structural analogues and some remote homologues is still challenging (kinch et al., ; sippl et ai., ) . such proteins might account for about % of all proteins encoded in a typical genome according to our studies (xu et ai., ; guo et ai., ) . these structures are theoretically modelable using comparative modeling techniques such as protein threading, but the predictions typically gave a low confidence level and the results may be wrong. second, even when a correct fold is identified, the accuracy ofthreading alignment has been about - % for proteins with less than % sequence identity with their template structures (venclovas et al., ) . novel ideas and new techniques are clearly needed now to make a major jump in improving the prediction capability of the existing threading methods; this has become quite clear based on the slow and incremental improvements in threading performance in the past few casp contests (venclovas et al., ) . the current energy functions are generally coarse gained mainly to achieve fast predictions. given the significant advances in computer hardware and algorithm development, it may be the time to use more sophisticated energy functions. for example, multibody interactions and more physical energy functions may help improve the threading prediction accuracy. although many theoretical studies have been carried out for threading algorithms, there is still significant room for further improving the computational efficiency of threading programs. better search methods against structural templates using advanced database techniques have not been explored thoroughly. more work can be done at the implementation level in a similar way to the implementation of blast, where many low-level operations were implemented in a highly efficient manner. in addition, algorithmic development needs to address new types of energy functions, such as new threading algorithms that could handle simultaneous backbone threading and side-chain packing to fully take advantage of more detailed energy function forms. threading algorithms that could handle multibody interactions and energy functions capturing more global properties (e.g. , compactness) ofproteins are clearly underdeveloped. existing confidence assessments are either too time-consuming in computation or not sufficiently accurate. more rigorous and faster assessment techniques for threading are clearly needed to achieve comparable performance to that of blast. assessments of different alignments using the same fold were basically not studied. furthermore, identification of"reliable" versus "unreliable" parts ofa threaded structure, and quantitative assessment of the structural deviations in terms of rmsd for regions of predicted structures have not been achieved. it has been found that using multiple fold recognition programs to build consensus of structural template is an effective way to increase the prediction accuracy (lundstrom et al., ) . furthermore, one can thread subdomain structures and use these substructures from different templates to build a new structure through a shotgun approach (fischer, (fischer, , . currently a consensus was built by a simple scheme of majority vote. much statistics can be done to do this in a more scientifically sound way. in addition, how to piece different substructures together to form a global protein structure is another challenging issue. further discussion on consensus building and subdomain threading can be found in chapter . as a structure prediction technique, threading potentially applies to at least % of all protein families. however, the application ofthreading to membrane proteins has been very limited due to the lack of available structural templates. threading techniques have been widely used for various purposes in biological studies, including (a) functional studies of proteins and experimental design (e.g., targeted mutagenesis) (madej et ai., ; xu et al., ; von grotthuss et al., ) , (b) genome annotation (xu et ai., ; mcguffin et al., ) , (c) helping solve experimental structures (ye et al., ) , (d) modeling protein complex structures (lu et al., ) , (e) prediction ofmisfolded structure (see chapter ), and (f) protein design (sorenson and head-gordon, ) . to further increase the utility of threading techniques to meet the needs for genome-scale protein structure prediction to keep up with the rate of genome sequencing and gene prediction, we clearly need a new generation of threading energy functions, threading algorithms, methods for assessing the statistical significance of threading results, and refinement of threaded structures. there are several comprehensive reviews and books on various aspects ofthreading. recent reviews related to threading include fetrow et al. ( ) and godzik ( ) . a number of books also provide some general coverage of threading and protein structure predictions (tsigelny, ; jiang et al. ; bourne and weissig, ) . for scoring function, the readers can find more information in chapters and ofthis book. for more information about general protein structure and structure-function relationship, we recommend branden and tooze ( ) and lesk ( ) . pdp: protein domain parser local alignment statistics gapped blast and psi-blast: a new generation of protein database search programs scop database in : refinements integrate structure and sequence family data domain combinations in archaeal, eubacterial and eukaryotic proteomes an insight into domain combinations linear time algorithms for np-hard problems restricted to partial k-tree the universal protein resource (uniprot) protein structure prediction and structural genomics a strategy for the rapid multiple alignment of protein sequences. confidence levels from tertiary structure comparisons flexible protein sequence patterns. a sensitive method to detect weak structural similarities a linear time algorithm for finding tree-decompositions of small treewidth structural bioinformatics a method to identify protein sequences that fold into a known three-dimensional structure introduction to protein structure fundamentals of algorithmics understanding protein structure: using scop for fold interpretation statistics of sequence-structure threading on the structural complexity ofa protein fold recognition with minimal gaps exploring the limits of precision and accuracy of protein structures determined by nuclear magnetic resonance spectroscopy on the use of chemically derived distance constraints in the prediction ofprotein structure with myoglobin as an example a unifold, mesofold, and superfold model of protein fold use homstrad: adding sequence information to structure-based alignments of homologous protein families cleavage inhibition ofthe murine coronavirus spike protein by a furin-like enzyme affects cell-eell but not virus-eell fusion smog: de novo design method based on simple, fast, and accurate free energy estimates . . methodology and supporting evidence identification of homology in protein structure classification multi-class protein fold recognition using support vector machines and neural networks the multiplicity of domains in proteins large macromolecular complexes in the protein data bank: a status report multi-domain proteins in the three kingdoms of life: orphan domains and other unassigned regions a study of combined structure/sequence profiles the protein folding problem: a biophysical enigma why do globular proteins fit the limited set of folding patterns? hybrid fold recognition: combining sequence derived properties with evolutionary information d-shotgun: a novel, cooperative, fold-recognition metapredictor protein fold recognition using sequence-derived predictions assessing the performance of fold recognition methods by means of a comprehensive benchmark assigning amino acid sequences to -dimensional protein folds planar graph decomposition and all pairs shortest paths structural genomics: bioinformatics in the driver's seat prediction of transcription regulatory sites in archaea by a comparative genomic approach can sequence determine function? a structural census ofgenomes: comparing bacterial, eukaryotic, and archaeal genomes in terms of protein structure how representative are the known structures of the proteins in a complete genome? a comprehensive structural census comparing genomes in terms ofprotein structure: surveys of a finite parts list fold recognition methods prospect-pspp: an automatic computational pipeline for protein structure prediction selection of representative protein data sets mapping the protein universe the fssp database: fold classification based on structure-structure alignment of proteins a hierarchical approach to all-atom protein loop prediction current topics in computational molecular biology protein secondary structure prediction based on position-specific scoring matrices genthreader: an efficient, and reliable protein fold recognition method for genomic sequences a new approach to protein fold recognition prospect ii: protein structure prediction program for genome-scale applications casp assessment of fold recognition target predictions the structure of the protein universe and genome evolution procheck: a program to check the stereochemical quality of protein structures the protein threading problem with sequence amino acid interaction preferences is np-complete introduction to proteinarchitecture: the structuralbiology ofproteins a unified statistical framework for sequence comparison and structure comparison emergence of preferred structures in a simple model of protein folding are protein folds atypical? designability of protein structures: a lattice-model study using the miyazawa-jernigan matrix a distance-dependent atomic knowledge-based potential for improved protein structure selection geometric cooperativity and anti-cooperativity of threebody interactions in native proteins multimeric threading-based prediction of protein-protein interactions on a genomic scale: application to the saccharomyces cerevisiae proteome protein distance constraints predicted by neural networks and probability density functions peons: a neuralnetwork-based consensus predictor that improves fold recognition threading analysis suggests that the obese gene product may be a helical cytokine comparative genomics ofthe archaea (euryarchaeota): evolution of conserved protein families, the stable core, and the variable shell how many species are there on earth improvement ofthe genthreader method for genomic fold recognition protein structure prediction by protein threading the genomic threading database: a comprehensive resource for structural annotations of the genomes from key organisms novel knowledge-based mean force potential at atomic level statistical significance of protein structure prediction by threading statistical significance of hierarchical multibody potentials based on delaunay tessellation and their application in sequence-structure alignment scop: a structural classification of proteins database for the investigation of sequences and structures protein superfamilies and domain superfolds cath-a hierarchic classification of protein domain structures a local alignment method for protein structure motifs threading with explicit models for evolutionary conservation ofstructure and sequence combination ofthreading potentials and sequence profiles improves fold recognition combinatorial optimization: algorithms and complexity new techniques in structural nmr-anisotropic interactions protein fold recognition through application of residual dipolar coupling data protein structure prediction using sparse dipolar coupling data the anatomy and taxonomy ofprotein structure graph minors . . algorithmic aspects of tree-width protein fold recognition by predictionbased threading definition of general topological equivalence in protein structures. a procedure involving comparison of properties and relationships through simulated annealing and dynamic programming an all-atom distance-dependent conditional probability discriminatory function for protein structure prediction fugue: sequence-structure homology recognition using environment-specific substitution tables and structuredependent gap penalties calculation ofconformational ensembles from potentials ofmean force. an approach to the knowledge-based prediction of local structures in globular proteins assessment of the casp fold recognition category structural genomics and its importance for gene function analysis defrosting the frozen approximation: prospec-tor: a new approach to threading confidence measures for protein fold recognition tree decomposition based protein threading redesigning the hydrophobic core of a model beta-sheet protein: destabilizing traps through a threading approach the cog database: a tool for genome-scale analysis of protein functions and evolution protein structure alignment nuclear magnetic dipole interactions in field-oriented proteins: information for structure determination in solution protein structure prediction: bioinformatic approach assessment of progress over the casp experiments mrna cap-l methyltransferase in the sars genome what if: a molecular modelling and drug design program application of computational biology in understanding emerging infectious diseases: inferring the biological function for s-m complex ofsars-co~in progress in bioinformatics pisces: a protein sequence culling server how many fold types of protein are there in nature protein fold recognition by threading: comparison of algorithms and analysis of results nucleation, rapid folding, and globular intrachain regions in proteins model for the three-dimensional structure ofvitroneetin: predictions for the multi-domain protein from threading and docking characterization of protein structure and function at genome scale with a computational prediction pipeline sequence-structure specificity of a knowledge based energy function at the secondary structure level a tree decomposition approach to protein structure prediction assessment of raptor's linear programming approach in cafasp raptor: optimal protein threading by linear programming. bioinform protein threading by linear programming a polynomial-time algorithm for a class of protein threading problems protein threading using prospect: design and evaluation a computational method for nmr-constrained protein threading protein threading by prospect: a prediction experiment in casp protein structure determination using protein threading and sparse nmr data protein domain decomposition using a graph-theoretic approach a practical method for interpretation ofthreading scores: an application of neural network an efficient computational method for globally optimal threading a graph-theoretic approach for the separation ofb and y ions in tandem mass spectra probabilistic cross-link analysis and experiment planning for high-throughput elucidation of protein structure high throughput protein fold identification by using experimental constraints derived from intramolecular cross-links and mass spectrometry similarities and differences between nonhomologous proteins with similar folds: evaluation of threading strategies estimating the number ofprotein folds scoring function for automated assessment of protein structure template quality distance-scaled, finite ideal-gas reference state improves structure-derived potentials of mean force for structure selection and stability prediction fold recognition by combining sequence profiles derived from evolution and from depth-dependent structural alignment offragments this research was sponsored in part by the u.s. department of energy's genomes to life program (www.doegenomestolife.org) under project "carbon sequestration in synechococcus sp.: from molecular machines to hierarchical modeling" (www genomesllife.orgj. yxandzjl'sworkwasalsosupportedinpartbynsfidbi- , nsf/itr-iis- , and a "distinguished cancer scholar" grant from the georgia cancer coalition. dx's work was also partially funded by nsf/eia- . key: cord- - cdi w authors: györkey, ferenc; sinkovics, joseph g.; györkey, phyllis title: electron microscopic observations on structures resembling myxovirus in human sarcomas date: - - journal: cancer doi: . / - ( ) : < ::aid-cncr > . .co; - sha: doc_id: cord_uid: cdi w human tumors of mesenchymal origin contain cytoplasmic structures resembling ribonucleoprotein strands of paramyxoviruses. similar structures have previously been reported in collagen diseases. the nature and function of these structures remain unresolved. h u m a n tumors of mesenchymal origin contain cytoplasmic structures resembling ribonucleoprotein strands of paramyxoviruses. similar structures have previously been reported in collagen diseases. the nature and function of these structures remain unresolved. t duced tumors of animals and naturally occurring tumors of man lacks valid criteria. one discrepancy concerns the viral etiology of animal and human malignant neoplasms. in certain animal tumors, such as murine leukemia, sarcoma, and mammary carcinoma, rna-containing type c and type b virus particles occur in large numbers. bioassays have irrefutably proved that these virus particles are the causative agents of these tumors. . , i n some other types of experimental tumors, no mature virions appear but there is oncogenic viral dna incorporated into the genome of the host cell. not only dna-but also rna-containing viruses may exist in the neoplastic cells in the form of subviral structures.lg cell-fusion, heterokaryon formation, and "rescue" by a helper virus may be needed for the maturation of the subviral structures into complete ~irions. . ~ most human tumors when examined in the electron microscope or tested in bioassays appear to be devoid of mature virions. extensive search is required to find a few elusive viruslike particles in human tumors. herpes, adeno-and reoviruses, as well as elementary bodies of mycoplasma, occasionally occur in human tumors probably as passengers and not -~ as etiologically important agents. ~ ~ occasionally, type c and related virus-like particles were found in neoplastic tissues deriving from human leukemia, lymphoma, and sarcoma. j t h e number of these particles rapidly diminishes in cells cultured in vitro; thus, these viruslike particles have never been identified as the human counterparts of oncogenic type c viruses of animals. on the contrary, culturing in vitro, particularly of lymphoid cells deriving from human lymphoma and from other lymphoproliferative entities, results in the rapid increase in the amount of a herpes-type ( e p stein-barr) virus. very little attention has been paid in the past to the existence of subviral structures in tumor cells of man. preliminary findings concerning such structures, however, have recently been reported by stewart who observed the occurrence of filamentous structures and budding type clike virus particles in tissue cultures of human liposarcoma and hodgkin's disease. i n the latter case, these structures were identified later as ribonucleoprotein strands of simian paramyxovirus . lymphoid cells grown in culture from patients with infectious mononucleosis or from idiopathic thrombocytopenic purpura occasionally contain lattice-like arrangements of short strands which, in cross section, appear as aggregates of small virus-like particles. the viral nature of these latter structures have been questioned; the structures were presented also as cellular elements. . thus, merely morphological studies failed to determine in most instances whether the filamentous structures represented viral nucleoprotein strands or cellular material. if the structures are of viral derivation, it remains cancer julie voi. unresolved whether these structures are components of a passenger virus or of a virus etiologically related to the induction of mesenchymal tumors of man. thus, the need for a complex study has become evident. such a study should deal with the occurrence and morphological features of these hypothetically subviral structures, and it should encompass attempts at isolation and identification of these structures as well as bioassays to establish whether the structures possess any oncogenic potency or not. t h e present paper is a preliminary report on morphological observations concerning structures that may be of viral derivation, as found in biopsies and tissue cultures of human tumors of mesenchymal origin. specimens of tumors: twelve tumor biopsies ( rhabdomyosarcomas, liposarcomas, chondrosarcomas, osteosarcoma, fibrosarcoma, neurofibrosarcoma, and kaposi's sarcoma) and primary tissue cultures o€ tumors ( rhabdomyosarcomas, chondrosarcomas, fibrosarcoma, and osteosarcoma) were examined electron microscopically. electron microjcopy: finely minced freshly biopsied tumor tissue or cultured cells dispersed by trypsinization or scraping were placed immediately in glutaralclehyde, postfixed in osmic acid, dehydrated in graded series of ethanol, embedded in epon araldite, cut with an lkb ultratome, stained with ur-any acetate followed by lead citrate, and examined in an rca g electron microscope. tissue cziztures: finely minced fresh tumor biopsy material was placed in glass t flasks under perforated cellophane membrane antl incubated in ham's fio medium containing % heat inactivated fetal calf serum, penicillin g ? u/ml, streptomycin pg/nil, antl lanamycin pg/ml. cultured cells were t h e discovery of the structures required extensive search; several sections of each specimens and several liiintlretls of fields were examined. t h e distribution of the structures was found to be quite irregular; in some sections, it was easy to find the structures, whereas other sections of the same specimen were devoid of them. small clusters of mature virions were seen only exceptionally in the specimens examined (fig. ) ; these findings will be reported elsewhere after lurtlier studies. normal kidney, skin, and leukocytes were examined for the presence of similar structures. extensive search involving a total of tissue specimens yielded negative results. morphological description of the stnrctures: t h e filamentous and tubular structures are located in the cytoplasm in aggregates of varying sizes. t h e aggregates may be membranebound. in the case of the kaposi's sarcoma, the structures were found between the nuclear membranes. t h e filaments measure - a in diameter and u p to a in length. in cross sections, the curved filaments appear as both electron-dense or lucent round bodies (figs. - ) . further studies are needed to determine the nature and significance of these structures. it is uncertain whether the structures as found in different tumors are of the same type. while some of these structures closely resemble unenveloped ribonucleoprotein strands of paramyxovir~i~es.~~ other slightly different, i.e., smaller, structures, particularly those appear-ing in cultured lymphoblasts,'s are thought to represent cellular material. jag during the replication of parainfluenza virus type in hela cells, cytoplasmic areas containing a dense network of filamentous structures without mature virions were seen. i t was postulated that the viral capsid is acquired when the viral nucleoprotein strands are assembled beneath the cell membrane and the virus particles pass through the cell membrane by a budding process.*q i t is compatible with this view that the filamentous structures described in this report occur at cytoplasmic sites where replication of viral nucleoprotein takes place. myxoviruses and type c oncorna viruses display morphological resemblances, thus some of the structures described herein may represent ribonucleoprotein strands of a human oncogenic virus of the type c or related class. type c viruses have recently been incriminated in the causation of human sarcomas. in tumors caused by rous sarcoma virus in primates, crystalline and filamentous cytoplasmic structures somewhat similar to those reported i n this paper have been found. t h e occurrence of quite similar structures in collagen diseases of man, particularly, as first shown by us, in systemic lupus erythematosus, , *, polymyositis, ~' sjogren's syndrome,:{o and discoid lupus, indicates that these structures are not confined solely to tumors. t h e ribonucleoprotein composition of tlie filmientous strands found in lupus was shown by digestion with rnaase. isolation of vii ' agents failed from biopsy materials containing the filamentous structures. thus, it was aljo proposed that similar strands represented cellular and not viral elements."sj'. even if these structures are of viral derivation, a we ha proposed, * a it remains to be determiiied whether they play the role of a passenger or of an initiator in collagen diseases arid in mesenchymal tumors. members a f the orthomyxo-and paramyxovirus groups may elicit or aggravate "autoimmune" reactions by endowing the host cells, in which viral components replicate with "neoantigenic" features; thus, subviral structures may be of great etiological significance in collagen diseases. paraniyxovirus and coronavirus-like structures have recently been found associated with viral hepatitis,l , *, but it remains to be determined whether these structures play the etiologic role or act only as passengers. autoimmune sequelae of viral hepatitis are well known; tlie harboring of subviral structures within parenchymal liver cells could explain snch "autoimmune" reactions. t h e structures described in this paper may represent unenvelopecl ribonucleoprotein strands of an ubiquitous virus which is widely spread in human populations and seldom reaches full maturity. it is evident that further morphological observations, in addition to virologic, serologic, and biologic studies, are needed in order to elucidate the significance of these filamentous structures in mesenchy-ma tumors of man. further isolations of reovirus type from cases of burkitt's lymphoma. brit. alerl. j . . chandra, s.: tlndulating tubules associated with endoplasmic reticullnu in pathologic tissue. lab. i nvest similarity between leukocyte cultures from cancerous and noncancerous human subjects: a n electron niicroscopic study t h e circulating lymphocyte. its role in infectious mononucleosis myxovirus-like structures accompanying nuclear changes in chronic polymyositis tubular structures associated with the endothelial endoplasmic reticulum in glomerular capillaries of rhesus monkey and nephritic man t h e mammary tumor system in mice, a brief review. i n viruses inducing cancer viral studies in human leukemia and lymphoma. i n proceedings of international conference on leukemia-lymphoma viral isolation studies of inclusion positive biopsy from human connective tissue diseases pathogenic effects of extracts of human osteosarcomas i n hamsters and mice systemic lupus erythernatosus antl myxovi- ' s discoid iupns crgthematosus. electron microscopic studies of paramyxovirns-like structures coronaviiuscs and viral hepatitis oncogenes of rna tumor viruses a s determinants of cancer cytoplasmic tubular sturctures in kidney biopics in systemic lupns erythematosus. arthritis xlreuin virus-like stuctures in lupus erythematosus electron mi-cro copic examination of cytoplasniic inclusion bodies in cells infected with parainflnenza virus type . viral- tumors intlnced in juvenile and adult primates by chicken sarcoma virus evidence for the in vitro transfer of defective rons sarcoma virus genome from hamster tumor cells to chick cells virus-like structures in sjogren's syndroms. i.nncet : working hypothesis: viral etiology of autoimmune diseases a comparison of murine and human leukemia a rapidly fatal case o'f systemic lupus erytheniatosus: structnres resemhling viral nncleoprotein strands in the kidney and activities of lymphocytes i n culture leukemogcnesis stemming from autoimmune disease. i n genetic concepts and neoplasia, t h e twenty-third annual symposium on fundamental cancer research, , t h e ljniversity of texas m. d. anderson hospital and l'nmor institute at houston search for viruses in childhood leukemia and rhabdomyosarcoma. i n leukemia in animals antl man t h e isolation of myxo type virus from two h u n~a n sarcomas viruses in human tumors. i. hodgkin's disease virus like particles in liver disease patients' sera. presentation a t the international congress of infectious diseases presence of particles other than the australia-sh antigen in a case of chronic active hepatitis with cirrhosis key: cord- - lnpujip authors: anthonsen, henrik w.; baptista, antónio; drabløs, finn; martel, paulo; petersen, steffen b. title: the blind watchmaker and rational protein engineering date: - - journal: j biotechnol doi: . / - ( ) -x sha: doc_id: cord_uid: lnpujip in the present review some scientific areas of key importance for protein engineering are discussed, such as problems involved in deducting protein sequence from dna sequence (due to posttranscriptional editing, splicing and posttranslational modifications), modelling of protein structures by homology, nmr of large proteins (including probing the molecular surface with relaxation agents), simulation of protein structures by molecular dynamics and simulation of electrostatic effects in proteins (including ph-dependent effects). it is argued that all of these areas could be of key importance in most protein engineering projects, because they give access to increased and often unique information. in the last part of the review some potential areas for future applications of protein engineering approaches are discussed, such as non-conventional media, de novo design and nanotechnology. nature has evolved using several types of random mutations in the genetic material as a fundamental mechanism, thereby creating new versions of existing proteins. by natural selection nature has given a preference to organisms with proteins which directly or indirectly made them better adapted to their environment. thus nature works like a blind watchmaker, trying out an endless number of combinations. this may seem to be an inefficient approach by industrial standards, but nevertheless nature has been able to develop some highly complex and sophisticated designs, simply by the power of natural selection over millions of years, occurring in a large number of parallel processes. by virtue of reproduction several copies of each organism have been able to test the effect of different mutations in parallel. it is quite probable that the mutation frequency was higher in ancient species (doolittle, ) , although it is still possible to find highly mutable loci in genes involved in adaptation to the environment (moxon et al., ) . enzymes have been used by man for thousands of years for modification of biological molecules. the use of rennin (chymosin) in rennet for cheese production is a relevant example. and with increased knowledge about proteins, genes and other biological macromolecules scientists started starting with a protein with known sequence and properties, we make a -d model of the protein from experimental structure data or by homology. by modelling and simulation we identify mutations that will modify selected properties of the protein (the design part of the process), these mutations are implemented at the dna level and expressed in a suitable organism (the production part of the process), and the success of the design is verified by experimental methods. to look at methods for making modified proteins with new or improved properties. at first this was done by speeding up nature's own approach, by increasing the number of mutations (e.g., by using chemicals or radiation) and by using a very strong selection based on tests for specific properties. with the introduction of new and powerful techniques for structure determination and site directed mutagenesis, it is now possible to do rational protein modification. rather than testing out a large number of random mutations, it has become feasible to identify key residues within the protein structure, to predict the effect of changing these residues, to implement these changes in the genetic material, and finally to produce large amounts of modified proteins. this is protein engineering. there are several reviews describing the fundamental ideas in protein engineering, see fersht and winter ( ) for a recent one. the basic protein engineering process is shown in fig. (see also petersen and martel ( ) ). in most cases it starts out with an unmodified protein with well-characterised properties. for some reason we want to modify this protein. in the case of an enzyme we may want to make it more stable, alter the specificity or increase the catalytic activity. first we enter the design part of the protein engineering process. based on structural data we create a computer model of the protein. by a combination of molecular modelling and experimental methods the correlation between relevant properties and structural features is established, and changes affecting these properties can be identified and evaluated for implementation. in more and more cases the effect of these changes can be simulated, and the modifications can be optimised with respect to these simulations. as soon as a new design has been es~tablished we may enter the production part of the process. the necessary mutations must be implemented in the genetic material, this genetic material is introduced into a production organism, and the resulting modified protein can (in most cases) be extracted from a bioprocess. this protein can be tested with respect to relevant properties, and if necessary it may be used as a basis for re-entering the design part of the protein engineering process. after a few iterations we may reach an optimal design. there are several examples of successful protein engineering projects. protein engineering may be used to improve protein stability (kaarsholm et al., ) , enhance or modify specificity (getzoff et al., ; witkowski et al., ) , adapt proteins to new environments (arnold, ; gupta, ) , or to engineer novel regulation into enzymes (higaki et al., ) . in some cases even de novo design of new proteins may be relevant, using knowledge gained from existing structures (kamtekar et al., ; shakhnovich and gutin, ; ghadiri et al., ; ball, ) . in a truly multidisciplinary project chymosin mutants with optimal activity at increased ph values compared to wild-type chymosin was designed and produced (pitts et al., ) . point mutations changing the charge distribution of superoxide dismutase have been used to increase reaction rate by improved electrostatic guidance (getzoff et al., ) . a project on converting trypsin into chymotrypsin has been important for understanding the role of chymotrypsin surface loops (hedstrom et al., ) , a serine active site hydrolase has been converted into a transferase by point mutations (witkowski et al., ) , and mutations in insulin aiming at increased folding stability have given an insulin with enhanced biological activity (kaarsholm et al., ) . an example of a rational de novo project (as opposed to the random approach used, e.g., in generation of catalytic antibodies) is the design of an enzymatic peptide catalysing the decarboxylation of oxaloacetate via an imine intermediate, in which a very simple design gave a three to four orders of magnitude faster formation of imine compared to simple amine catalysts . in some cases it may also be an interesting approach to incorporate nonpeptidic residues into otherwise normal proteins (baca et al., ) , or to build de novo proteins by assembling peptidic building blocks on to a nonpeptidic template (tuchscherer et al., ) . it has been shown that incorporation of nonpeptidic residues into e-turns of hiv- protease gives a more stable enzyme (baca et al., ) . the main problem with this approach is how to incorporate the non-standard residues. in the hiv- protease case solid-phase peptide synthesis combined with traditional organic synthesis was used, others have suggested that the degeneracy of the genetic code may be used to incorporate novel residues via the standard protein synthesis machinery of the cell (fahy, ) . in the present review we will look at the design part of the protein engineering process, with emphasis on some of the more difficult steps, especially homology based modelling in cases with very low sequence similarity, nuclear magnetic resonance (nmr) of very large proteins and modelling of electrostatic interactions. in the last part of the review we will discuss some possible future directions for protein engineering and protein design. any protein engineering project is based on information about the protein sequence. this information may stem from either direct protein sequencing or a deducted translation of the dna/rna sequence. the amount of information on protein and nucleic acid sequences, as well as on relevant data like -d structures and disease-related mutations, is growing at a very rapid pace, and novel databases and computer tools give increased access to these data (coulson, ) . it is very reasonable to expect that projects like the human genome project will succeed in providing us with sequence information about every single gene in our chromosomes within the next decade. this information will be after transcription, the mrna may be edited, a process that now has been reported in man, plants and primitive organisms (trypanosoma brucei). the mrna is then translated into a protein sequence. this protein sequence can subsequently be modified, leading to n-or o-glycosylation, phosphorylation, sulfation or the covalent attachment of fatty acid moieties to the protein. at this stage the protein is ready for transport to its final destination -which may be right where it is at the time of synthesis, but the destination may also be extracellular or in secluded compartments such as the mitochondria or lysosomes. in this case the protein is equipped with a signal sequence. after arrival to its destination the protein is processed, often involving proteolytic cleavage of the signal sequence. shorter routes to the functional protein with fewer steps undoubtedly exist as well as routes with interchanged steps of processing. finally, the catabolism of the protein is also part of the process, but has been left out in the figure. of key importance for our understanding of the biology, development and evolution of man. it should, however, be kept in mind that the sequence itself may give us little information about regulation of gene expression, i.e., under what conditions genes are expressed, if they are expressed at all. most protein sequences have been deducted from gene sequences. it is in most cases a priori assumed that a trivial mapping exists between the two sets of information. however, this may not necessarily be the case. in fig. , the various steps currently recognised as being of importance for the production of the mature enzyme are shown, and several of these steps may affect the mapping from gene to protein. posttranscriptional editing is modifications at the mrna level affecting the mapping of information from gene to protein, often involving modification, insertion or deletion of individual nucleotides at specific positions (cattaneo, ) . currently only speculative models exist for the underlying molecular mechanism(s) for posttranscriptional editing. in the case of mammalian apolipoprotein b two forms exist, both originating from a single gene. the shorter form, apo b , arises by a posttranscriptional mrna editing whereby cytidine deamination produces an uaa termination codon (teng et al., ) . in the ampa receptor subunit glur-b mrna editing is responsible for changing a glutamine codon (cag) into a arginine codon (cgg) (higuchi et al., ) . this editing has a pronounced effect on the ca + permeability of the ampa receptor channel, and it seems to be controlled by the intron-exon structure of the rna. similar mrna editing has been reported in the related kainate receptor subunits giur- and giur- , where two additional codons in the first trans-membrane region are altered (sommer et al., ; k hler et al., ) . it is also interesting in this context that certain human genetic diseases have been related to reiteration of the codon cag (green, ) . mrna editing in plant mitochondria and chloroplasts has also been reported (gray and covello, ) . here the posttranscriptional mrna editing consists almost exclusively of c to u substitutions. editing occurs predominantly inside coding regions, mostly at isolated c residues, and usually at the first or second position of the codons, thus almost always changing the amino acid compared to that specified by the unedited codon. in trypanosoma brucei some extensive and well-documented posttranscriptional cases of editing have been reported (read et al., ; harris et al., ; adler et al., ) . the editing takes place at the mitochondrial transcript level where a large number of uridine nucleic acid bases are added or deleted from the mrna, which then subsequently is translated. several non-editing processes affecting the transcription/translation steps are also known. although the ribosomes in an almost perfect manner translate the message provided by the mrna (with error rates less than x - per amino acid incorporated), it appears as if the mrna in certain cases contain information, that forces the ribosome to read the nucleic acid information in a non-canonical fashion (farabaugh, ) . a special case, that may deserve some attention as well, is the seleno proteins, were seleno cystein is introduced into the protein by an alternative interpretation of selected codons (b ck et al., ; yoshimura et al., ; farabaugh, ) . translational frameshifting has been found in retroviruses, coronaviruses, transposons and a prokaryotic gene, leading to different translations of the same gene. two cases of translational 'hops' have been reported, where a segment of the mrna is being skipped by all ribosomes, in the two cases and nucleotides were skipped, respectively (farabaugh, ) . to our knowledge posttranscriptional editing and related processes are uncommon but definitely present in humans. it is, therefore, important to understand precisely how these mechanisms work, in order to correctly deduct the protein sequence from the gene sequence. the most common posttranslational modifications are side chain modifications like phosphorylations, glycosylations and farnesylations, as well as others. however, some modifications may also affect the (apparent) gene to protein mapping. posttranslational processing may involve removal of both terminal and internal protein sequence fragments. in the latter case an internal protein region is removed from a protein precursor, and the external domains are joined to form a mature protein (hodges et al., ; xu et al., ) . interestingly, all intervening protein sequences reported so far have sequence similarity to homing endonucleases (doolittle, ) , which also can be found in coding regions of group i introns (grivell, ) . posttranslational modifications like phosphorylation, glycosylation, sulfation, methylation, farnesylation, prenylation, myristylation and hydroxylation should also be considered in this context. they modify properties of individual residues and of the protein, and may thus make surface prediction, dynamics simulations and structural modelling in general more complex. the residues that are specifically prone to such modifications are tyrosines (phosphorylation and sulfation), serine and threonine (o-glycosylation), asparagine (nglycosylation), proline and lysine (hydroxylation) and lysine (methylation). in addition glutamic acid residues can become y-carboxylated leading to high affinity towards calcium ions (alberts, ) . specific transferases are involved in the modification, e.g., tyrosylprotein sulfotransferases (suiko et al., ) and farnesyl-protein transferases (omer et al., ) . phosphorylation of amino acid residues is an important way of controlling the enzymatic function of key enzymes in the metabolic and signalling pathways. tyrosine kinases phosphorylate tyrosine residues -thus introducing an electrostatic charge at a residue, which under normal physiological ph is uncharged. phosphorylation is central to the function of many receptors, such as the insulin and insulin-like growth factor i receptors. given the possibility that several modifications may be introduced in the sequence when we move from gene to mature protein, the task of deducting a protein sequence from the gene sequence may be more complex than we normally assume. although the protein sequence itself is a valuable starting point, the optimal basis for a rational protein engineering project will be a full structure determination of the protein. in many cases this turns out to be an expensive and timeconsuming part of the project. most structure determinations are based on x-ray crystallography. this approach may give structures of atomic resolution, but is limited by the fact that stable high quality crystals are needed. many proteins are very difficult to crystallise, in particular many structural and membrane-associated proteins. a large number of important x-ray structures have been published over the last few years, and the structures of the hhai methylase (klimasauskas et al., ) , the tbp/tata-box complex (kim et al., a; kim et al., b) and the porcine ribonuclease inhibitor (kobe and deisenhofer, ) are mentioned as examples only. nmr may be an alternative in many cases, as the proteins can be studied in solution, and for some experiments they can even be membrane associated. however, nmr is limited to relatively small molecules, and even with incorporation of labelling in the protein the upper limit for a full structure determination using current state of the art methods seems to be close to kda. some novel techniques for studying structural aspects of larger proteins will be discussed (vide infra). representative examples of important nmr structures may be interleukin / (clore et al., a) , the glucose permease iia domain (fairbrother et al., ) and the human retionic acid receptor-/ dna-binding domain (knegtel et al., ) . cryo electron microscopy (cem) is a relatively new approach to protein structure determination. the resolution of the structures are still lower than the corresponding x-ray structures, and a -dimensional crystal is a prerequisite. however, despite this cem appears to be a very promising approach to structure determination of membrane associated proteins that can form -dimensional crystals. cem has been used to study the nicotinic acetylcholine receptor at .~ resolution (unwin, ) and the atp-driven calcium pump at a resolution (toyoshima et al., ) , and in a combined approach using high resolution x-ray data superimposed on cem data the structure of the actin-myosin complex (rayment et al., ) and of the adenovirus capsid (stewart et al., ) has been studied. the recent structure by kiihlbrandt et al. ( ) of the chlorophyll a/b-protein complex at . a resolution shows that the resolution of cem rapidly is approaching the resolution of most x-ray protein data. scanning tunnelling microscopy (stm) is another new approach for studying protein structures (amrein and gross, ; lewerenz et al., ; haggerty and lenhoff, ) . the method is interesting because of a very high sensitivity, as individual molecules may be examined. the method will give a representation of the surface of the molecule, rather than a full structure determination. however, it is possible that both cem and stm can be used for identification of protein similarity. if data from these methods show that the overall shape of a protein is similar to some other known high resolution protein structure, then the known structure may be evaluated as a potential template for homology based modelling. we believe that such a model can either be used as an improved starting point for a full structure determination (i.e., for doing molecular replacement on x-ray data), or as a low resolution structure determination by itself. in homology based modelling a known structure is used as a template for modelling the structure of an homologous sequence, based on the assumption that the structures are similar. this is a very simple and rapid process, compared to a full structure determination. the sequences may be homologous in the strict sense, meaning that there is an evolutionary relationship between protein data banks the sequences. the same approach may obviously also be used for sequences that are similar, but not necessarily evolutionary related, and in that case we probably should talk about similarity based modelling. however, in this paper we will use homology based modelling as a general term, especially since the distinction between homology and similarity may be difficult in many cases. homology based modelling may turn out to be essential for the future of protein engineering. in fig. , the number of entries in the swissprot protein sequence database (bairoch and boeckmann, ) and the brookhaven protein structure database (bernstein et al., ; abola et al., ) are shown as a function of time. as we can see, there is a very significant gap between the number of sequences and the number of structures. this gap is in fact even larger than shown in fig. , as not all entries in the brookhaven database are unique structures. a large number of entries are mutants of other structures or identical proteins with different substrates or inhibitors. there has been an exceptional growth in the number of protein structures over the last - years. however, it is unrealistic to assume that we will be able to get high resolution experimental structures of all known proteins. the structure determination process is too time consuming, and the sequence databases are growing at a far faster pace, as shown in fig. , especially as a consequence of several large-scale genome sequencing projects. on the other hand, it may not really be necessary to do experimental structure determination of all proteins (ring and cohen, ) . the assumption that similar sequences have similar structures (see fig. ) has been proved valid several times and it seems to be true even for short peptide sequences as long as they come from proteins within the same general folding class ). an interesting case which is to some degree an exception to this rule is the structure of hiv- reverse transcriptase (kohlstaedt et al., ) . two units with identical sequence have similar secondary structure, but very different tertiary structure. however, this seems to be a rather exceptional case. new approaches to general structure alignment (orengo structure distance sequence distance fig. . sequence and structure similarity. in most cases similar sequences have similar structures (region ), and dissimilar sequences (i.e., measured by a standard mutation matrix) have dissimilar structures (region ). in several cases quite dissimilar sequences have been shown to have very similar structures, at least with respect to individual domains (region ). in very special cases we may have similar sequences with different structures (region ), at least with respect to tertiary structure, showing that environment and binding to other proteins may be essential for the final conformation in some cases. however, in most cases it seems to be safe to assume that structures can be found in the lower grey triangle of this graph, indicating that structure is better conserved than sequence. holm et al., ; alexandrov and go, ; lessel and schomburg, ) have made it possible to search for structurally conserved domains in proteins with very low sequence similarity (swindells, ) . this is an important approach, as structure normally is better conserved than sequence (doolittle, ) . several cases have been identified where the sequences are very different (at least by traditional similarity measures), whereas the three-dimensional structures are surprisingly similar. the identification of a globin fold in a bacterial toxin (holm and sander, ) , and the similarity between the dsba protein and thioredoxin (martin et al., ) are relevant examples. recently the structure of the human serum amyloid p component was shown to be similar to concanavalin a and pea lectin, despite only % sequence identity (emsley et al., ) , and the similarity between hen egg-white lysozyme and a lysozyme-like domain in bacterial muramidase "is remarkable in view of the absence of any significant sequence homology", as noted by thunnissen et al. ( ) . this shows that there probably is a limited number of protein folds, and this number must be lower than the number of sequence classes, defined as groups of similar protein sequences. recent estimates show that this number probably is close to different protein folds (chothia, ) , and approx. of these folds are known so far (burley, ; orengo et al., ) . this means that rather than full structure determination of a very large number of proteins, it may be sufficient to do structure determination of only a few selected examples of each protein fold, and use this as a basis for homology based modelling of other proteins shown to have the same fold. homology based modelling of the -d structure of a novel sequence can be divided into several steps. first, one or more templates must be identified, defined as known protein structures assumed to have the same fold as the trial sequence. then a sequence alignment between trial sequence and template is defined, and based upon this alignment an initial trial model can be built. this initial model must be refined in several steps, taking care of gap splicing, loops, side chain packing etc. the final model can be evaluated by several quality criteria for protein structures. an example of homology based modelling is the modelling of cinnamyl alcohol dehydrogenase based on the structure of alcohol dehydrogenase (mckie et al., ) . the protein folding problem is a fundamental problem in structural biology. this problem can be defined as the ab initio computation of a protein's tertiary structure starting from the protein sequence. this problem has not been solved and appears to be extremely difficult. if we want to solve the problem by computing an energy term for all conformations of a protein, defined by rotation around the ~b and ~o backbone angles of n residues in degree steps, we have to evaluate (n-d alternatives, even without considering the side chains. for a peptide with residues this corresponds to conformations. a hypothetical computer with processors, each processor running at hz (the frequency of uv light) and completing the energy evaluation of one conformation per cycle would need x years in order to test all conformations. the estimated age of the universe is x ~ years. a more realistic approach is the use of molecular dynamics or monte carlo methods for simulation of protein folding. however, it is still very difficult to use this as an ab initio approach, both because folding is a very slow process compared to a realistic simulation time scale, and also because it is very difficult to distinguish between correctly and incorrectly folded structures using standard molecular mechanics force fields (novotny et al., ) . a possible alternative approach may be to generate potential folds on a simplified lattice representation of possible residue positions (covell and jernigan, ; crippen, ) . however, this approach is still very experimental. some progress has been achieved in the area of secondary (rather than tertiary) structure prediction (benner and gerloff, ) . studies of local information content indicate that % match may be an upper limit for single-sequence prediction methods (rao et al., ) , whereas methods taking homology data into account may probably raise this limit to approx. %. methods based on neural networks and combinations of several prediction schemes seem to give good predictions, and especially methods using homology data from multiple alignments may give predictions at % match or better in many cases (salzberg and cost, ; boscott et al., ; rost and sander, a; rost et al., ; levin et al., ) . also methods taking potential residue-residue interactions into account, like the hydrophobic cluster analysis (hca), may be used for identification of potential secondary structure elements (woodcock et al., ) . it has been shown that by restricting the prediction to a consensus region with stable conformation it is possible to make very reliable predictions (rooman and wodak, ) . in one case, neural networks were shown to be capable of returning a limited amount of information on the tertiary structure (bohr et al., ) . . structure retrieval by secondary structure. a flow chart for structure retrieval by secondary structure (right side) compared to retrieval by sequence (left side). please see the text for details. in this example the secondary structure library was generated using dssp (kabsch and sander, ) , the secondary structure was predicted with the phd program (rest et al., ), and fasta (pearson, ; pearson and lipman, ) was used to search the secondary structure library and the nrl- -d databases (namboodiri et al., ; george et al., ) . only the secondary structure based method was able to identify the hla class i structure as similar to the class ii structure. the ribbon representation of the hla class i antigen binding region used in this figure was generated with molscript (kraulis, ) . be inconsistent, compared to the more sophisticated classification which can be achieved by a trained expert. recent studies show that the average agreement between alternative assignment methods used on identical structures is close to % for three methods (colloc'h et al., ) , or % if only two methods are compared (woodcock et al., ) . vadar is a new classification method which is aiming at a better agreement between manual and automatic assignment (wishart et al., ) , to what degree this may have influence on prediction systems remains to be seen. over the last few years it has been realised that the inverse folding problem is much easier to solve (bowie et al., ; blundell and johnson, ; bowie and eisenberg, ) . the inverse folding problem can be defined as follows: given a known protein structure, identify all protein sequences which can be assumed to fold in the same way. a large number of protein structures must be available in order to use this as a general approach, as the relevant protein fold has to be represented in the database in order to be identified. however, with a limited number of possible folds actually used by nature, a complete database of all folds appears to be possible. some information about possible folding classes can be derived from experimental data. circular dichroism can be used as a crude way of measuring the relative amounts of secondary structure in a protein. classification methods based on amino acid composition can be used for classification of proteins into broad structural classes zhou et al., ; chou and zhang, ; dubchak et ai., ) . this information may limit the number of different folds which have to be evaluated. it is also possible that such information may be used to improve the performance of other methods, although data on secondary structure prediction of all-helical proteins seems to indicate that the gain may be small (rost and sander, b) . however, for a unique identification of folding class more sensitive methods are needed, and the most useful one is probably some kind of protein sequence library search. in order to identify the folding class we have to search a database of known protein structures with our trial sequence. the problem is that standard methods for sequence retrieval may not be sensitive enough in all cases. if the sequences are similar, then retrieval is trivial. however, we know that there are cases where structures are known to be similar despite very different sequences. how can these cases be identified in a reliable way? the most promising approaches are based on methods for describing the environment of each residue (bowie et al., ; eisenberg et al., ; overington et al., ; ouzounis et al., ; wilmanns and eisenberg, ; liithy et al., ) . this description can be used for generating a profile, showing to what degree each residue is found in a similar environment in other structures, and this profile can be used as a basis for sequence alignment and library searches. similar property profiles can also be used for searching database systems of protein structures (vriend et al., ) . a very simple approach can be used if we accept the hypothesis that protein sequences representing structures with a similar linear distribution of secondary structure elements may fold in a similar way. we can then create a sequence type library of known structures where the residues are coded by secondary structure codes rather than residue codes (see fig. ). given the sequence of a protein with unknown -d structure, we can use a secondary structure prediction method and translate the sequence into a secondary structure description. if we define a suitable 'mutation' matrix describing the probability of inter conversion between different secondary structure elements, then a standard library search program like fasta (pearson and lipman, ; pearson, ) can be used in order to identify potential template structures. the example shown in fig. is the identification of hla class i as a suitable candidate for homology modelling of hla class ii. the sequence similarity is very low, % sequence identity in the antigen binding region (based on alignment of the structures), and especially for this region most sequence based methods will retrieve a large number of alternative sequences before any of the class i molecules. bly improve the performance to use a positiondependent gap penalty, where most gaps are placed in loop regions rather than in helices or strands. however, the method is very simple to implement and test, as necessary tools and data already are available in most labs. however, for the secondary structure based approach the hla class i sequences are retrieved as top candidates. the structure prediction did not include any information about the hla class ii structure, which recently has been published (brown et al., ) . it should be mentioned that the % sequence identity score is not significantly higher than the score from a random alignment of sequences. if we, for each sequence in the swissprot protein sequence library, align it against a sequence selected at random from the same library (alignment without gaps, using the full length of the shortest sequence, and start the alignment at a random position within the longest sequence), then the average percentage of identical residues is ( _+ )% at standard deviations. the identification method using secondary structure is based on an assumption which has to be examined more closely, and the implementation of it is very crude. much work can be done on the secondary structure prediction, the 'mutation' matrix and the search method. it will proba- . . sequence alignment pip _rat as described in the introduction a crucial feature in molecular evolution has been the parallel exploration of several different mutations. and although mechanisms like horizontal gene transfer and intragenic recombination may have been important as key steps in the evolution of new proteins, the most common mechanism seems to have been gene duplication followed by mutational modification (doolittle, ) . this means that especially multiple sequence alignment can give essential information about the mutation studies already performed by nature. conserved residues are normally conserved because they . multim alignment. alignment of inositol triphosphate specific phospholipase c / from rat (pip rat) against three other pip sequences from rat. each horizontal bar represents a sequence, marked in residue intervals. black lines connecting the bars represent well conserved motifs found in all sequences, in this case subsequences of residues where at least residues are completely conserved in all sequences. it is very easy to identify two well conserved regions, annotated as region x and y in the swissprot entries, despite a residue insertion in two of the sequences. this insertion contains sh and sh domains (pawson, ) . it is an interesting observation that the extra c-terminal domain of the pipi_rat sequence shows a weak similarity to myosin and tropomyosin sequences. have an important structural or functional role in the protein, and identification of such residues will thus give vital information about structure and activity of a protein. several tools have been developed for multiple alignment. a very attractive one is macaw (schuler et al., ) , which will generate several alternative alignments of a given set of regions, and in a very visual way help the user to identify a reasonable combination of (sub)alignments. an even more general tool is muitim (drablcs and . here all possible alignments, based on short motifs, are shown simultaneously, and the user is free to identify potential similarities even in cases with low sequence identity and very disperse motifs. this is possible because of the superior classification potential of the human brain compared to most automatic approaches. the method includes an option for probability based filtering of motifs, and an example of a multim alignment is shown in fig. . however, it is important to realise that in standard sequence alignment we are trying to solve a three-dimensional problem (residue interactions) by using an essentially one-dimensional method (alignment of linear protein sequences). as a consequence important conserved throughspace interactions may not be evident from a standard sequence alignment. a good example can be found in the alignment of lipases (schrag et al., ) . in fig. , the sequence alignment of residues in a structurally conserved core of three lipases (rhizomucor miehei lipase (derewenda et al., ) , candida antarctica b lipase (a. jones, personal communication) and human pancreatic lipase (winkler et al., ) is shown. the active site residues, ser (s), asp (d) and his (h), are shown as black boxes. the ser and his residues are at identical positions. however, the asp residue of the pancreas lipase is at a very different sequence position compared to the other two lipases. it would be very difficult to identify this as the active site asp from a sequence alignment. if we look at the structural alignment in fig. , we see that the positions are structurally equivalent, it is possible for all three lipases to have highly similar relative orientation of the active site atoms, despite the fact that the alternative asp positions are located at the end of two different / -strands. an improved alignment may be generated if we can incorporate -d data for at least one of the sequences in the linear alignment (gracy et al., ) . however, in order to get a reliable alignment of sequences with low sequence similarity, we have to take true three-dimensional effects into account. this means that if we are able to identify a known -d structure as a potential basis for modelling, then the sequence alignment should be done in -d using this structure as a template. this can be done by threading the sequence through the structure and calculating pairwise interactions (jones et al., ; bryant and lawrence, ) . as soon as a template has been identified, and an alignment between this template and a sequence has been defined, a -d model of the protein can be generated. we can either use the template coordinates directly, combined with dif- fig. . sequence alignment of lipases. alignment of structurally conserved regions of three lipases. for each lipase the solvent accessible surface in % compared to the gxg standard state (grey scale, white is buried and black is exposed), the secondary structure as defined by the dssp program, and the sequence is shown. the position of each subsequence in the full sequence is also shown. the active site residues are shown in white on black. please observe the shift of the active site asp (d) between two very different positions. the alignment was generated using alscript (barton, ) . ferent modelling approaches for the ill-defined regions, or the template can be used as a more general basis for folding the protein by distance geometry (srinivasan et al., ) or general molecular dynamics methods. loop regions are often highly variable, and must be treated with special approaches (topham et al., ) . it is also necessary to consider the orientation of side chains. although the backbone may be well conserved, many residues especially at the protein surface will be mutated, as shown in fig. . the stability of a protein depends upon an optimal packing of residues, and it is important to optimise side chain conformation if we want to study protein stability and complex formation. a very common approach is the use of rotamer libraries combined with molecular dynamics refinement. recent studies show that this step of the modelling in fact may be less difficult than has been assumed . and important interactions, and exposed regions may to some degree be identified by using antibodies. however, in many cases the rational for modelling by homology is the very lack of experimental data related to structure, and we have to use other more general methods for evaluation of models. some of the approaches we already have described for sequence alignment can obviously also be used for evaluation of models. in general, model evaluation can be based on -d profiles (lfithy et al., ) , contact profiles (ouzounis et al., ) or more general energy potentials (hendlich et al., ; jones et al., ; nishikawa and matsuo, ) . some of these approaches have been implemented as programs for evaluation of structures or models, like procheck (laskowski et al., ) and prosa (sippl, a, b) . however, in general no model (or even experimental structure) should be trusted beyond what can be verified by experimental methods. a protein model based on homology (or similarity) has to be verified in as many ways as possible, and experimental methods should always be preferred. mutation studies may give valuable information about active site residues a prerequisite for rational protein engineering is -d structure information about the protein. in fig. , including parts of the sequences connecting the core regions. the active site asp is able to maintain a similar relative orientation, despite very different sequence positions. the alignment was generated using insight (biosym technologies). adddition to x-ray crystallography, nmr is the most important method for protein structure determination. x-ray crystallography has several advantages when compared to nmr. solving the crystal structure by x-ray crystallography is usually fast as soon as good crystals of the protein are obtained (even if it may not be so easy to obtain these crystals). it is also possible to determine the structure of very big proteins. the major disadvantage of x-ray crystallography is that it is the crystal structure that is determined. this implies that crystal contacts may distort the structure (chazin et al., ; wagner et al., ) . since active sites and other binding sites usually are located on the surface of the proteins, very important regions of the protein may be distorted. some structures even show large differences between nmr and x-ray structure (frey et al., ; klevit and waygood, ) the advantage of nmr is that it is dealing with protein molecules in solution, usually in an environment not too different from its natural one. it is possible to study the protein and the dynamical aspects of its interaction with other molecules like substrates, inhibitors, etc. it is also possible to obtain information about apparent pk a values, hydrogen exchange rates, hydrogen binding and conformational changes. all nuclei contain protons, and therefore they carry charge. some nuclei also possess a nuclear spin. this creates a magnetic dipole, and the nuclei will be oriented with respect to an external magnetic field. the most commonly studied nuclei in protein nmr ( h, c and n) have two possible orientations, representing high and low energy states. the frequency of the transition between the two orientations is proportional to the magnetic field. at a magnetic field of . tesla the energy difference corresponds to about mhz for protons. in an undisturbed system there will be an equilibrium population of the possible orientations, with a small difference in spin population between the high and low energy orientation. the equilibrium population can be perturbed by a radio frequency pulse of a frequency at or close to the transition frequency. in addition, the spins will be brought into phase coherence (concerted motion) and a detectable magnetisation will be created. the intensity of the nmr signal is proportional to the population difference between the levels the nuclei can possess. nuclei of the same type in different chemical and structural environments will experience different magnetic fields due to shielding from electrons. the shielding effect leads to different resonance frequencies for nuclei of the same type. the effect is measured as a difference in resonance frequency (in parts per million, ppm) between the nuclei of interest and a reference substance, and this is called the chemical shift. in molecules with low internal symmetry most atoms will experience different amounts of shielding, the resonance signals will be distributed over a well-defined range, and we get a typical nmr spectrum. the process that brings the magnetisation back to equilibrium may be divided into two parts, longitudinal and transverse relaxation. the longitudinal or t relaxation describes the time it takes to reach the equilibrium population. the transverse or t relaxation describes the time it takes before the induced phase coherence is lost. for macromolecules the t relaxation is always shorter than the t a relaxation. short t relaxation leads to broad signals because of poor definition of the chemical shift. most molecules have dipoles with magnetic moment, and the most important cause of relaxation is fluctuation of the magnetic field caused by the brownian motion of molecular dipoles in the solution. how effective a dipole may relax the signal depends upon the size of the magnetic moment, the distance to the dipole, and the frequency distribution of the fluctuating dipoles. a nucleus may also detect the presence of nearby nuclei (less than three bonds apart), and this will split the nmr signal from the nucleus into more components. several nuclei in a coupling network is called a spin system. by applying radio frequency pulses it is possible to create and transfer magnetisation to different nuclei. it is, as an example, possible to create magnetisation at one nucleus, and transfer the magnetisation through bonds to other nuclei where it may be detected. the pulses are applied in a so-called pulse sequence (ernst, ; kessler et al., ) . the methodology for determination of protein structure by two-dimensional nmr is described in several textbooks and review papers (wagner, ; wiithrich, ; wider et al., ) . the standard method is based on two steps, sequential assignment: assignment of resonances from individual amino acids, and distance information: assignment of distance correlated peaks between different amino acids. the first step involves acquiring coupling correlated spectra (cosy, tocsy) in deuterium oxide to determine the spin system of correlated resonances. some amino acids have spin systems that in most cases make them easy to identify (gly, ala, thr, ile, val, leu). the other amino acids have to be grouped into several classes, due to identical spin systems, even though they are chemically different. the spin systems can be correlated to the nh proton by acquiring cosy and tocsy spectra in water. the assigned nh resonance is then used in distance correlated spectra (noesy) to assign correlations to protons (nh, h,, h~) at the previous amino acid residue (fig. ) . by combining the knowledge of the primary sequence (which gives the spin system order) with the nmr data collected it is possible to complete the sequential assignment. when the sequential assignment is done the assignment of short range noe (up to four residues) will give information about secondary structure (a-helix, fl-strand). long-range correlations will serve as constraints (together with scalar couplings) to determine the tertiary structure of the protein. excellent procedures describing these steps are available (roberts, ; wiithrich, ) . with large proteins there will be spectral overlap of resonance lines. the problem is partially solved by labelling the protein with c and n isotopes. triple resonance multidimensional nmr methods (griesinger et al., ; kay et al., ) may then be applied. the resonances will then be spread out in two more dimensions ( c and ~sn) and the problem with overlap is reduced. these methods depend upon the use of scalar couplings to perform the sequential assignment, the sequential assignment procedure will then be less prone to error. the noesy spectra of such large proteins are often very crowded, but four-dimensional experiments like the c-~ c edited noesy spectrum (clore et al., b) have been designed. such experiments will spread the proton-proton distance correlated peaks by the chemical shift of its corresponding c neighbour and reduce the spectral overlap. secondary structure elements may also be predicted from the chemical shift of h and c (spera and bax, ; williamson and asakura, ; wishart et al., ) . obtaining nmr-spectra of proteins has some aspects that should be considered. spectral overlap. as we move to larger proteins the probability of overlap of resonance lines increases. at some point it will become impossible to do sequential assignment due to this overlap. application of -d and -d multiresonance nmr has made it possible to assign proteins in the kda range (foght et al., ; stockman et al., ) . fast relaxation. as the size of the protein is increased the rate of tumbling in solution is re-duced. this leads to a reduced transverse relaxation time (t ), and broadening of the resonance lines in the nmr spectra. the intensities of the peaks are reduced and they may be difficult to detect. the short transverse relaxation time will also limit the length of the pulse sequences it is possible to apply (because there will be no phase coherence left), and multidimensional methods become difficult. it is possible to determine a -d structure by nmr or x-ray crystallography are probably a subset of all proteins (wagner, ) . proteins may have regions with mobility and few cross peaks. the effective size of a protein is often increased by aggregation. the amount of aggregation can often be reduced by reducing the protein concentration. thus, very often the degree of aggregation will determine whether it is possible to assign and solve a protein structure by nmr, by limiting the maximum concentration that may be used. the stability of the proteins is also a major issue. a sample may be left in solution for days, often at elevated temperatures, so denaturation may become a problem. photo-cidnp (chemically induced nuclear polarisation) is an interesting technique for the study of surface positioned aromatic residues in proteins (broadhurst et al., ; cassels et al., ; hore and kaptain, ; scheffier et al., ) . by introducing a dye and exciting it with a laser, it is possible to transfer magnetisation to aromatic residues, where it can be observed. in addition to high-resolution nmr, solid state nmr has also been applied to studies of proteins. studies of active sites and conformation of bound inhibitors yields interesting information. the stability of proteins may be monitored under different conditions by detecting signals from transition intermediates bound to the active site (burke et al., ; gregory et al., ) . structural constraints on transition state conformation of bound inhibitors can be obtained (auger et al., ; christensen and schaefer, ) . structural constraints of the fold and conformation of the amino sequence may be gathered by setting upper and lower distances for lengths between specific amino acids (mcdowell et al., ) . using solid-state nmr it is also possible to study membrane proteins and their orientation with respect to their membrane (killian et al., ; ulrich et al., ) . we expect such studies to give insight into ion channels in membranes (woolley and wallace, ). an important mechanism for relaxation in high-resolution nmr is dipolar relaxation. usually this is induced by the spin of nuclei in the immediate vicinity, and it is a function of the size of the dipole. the electron is also a magnetic dipole, and the magnitude of this dipole is about -times that of a proton. paramagnetic compounds have an electron that will interact with . the paramagnetic relaxation method. outline of the paramagnetic relaxation method. the protons located at the protein surface will be closer to the dissolved paramagnetic relaxation agent than the protons located inside the protein core, hence the resonance lines from protons at the surface will be broadened more than resonance lines stemming from protons located inside the protein. nearby protons and increase the relaxation rate of these protons. the widest use of paramagnetic compounds has been of gd ÷ bound to specific sites in a protein , but also other compounds have been used (chang et al., ; hernandez et al., a, b ). this will make it possible to identify resonance lines from residues in the vicinity of the binding site. it is also possible to calculate distances from the paramagnetic atom as the relaxation effect is distance dependent. the paramagnetic broadening effect can also be used with a compound moving freely in solution (drayney and kingsbury, ; esposito et al., ; petros et al., ) . in this way residues located on or close to the protein surface will give broadened resonance lines compared to residues in the interior of the protein. this method can be used to measure important noe and chemical shifts inside the protein directly, or it can be used as a difference method to identify resonances at the surface by comparing spectra acquired with and without the paramagnetic relaxation agent (fig. ) . we have used the paramagnetic compound gadolinium diethylenetriamine pentaacetic acid (gd-dtpa) as a relaxation agent. gd-dtpa will increase both the longitudinal and the transverse relaxation rates of protons within the influence sphere. suitable nmr experiments to highlight the relaxation effect may be noesy, roesy and tocsy (bax and davis, ; braunschweiler and ernst, ) gd-dtpa is widely used in magnetic resonance imaging (mri) to enhance tissue contrast. it is assumed to be non-toxic and we do not expect it to bind to proteins. we used the wellstudied protein hen egg-white lysozyme as a test protein. both the structure and the nmr spectra of this protein are known (diamond, ; redfield and dobson, ) , and the protein is extremely well suited for nmr experiments. in fig. , the -d ih-nmr spectrum recorded in the presence and absence of gd-dtpa is shown. although it is evident that there is a selective broadening in the -d spectrum, it is also clear that there are problems with overlapping spectral lines. we therefore applied two-dimensional nmr methods, and shown in fig. is the low field region of a noesy spectrum of lysozyme. the region corresponds to the same region as shown in fig. . from fig. we see that the signals from w , w and w disappear with addition of gd-dtpa, while the signals from w , w and wlll still are observable. by examination of the solvent accessible surface of lysozyme it is evident that the indole nh of w , w and w is exposed to solvent, while the indole nh of w , w and wlll is not exposed. this shows that the changes in the spectrum are as expected from the structure data. the appearance of the nh-nh region of the spectrum (fig. ) also shows the reduction in the number of signals in the gd-dtpa exposed spectrum. this shows that the paramagnetic broadening effect can be used for selective identification of signals from solvent exposed residues in a protein. one of the fundamental steps in the protein engineering process shown in fig. is the design step, where a correlation between structure and properties is established in order to select potential structural candidates that match new functional profiles. the understanding of this correlation implies a realistic modelling of the physical chemical properties involved in the functional features to be engineered. these features are basically of two types: diffusional and catalytic. any ligand binding to a protein, whether ligandreceptor or substrate-enzyme, is essentially a diffusional encounter of two molecules. electrostatic interactions are the strongest long-range forces at the molecular scale and, thus, it is not surprising that they are one of the determinant effects in the final part of the encounter (berg and von hippel, ) . in the case of substrateenzyme interactions the catalytic step that follows the binding of the substrate seems to be possible mainly by the presence of electrostatic forces that stabilise the reaction intermediates in the binding site (warshel et al., ) , from which the product formation may proceed. another and much more basic necessary condition for a successfully engineered protein is that a functional folded conformation is maintained. solvation of charged groups is one of the determinants in protein folding (dill, ) , so that even the conformation of the protein is electrostatically driven. given the ubiquitous role of electrostatic interactions, it is then obvious that their accurate modelling is an essential prerequisite in the design of engineered proteins. several good reviews exist on protein electrostatics (warshel and russel, ; matthew, ; rogers, ; harvey, ; davies and mccammon, ; sharp and honig, ) . this section intends to give a brief overview of the subject. we start by presenting the methods one can use to model electrostatic interactions. the most familiar methodology in biomolecular modelling is certainly molecular mechanics (mm) (either through energy minimisations or molecular dynamics (md)). we point out some of the limitations of mm in the treatment of electrostatic interactions, and the need to use alternative ways of describing the system, such as continuum methods. the computation of ph-dependent properties and some potential extensions of mm are also discussed. finally, we refer some applications of electrostatic methods relevant to protein engineering. in mm simulations, electrostatic interactions are usually described with a pairwise coulombic term of the form qlq /dr ,were ql and q are the charges of the pair of atoms, r their distance, and d the dielectric constant. d is usually set equal to when the solvent is included. a complete simulation in a sufficiently big box with water molecules should, in principle, give a realistic description of the protein molecule (harvey, ) . this would be specially true if a force field including electronic polarizability effects (see . .) was available for use with biomolecular systems, which unfortunately is not the case (harvey, ; davis and mccammon, ) . we use the term force field in this context as including both the functional form and parameters describing the energetics of the system, from which the forces are derived. simulations where solvent molecules are not treated explicitly are naturally appealing, since the computation time increases with the square of the number of atoms. several methods have been proposed that attempt to account for solvent effects. the more popular approach is an ad hoc dielectric 'constant' proportional to the distance (e.g., mccammon and harvey, ) but different distance dependencies can be used (e.g., solmajer and mehler, ) . a variety of more elaborated methods were also suggested (northrup et al., ; still et al., ; gilson and honig, ) . all these methods should be viewed as attempts of including solvent screening effects in a simplified way. they can be useful when inclusion of water is computationally prohibitive, but they cannot substitute for an explicit inclusion of solvent since, e.g., the existence of hydrogen bonding with the solvent is not properly described by these approaches. mm of biomolecules has, in general, heavy computation needs. the number of water molecules that should be included in order to simulate a typical protein in a realistic way is quite large, especially if one wants to perform md. also, each pair of atoms has its own electrostatic interaction and the number of pairs cannot be lowered by a short cut-off distance (e.g., a) as in van der waals interactions, since electrostatic interactions are very long range, typically up to ~,. mm simulations have also some limitations on the description of the system, since ph and ionic strength effects usually are difficult or impossible to include. the only way to include ph effects is through the protonation state of the residues. each titrable group (in asp, glu, his, tyr, lys, arg, c-and n-terminal) in the protein have two states, protonated or unprotonated. thus, a protein with n titrable groups will have n possible protonation charge sets. the best we can do is to choose the set corresponding to the protonation states of model compounds at the desired ph. free ions can be included in md simulations of proteins (levitt, ; mark et al., ) , but it is not clear if the simulated time intervals are long enough to realistically reflect ionic strength effects. another problem with mm is that the understanding it provides of the system (through energy minimisation or md) does not include entropic aspects explicitly, i.e., it does not give free energies directly. there are methods to calculate free energies based on mm potentials (beveridge and dicapua, ) , but even though several applications have been made on biomolecular systems (for a review see beveridge and dicapua, ) , they are still too demanding for routine use in systems of this size. then, when the properties under study are related to free energies rather than energies (which is often the case), mm by itself can only be seen as a first approach. in summary, although mm simulations can provide some unique information on the structural and dynamical behaviour of biomolecular systems, some limitations exist due to both conceptual and practical reasons, in particular regarding the treatment of electrostatic interactions. fortunately, other methods exist that can provide insight on aspects whose modelling is poor or absent in mm simulations, although at the cost of the atomic detail in the description. there is no 'best' modelling method and we should resort to the several methods available in order to gain an understanding of the system that is as complete as possible. the so-called continuum or macroscopic models assume that electrostatic laws are valid at the protein molecular level and that macroscopic concepts such as dielectric properties are applicable. protein and solvent are treated as dielectric materials where charges are located. these charges may be titrable groups (whose protonation state may vary), permanent ions (structural and bound ions, etc.) or, more recently, permanent partial charges of polar groups. given the dielectric description of the system and the placement of the charges, the problem can be reduced to the solution of the poisson equation (or any equivalent formulation), as can any problem of electrostatics (e.g., jackson, ) . the electrostatic potential thus obtained can be used to study diffusional processes or visually compare different molecules (see . .). the simplest continuum model assumes the same dielectric constant inside and outside the protein. typically, a value somewhere between the protein and solvent dielectric constants has been used (sheridan and allen, ; koppenol and margoliash, ; hol, ) . this approach completely ignores the effects of having two very different dielectric regions, but can be used for a first qualitative computation. the more common continuum models treat the protein as a low dielectric cavity immersed in a high dielectric medium, the solvent. the way the charges are placed in this cavity and the way the electrostatic problem is solved vary with the particular method. analytical solutions can be obtained for the simplest shapes, such as spheres, but in general the more complex shapes require numerical techniques. in the first cavity model the protein was assumed to be a sphere with the charge uniformly distributed over its surface (linderstr~m-lang, ) . tanford and kirkwood ( ) proposed a more detailed model in which each charge has a fixed position below the surface. assuming a spherical geometry allows for a simple solution to the electrostatic problem. it is even possible to include an ionic atmosphere that accounts for ionic strength effects (leading to the poisson-boltzman equation). the effect of ph occurs naturally in the formalism. the energy cost of burying a charge inside the low-dielectric protein (self-energy) is taken to be the same as in small model compounds, since at the time when this method was developed (before protein crystallography) charges were believed to be restricted to the protein surface. this limits the method to proteins without buried charges, unless we have some estimate on the self-energy. there are, obviously, some problems in fitting real, irregularshaped proteins to a spherical model. some solutions to this problem were proposed, including an ad hoc scaling of interactions based on solvent accessibility (shire et al., ) , and the placing of more exposed charges in the solvent region (states and karplus, ) . the inclusion of non-spherical geometries im-plies the use of numerical techniques, as referred above. warwicker and watson ( ) and used the finite differences technique to solve, respectively, the poisson and poisson-boltzman equations. self-energies can be included (gilson and honig, ) , such that the method is fully applicable when buried charges exist. the intrinsic discretization of the system in the finite differences technique, makes these methods readily applicable to any kind of spatial dependency on any of the properties involved. the inclusion of a spatially-dependent dielectric constant, for instance, will be relatively simple. other extensions such as additional dielectric regions (ligands, membranes, etc.), eventually with charges, should also be possible. alternative numerical techniques for solving the poisson or poisson-boltzman equations have also been used, including finite elements (orttung, ) and boundary elements (zauhar and morgan, ) . the dielectric constant in a region comes from the existence of dipoles in that region, permanent or induced. permanent dipoles are due to atomic partial charges (e.g., water dipole, peptide bond dipole). induced dipoles are due to the polarizability of electron clouds. warshel and levitt ( ) represented this electronic polarizability by using point dipoles in the atoms. as pointed out by davies and mccammon ( ) this representation is roughly equivalent to a spatially-dependent dielectric constant. this approach is usually combined with a simplified representation ot water by a grid of dipoles (warshel and russel, ) . ionic strength and ph effects are not considered. all the above methods deal with a particular charge set (see . .), even when ph effects are considered. however, a protein in solution does not exist in a single charge set. we are usually interested in the properties of a protein at a given ph and ionic strength, not at a particular charge set. moreover, if we want to test the available methods, we have to test them against experimental results which usually do not correspond to a specific charge set. a common test on the accuracy of electrostatic models is their ability in predicting pk a values of titrable groups in a protein (see . .), obtained via titrations, nmr, etc. these values can be quite different from the ones of model compounds, due to environment of the groups in the protein. this difference (pk a shift) can be of several pk units. the experimentally determined apparent pka (pkap p) is determined as the ph value at which half of the groups of that residue are protonated in the protein solution, i.e., when its mean charge is / (thus, the equivalent notation pk / ). then, if we can devise a method to compute the mean charge of the titrable groups at several ph values, we can predict their pkap p values. as mentioned above (see . .), we have n possible charge sets. any structural property can, in principle, be computed through a boltzman sum over all those sets, with each one contributing according to its free energy (taken as the electrostatic energy) (tanford and kirkwood, ; bashford and karplus, ) . the property thus computed is characteristic of the chosen ph value (and ionic strength, if considered) instead of a specific charge set. we are particularly interested in computing the mean charges at a given ph (see last paragraph). a sum with n terms is not, however, a trivial calculation in terms of computer time. tanford and roxby ( ) avoided the boltzman sum by placing the mean charges directly on the titrable groups, instead of using one of the integer sets. this corresponds to considering the titration of the different groups as independent (a mean field approximation; bashford and karplus, ) . other alternatives to the boltzman sum are the monte carlo method (beroza et al., ) , less drastic mean field approximations (yang et al., ; gilson, ) , the 'reduced site' approximation (bashford and karplus, ) , or even assume that the predominant charge set is enough to describe the system (gilson, ) . since electrostatic interactions in proteins are typically dominated by titrable groups whose charge is affected by ph, no electrostatic treat-ment can be complete without taking this effect into account. a simple, although effective, way of doing this is to: (i) compute the electrostatic free energies (e.g., by a continuum method); (ii) compute the mean charge of each titrable group at a given ph (e.g., by a mean field approximation); (iii) use those charges to compute the electrostatic potential (e.g., by a continuum method), which can be displayed together with the protein structure (see the human pancreatic lipase example in section . .). in this way a ph-dependent electrostatic model of the protein can be obtained, which is not possible with usual mm-based modelling techniques. as stated above (see . .), electronic polarizability is not explicitly considered in common force fields. van belle et al. ( ) included the induced dipole formalism (warshel and levitt, ) in mm calculations. the electrostatic interactions in the applied force field were simply 'corrected' with additional terms due to inducible dipoles. however, it should be noted that a force field fitted to experimental data without polarizability terms, should be fitted again if those terms are included. the protein conformation used in molecular modelling is usually an experimentally based (xray, nmr) mean conformation, characteristic of those particular experimental conditions. that conformation may, however, be inadequate for modelling the protein properties at different conditions. in particular, proteins are known to denaturate at extreme ph conditions. thus, ph-dependent methods such as the continuum methods may give incorrect results when using one single conformation over the whole ph range. actually, md simulations have shown that the results can be highly dependent on side chain conformation (wendoloski and matthew, ) . although overall properties like titration curves did not seem to be very sensitive, individual pka's showed variations up to . pk units. as mentioned in section . , mm has the problem of what charge set to use in simulations. instead of using a charge set corresponding to model compounds at the intended ph, one may use the predominant charge set of the protein, determined, e.g., by a continuum method, as suggested by gilson ( ) . a different approach to this problem would be to devise a way of including the averaged effect of all charge sets in the mm simulation. we have recently developed a method where a force field is derived which includes the proper averaged effect of all charge sets (a potential of mean force) (to be published). the method depends on the calculation of electrostatic free energies obtained from, e.g., a continuum method. the electrostatic potential, computed in some of the referred methods, can help to understand the contribution of electrostatic interactions in the diffusional encounters of proteins with ligands (substrates or not). the diffusional process driven by the electrostatic field can be simulated through brownian dynamics (bd) and diffusion rates may be computed (for references see, e.g., davies and mccammon, ) . the effect of mutations on the diffusion of superoxide ion into the active site of superoxide dismutase has been studied by this technique (sines et al., ) and faster mutants showing - -fold increase in reaction rate could be designed (getzoff et al., ) , although this enzyme usually is considered to be 'perfect'. electrostatically driven bd simulations can help to reveal steric 'bottlenecks' (reynolds et al., ) and orientational effects (luty et al., ) . this method can also be applied to study the encounter of two proteins (northrup et al., ) . visual comparison of electrostatic fields can also provide useful information. soman et al. ( ) showed that rat and cow trypsins have similar electrostatic potentials near the active site, despite a total charge difference of . units. as an illustration of such type of comparisons, using ph-dependent electrostatics, we have applied the solvent accessibility-modified tanford-kirkwood method (see . .) to the human pancreatic lipase structures with both closed (van tilbeurgh et al., ) and open lid (van tilbeurgh et al., ) , as shown in fig. a and b. fig. c-f shows surfaces corresponding to an electrostatic potential equal to + . kt/e (where k is the boltzman constant, t the absolute temperature and e the proton charge). these surfaces correspond to regions were the electrostatic interactions on a charge are roughly of the same magnitude as the thermal effects due to the surrounding solvent, i.e., where charged molecules in solution start to feel electrostatic steering or repulsion. at ph clear differences exist between the closed and open forms, the latter showing a dipolar groove in the presumed binding site region. at pi-i the molecule is strongly positively charged and most electrostatically differentiated regions have disappeared. given the role of electrostatic interactions on molecular orientation and association (see the beginning of this section ( )), this is expected to markedly affect the interaction with the lipid-water interface. for enzymes the catalytic activity involving a charged residue can be modulated by shifting the pk a of that residue. the pk a shifts of the active site histidine has been successfully predicted for a number of mutants of subtilisin loewenthal et al., ) . one of the main reasons why enzymes are good catalysts is because they stabilise the transition state intermediate (fersht, ) . for enzymatic reactions that are not diffusion limited, engineering leading to an enhanced stabilisation of the intermediate will result in an increased activity. the induced dipole method was used to compute the activation free energy for different mutants of trypsin and subtilisin (warshel et al., ) , with some qualitative agreement with the experimental results. the prediction of changes introduced by mutations on redox potentials could also be of interest to protein engineering. prediction of redox potentials has been made with some success (rogers et al., ; durell et al., ) . in plastocyanin the effect of chemically modifying charged groups was also considered (durell et al., ) . the effect of mutations could also be analysed, as has been done for pk a shift calculations (see above). the above examples clearly show that, whatever the particular method used, the modelling of a c e d t electrostatic interactions in proteins has an important role to play in protein engineering. a highly relevant example is the design of a faster 'perfect' enzyme (getzoff et al., ) , which also illustrates the combination of different methods (bd and electrostatic continuum methods) that can sometimes be determinant in a modelling study. the science of protein engineering is advancing rapidly, and is emerging in many new contexts, such as metabolic engineering. rational protein engineering is a complex undertakingand only the groups with sufficient understanding of sequences and -d structures can handle the complex underlying problems. predicting protein structure may be difficult -but predicting future developments in a very active branch of science can be hazardous at the best. however, we will review a few of the more recent research aspects that we are convinced will be of key importance in the future development of protein engineering. often the substrates or products in an enzymatic process are poorly soluble in an aqueous medium. this may lead to poor yields and difficult or expensive purification steps. the potential of using other solvents, either pure or in mixture, where substrates and/or products may be soluble has attracted a great deal of attention (tramper et al., ; arnold, ) . dissolving the protein in organic solvents will alter the macroscopic dielectric constant and lead to a much less pronounced difference between the interior and exterior static dielectric behaviour. protein function in such media may be altered and is poorly understood; we can expect a significant development in the future. despite the often dramatic change in dielectric constant when changing the solvent from, e.g., water to an organic substance, the protein -d structure can remain virtually intact, as has been documented in the case of subtilisin carlsberg dissolved in anhydrous acetonitrile (fitzpatrick et al., ) . the hydrogen bonding pattern of the active site environment is unchanged, and of the enzyme-bound structural water molecules are still in place. one-third of the enzymebound acetonitrile molecules reside in the active site. many enzymes remain active in organic solvents and in the case of enzyme reactions where the substrate has very poor water solubility, a change to organic solvent can be of major importance (gupta, ). an extreme case of a non-conventional medium for enzymatic action is the gas phase. certain enzymes, immobilised on a solid bed, have been shown to be active at elevated temperatures towards selected substrates in the gas phase (lamare and legoy, ) . obviously the range of substrates that potentially can be used is limited to those that actually can be brought into the gas phase under conditions where the enzyme is still active. enzymes for which such reactions have been studied include hydrogenase, alcohol oxidase and lipases. the fact that even interfacially activated lipases (such as the porcine pancreatic and the candida rugosa lipases) function with gas phase carried substrate molecules opens up the interesting possibility of studying the role of water in this reaction. protein engineering may be used to enhance enzyme activity in organic solvents (arnold, ; fig. . electrostatic maps of hpl with closed and open lid. ribbon models of human pancreatic lipase with colipase are shown with closed (left: a,c,e) and open (right: b,d,f) lid. the colipase is shown in blue and the mainly a-helical 'lid' region is highlighted in cyan. the residues of the active site are shown in green. access to the active site pocket seems to be controlled by the conformational st'ate of the lid. electrostatic isopotential contours of + . kt/e are shown at ph (c,d) and ph (e,f). the negative surfaces are represented in red and the positive surfaces in blue. the models and isopotentiai contours were produced with insight h and delphi (biosym technologies, san diego). the ph-dependent charge sets were computed with titra (to be published). chen and arnold, ) . when dissolving subtilisin e in % dimethylformamide (dmf) the kcat/k m for the model substrate suc-ala-ala-pro-met-p-nitroanilide drops -fold. after ten mutations were introduced, the activity in dmf was restored almost to the level of the native enzyme in water. all metabolic conversions in micro-organisms are carried out directly or indirectly by proteins. our ability to manipulate single genes has opened up for the actual control of such processes. we may alter the efficacy of a certain pathway or we may introduce totally new pathways. thus, escherichia coli can be modified in such a way that one can use i>glucose in the e. coli based manufacture of hydroquinone, benzoquinone, catechol and adipic acid (dell and frost, ; draths and frost, ; frost, ) . presently such compounds are produced through organic chemical synthesis using aromatics as one of the reactants. the prospect of producing the same compounds using only microbes and glucose thus has some obvious environmental benefits. we expect to see a virtual surge in the engineering of microorganisms towards the production of rare chemical or biochemical compounds or compounds for which the current synthetic route is costly either economically or from an environmental perspective. the perspective of designing and producing functional protein molecules from scratch is extremely attractive to many visionary scientists. some central questions arise: do we know enough to undertake such tasks, and what goals can we define? screening mutation studies of protein interfaces show that the majority of mutations reduce activity or binding affinity (cunningham and wells, ) , indicating that most proteins already represent highly optimised designs. the groups active in this area have aimed at constructing certain -dimensional folds such as the four helix bundle (felix) (hecht et al., ) and histidine-based metal binding sites (arnold, ) and even the observation of limited enzymatic activity is regarded as a successful result . protein de novo design of helix bundles may even follow a very simple binary pattern of polar and nonpolar amino acids as was concluded in a study of four-helix bundle proteins (kamtekar et al., ) . the helix-helix contact surfaces are mainly hydrophobic, whereas the solvent exposed regions are hydrophilic. many variants conforming to this hydrophobic pattern were generated and two of these proteins were stabilised with . and . kcal tool -~ relatively to the unfolded form, thus approaching what is found for many natural proteins. the authors suggest that such a binary pattern may have been important in the early stages of evolution. in our laboratory we have results supporting this conclusion for the trypsin family of proteins, which is predominantly in a / -strand based fold . fusion and hybrid proteins may be produced by fusing the genes or gene fragments including a proper linking region between the two genes (argos, ) . this in principle may allow for combining properties from two different proteins. thus artificial bifunctional enzymes have been produced by fusing the genes for the proteins, e.g., / -galactosidase and galactokinase (bulow, ). in a recent paper an elegant hybrid protein concept is described. a hybrid antibody fragment was designed to consist of a heavy-chain variable domain from one antibody connected through a linker region of - residues to a short lightchain variable domain from another antibody (holliger et al., ) . the antibody fragments displayed similar binding characteristics as the parent antibodies. the prospect of engineering multifunctional antibodies for medical applications is imminent. a hybrid protein between the glucose transporter and the n-acetylglucosamine transporter of e. coli have been produced. the two proteins displayed % residue identity. the hybrid protein consisted of the putative transmembrane do-main from the glucose transporter and the two hydrophilic domains from the n-acetylglucosamine transporter. the hybrid protein was, somewhat surprisingly, still specific for glucose (hummel et al., ) . interestingly, several naturally occurring proteins themselves seem to have originated through gene fusion. in the case of hexokinase it is proposed that it originated from a duplication of the glucokinase gene maintaining even the gene organisation (kogure et al., ) . several other proteins such as receptor proteins of the insulin family can best be understood as gene fusion products of a kinase domain onto the rest of the receptor (which in itself may consist of several fragments). with potential medical applications, proteinnucleic acid hybrids have been constructed, where the nucleic acid fragment complemented the sequence of a fragment of mrna that the rnase should be targeted towards. the results obtained confirmed that this approach indeed worked (kanaya et al., ) . the potentials for generating anti-viral agents against, e.g., hiv are obvious. as a consequence of the enormous growth in our understanding of molecular biology and material technology, a new technological sector is emerging which takes aim at exploring the possible advantages in creating micro-machines and switchable molecular entities. this concept is currently known as nano technology (birge, ) . two concepts that we find particularly interesting are described briefly below. rhodopsin is a very ancient molecular construct -we find rhodopsin like molecules in a range of roles, all of them associated with its membrane location. proton transport and receptor functions are particularly interesting. bacteriorhodopsin from halobacterium halobium maintains a large ph gradient across the bacterial membrane. this protein complex is coloured, and its colour can be changed by exposing the protein to light of an appropriate frequency. the lifetime of the excited state can be adjusted by adjusting the physical chemical parameters of the medium the rhodopsin is embedded in (birge, ) . this protein can be used as a molecular switch in a very broad sense, e.g., as part of a high density memory device. however, changing the colour of a protein molecule is just one example that could be considered. another molecular based switch concept involves the transfer of a molecular ring (paraquat-derived rotaxane ring) between two binding sites (bradley, ) . currently the transfer is induced by a solvent change, but it is believed that an electrochemical transfer mechanism can be developed as well. similar concepts can probably also be developed for proteins. the present paper reviews some of many new developments in protein engineering. the review is not exhaustive -it is simply not possible to do this properly within the limits of this paper. we have tried to review some selected scientific areas of key importance for protein engineering, such as the validity of protein sequence information as well as structural information. sometimes the translation of a gene sequence to amino acid sequence is not trivial -a range of posttranscriptional editing and splicing events may occur, leading to a functional protein, where the amino acid sequence cannot be directly deducted from the gene sequence. in addition, posttranslational modification may provide triggers for other parts of the cells molecular machinery. we are thus in a situation where the full benefits and profits from projects such as the human genome project may escape us for a while. we have covered some of the recent developments in the modelling of protein structure by homology, which we regard as one of the most strategic areas of development. we will be flooded with sequence information deducted from gene sequences, and in the cases where the deducted amino acid sequences are assumed valid, we have to use homology based structure prediction in most cases. given that the number of protein structure families is expected to be limited the task is durable. here we should again caution the reader. we have no a priori reason to assume that non-soluble proteins, such as structural proteins, have structures that can be predicted from our limited library of mostly globular, soluble proteins. some structural proteins are gigantic, the cuticle collagen in the riftia worms from deep sea hydrothermal vents have a molecular mass of . kda (gaill et al., ) . it is extremely unlikely that a -d structure at atomic resolution of such a protein will ever be determined using methods we have available today. nmr has emerged with surprising speed as a structure determination tool. many excellent reviews have been written on this topic. we have decided to direct the readers attention to some recent developments that we believe will be of significant importance to the usage of nmr in protein engineering projects. the potential of using nmr to study the solvent exposed outer shell of larger proteins, that by far exceed the kda limit mentioned earlier is intriguing. this is particularly so, since most functionality of a protein is a feature of exactly the residues in the outer shell. thus, we can 'peel' the protein, and thereby isolate the spectral information that pertains to the surface only. this simplifies the spectra, and in some cases even allows for a partial assignment of specific residues. recent developments in ph-dependent protein electrostatics have been given special attention here. the similarities and differences within a family of structurally related proteins can only be understood if we are capable of interpreting the consequences of the substitutions, insertions and deletions that mostly occur at the surface of the proteins. when such changes are found and they involve charged residues, this will effect the extent or polarity of the electrostatic fields that the protein molecule is embedded in. we believe that the consequences of charge mutations to a large extent can be predicted through the use of ph-dependent electrostatics although practical examples are still lacking. to our knowledge the results on the electrostatic consequences of the lid motion in the human pancreatic lipase (vide supra) are among the first such reported. the story of molecular biology is continuously unfolding -and our understanding of our own biology, development and evolution is becoming 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structural class from amino acid composition we want to thank christian cambillau, cnrs, marseille, for kindly providing us with pre-release -d data of human pancreatic lipase, jerry h. brown, harvard university, for sending us a prerelease dataset for the hla ii structure, alwyn jones, uppsala university, for pre-release -d data of candida antarctica b lipase, and johnmccarthy, brookhaven national laboratory, for helping us with data on previous pdb releases. the french norwegian foundation (fns ) and the norwegian research council (bp ) have contributed with financial support to some of the research activities described in this paper. a.b. and p.m. thank junta nacional de investi-ga~o cientlfica, portugal, for their grants. key: cord- - rchp yy authors: taufer, michela; leung, ming-ying; solorio, thamar; licon, abel; mireles, david; araiza, roberto; johnson, kyle l. title: rnavlab: a virtual laboratory for studying rna secondary structures based on grid computing technology date: - - journal: parallel computing doi: . /j.parco. . . sha: doc_id: cord_uid: rchp yy abstract as ribonucleic acid (rna) molecules play important roles in many biological processes including gene expression and regulation, their secondary structures have been the focus of many recent studies. despite the computing power of supercomputers, computationally predicting secondary structures with thermodynamic methods is still not feasible when the rna molecules have long nucleotide sequences and include complex motifs such as pseudoknots. this paper presents rnavlab (rna virtual laboratory), a virtual laboratory for studying rna secondary structures including pseudoknots that allows scientists to address this challenge. two important case studies show the versatility and functionalities of rnavlab. the first study quantifies its capability to rebuild longer secondary structures from motifs found in systematically sampled nucleotide segments. the extensive sampling and predictions are made feasible in a short turnaround time because of the grid technology used. the second study shows how rnavlab allows scientists to study the viral rna genome replication mechanisms used by members of the virus family nodaviridae. ribonucleic acid (rna) is made up of four types of nucleotide bases: adenine (a), cytosine (c), guanine (g), and uracil (u). a sequence of these bases is strung together to form an rna molecule that, unlike deoxyribonucleic acid (dna), is often single-stranded. rna molecules vary greatly in size, ranging from about nucleotide bases in micrornas to long polymers of over , bases comprising entire viral genomes [ ] . among the four nucleotide bases, c and g form a complementary base pair by hydrogen bonding, as do a and u and, less frequently, g and u. although a single-stranded rna molecule is a linear polymer, it tends to fold back on itself to form a three dimensional ( d) functional structure mostly by pairing between complementary bases. the d structure of an rna molecule is often the key to its function. because of the instability of rna molecules, experimental determination of their precise d structures is a time consuming and rather costly process. however, useful information about an rna molecule can be gained from knowing its secondary structure, which refers to the collection of hydrogen bonded base pairs in the molecule. essentially, all rna secondary structures are made up of elements - /$ -see front matter Ó elsevier b.v. all rights reserved. doi: . /j.parco. . . that can be classified into two basic categories: stem-loops and pseudoknots (see fig. ). both kinds of secondary structure elements have been implicated in important biological processes such as gene expression and regulation for stem-loops [ ] and pseudoknots [ , ] . we also note that in both stem-loops and pseudoknots, it is necessary to have a stretch of nucleotide sequence (accguc in fig. a and b) followed by its inverted complementary sequence (gacggu) downstream. for simplicity, we shall refer to these kinds of patterns as close inversions. the development of mathematical models and computational prediction algorithms for stem-loop structures based on thermodynamic models started in the s [ , ] . pseudoknots, because of the extra base pairings, must be represented by more complex models and data structures. despite the computing power of supercomputers and emerging advanced technologies, e.g., multi-core architectures, the prediction of secondary structures of long rna sequences (on the order of thousands of nucleotides) based on thermodynamic methods, e.g. [ ] , is still not feasible, especially if the structures include complex secondary structures like pseudoknots. the time and space required for accurate predictions of pseudoknots based on energy minimizations grow very rapidly with the sequence length. fig. shows the time and memory (in logarithmic scale) allocated for the prediction of rna pseudoknots with various lengths using one of the most accurate prediction programs, pknots-re [ ] . the algorithm underlying pknots-re has a runtime and memory demand in the order of n and n , respectively, where n is the length of the input sequence [ ] . the program conducts an exhaustive search for the optimal structure with the lowest free energy and has the capability to predict rather complex structures, even some non-planar structures, for short rna segments up to nucleotides in length. to overcome the tremendous demand on computing resources needed by pseudoknot prediction, alternative algorithms have been proposed (e.g., pknots-rg [ ] , ilm [ ] , and hotknots [ ] ) that tend either to restrict the types of pseudoknots or the length of the secondary structure to be predicted to keep runtime and memory size under control. for instance, the program pknots-rg [ ] limits the types of pseudoknots to simpler structures for longer segments, up to nucleotides. however, a large variety of pseudoknots have been shown to occur in nature. their omission from computational methods may significantly affect the prediction accuracy. furthermore, even the simplified programs are not able to predict secondary structures on the order of thousands of nucleotides. our analysis of the length of pseudoknots in a widely used database known as pseudobase [ ] , which collects pseudoknots, shows that most pseudoknots known to date are formed by rna segments whose lengths are less than nucleotides, i.e., % of the segments in the database have lengths that range between and nucleotides. the range of lengths between (lower quartile) and . (upper quartile) nucleotides covers % of all segments. this observation leads to the idea of overcoming the computing constraints presented above by developing a strategy for cutting long rna sequences into segments not longer than bases in length and distributing the task of structure prediction of each segment to be done simultaneously on different computers. ideally, if two segments that are cut from the same rna sequence overlap each other, then the predicted structures of their overlapping part should be consistent with one another. such consistency is important for the final structure assembly. in preliminary work, we observed that arbitrarily cutting the rna sequence into overlapping segments is not advantageous for consistency. it is well conceivable that when an arbitrary cut goes through the middle of a close inversion, the bases forming the pairings do not get into the same segment, resulting in the omission of the structure from that prediction. for instance, consider a base segment of the severe acute respiratory syndrome (sars) coronavirus genome, which is one of the coronavirus genomes analyzed by chew et al. in [ ] , from position , to , and another segment from position , to , . when the program pknots-re is applied to these segments, two predictions are produced which are shown in fig. . note that, over the stretch of the bases that the two segments overlap one another, the two predictions are different. this kind of inconsistency poses a serious problem when the predicted structures of the segments need to be assembled. if the prediction of secondary structures for long rna sequences is not feasible with thermodynamic methods even with powerful supercomputers, arbitrarily cutting an rna sequence into shorter overlapping segments makes the single segment predictions feasible but not advantageous for consistency, unless the cutting algorithm uses the locations of high concentration of close inversions. in addition, once motifs are predicted from sampled segments, they have still to be assembled together into complete secondary structures. both predictions and rebuilding can significantly benefit from the combined prediction capability of different codes, as opposed to using the single codes separately [ ] . prediction of large numbers of short, overlapping segments is still computationally demanding but it also allows massive task parallelism that can be addressed with grid computing resources. of course, the scientist who uses such an approach of sampling and rebuilding from segments to predict longer secondary structures has to benefit from the computing capabilities of such a framework without being required to cut and paste results from one code output to another, redirecting or reformatting output files (e.g., from fasta to embl format) before forwarding them to the next step in the analysis, or dealing with distributed computer systems. therefore, the ideal computing environment for the scientist should integrate key services and functionalities by combining different mechanisms and programs in an automated, computationally powerful, and reliable cyberinfrastructure accessible through an easy-to-use, familiar interface. these overall needs and key services are integrated in rnavlab (rna virtual laboratory), a virtual laboratory for the study of rna secondary structures. rnavlab addresses the challenges above by combining sampling of nucleotide sequences, predictions based on different codes and supported by grid computing technology, and analysis of large sets of secondary structures with different scientific scopes. scientific scopes include associating stem-loop and pseudoknot types with functions in genomes. in this paper two important case studies using rnavlab are presented: to rebuild secondary structures in longer sequences by systematically sampling nucleotide segments from an rna molecule and assembling the significant motifs found in the secondary structures of the segments (i.e., stem-loops and pseudoknots). the extensive and systematic sampling of nucleotide segments is vital for overcoming the inconsistency outlined above; the computing power needed for the prediction of the numerous segments is provided by grid technology. motifs are identified in the secondary structures of each segment and assembled in a single structure based on their recurrences and statistical insights. to study the association of stem-loop and pseudoknot structures with the viral rna replication mechanisms for the genomes of members of the virus family nodaviridae. in other words, rnavlab helps scientists to address questions such as ''what are the mechanisms of nodavirus rna replication?". the association of predicted secondary structure near the terminus of the rna genome segments from seven members of the nodavirus family with their rna replication mechanisms is targeted. the nodavirus genomes provide an excellent model system for the study of rna replication due to their genetic simplicity, the robust yield of rna replication products, and the ability of the rnas to replicate in cells from a wide variety of organisms. the rest of this paper is organized as follows: section discusses significant related work in the field. section presents the rnavlab virtual laboratory and its components. section shows how to use rnavlab for rebuilding longer rna secondary structures from rna segments and to assist in the study of nodavirus rna replication. section concludes and briefly presents future work. when dealing with rna secondary structures, scientists have several sources of data and tools available. however, to retrieve pieces of information from these sources as well as to sort and elaborate the data with these tools, scientists have to do significant handwork by sorting, computing, merging, and comparing results as well as extrapolating conclusions. for instance, when dealing with pseudoknots, scientists need to access databases such as pseudobase [ ] , which are not always provided with advanced search engines. the data from the database has to be copied into files of different format (e.g., fasta and embl). ultimately the scientists have to download and install codes on platforms that do not always fully support the code execution. even if portable, some of these codes require significant amount of computing power that is not always available on the scientist's pc. an alternative is to submit the retrieved data to portals that provide prediction and visualization functions. however, the portals provide single prediction approaches that force the scientists to find multiple portals for their prediction. ideally scientists should be led through the different steps by a unified, easy-to-use environment that screens them from database issues providing them with powerful tools for search, prediction, analysis, and visualization. while past efforts have been focused on increasing prediction accuracy of sequential rna prediction programs [ , ] and prediction efficiency has been improved by using massively parallel high performance machines [ ] or local clusters [ ] , not much is known about rna prediction systems based on grid computing technology (i.e., heterogeneous, volatile computers, ranging from supercomputers to clusters and pcs connected to the internet, spread out in different locations). previously, grid technology was applied successfully to protein structure prediction [ , ] and similar achievements are expected for rna secondary structure prediction. with their significant computing power, these computing systems allow the scientists to explore larger spaces of rna secondary structures. among the several tools based on thermodynamics that are available for rna secondary structure predictions and analysis, the vienna package [ ] is one of the most well-known packages. it consists of a c code library and several stand-alone programs for the prediction and comparison of rna secondary structures. the stand-alone programs are not integrated in a unified environment and do not address multiple prediction approaches but instead deploy the zuker algorithm [ ] . therefore, the vienna package does not include the prediction and analysis of pseudoknots. last but not least, the package does not integrate grid technology. as an alternative to thermodynamic methods for rna secondary structure prediction, stochastic context free grammars (scfg) have been proposed for secondary structure prediction [ ] . these approaches rely on estimating probability distributions over a set of transformation rules that define how the fold is formed. scfgs have the ability to learn the parameters of a generative model by observing a set of sequences with their corresponding secondary structures. in general, scfgs are outperformed by physics based approaches, although recently do et al. proposed a generalization of scfgs where a flexible and richer feature set allows to include free energy parameters more akin to thermodynamic models [ ] . however, the complexity of the rna secondary structures predicted by these methods is restricted by the expressibility of their grammars, thus highly complex structures, such as pseudoknots, cannot be predicted by scfgs. in addition, the above-mentioned tools are single purpose; they can only be used for secondary structure prediction. for finding consensus motifs that can be associated with rna functionalities, most of the previous work takes as input a set of primary sequences and generates as output the set of structural motifs identified, and the differences lie in the search strategy for identifying common motifs. for instance, work presented in [ , ] uses suffix arrays for efficiently exploring the space of valid secondary structures in their seed method. in seed, the search space is constrained by the seed sequence, which is just one of the sequences in the set used to instantiate valid motifs. seed ranks motifs using a metric that combines the entropy of the segment with the free energy of the secondary structure, as computed by mfold [ ] . this ranking function reached good results and the top motifs had also the highest matthews correlation coefficient [ ] . a drawback of seed is the fact that it is limited to finding patterns in stem regions only, i.e., no loops or pseudoknots can be identified by the seed method. ashlock and schonfeld propose a depth annotation scheme to identify common motifs that uses an evolutionary algorithm to cluster folds by projecting them in a two dimensional euclidean space [ ] . this method can identify pseudoknots by assigning a unique identifier to stems. the intuition behind this approach is that similar folds will be placed closer by the projection algorithm. to identify motifs, we need to analyze the output of the projection. since the method provides a visual representation of the similarity between bricks, it is simple to identify motifs by just looking for clusters. however, as the number of bricks increases, spotting the clusters become less straightforward and we need the help of a clustering algorithm. another shortcoming of this method is that the distances between the pairs of depth annotations depend on a specific size of segment. thus prior knowledge of the sequences is needed in order to define an appropriate window size. there are other approaches to motif finding, see for example [ ] , but most of them give the desired results provided the secondary structure is not complex (no loops or pseudoknots are included), or provided that we have enough prior knowledge regarding the identity of the motifs. on the contrary, our automated method targets motifs that are as general as possible and exhaustively explores the search space of all the sequences of nucleotides. it is a strictly structural method in the sense that, for the experiments presented here, we only looked at the secondary structure predicted by pknots-rg. our preliminary results show that our method can find motifs as simple as small stems and as complex as pseudoknots and loops. with reference to the deployment of rnavlab proposed in this paper, to our knowledge, these existing tools have not been used for such an exhaustive survey of the link between rna secondary structure prediction and nodavirus genome replication. lastly the highly modular design of rnavlab makes it easy to add new tools. the tools described above can then be integrated into the array of tools that already exist in rnavlab. rnavlab has a modular structure that makes it easy to integrate new features. as shown in fig. , rnavlab has three major components: ( ) a segment sampler component (sampling) to sample rna sequence segments guided by simple heuristics and more sophisticated mathematical methods capable of identifying palindrome distributions; ( ) a structure prediction component (prediction) to predict the structures of the sampled segments using different programs on heterogeneous computers; and ( ) a structure analysis component (analysis) to compare and contrast predictions with observed structures as well as identify similarities and other characteristics across predictions. each component, described in more detail in this section, is shown in fig. . rnavlab also includes a database of pseudoknot structures, pseudobase++ and an easy-to-use interface; both the database and the interface are also described in this section. the segment sampler component (sampling) samples overlapping segments in rna sequences and passes them to the structure prediction component for the search of significant motifs. generally, rna segments containing a high concentration of close inversions have greater tendency to form local secondary structures because the symmetry facilitates base pairing required in the formation of stem-loops and pseudoknots [ , ] . currently rnavlab includes two sampling strategies: a windowing strategy and a progressive segmentation strategy. in the windowing sampling approach, each set of segments has a fixed size (window size) and a fixed sliding step (window step). the segments in a set are generated by progressively sliding the fixed-size window forward for a fixed number of steps. at each step, the nucleotides within the window form a segment. for an extensive sampling, this procedure is repeated to generate several sets by increasing the window sizes and/or the window steps, each time generating a new set of segments. in our experiments the window sizes are increased by five bases. the maximum length of a window is n= , where n is the length of the rna sequence we want to rebuild the secondary structure for. window steps range from base to w À bases, where w is the window size. in the progressive sampling approach the user defines a starting point, ending point, and a ''step size"; the sampler generates a series of segments by progressively removing ''step size" bases from the original segment, whose length is defined by the beginning and ending points ( and termini, respectively) given by the user, starting from the beginning point and progressing in a -to- direction. the series of segments with progressively decreasing lengths are forwarded to the prediction component for prediction. segments can be inverted before being forwarded. the extension of this component to employ more sophisticated statistics-based sampling methods using the distributional properties of close inversion on random rna sequences is work in progress. the structure prediction component (prediction) harnesses heterogeneous computing resources across the university of texas at el paso (utep) campus to rebuild rna secondary structures from the segments generated by sampling, using a variety of prediction programs. currently rnavlab supports the following prediction programs: pknots-re [ ] , pknots-rg [ ] , and nupack [ ] . the structure prediction component links the segment sampler component to the structure analysis component. its main functionalities are: ( ) to dispatch jobs provided by the segment sampler to available computing resources across the utep campus and ( ) to identify completed jobs so that results can be forwarded to the structure analysis component. to setup a grid computing environment, rnavlab relies on the condor framework [ ] , for a variety of reasons. condor provides all of the functionalities needed to implement a seamless grid layer that allows for faster prediction of rna secondary structures by harnessing the idle cycles of computers, i.e., workstations and clusters, across the campus. the pool of machines on which rnavlab is currently relying consists of single-, double-, and quad-processor -and -bit machines, and while all these machines run linux (i.e., fedora, kubuntu, suse), condor can also be installed on unix, windows (e.g., , xp), and macintosh (os x) machines, among others. condor handles all the details of sending executable and data files to computing resources and retrieving the computation results. furthermore, condor provides other useful features, such as checkpointing and job migration that only require re-linking the prediction programs used by rnavlab with condor libraries. these features can be very helpful, especially with predictions that take a long time: if the application is interrupted, checkpointing saves the computation's state so it can be resumed later (instead of starting from scratch), and job migration allows a saved state to resume execution in a different machine. rnavlab successfully re-links pknots-re and nupack, but not pknots-rg, due to its use of pthreads. therefore, pknots-rg cannot use checkpointing and job migration, but it can still be dispatched to the computing resources. pknots-rg is usually the fastest of all three programs and therefore checkpointing may not be necessary or even helpful. to interface rnavlab with condor and dispatch jobs, an xml file describing the submitted jobs is generated when the user invokes the use of global resources through the rnavlab interface. each job consists of a unique identifier, the name of the prediction program to be used (e.g., pknots-re, pknots-rg, and nupack), and the command-line parameters (i.e., the input files with the rna sequences). the xml format is converted into a condor submit file format, and condor is used to submit the jobs to the pool. condor also provides the functionality needed to check whether a submitted job has completed execution: this functionality is blocked while the job is running and only returns when the job is completed. once all the jobs are submitted through condor, rnavlab sequentially checks for the completion of jobs. if rnavlab stalls because a job is not finished, it does not stall the other jobs, since they are already on the queue. once jobs are completed, their results are forwarded to the structure analysis component and visualized, if required by the user, on the interface. although the current pool of machines used by rnavlab consists only of linux machines, future work includes its extension to clusters available across the campus as well as the integration and support of boinc (berkeley open infrastructure for network computing) [ ] to allow researchers to deploy desktop and laptop pcs owned by students or administration personnel outside the campus when these computers are idle. work in [ ] shows that adding idle cycles of pcs significantly increases available computing power. the structure analysis component (analysis) evaluates the consistency of the various predictions collected by the structure prediction component. currently a set of tools allows the end-user to perform secondary structure classifications, comparisons, alignments, and motif identification. in general, a motif is a repeated pattern in a set of sequences of nucleotides (primary structure), or in a set of secondary structures. an innovative aspect of this component is that rnavlab deals with secondary structure rather than nucleotide sequences for classification, comparison, alignment, and identification. secondary structures are considered in terms of strings of brackets, i.e., ''(" and '')", ''[" and '']", ''{" and ''}", dots ''.", and colons '':". two paired nucleotides are represented with a pair of brackets collocated in the string at the same positions as the related nucleotides in the input segment. with reference to the classification of secondary structures and more in particular of pseudoknots, the pseudoviewer classification tool deploys the classification of pseudoknots in [ ] that clusters them into six different simple types, i.e., h-, hh-, hhh-, hl_out-, hl_in-, and ll_in-type. note that ''h" means hairpin loop, ''l" means bulge loop, ''in" means internal loop or multiple internal loops, and ''out" means external loop or multiple external loops. the tool for classification works on the string of brackets to extract the proper type. fig. shows the six pseudoknot types. comparisons of observed and predicted structures, or across predicted structures using different programs are performed on aligned or non-aligned strings of brackets. three different algorithms can be used for comparisons: a variant of the hamilton algorithm (hamiltonian comparison) -the algorithm assigns each kind of nucleotide bond a numerical tag. bonds gc, cg, ua, and au are assigned tags from to , respectively. the closures of the bonds are all assigned . the resulting numerical strings are compared and when two non-zero numbers and their respective closing match, it is counted as a true pair. this approach is useful when the types of nucleotide bonds are important. fig. shows an example of comparison of a predicted string (fig. a) with an observed string (fig. b ) by using the variant of the hamilton algorithm. the two strings of brackets associated with the prediction and observed secondary structures are converted to two strings of numbers in fig. c . the strings of numbers are compared and the related sensitivity and selectivity are computed. here, high sensitivity expresses the ability to predict all observed pairs and high selectivity expresses the ability to only predict observed pairs. a stack based algorithm (comp. stack) -the algorithm uses stacks to keep track of the positions of open parenthesis and brackets in secondary structures with and without pseudoknots. when an open bracket or parenthesis is encountered, its position is pushed into a stack associated to a stem-loop. pseudoknots are considered to be a combination of two stemloops and therefore use two stacks. when a closed bracket or parenthesis is encountered, the position is popped from the associated stack. if a bracket or parenthesis is encountered at the same time in both structures and the position popped from both stacks is the same, this is counted as a true pair. this approach is useful when the identification of exactly alike structures is important. fig. shows two examples of comparison with this approach: in fig. a the secondary structures are very similar but the bonding nucleotides are shifted and therefore, the comparison has sensitivity and selectivity equal to zero. in fig. b , three bonds in the first string have the same position as three bonds in the second string and this matching results in a higher score for this example in terms of sensitivity and selectivity. a lenient algorithm (comp. oc -open-close parenthesis) -the algorithm uses simple string comparisons that allows for similar yet shifted structures to receive high comparison scores. the algorithm traverses two bracket strings and counts how many times an open bracket or parenthesis is in the same position and how many times a closed bracket or parenthesis is in the same position for the strings, without considering the type of nucleotides involved. the smaller of these two values is the amount of true pairs. fig. compares the two strings already compared in fig. a with the stack based algorithm. by using the lenient algorithm, higher sensitivity and selectivity are achieved. the alignment of two or more secondary structures is performed by aligning their bracket strings using variants of wellknown alignment algorithms such as the smith-waterman [ ] and needleman-wunsch [ ] algorithms. unlike the aforementioned algorithms that align string of nucleotides, i.e., ''a", ''u", ''c", and ''g", the variant algorithms align strings of brackets, i.e., '':", ''(", and ''[". shifts are indicated with an underscore, i.e., ''_". the alignment of secondary structure is very important to identify secondary structures that are similar in their shape but are shifted: this can happen when e.g., a predicted structure is compared with an experimentally observed structure or when the genome structures of two viruses belonging to the same family are compared. fig. shows an example of how the alignment with the variant of the smith-waterman algorithm can improve comparison sensitivity and selectivity. fig. a shows the comparison of two strings that have not been a priori aligned; fig. b shows the comparison of the same strings once they have been aligned. the alignment allows for the identification of the two similar structures and affects the sensitivity and selectivity by increasing their final values. the motif identifier tool performs the identification of common motifs that can be ultimately used for: ( ) rebuilding large secondary structures from smaller ones belonging to overlapping rna segments of the same virus; ( ) quantifying the capability of the several prediction programs to capture secondary structures that have been experimentally observed; and ( ) classifying unknown viruses by matching common motifs present in their rna with similar motifs in viruses belonging to a known family. the tool proceeds as follows: first it identifies all the valid secondary structures, from the most simple (e.g., a hairpin comprising a few base pairs) to the most complex (e.g., pseudoknots), that can be generated from the input of secondary structures. then by using an associative array of linked lists, the tool finds and stores the locations of each substructure generated in the previous step. to narrow down the number of motifs and identify the most relevant ones, ranking techniques are applied. ranking criteria include: the frequency of the motif over the maximal number of possible occurrences, the number of bonding nucleotides, the length of the secondary structure, and the motif location in the rna segment. other possible ranking criteria can include information of the primary structure such as the percentage of bases correctly matched, and/or free energy of the structure as in [ ] . in rnavlab, we score motifs based on their frequency ðf Þ, number of base pairs ðsÞ, and the length of the overlapping region ðoÞ: the simple intuitive motivation behind this scoring function is that more accurate secondary structures are more likely to be predicted in overlapping segments with higher frequency. to assemble the significant motifs and rebuild the final secondary structure out of the segment motifs, we use the scoring function above. we project motifs, in descending order according to their score, into a final structure until there are no more mutually exclusive motifs in the set. in other words, we only project different motifs found in segments when they do not overlap with each other. as part of the rebuilding algorithm, we also define the minimum frequency that a motif present in overlapping segments has to meet in order to be projected in the resulting rebuilt sequence (threshold). threshold values can range from to . finally, we compute the energy of the rebuilt structures as a whole by using the same energy algorithm used in pknots-rg and nupack. fig. shows the pseudocode of the tool. in contrast to other approaches for finding common motifs described in section , the motif identifier tool finds strictly structural motifs, but it only looks for motifs composed of the folds defined in the initial set: the search space explored is defined by all the valid secondary structures in the set and the tool does not use any information from the primary structures. the algorithm in fig. was inspired by previous work such as [ , , ] . a key difference is the lack of an ad hoc design: no prior knowledge is assumed about existing motifs and the goal is to discover the motifs that are more likely to be part of the native structure. the algorithm of the motif identifier is simple but particularly powerful because it allows identifying complex structures such as pseudoknots. lastly, since the design is modular new features such as different rank- ing functions can be easily added. the different tools in the analysis component can be combined to perform more complex operations on the secondary structures. for instance, motifs that have been identified by the motif identifier can be aligned and compared by any of the alignment and comparison tools. information on the consistency of the prediction results collected can be fed back to the database and made available to the segment sampler component to adjust the sampling strategy and adaptively identify new rna segments for predictions. pseudobase++ (http://pseudobaseplusplus.utep.edu) is part of rnavlab and includes a database of pseudoknots, a search engine to access the data, and a user interface to select, visualize and insert new data through any web-browser. the database is an extension of pseudobase [ ] : it contains the data related to pseudoknots that is already provided in pseudobase and other additional data that enriches the information associated to each pseudoknot entry. pseudobase++ is currently focusing on pseudoknot structures. the primary source of data in pseudobase++ is pseudobase: structures are from this database. the rnavlab user interface is designed and implemented around the rnavlab computational environment with a strong focus on compatibility. the implementation idea is also derived from the key concept of encompassing high functionality within a simple but comprehensive interface. by maintaining these two design concepts throughout the process of implementation, the resulting application successfully supports this easy-to-use interface with the rich functionality provided by rnavlab. the interface is developed in java, thus in nature, preventing any operating system dependencies. the rnavlab interface is designed to accommodate the visual structure of a basic media player. this is to create an immediate familiarity for the users as well as maintain a simple comprehensive infrastructure. the interface includes four tabs: database, tools, full results, and previous results. the database tab is a representation of rna secondary structures in the database. the tools tab is divided into two main sections. one of the sections maintains total functionality with visual representations for the database of sequences, results pertaining to the user's requests, and previously obtained results. this section is conveniently organized with a tabled panel allowing quick access to any sub-section (fig. , section on the left). the other main section of the interface provides a constant representation of all concurrent processes being performed by the user as well as a list of each of these processes once they are completed (fig. , section on the right). by selecting any of these processes, the results pertaining to that particular process are displayed conveniently on an information panel located just below in the section on the right in the information window. though the results are saved and displayed in the full result tab and previous results tab, the visualization in the information window is implemented to allow direct comparisons to several outcomes simultaneously. the interface is capable of supporting two different types of processes through the tools tab, one of sampling and prediction and the other of analysis. each process has its own convenient selection section with easy checkbox and radio-button options. each section maintains a copy of the database of sequences with independent sub-lists designated to separate desired sequences or groups of sequences. the first section on the top of the tools tab allows the sampling and prediction with pknots-re, pknots-rg, and nupack either locally (on the local machine) or globally (across the utep campus machines) in accordance to the particular selected sequences or groups of sequences. reversing and sampling the resulting sequences and classifying the resulting pseudoknot types are also sub-options included in this section. the second section on the bottom of the tools tab allows the user to analyze selected secondary sequences using the tools described in section . . the tools can be used simultaneously in different combinations. we present two case studies that address the potentials of rnavlab from two different points of view. in section . , we present a study in which we address two important analysis components of rnavlab. first, we quantify its capability to capture the secondary structure observed experimentally. we compare performance and accuracy (in terms of sensitivity and selectivity) of the rnavlab rebuilding algorithm based on nucleotide segments against a traditional algorithm using the same prediction code and the entire sequence. second, we statistically quantify the effectiveness of the rnavlab naive approach for sampling nucleotide segments and we measure whether the extensive sampling and predictions can compensate for the fact that no attention is paid to the type of nucleotides in the segments, i.e., whether or not there are palindromic sequences. in section . , we present a second case study in which we show how rnavlab can be used for studying the correlation between viral rna replication mechanisms used by members of the nodavirus family and the secondary structures adopted by the ends of their rna segments, which are hypothesized to play a role in the initiation of complementary strand synthesis during rna replication. secondary structures for long rna sequences i.e., on the order of thousands of nucleotides, that have been experimentally validated are rare. when available, our method can deal with the prediction of these sequences but other methods that predict secondary structures using the entire sequence as a whole cannot, making a comparison between the two approaches infeasible. therefore, for our analysis in this paper we used the longest nucleotide sequences from group a in [ ] that have lengths ranging from to bases and are still predictable as a whole by the pknots-rg code. note that since we are not considering the exact same set as in [ ] , we cannot perform a direct comparison against those results. the sampling, motif identification, and rebuilding were executed on the rnavlab server. window sizes, window steps, and thresholds used in the experiments are defined in the previous section. the predictions were performed on a -node cluster (each node consists of amd opteron processors running at ghz with gbyte of ram and a local gbyte hard disk) that is part of the on-campus grid resources of rnavlab. the accuracy of predictions is measured in terms of sensitivity (i.e., ability to predict all true pairs) and selectivity (i.e., ability to only predict true pairs). predictions are compared with the experimental secondary structures provided in [ ] . in fig. , we present a summary of the accuracies: the oracle or upper bound on sensitivity and selectivity for our method (rebuilt sen. and rebuilt sel.) is compared with the sensitivity and selectivity of pknots-rg when considering the entire sequence for prediction (pred. sen. and pred. sel.) as well as the sensitivity and selectivity achieved by our algorithm when selecting those structures with the lowest free energy (min en., min en. sen., and min en. sel.). since we are using pknots-rg for the prediction of the segments, intuitively we would expect our algorithm to achieve results that are at most equally accurate as those achieved by this prediction code when predicting the whole sequence. however, because we are allowing the prediction of segments starting at different positions in the primary structure, our method can find structures that are very different from those predicted by the code on the entire sequence. out of the total sequences presented in the figure, the oracle outperformed pknots-rg on sensitivity and/or selectivity for sequences (see bold values in the figure) . however, the selection criteria based on the minimum free energy (min en. sen. and min en. sel.) are not as accurate: only in out of the cases did these criteria yield better or equal results than pknots-rg. the results of our rebuilding algorithm are promising, especially considering the potential of our approach in overcoming the limitations of current prediction methods on the length and complexity of the sequences. currently, the most salient weakness of our method involves the selection of the final rebuilt structure. the minimum free energy is not by itself a good factor for selection, probably due to what is already common belief that native structures will often be near-optimal in terms of the minimum free energy. in cutting an rna sequence into segments of overlapping sequences, we experimented with various window sizes and window step sizes. in rebuilding the overall structures from the segments, different threshold values were used. we noticed that as values of these parameters vary, the overall accuracy, measured by sensitivity and selectivity of the rebuilt structures, also changes. in order to check whether any significant systematic relationship exists between the accuracy of the rebuilt structures and the parameters, we carried out a multiple regression analysis on each of the sequences in table with sensitivity and selectivity as response variables and (window size, window step, threshold) as prediction variables. in all except one sequence, both sensitivity and selectivity are significantly (p-value < . ) related to the three prediction variables. both response variables correlate positively with window size, but negatively with window step and threshold. the positive correlation with window size agrees with our expectation that having a larger sequence segment, which constitutes a larger portion of the whole rna molecule, in a single prediction should generally be beneficial to the accuracy of the rebuilt structure. on the other hand, a larger window step would mean that successive sequence segments overlap less with each other, so that it is easier to miss those secondary structures spanning both segments but not captured within either one, resulting in the negative correlation with the window step parameter. the negative correlation of threshold with structure accuracy implies that every motif detected in a sequence segment should be taken into account in the rebuilt structure. a very strong positive correlation between sensitivity and selectivity (correlation coefficient > . ) has been detected in each of the sequences while the prediction variables are being varied. this suggests that our structure rebuilding approach can be made highly effective simultaneously in both measures of accuracy. it is also interesting to note that the minimum free energy of a rebuilt structure generally shows a negative correlation with sensitivity, but a positive correlation with selectivity, suggesting that the minimum free energy does not necessarily reflect the accuracy of the rebuilt structure. while the minimum free energy is the quantity used pervasively in many secondary structure prediction algorithms for determining the identity of the optimal structure, there seems to be a necessity for seeking an alternative measure. for testing the usability of rnavlab, we considered as our biological system several members of the virus family nodaviridae collectively known as the nodaviruses, a family of tiny, icosahedral viruses with bipartite, single-stranded rna genomes. the abundant replication and small genomes of these viruses have made them attractive models for the study of virus structure, virus assembly, and rna replication. the family nodaviridae is comprised of two genera: alphanodaviruses and betanodaviruses. while the betanodaviruses have been isolated only from fish, the alphanodaviruses infect predominantly insects; the alphanodavirus nodamura virus (nov) also infects mice. other members of the alphanodavirus genus include black beetle virus (bbv), boolarra virus (bov), flock house virus (fhv), and pariacoto virus (pav). the betanodavirus genus includes many members, including the type species of the genus, striped jack nervous necrosis virus (sjnnv), and greasy grouper nervous necrosis virus (ggnnv). these seven viruses were selected here for further study on the basis of the availability of cdna clones of their genomic rnas, reagents that will enable us to perform functional assays to determine whether the predicted rna structures play a role in the viral life cycle. table shows the abbreviation of the seven viruses, their lengths in terms of nucleotides (nt), and the hosts from which the viruses were isolated, i.e., nov [ ] , bbv [ ] , bov [ ] , fhv [ ] , pav [ ] , sjnnv [ ] , and ggnnv [ ] . the nodavirus genome is divided into two segments of positive-strand rna: rna encodes the viral rna-dependent rna polymerase (rdrp) that replicates both genomic segments, while rna encodes the precursor to the protein that comprises the viral outer coat (capsid) [ ] . a small subgenomic rna , which is not encapsidated into viral particles, encodes a protein that suppresses host defense mechanisms like rna interference. during viral rna replication, the genomic rna is copied first into a complementary negative strand, which is then used as a template for further positive-strand synthesis. the role of rna secondary structure in the genome replication of other rna viruses, e.g., members of the plant tombusvirus, potexvirus, and bromovirus families and the animal picornavirus, coronavirus, and flavivirus families, has been well established in the literature. for example, the rdrp of brome mosaic virus initiates negative strand synthesis at a trna-like structure at the end of the positive-sense rna template [ ] . for the nodavirus family, kaesberg et al. [ ] had previously used the method of zuker and stiegler [ ] to perform rna secondary structure analysis on the noncoding regions of genomic rna and rna of four nodaviruses (bbv, fhv, nov, and bov). this method was able only to predict simple stem-loop structures and not pseudoknots. these authors predicted the presence of stem-loop structures near the terminus of rna for each of these viruses. however, the cloning and sequencing of three additional nodavirus genomes allowed us to revisit the issue of nodavirus rna secondary structures and technical advances in the field allow us to test the effect of secondary structure on genome replication in cultured cells. the role of secondary structure on nodavirus rna replication has been studied previously for only one member, flock house virus. a long-range interaction between two regions of rna is required for synthesis of subgenomic rna [ ] . the results of genetic experiments suggest that a similar long-range interaction may be also required for synthesis of the rna of another member of the family, nodamura virus (nov) as well [ ] . however, the role of rna secondary structure in replication of nodavirus genomic rnas remains unclear. defining this role is crucial to understanding the mechanism of nodavirus rna replication. the predictive approaches used in rnavlab will greatly facilitate our molecular studies by providing a ''road map" to elements of possible structural importance, allowing these sequences to be targeted by site-directed mutagenesis. previous studies with fhv showed that sequences at the end of rna , particularly within the terminal nucleotides, were critical for rna replication, and could direct replication of chimeric rnas that contained heterologous core sequences flanked by rna sequences [ ] . by replacing the center of rna with the same heterologous sequence, the work in [ ] created a family of rna molecules that differed only at their termini. the different properties of these molecules could be therefore confidently attributed to these termini. this system established a uniform assay for the different rnas, using a single probe to the common central core region for northern blot hybridization experiments. since such chimeric rna molecules replicate efficiently, they provide an ideal model substrate for secondary structure prediction and analysis. we used rnavlab to computationally predict and identify secondary structure motifs in the terminal nucleotides in the end of rna that could potentially be critical for rna replication [ ] . we analyzed predicted rna secondary structures of progressively shorter lengths from the end of rna from the seven members of the nodavirus family presented in table . our goal was to identify common motifs across samples, code predictions, and viruses. due to the dynamic nature of the prediction programs, the final secondary structures are heavily dependent on neighboring structures: having a certain substructure present in all the predictions, independently from the starting and ending points of the segments, may indicate a strong base pairing that ultimately may be present in nature. for each virus, the terminal nucleotides of the end of rna were sampled using the progressive segmentation strategy with a step size of nucleotides. the three different prediction programs currently available in rnavlab were used, i.e., pknots-rg, pknots-re, and nupack. all the final predictions, obtained from genome segments from different viruses, with different lengths, and different prediction programs, were processed by the tool in the structure analysis component that allows for identifying common motifs, i.e., pseudoknots or stem-loops, and then were aligned for identification of shifted and overlapping structures. the prediction time for the several secondary structures ranged from several hours for long segments predicted using pknots-re to a couple of seconds for short segments predicted using pknots-rg and nupack. the predictions were performed in parallel across the pool of machines managed by condor. for each of the seven viruses, sample segments per virus were processed for prediction using the three codes, for a total of predictions. within the predictions, common motifs were found, ranging from simple motif with a single bond to more complex structures such as pseudoknots. to reduce the number of motifs and identify the most significant ones, overlapping motifs were merged into canonical motifs. overlapping motifs are those contained within larger motifs in terms of nucleotide length and number of bonds but with the same prediction frequency for the same prediction codes and viral genomes. moreover, simpler motifs, i.e., hairpins with fewer than base pairs, and less frequent motifs, i.e., motifs that were predicted by a single code or had a frequency below %, were not considered. the frequency of a motif is measured as the number of times the motif was indeed predicted over the maximum number of times the motif could be predicted by the three codes in samples with a suitable length to accommodate the motif length and its starting portion. this (((((((::::::::::))))))) bbv . , , (((((::::))))) bbv . , , (((((::::::))))) bov . , , ((((((::::)))))) bov approach reduced the number of common motifs to a set of motifs shown in table . for each virus, table presents the motif length, the number of nucleotide bonds, the frequency of the predictions across codes and samples, the number of predictions per code (where e stands for pknots-re, r stands for pknots-rg, and n stands for nupack), and the motif secondary structures in terms of brackets. the alignment of the motifs allows for rebuilding larger motifs. figs. - show the output of the rnavlab analysis (a) and the pseudoviewer representation of the rna terminal part for each virus with the common motif that has resulted from the analysis of the several predictions (b). for nov (fig. ) and bbv (fig. ) , rnavlab consistently predicts the presence of stem-loop structures near the end of rna . in particular, for nov a hairpin is predicted from nt to nt , within the last nucleotides of rna . for segments shorter than nucleotides, the stem-loop is no longer predicted. for bbv, we predict two hairpins within the last nucleotides: from nt to nt and from nt to nt , respectively. for both sjnnv (fig. ) and ggnnv (fig. ), rnavlab identifies a pseudoknot followed by a hairpin at the terminal part of their rna segment: while the pseudoknot is identical, the terminal hairpin in sjnnv resembles the hairpin in ggnnv but it has an additional pair of nucleotides. a similar pseudoknot followed by a hairpin is also found at the end of bov (fig. ) and at the terminal part of pav (fig. ) , although for the latter virus, this structure is followed by a tail of nucleotides that is predicted to fold into a variety of hairpins of different sizes that vary with the program used. a pseudoknot is found at the very end of fhv (fig. ) together with a hairpin that is located inside the pseudoknot to form an hl-out type pseudoknot. these results found with rnavlab are consistent with the work conducted experimentally in [ ] . we are currently studying whether the pseudoknots and stem-loops are indeed critical structures that drive the genome replication of these viruses. to address this critical question we are combining computational and experimental methods. driven by the computational results presented in this paper, we will ultimately address the question of whether the biological relevance of the pseudoknots and stem-loops can indeed be experimentally verified. this will be accomplished using two approaches. first, to determine whether the predicted structures can be verified in solution, we will generate a nuclease map using single-and double-strand-specific ribonucleases (rnases) as described by tuplin et al. in [ ] . viral rna will be transcribed in vitro and treated with rnases a, t , and v , to cleave the end of single-stranded uracils and cytosines, the end of single-stranded guanines, and double-stranded nucleotides, respectively. a labeled primer will be used for reverse-transcription of the digested rnas and sequencing of the undigested cdna transcription templates. this technique will allow us to confirm or refute predicted structural elements. second, to test the role of these predicted structures in the viral life cycle, we will use a process called reverse genetics. because it is technically difficult to make changes to rna directly, we will use cloned complementary dna (cdna) copies of the viral genomic rna instead. we will use site-directed mutagenesis to make specific deletions and substitutions of nucleotides within the predicted structural elements. the altered cdnas will be introduced into cultured cells, where they will be transcribed into rna by cellular dna-dependent rna polymerases. these primary rna transcripts will then be able to replicate in the presence of the viral rna-dependent rna polymerase (rdrp). therefore, we will test the effect of the rna mutations by transfecting the mutant form into cells together with rna as a source of rdrp. following incubation to allow the viral rna to replicate, we will isolate total cellular rna from the cells and assay for the presence of rna replication products (negative strand intermediates of rna and rna , together with production of subgenomic rna ) using strand-specific northern blot hybridization. for example, to test the function of the predicted stem-loop near the end of nov rna , we could delete it altogether or change specific nucleotides to disrupt base pairing interactions within the predicted stem. if these structures are important for viral rna replication, we will observe a decrease in the level of replication products detected (relative to wild-type rna controls). together these methods will allow us to experimentally test whether the structures we predict in the rnavlab are physically present in the viral rnas and whether they have functional significance in the viral life cycle. rnavlab is a virtual laboratory that facilitates the study of rna secondary structures, i.e., prediction, alignment, comparison, identification, and classification of common secondary structures motifs across viruses, through an automated, compu- tationally powerful approach: the scientist's intervention is minimized and grid computing technologies are used to address computing intensive tasks such as the prediction of long rna secondary structures including pseudoknots. in this paper, rnavlab is used for rebuilding long secondary structures from significant motifs in rna segments as well as the computational study of mechanisms that guide rna replication in the virus family nodaviridae. for of the rna sequences with different lengths that we used for the validation of our rebuilding approach, we obtained more accurate (either in terms of sensitivity or selectivity or both) rebuilt structures than those predicted by the same code applied to the whole sequences. the regression analysis results indicated that ( ) there is a significant relationship between the accuracy of the rebuilt structure (in terms of sensitivity and selectivity) and the sampling factors (i.e., window size, window step, and threshold values); ( ) our method equally targets both the measurements of accuracy, i.e., sensitivity and selectivity; and ( ) the minimum free energy cannot be trusted by itself as a quality measure of the secondary structure. by predicting rna secondary structures of progressively shorter lengths from the end on the nodamura virus rna , rnavlab indicates that, across prediction programs and with different sampled segments, a hairpin structure from nt to nt is consistently predicted in the terminal segment of the rna for the nodamura virus and two hairpins are predicted for the black beetle virus, from nt to nt and from nt to nt , respectively. these results for nov and bbv are consistent with the prediction described in [ ] except that their prediction included a different fold for bbv nt - . similar secondary structures, i.e., a pseudoknot followed by a hairpin, are predicted in the other members of the family, i.e., boolarra virus, pariacoto virus, striped jack nervous necrosis virus, greasy grouper nervous necrosis virus. only in the flock house virus the pseudoknot includes the hairpin. ongoing work includes studying whether the computational findings in both the case studies can indeed be experimentally verified. if this is the case, rnavlab will be a powerful tool to drive molecular studies by providing a ''road map" to elements of possible structural importance, allowing these sequences to be targeted by site-directed mutagenesis. mechanisms and enzymes involved in sars coronavirus genome expression stem-loop structures in prokaryotic genomes an atypical rna pseudoknot simulator and an upstream attenuation signal for À ribosomal frameshifting of sars coronavirus a pseudoknot in the non-core region of the glms ribozyme enhances self-cleavage activity simultaneous solution of the rna folding, alignment, and protosequence problems computer prediction of rna structure algorithms and thermodynamics for rna secondary structure prediction: a practical guide a dynamic programming algorithm for rna structure prediction including pseudoknots design, implementation, and evaluation of a practical pseudoknot folding algorithm based on thermodynamics ilm: a web server for predicting rna secondary structures with pseudoknots hotknots: heuristic prediction of rna secondary structures including pseudoknots pseudobase: a database with rna pseudoknots palindromes in sars and other coronaviruses comppknots: a framework for parallel prediction and comparison of rna secondary structures with pseudoknots an algorithm for comparing multiple rna secondary structures comparing multiple rna secondary structures using tree comparisons predictor@home: a protein structure prediction supercomputer based on global computing simulation of folding of a small alpha-helical protein in atomistic detail using worldwide distributed computing vienna rna secondary structure server rna secondary structure prediction using stochastic context-free grammars and evolutionary history contrafold: rna secondary structure prediction without physics-based models exploring the space of rna secondary structure motifs using suffix arrays identification of consensus rna secondary structures using suffix arrays comparison of the predicted and observed secondary structure of t phage lysozyme depth annotation of rna folds for secondary structure motif search rna motif search using the structure to string str method nonrandom clusters of palindromes in herpesvirus genomes nupack: a software suite for the analysis and design of nucleic acids distributed computing in practice: the condor experience boinc: a system for public-resource computing and storage pseudoviewer : visualization of rna pseudoknots of any type identification of common molecular subsequences a general method applicable to the search for similarities in the amino acid sequences of two proteins an approach to selecting putative rna motifs using mdl principle nodamura virus from japan: a new and unusual arbovirus resistant to diethyl ether and chloroform a small rna virus with a divided genome from heteronychus arator (f.) [coleoptera: scarabaeidae boolarra virus: a member of the nodaviridae isolated from oncopera intricoides (lepidoptera: hepialidae) flock house virus: a nodavirus isolated from costelytra zealandica (white) (coleoptera: scarabaeidae) a noda-like virus isolated from the sweet potato pest spodoptera eridania (cramer) (lepidoptera: noctuidae) properties of a new virus belonging to nodaviridae found in larval striped jack (pseudocaranx dentex) with nervous necrosis determination of the complete nucleotide sequences of rna and rna from greasy grouper (epinephelus tauvina) nervous necrosis virus, singapore strain virus taxonomy, eighth report of the international committee on taxonomy of viruses minus-strand initiation by brome mosaic virus replicase within the trna-like structure of native and modified rna templates structural homology among four nodaviruses as deduced by sequencing and x-ray crystallography optimal computer folding of large rna sequences using thermodynamics and auxiliary information long-distance base pairing in flock house virus rna regulates subgenomic rna synthesis and rna replication recovery of infectivity from cdna clones of nodamura virus and identification of small nonstructural proteins the cis-acting replication signal at the end of flock house virus rna is rna -dependent detailed mapping of rna secondary structures in core and ns b-encoding region sequences of hepatitis c virus by rnase cleavage and novel bioinformatic prediction methods key: cord- - oq n af authors: liu, zhi-ping; wu, ling-yun; wang, yong; zhang, xiang-sun; chen, luonan title: bridging protein local structures and protein functions date: - - journal: amino acids doi: . /s - - - sha: doc_id: cord_uid: oq n af one of the major goals of molecular and evolutionary biology is to understand the functions of proteins by extracting functional information from protein sequences, structures and interactions. in this review, we summarize the repertoire of methods currently being applied and report recent progress in the field of in silico annotation of protein function based on the accumulation of vast amounts of sequence and structure data. in particular, we emphasize the newly developed structure-based methods, which are able to identify locally structural motifs and reveal their relationship with protein functions. these methods include computational tools to identify the structural motifs and reveal the strong relationship between these pre-computed local structures and protein functions. we also discuss remaining problems and possible directions for this exciting and challenging area. dna sequences can be called 'the blueprint of life', while proteins represent the fulfillment of this blueprint in terms of structures and functions. a fundamental goal of functional genomics research is to understand how proteins carry out functions in a living cell (eisenberg et al. ; brenner ; goldsmith-fischman and honig ) . in addition to experimental methods, computational methods have been extensively applied with the aim of developing hypotheses in terms of assigning specific functions to specific proteins and providing valuable biological insights. the basic rationale behind such research is that the gene sequence determines the amino acid sequence, and the amino acid sequence determines the protein structure, which, in turn, determines the protein function (whisstock and lesk ) . many proteins, even among those in the protein data bank (pdb), have not yet been annotated, although we have succeeded in deriving their structures watson et al. ) . we review here the in silico annotation methods currently used to determine protein function from protein local structures. generally speaking, proteins are the main catalysts, structure components, signal transfers and molecular machines in a biological organism. as such, they are the basic elements of functions. however, the definition of function means different things to different people since it is an evolving concept associated to an abundance of interpretations. in general, these functions can be described at many levels, ranging from the biochemical functions at the molecular level (e.g. catalytic or binding activities) to biological processes at the level of biomolecular cooperation (e.g. signal transduction or cellular physiological process) to the cellular components at the cell level of an organ (e.g. nucleus or rough endoplasmic) (devos and valencia ; watson et al. ) . several schemes/tools/ databases have been developed in recent decades for measuring protein functions in a systematic model with the aim of annotating the functions of proteins ; these include ec (barrett ) , mips (ruepp et al. ) , go (the gene ontology consortium ; camon et al. ) and kegg (kanehisa and goto ) , as shown in table . using the existing function annotations as 'gold standard' data, researchers have been able to develop many protein function annotation methods in recent years based on protein relationships. we summarize the existing function annotation methods in the framework of fig. , which shows the basic tendency for the functional inference methodology-i.e. to explore sequence similarity, structure similarity, protein interaction and their integration. we briefly review these in the following list: • using sequence information. the methods in this category often utilize a blast, fasta or psi-blast score to detect the sequence similarity and annotate the functions to a target protein from its homologous protein (whisstock and lesk ; watson et al. ) . in the safe zone (rost ) of sequence similarity, the sequence-based methods can provide putative annotations with high confidence (wilson et al. ) . a number of papers have tested the global performance between the relationship of the sequence similarity and function similarity. shah and hunter ( ) tested the sequence similarity among enzymes in many ec classes at various thresholds and concluded that the functional similarity could not be detected perfectly when the sequences are not similar enough. wilson et al. ( ) and devos and valencia ( ) obtained similar results. joshi and xu ( ) presented a systematic analysis on the sequencefunction relationships in four model organisms. • using structure information. protein structures are more conserved than protein sequences (orengo et al. ; hou et al. ) . a number of methods have been developed with the aim of assessing protein structure similarity (kolodny et al. ) ; these can be grouped as coordinate-based [such as structal (gerstein and levitt ) , samo , tm-align (zhang and skolnick ) and prosup (lackner et al. ) ], distance-matrix-based [such as dali (holm and sander ) , ce (shindyalov and bourne ) , fatcat (ye and godzik ) , ssap (orengo and taylor ) ] and secondary-structure-based [such as vast (gibrat et al. ) , ssm (krissinel and henrick ) , lock (singh and brutlag ) and fast (zhu and weng ) ]. classifying the proteins into different classes or families based on global structure similarity will assist researchers in determining the relationships among different proteins and provide a foundation of functional organization (brenner ) . scop (murzin et al. ) , cath (orengo et al. ) and fssp (holm and sander ) comprehensively cluster all proteins with known structures. based on the safe zone means that pairwise sequence identity is higher than %, the twilight zone, about - %, the midnight zone below % those clusters, the functional relationships among the proteins can be roughly detected. • using interactome information. proteins always interact with other molecules to carry out their functions (sharan et al. ). information on protein-protein interactions or other interaction maps among molecules, such as dna binding with protein, can be explored to annotate the protein functions from complexes and pathways of the biochemical processes. the network-based methods extend the functional inference from the single molecular level to a systematic level by considering interactions among genetic components and transferring functions among them (vazquez et al. ; barabasi and oltvai ; zhang et al. ). sharan et al. ( ) cataloged the methods to direct methods and module-assisted methods individually. • using integrated information. another sensible strategy is to use many different data sources to increase the chances of obtaining function annotations for any given protein. for example, in marcotte et al. ( ) , proteins are grouped by experimental data, such as metabolic function, phylogenetic profiles, rosetta stone results and correlated messenger rna expression patterns to determine the functional relationships among proteins of the yeast. in fact, many methods are in this framework (sanishvili et al. ; george et al. ; pal and eisenberg ; zhao et al. a, b) , especially when data integration becomes the focus of the systems biology study. in this review we highlight the relationships between protein local structures and protein functions since it is commonly believed that local regions on the structures are responsible for the performance of the particular functional tasks (russell ; ferre et al. ) . well-known examples include the ser-his-asp triad in enzymes and other known special structural frameworks that carry out certain functions of catalysis (torrance et al. ) . it is now widely recognized that some fold similarities suggest an 'analogous' rather than a 'homologous' relationship (russell ) . proteins can adopt similar tertiary folds while performing different functions at different binding site locations. given the existing status that the midnight zone functional linkages escape from the sequence and global structure similarity, only the local structures can be used to analyze detailed relationships with functions by determining the protein-protein interaction, protein-dna interaction or other global performance from the physical perspective. also, the local structures of protein provide more detail information on protein function not only from the single targeted action of that protein, but also from the integrative process due to the detailed components and the three-dimensional architecture. the local structures are also important in the design of drugs and bioengineering. in an interesting paper, schnell and chou ( ) convincingly provided nuclear magnetic resonance (nmr) data showing that the m proton channel of influenza a virus is typically controlled by the local conformational change with a phgated mechanism. the discovery provides sound evidence that the local structures are crucial for determining protein function, and it is vitally important in the search for effective anti-influenza drugs (borman ) . bridging protein local structures and protein functions can timely provide useful information for structure-based drug design [e.g. see the methods in chou et al. ( ) and wang et al. ( a) against severe acute respiratory syndrome (sars), and that in du et al. ( ) against chicken influenza a virus h n , as well as a review paper (chou ) ]. thus, it a key task of researchers in this field is to investigate the relationships between protein functions and protein local structures. this review is organized into four parts. first, we will describe the main molecular functions related to protein local structures. this is followed by a description of existing definitions and methods for detecting similarities in local structures. in the third part, the detailed methodologies to bridge local structures with functions are reviewed. some discussion and future directions are summarized in the last part. to bridge the relationship between local structures and functions, we first catalog the molecular functions of proteins strongly related to local structures. the local structures are often regarded as the protein-protein interfaces, catalytic sites, ligand-binding sites, metal-binding sites, post-translational modification sites or other miscellaneous active sites. table lists some of the important functional categories (chakrabarti and lanczycki ) . a protein generally interacts with other proteins in performing and regulating many processes in a cell. the pace of discovery of protein-protein interactions has recently accelerated due to rapid advances in new technologies (salwinski and eisenberg ; chou and cai ) . the basis of protein-protein interactions often lie in local planar patches on the protein surface. the factors that influence the formation of protein-protein complexes can be cataloged into four different types-i.e. homodimeric protein, heterodimeric proteins, enzyme-inhibitor complexes and antibody-protein complexes (jones and thornton ) . from the structural perspective, structural characterization of macromolecular assemblies usually poses a more difficult challenge than structure determination of individual proteins (russell et al. ) . effective approaches for the prediction of protein-protein interactions at physical interaction levels are also strongly in demand (wodak and mendez ) . zhou and qin ( ) reviewed the methods currently being applied for interface prediction. the characteristics between interface and noninterface portions of a protein surface, such as sequence conservation, proportions of amino acids, secondary structure, solvent accessibility and side-chain conformational entropy, are often used to distinguish the specificity of local structures relating to protein binding function. in the transcription and translation process, proteins always bind to dna and rna to fulfill various functions. proteinnucleotide binding is a fundamental function of proteins. luscombe et al. ( ) classified the dna-binding proteins into eight different structural/functional groups. the helix-turn-helix (hth) motif is one of the most common structures used by proteins to bind dna, while protein-rna binding involves a number of different structure specificities. a comparison between protein-rna and protein-dna complexes revealed that while base and backbone contacts (both hydrogen bonding and van der waals) are observed with equal frequency in protein-rna complexes, backbone contacts are more dominant in protein-dna complexes (jones et al. ) . the positively charged residue, arginine, and the single aromatic residues, phenylalanine and tyrosine, all play key roles in the sites for the rna-binding function. ligand binding is a key aspect of protein functions. proteins recognize their natural ligands for transportation, signal transduction or catalysis (campbell et al. ) . the cleft volumes in proteins have strong relationships with their molecular interactions and functions. the ligands are always bound in the largest clefts (laskowski et al. ) . metal ions have a role in a variety of important functions, including protein folding, assembly, stability, conformational change and catalysis (barondeau and getzoff ) . in order to leverage the wealth of native metalloprotein structures into a deep understanding of metal ion site specificity and activity, high-resolution analyses of metal site structures and metalloprotein design are increasingly being performed. one of the most ubiquitous zinc-binding motifs is the c h zinc finger motif, which was first identified in transcription factors (ebert and altman ) . another broad concept for protein local structures is the active site. active sites of a protein are comprehensively related to functionally important local regions of the protein. the special features of functional local structure are to provide deep insights into the relationship between structure and function. for example, the catalytic triads provide a target of structure for finding the catalytic function of the proteins. to date, many different types of local structures have been defined or identified based on the geometry of the local regions, protein surface patterns, chemical groups or the electronic features. local structure features are believed to be the factors related to concrete functions. at the sequence level, the local regions may be scattered on the primary sequence, forming special motifs. alternatively, at the folding level, they form locally spatial shapes. we can simply catalog the types of methods used to identify the local structures as follows: methods to detect profiles of sequences with special local shapes, and methods to detect the substructures with special features based on folding. the primary sequence of a protein consists of (combinations of) different amino acids, which fold and pack together to constitute a special three-dimensional structure. sequence motifs are conserved segments in protein primary sequences. multiple sequence alignment is often used to identify the common patterns in several protein sequences, especially in the homology family. more advanced sequence comparison algorithms can detect the profiles of the functional residues in the primary sequence. of these algorithms, one of the most common methods is the hidden markov model (hmm). there are a number of important sequence pattern databases, which are publicly available from the internet (table ) . local three-dimensional structural patterns, such as the surface cavities of protein (e.g. the clefts and pockets) also have conserved structural features. table lists a number of methods currently used to identify local structure patterns. the procedure of recognition can be generally divided into two parts. the first is to construct the local structures. the geometric structure patterns and biochemical properties can be used to segment the protein architecture into small substructures. the second is to search the annotated sites from the literature and databases. the analysis of the protein surface is an active area of research in terms of the study of local structures. to date, two aspects of protein surface patches have attracted the most attention. the first is based on the defined features, such as surface curvature, surface cavities, electrostatic potential and hydrophobicity. castp (binkowski et al. b ) uses the weighted delaunay triangulation and the alpha complex for shape measurements. the local regions are defined by computational geometry, which identifies and measures surface accessible pockets as well as interior inaccessible cavities for proteins and other molecules. computational geometry also measures analytically the area and volume of each pocket and cavity, both in solvent accessible surface (sa, richards' surface) and molecular surface (ms, connolly's surface). castp provides an online resource for locating, delineating and measuring concave surface regions on the three-dimensional structures of proteins. these include pockets located on protein surfaces and voids buried in the interior of proteins. pvsoar (binkowski et al. ) provides an online resource to identify similar protein surface regions. kinoshita and nakamura ( ) provided a molecular surface database of proteins' functional sites, named the ef-site. the method displays the electrostatic potentials and hydrophobic properties of proteins together on the connolly surfaces of the active sites for analysis of the molecular recognition mechanisms. the connolly surfaces are made by using the molecular surface package program, and the electrostatic potentials are calculated by solving poisson-boltzmann equations with the self-consistent boundary method. the second aspect of protein surface patches is based on a predefined segmentation size of the surface. the method uses a segmentation procedure to divide the surface into small segmentations that correspond to certain physical modules of the surface. surfnet (laskowski ) generates molecular surfaces and gaps between surfaces from three-dimensional coordinates supplied in a pdbformat file. the gap regions can correspond to the voids between two or more molecules or to the internal cavities and surface grooves within a single molecule. the program visualizes molecular surfaces, cavities and intermolecular interactions by segmenting the surfaces. based on the surfnet algorithm, surface (ferre et al. ) identifies clefts and explores the cleft boundaries called the surface patch. a non-redundant set of protein chains is then used to build a database of protein surface patches. lig-site (hendlich et al. ) is a program for the automatic and time-efficient detection of pockets on the surface of proteins that act as binding sites for small molecule ligands. pockets are identified with a series of simple operations on a cubic grid. the special features of catalytic sites or other types of functional sites are also detected as local structures. some functional annotations of residues can be found in databases and the literature, and the location of these residues can be represented as potential structural motifs. although it is difficult to define just precisely what is the active site in protein structures, there are a number of methods for identifying active sites or functionally important residues. wallace et al. ( ) described a geometric hashing algorithm, called tess, to derive three-dimensional coordinate templates for motifs. tess has been used to create a database of enzyme active site templates called procat (wallace et al. ) . procat provides facilities for interrogating a database of three-dimensional enzyme active site templates. it has been superseded by the catalytic site atlas (csa). the csa (porter et al. ; torrance et al. ) is a database documenting enzyme active sites and catalytic residues in enzymes with a threedimensional structure. it contains the original annotated entries derived from the primary literature by hand and the homologous entries found by the psi-blast alignment. a hetatm and all annotated sites in the pdb also provide patterns of protein local structures strongly related to protein functions. stark and russell ( a) reported patterns in non-homologous tertiary structures (pints) that can be used to uncover the recurring three-dimensional side-chain patterns based on the algorithm in stark et al. ( c) . sitebase (gold and jackson a) is a database of known ligand-binding sites within the pdb. the search for an annotated position in the pdb constructs the location information of the ligand-binding sites. a collection of known sites from mining the annotations in the pdb has been designated as the pdbsite (ivanisenko et al. ) , which collects amino acid content structure features calculated by spatial protein structures, and physicochemical properties of sites and their spatial surroundings. the pdbsitescan (ivanisenko et al. ) provides an automatic search of three-dimensional protein fragments similar in structure to known functional sites. a comparison of local structures in the pdb also provides valuable information for constructing the structural motifs. kleywegt ( ) presented two programs, spatial arrangement of side-chains and main-chains (spasm) and rigor, for recognizing spatial motifs in protein structure. spasm can be used to find matches in the structural database for any user-defined motif. the program also has a unique capability to carry out ''fuzzy pattern matching'' with relax requirements on the types of some or all of the matching residues. rigor, on the other hand, can compare a database of pre-defined motifs against a perhaps newly determined structure. rigor scans a single protein structure for the occurrence of the pre-defined motifs from a database. zemla ( ) presented a method for finding three-dimensional similarities in protein structure. this algorithm is able to generate different local superpositions between pairs of structures and to detect similar fragments. it allows the clustering of similar fragments and the use of such clusters to identify sequence patterns that represent local structure motifs. sumo (jambon et al. ) can detect the common site, which corresponds to the catalytic triad. the general procedure of bridging the local structures with functions lies in constructing a candidate pool of local structures, identifying important features of function-related local structures and validating their functional importance. the existing methods can be grouped into two categories, i.e. unsupervised and supervised methods, as shown in fig. . the unsupervised methods directly mine those local structures with special features and then detect their functional implications. the supervised methods use known function-related structures as the templates and match these similar patterns by comparison. there are strong relationships between the two kinds of methods. most of the proposed methods are based on physical and/or biochemical patterns of the protein, and some particular patterns of local structures are strongly related to functions. in the unsupervised methods, the patterns are derived directly from a group of local structures without known functions. their functional importance and characteristics are identified by analyzing the conserved factors in the common features of local structures. the identified function-related local structures can then be used to enlarge the pool of functional templates, which in turn can be used to measure the potential functional importance of the new substructures. figure a shows these relations. these functionally important local regions can be referred to as functional motifs. the functional motif is the particular local structure pattern with factors that are the determinations of performing particular functions. note that the functional motif is very important for studying the relationship between structure and function in theory, and it is of practical importance to the protein design of drug targets and other bioengineering fields. we can investigate the functional patterns of the local structures in multiple ways. more specifically, we group existing methods to bridge protein local structure and function into three categories based on the hierarchical perspective, as shown in fig. b . . element-based methods. these identify the local structures from sequence, structure and/or other important amino acid residues information. the methods detect the common or conservation patterns in these elements of proteins and bridge the gaps between the local structures and functions at the micro level. during the bridging process, if prior knowledge is used to identify the functional importance or guide the detection, the method belongs to the supervised category, otherwise it belongs to the unsupervised division. . feature-based methods. these investigate the putative features between the local structures and functions. this category can be further divided into two subcategories-i.e. scoring methods and learning methods. the identified functional features of local structures provide templates of functional motifs. in the scoring methods, the features of local structures are scored by a defined function, and then the scores are used to decide whether the targets are functionally important. thresholds are often then chosen to provide guidance for detecting the importance of target local structures. in the learning methods, some features are chosen and learned from the known function-related local structures. the learned features in the trained machines can be used as the classifier to decide whether the testing targets are strongly related to the function. these methods belong to the supervised division. . network-based methods. these are based on graph theory and network topology. the methods can be divided into two subcategories. the first is at the individual level and the second is at the mapping level. at the individual level, the protein can be represented as an interactive graph of the residues, with linkages representing the close distance among them. cliques of the graph, hub residues and residues with other special topology measures may correspond to functionally important regions and residues. at the mapping level, a network represents the similarity relations among the local structures. the functional motifs are mined from informative subgraphs. this approach lies in between the other two methods mentioned above and can be regarded as being semi-supervised because it uses some heuristic knowledge. element-based methods are based on a basic intuition that the conserved part of a sequence and structure is an important functional motif (aloy et al. ; jones and thornton ) . the first step is a discovery process, which mines similar local structures from the sequences or structures of the target proteins. when similar local patterns of structures in some proteins are identified, the identified structure features of local regions will be the determinants of similar functions among the proteins. the second step is to match the process by comparing the target to the known functional templates. based on the similarity between these, the function relationship is inferred. this method is also a basic tool for developing more advanced techniques to bridge the relationship between local structures and functions. the sequences, structures or other elements of the proteins are considered in the comparison. table lists the main methods that are currently being used. depending on whether or not some prior knowledge is used in the assessment, the method is classified as being supervised or unsupervised. similar patterns of local structures can be identified in different proteins, even in proteins of the midnight zone with neither sequence homology nor structure homology. in this case, the alignment of the sequences and/or structure ( ) tertiary side-chain patterns subgraph-isomorphism matching assam artymiuk et al. ( ) segments can imply similar functions of the local structures. these similar local structures of the proteins are important prognostic factors of their similar functions. multiple sequence alignment ma et al. ( ) used ten protein interface families selected from two-chain interface entries in pdb, identified surface residues and filtered out contact residues. the alignment results of the residue properties revealed that polar residue hot spots occur frequently at the interfaces of macromolecular complexes, thereby distinguishing binding sites from the remainder of the surface. using multiple structure alignment, these authors also showed the correspondence between energy hot spots and structurally conserved residues. three residues (trp, phe and met) were observed to be significantly conservative in binding sites. these identified local structures are linked with binding functions. all residues in a protein are not equally important. some are essential for certain structures or functions, whereas others can be readily replaced. conservation analysis is one of the most widely used techniques for predicting these functionally important residues in protein sequences. capra and singh ( ) proposed a method focusing on the analysis of a multiple sequence alignment of the homologous sequences in order to find columns that are preferentially conserved. the results show that conservation is highly predictive in identifying catalytic sites and residues near bound ligands, while it is much less effective in identifying residues in protein-protein interfaces. structure alignment: geometric hashing rosen et al. ( ) proposed a surface comparison algorithm in search of active sites and functional similarity. these authors first represents the surface by a face-center critical point technique and then derive active sites using geometric hashing to match the two surfaces. finally, a clustering process is used to obtain the functional active sites. this method addresses the question of the usefulness of geometric comparisons and concludes that pure geometric surface matching is capable of obtaining biological meaningful solutions. based on the geometric hashing algorithm, leibowitz et al. ( ) presented a multiple structural alignment algorithm to detect a recurring substructural motif. given an ensemble of protein structures, the algorithm automatically finds the largest common substructure (core) of c a atoms that appears in all of the molecules in the ensemble. the detection of the core and the structural alignment are carried out simultaneously. fischer et al. ( ) also presented an approach using geometric hashing to compare spatial, sequence-order independent atoms. it automatically detects a recurring three-dimensional motif in protein molecules without any predefinition of the motif. pairwise alignment of constructed local structures there are several methods that detect the functional relationship between local structures by structure alignment in an allagainst-all manner. pazos and sternberg ( ) presented an automatic method to extract functional sites (residues associated to functions). the method relates proteins with the same go functions through structural alignment in an all-against-all manner and extracts three-dimensional profiles of conserved residues. based on the identified local structures derived from geometry or physicochemical features, the functional relationship of these local regions can be detected and the comparison result is stored in a database. when querying a local structure, similar hits imply functional relationships. binkowski et al. ( a binkowski et al. ( , described such an approach for inferring functional relationships of proteins based on the pvsoar by detecting sequence and spatial patterns of the functional relationship of pockets on protein surfaces. the pvsoar database provides a pairwise comparison of the pockets in the pocket database castp. similar pockets in different match degrees are searched for in an advanced analysis of the function relationship among the local structural motifs. with respect to the pockets on the protein surface, schmitt et al. ( ) developed a similar method based on a clique detection algorithm by comparing the query against the whole database. kinoshita and nakamura ( ) also provided an analogous method for comparing molecular surface geometries and electrostatic potential on the surfaces based on ef-site. their method bridges the protein surface electronic features of the local region with the specific functions. jambon et al. ( ) designed a new but similar approach for finding similarities using pairwise matching to detect common three-dimensional sites in proteins. the basis for their method is a representation of the protein structure by a set of stereochemical groups. protein surface regions with similar physicochemical properties and shapes may perform similar functions and bind similar partners. shulman-peleg et al. ( ) constructed two web servers and software packages for use in recognizing the similarity of binding sites and interface-siteengine and interface-to-interface (i i)-siteengine. the input into the two methods is two protein structures or two protein-protein complexes; the output is the surface of the proteins for a region similar to the binding sites or the interfaces. the methods are efficient for large-scale database searches of the entire pdb. obviously, the two locally identified structures are related to functions by searching similar local regions of their protein structures. pairwise alignment of annotated local structures information on functional sites obtained from databases or the literature can be used to construct the function-related local structure database, while the pairwise alignment method is used to detect the functional relationships. stark and russell ( a) developed pints to uncover the recurring three-dimensional side-chain patterns based on the algorithm in stark et al. ( c) . their method queries the structural motif database constructed from the annotation mining from pdb to find similar three-dimensional motifs by a recursive, depth-first search algorithm, i.e. to find all possible groups of identical amino acids common to two protein structures independent of sequence order (russell ). the search is conducted with distance constraints by ignoring those amino acids unlikely to be involved in the protein function. stark et al. ( b) identified some functional sites and compared these with procat and rigor. moreover, pints provides a measure of statistical significance based on a rigorous model for the behavior of rmsd (stark et al. c) . sitebase (gold and jackson a ) is a database of known ligand-binding sites within the pdb. gold and jackson ( a) provided a method that automatically identifies ligand-binding sites by searching for hetatm keywords in pdb files and constructing a database by excluding protein/peptide ligands and treating het-groups as individual ligand-binding sites. protein atoms within a -Å radius of any ligand atom were defined as its binding site in this work, and the ligand-binding was identified by comparison in an all-against-all way with geometric hashing. similar functions of binding sites were detected regardless of the sequence and folding similarity (gold and jackson b) . pdbsitescan (ivanisenko et al. ) provides an automatic search of three-dimensional protein fragments that are similar in structure to known functional sites. a collection of known sites has been designated as the pdbsite (ivanisenko et al. ) , which is a database of amino acid content, structure features calculated by spatial protein structures and the physicochemical properties of sites and their spatial surroundings. protein-protein interaction sites are also generated by an analysis of contact residues in heterocomplexes. the algorithm is developed based on an exhaustive examination of all possible combinations of protein positions. the bid (fischer et al. ) database searches the primary scientific literature directly for detailed data on protein interfaces by text mining and stores the characterization of protein-protein binding interfaces at the amino acid level. the bid also organizes protein interaction information into tables, graphical contact maps and descriptive functional profiles. evolutionary tracing protein functional sites have a number of similar and unique features. in order to explore the information fully, one can incorporate both sequence and structure data in a functional site prediction method. the evolutionary trace (et) method is one such method that relies on both sequence and structure information. the most basic form of the algorithm requires a multiple sequence alignment of a protein family and an evolutionary tree, based on sequence identity, which can approximate the functional classification of the protein sequences (lichtarge and sowa ) . yao et al. ( ) proposed an automatic et method that ranks the evolutionary importance of amino acids in protein sequences. this was the first method to quantify the significance of the overlap observed between the best-ranked residues and functional sites. the information inherent in a phylogenetic tree is added to the analysis of conserved sequences, often revealing the more subtle aspects of protein function. starting with a multiple sequence alignment, a representative structure and a phylogenetic tree, this method evaluates conservation at each position in the alignment for different sequence similarity cut-offs. in its original implementation, residues were classified as variable, conserved or a group-specific set that is specific to one branch of the phylogenetic tree. this analysis can be further expanded by the use of amino acid substitution matrices to evaluate conservation. in either case, a representative structure is used to visualize the distribution of scores at the end of the analysis. based on the et method, landgraf et al. ( ) presented a three-dimensional cluster analysis that offers a method for predicting functional residue clusters. this method requires a representative structure and a multiple sequence alignment as input data. individual residues are represented in terms of regional alignments that reflect both their structural environment and their evolutionary variation, as defined by the alignment of homologous sequences. the overall and regional alignments are calculated from the global and regional similarity matrices, which contain scores for all pairwise sequence comparisons in the respective alignments. three-dimensional clustering analysis is an easily applied method for the prediction of functionally relevant spatial clusters of residues in proteins. armon et al. ( ) proposed the consurf method, which takes into account the evolutionary relationships among the sequence homologues by closely approximating the evolutionary process and by considering the phylogenetic relationships among the sequences and the similarity between amino acids. consurf maps evolutionary conserved regions on the surface of proteins with a known structure; it also aligns sequence homologues of the protein and uses the alignment to construct phylogenetic trees. the trees are then used to infer the presumed amino acid exchanges that occur throughout the evolution. each exchange is then weighted by the physicochemical distance between the exchanged amino acid residues. the results show that the patches of conserved residues correlate well with the known functional regions of the domains and are more sensitive than the et method. to obtain an indication of the validity of functional inheritance, aloy et al. ( ) proposed a method to evaluate the reliability by exploiting the conservative functional sites predicted by the et method. their method first used a fully automatic procedure to carry out the et method, and then was benchmarked in terms of required sequence divergence and the resultant selectivity and specificity of the prediction. finally, the results that were obtained using the prediction of location of functional sites to assist in filtering putative complexes were evaluated. the functional importance of local structures can be detected by empirical methods or by computational methods. the identified functional motif can then be used as the structure template to detect the functional regions in other protein structures. the chosen method often consists of a comparison process, and the structure and physicochemical features can be considered in the comparison to the templates. in addition, a measurement of the similarity to the template is used to assess the functional importance of the testing of local structures. wallace et al. ( ) described a three-dimensional template matching method based on geometric hashing for automatically deriving three-dimensional templates from the protein structures deposited in pdb. in their paper, these researchers described a template derived for the ser-his-asp catalytic triad. their results showed that the resultant template provides a highly selective tool for automatically differentiating between catalytic and noncatalytic ser-his-asp associations. goyal and mande ( ) described the generation of three-dimensional structural motifs for metal-binding sites from known metalloproteins. using three-residue templates and four-residue templates, the method scans all available protein structures in the pdb database for putative metalbinding sites. the search of the whole pdb database predicted many novel metal-binding sites, which are the identified functional motifs. chakrabarti and lanczycki ( ) recently performed a detailed survey of compositional and evolutionary constraints at the molecular and biological functional levels for a large set of known functionally important sites extracted from a wide range of protein families. they compared the degree of conservation across different functionally important sites. the compositional and evolutionary information at functionally important sites was compiled into a library of functional templates. in their paper, these researchers developed a module that predicts functionally important columns of an alignment based on the detection of a significant 'template match score' to a library template. benchmark studies showed good sensitivity/ specificity for the prediction of functional sites and high accuracy in attributing correct molecular function type to the predicted sites. the comparison between potential sites and the templates is very important in these kinds of methods. artymiuk et al. ( ) developed a program called assam, which represents a motif-by-distance matrix between pseudo-atoms and uses the subgraph-isomorphism algorithms to find matches. this is an elegant method for the detection of common tertiary side-chain patterns based on the use of the ullman subgraph isomorphism algorithm. singh and saha ( ) formulated the problem of identifying a given structural motif (pattern) in a target protein and discussed the notion of complete and partial matches. they described the precise error criterion that has to minimized and also discussed different metrics for evaluating the quality of partial matches. they also presented a novel polynomial time algorithm for solving the problem of matching a given motif in a target protein. the functions of a protein are strongly related to the physicochemical features of that protein. the physical features (such as geometry, size, depth and shape) and the chemical features (such as energy, hydrophobicity, amino acid propensity and conservation) of the local structure are often measured by a score function or learned by a machine learning algorithm. the functional importance and specificity of a protein can be identified from the evaluation score or the trained standards of features. the main methods are listed in table . the scoring method can often calculate an explicit value for the features, while the learning method can reveal the patterns inexplicitly. the properties of local structures are believed to be conserved in terms of determining their functions. the identified local regions of structure are analyzed based on the variations in their properties, which are investigated using the identified functionally important sets of local structures. the method to predict the functions of the local structures is often based on a scoring scheme that is used to analyze the properties of the targets. in particular, the scores of the features are used as the measurements to determine whether the local structure has functional importance, for example, for a particular function. scoring by physical features first, the physical features of the local structures, such as size, depth and shape, are ( ) considered for scoring the function-related features. the shape features alone may provide basic information for the analysis of the functional features related to the protein function. siggers et al. ( ) introduced a new method to structurally align interfaces observed in protein-dna complexes. their method is based on a procedure that describes the interfacial geometry in terms of the spatial relationships between individual amino acid-nucleotide pairs. they subsequently provided a yet newer method to study the determinants of binding specificity. kawabata and go ( ) proposed a new definition for pockets using two explicit adjustable parameters, the radii of small and large probe spheres, which correspond to the two physical properties, 'size' and 'depth'. a pocket region was defined as a space into which a small probe can enter, but a large probe cannot. based on the geometric standards of large probe spheres, this method identified the binding site positions. from the geometrical viewpoint, the methods described above need further improvement to describe or compare the global shape and the local structures. morris et al. ( ) presented a novel technique for capturing the global shape of a protein's binding pocket or ligand. this method uses the coefficients of a real spherical harmonics expansion to describe the shape of a protein's binding pocket. shape similarity is computed as the l distance in coefficient space. kahraman et al. ( ) used a recently developed shape matching method to compare the shapes of protein-binding pockets to the shapes of their ligands. their results indicate that pockets binding the same ligand show greater variation in their shapes than those which can be accounted for by the conformational variability of the ligand. this result suggests that geometrical complementarity in general is not sufficient to derive molecular recognition. scoring by chemical features chemical features of local structures are very important for determining their functional specificity. these feature scores of local structures can be used as standards to determine their functions. the structural locations of functional sites are conserved between homologous proteins because functionally important residues tend to cluster together in space, forming threedimensional residue clusters or surface patches. panchenko et al. ( ) presented a method to assign each residue a score that depends on its own conservation in homologs and the conservation of residues in its spatial neighborhood. the high-scoring sites are more likely to be involved in specific binding or catalysis. functionally important residues in a protein are known to be those computed to have energy among experimentally destabilized residues. elcock ( ) proposed a method to predict functionally important residues based solely on the computed energetics of a protein structure. the energetic properties of binding surfaces in proteinprotein interfaces and protein-ligand sites were shown to be different (burgoyne and jackson ) . the pockets from qsitefinder (laurie and jackson ) were ranked by the scores of these properties-i.e. hydrophobicity, desolvation, electrostatics and conservation-which are used to determine binding sites. jones et al. ( ) developed a method to detect dnabinding sites on a protein surface. the surface patches and the dna-binding sites were initially analyzed for accessibility, electrostatic potential, residue propensity, hydrophobicity and residue conservation. in general, dnabinding sites are among the top % of patches with the largest positive electrostatic scores. this knowledge was used to make predictions. jones et al. ( ) presented a similar computational analysis of protein-rna interactions. there are a number of differences between dnabinding sites and rna-binding sites. for the rna-binding sites, van der waals contacts play a more important role than hydrogen bond contacts. as to the protein-dna binding local structures, luscombe et al. ( ) investigated hydrogen bonds as well as van der waals contacts and water-mediated bonds to assess whether there are universal rules that govern amino acid-base recognition. in a subsequent study, luscombe and thornton ( ) also identified the amino acid conservation and the effects of mutations on binding specificity. in liang et al. ( ) , an empirical score function consisting of a linear combination of the energy score, interface propensity and residue conservation score is used to predict interface residues. the top-ranked patches are predicted to be the potential interface sites. the accuracy of prediction has been improved significantly, relative to any single or pairwise combination, by combining the three terms. cheng et al. ( ) presented a method to predict protein function site using sequence alignment information as well as rosetta protein design and rosetta free energy calculations. logistic regression with the generalized linear model has been used to the determine weights of the sequence conservation, natural/designed sequence profile difference and natural/optimal residue free energy gap, all of which optimize the separation between functional and non-functional residues. innis et al. ( ) presented conserved functional group (cfg) analysis to predict function sites in proteins. the method relies on a simplified representation of the chemical groups found in amino acid side-chains to identify functional sites from a single protein structure and a number of its sequence homologs. scoring by physicochemical features those features based only on physical geometry or chemical energy often can not represent functional features comprehensively. most of the methods are used to integrate several important features together and then score these features for bridging the gaps between local structures and functions. the ligsite algorithm is based only on the geometry. huang and schroeder ( ) presented an extension and implementation method, ligsite csc , which is based on the notion of surface-solvent-surface events and the degree of conservation of the involved surface residues. the use of the connolly surface has led to slight improvements, whereas the prediction re-ranking significantly improved the binding site predictions. glaser et al. ( ) improved previous approaches by combining two known measures of 'functionality' in proteins, i.e. cleft volume and residue conservation, to develop a method for identifying the location of ligand-binding pockets in proteins. neuvirth et al. ( ) proposed a structure-based algorithm to identify the location of protein-protein interaction sites. the sites are defined based on connolly's molecular dot surfaces. the method defines an interface score that combines the chemical and geometry features of the interaction sites. interfacial residues are considered to be those with the % highest scores. geometry and energy properties have also been used to analyze the pocket functions for docking (li et al. ). hoskins et al. ( ) considered the use of solvent accessibility, residue propensity and hydrophobicity in conjunction with secondary structure data as prediction parameters to predict proteinprotein interaction sites. the influence of residue type and secondary structure on solvent accessibility is analyzed, and a measure of relative exposedness is defined. the highscoring residues are clustered as a basis for predicting interaction sites. tsuchiya et al. ( ) provided a method for analyzing protein-dna complexes, focusing on the shape of the molecular surface of the protein and dna, along with the electrostatic potential on the surface, and calculated a new evaluation score. based on the score, the method was used to classify dna-binding from non-dna-binding proteins. taroni et al. ( ) provided an analysis of the characteristic properties of sugar-binding sites. for each site, six parameters were evaluated-i.e. solvation potential, residue propensity, hydrophobicity, planarity, protrusion and relative accessible surface area (asa). three of the parameters were found to distinguish the observed sugarbinding sites from the other surface patches. these parameters were then used to calculate the probability of a surface patch being a carbohydrate-binding site. the total score of the properties was used to determine whether the surface patch was a carbohydrate-binding site. the features of the local structures play crucial roles in predicting protein function. to identify the relationship between protein local structure and protein function, the structural and/or physicochemical features can be learned implicitly using machine learning methods, such as the support vector machine (svm) and neural network. the support vector machine uses a linear model to implement nonlinear class boundaries through the input of a number of nonlinear mapping vectors into a high-dimensional feature space. it is based on mathematics theory and has many successful applications in statistical learning fields (vapnik ). these methods have been confirmed to be able to learn the features of local structures with functional importance. the features can first be investigated in the learning process and used to detect whether these features relate some specific functions. koike and takagi ( ) proposed an svm method to identify protein-protein interaction sites. the profiles of sequentially/spatially neighboring residues, plus additional information, constitute a feature vector, and the interaction site ratios are calculated by svm regression. the predictive performance is evaluated and compared in different quantitative features. cai et al. ( ) proposed an svm algorithm to predict the catalytic triad of the serine hydrolase family. bordner and abagyan ( ) proposed a similar svm to predict protein-protein interfaces. the local surface properties with a combination of an evolutionary conservation signal were used to train the machine on a large nonredundant data set of protein-protein interfaces. an svm learning protocol was provided by bhardwaj et al. ( ) for the prediction of dna-binding proteins. the characteristics, including surface and overall composition, charge and positive potential patches on the protein surface, were derived, and the svm was trained as a classifier to detect the dna-binding proteins. the high accuracy value has been achieved in a large set of testing proteins regardless of their sequence or structure homology. chung et al. ( ) recently exploited the svm approach to detect whether identified potential proteinbinding sites interact with each other. the information related to sequence and structural complementary across protein interfaces were extracted from the pdb. this work also built a pipeline to predict the location of binding sites. neural network the neural network is a learning method which adapts the relationships of neurons; as such, it is a simplified model of the neural processing of the human brain (zhang ) . based on the analysis of the both structures and sequences, gutteridge et al. ( ) used a neural network to identify catalytic residues in enzymes. the locations of the active sites were predicted by the neural network output and spatial clustering of the highest scoring residues. in most testing cases, the likely functional residues were identified correctly, as were a number of potentially novel functional groups. ofran and rost ( ) described a neural network to identify protein-protein interfaces from sequences. since the compositions of contacting residues of the interaction sites were believed to be unique, the features of this known interaction sites were used to train the neural network. zhou and shan ( ) trained a neural network to predict protein-protein interactions. their method combines conservation and structural properties of individual residues. fariselli et al. ( ) reported a neural network-based system using information on evolutionary conservation and surface disposition. chen and zhou ( ) also provided a neural network method to predict interface residues in a protein-protein complex. there are also neural network methods for predicting nucleic acid-binding (na-binding) sites. stawiski et al. ( ) presented an automatic neural network approach to predict na-binding proteins, specifically dna-binding proteins. this method uses an ensemble of features extracted from characterization of the structural and sequence properties of large, positively charged electrostatic patches. structural and physical properties of dna provide important constraints on the binding sites formed on the surfaces of the dna-targeting proteins. the characteristics of dna-binding sites may form the basis for predicting dna-binding sites from the structures of proteins alone. tjong and zhou ( ) used a representative set of protein-dna complexes from the pdb to analyze characteristics and to train a neural network predictor of dna-binding sites. the input to the predictor consists of psi-blast sequence profiles and solvent accessibility of each surface residue and of its closest neighboring residues. ferrer-costa et al. ( ) provided a web-based method to detect if a protein structure contains a dnabinding helix-turn-helix (dbhth) motif. the method uses a neural network with no hidden layers, i.e. a linear predictor, to classify whether a protein is dna-binding with the hth motif. the linear predictor was trained on a nonhomologous set of structures of protein chains with a dbhth motif and without the motifs. sodhi et al. ( ) used a neural network to predict metal-binding sites residues in low-resolution structural models. the method involves sequence profile information combined with approximate structural data. several neural networks were proposed to distinguish the metal sites from non-sites and then to detect these functionally important regions. in keil et al. ( ) , the patches of the molecular surface were segmented into overlapping patches. the properties of these patches were calculated based on the physical and chemical properties. a neural network strategy was then used to identify possible binding sites by classifying the surface patches as protein-protein, protein-dna, protein-ligand or nonbinding sites. kuznetsov et al. ( ) applied an svm method to predict dna-binding sites using the features including amino acid sequence, profile of evolutionary conservation of sequence positions, and low-resolution structural information. the results indicate that an svm predictor based on a properly scaled profile of evolutionary conservation in the form of a position specific scoring matrix (pssm) significantly outperforms a pssm-based neural network predictor. such results imply that the combination of the two methods may improve the accuracy. passerini et al. ( ) introduced a two-stage learning method for identifying histidines and cysteines that participate in binding of several transition metals and iron complexes. the first stage is an svm, which is trained to locally classify the binding state of single histidines and cysteines. the second stage is a neural network trained to refine local predictions. the methods use only sequence information by utilizing position-specific evolutionary profiles. statistical methods statistical learning also provides an effective way to link the features of local structures with their functional implication. liang et al. ( a) provided a supervised learning algorithm, feature, for the automatic discovery of physical and chemical descriptions of protein microenvironments. the calculated feature vectors were used to predict functional motifs based on bayesian inference. the method has also been proposed as an interactive web tool, webfeature, for identifying and visualizing functional sites . bradford et al. ( ) developed a method to predict both protein-protein binding site location and interface type (obligate or non-obligate) using a bayesian network in combination with surface patch analysis. two bayesian network structures, naive and expert, were trained to distinguish interaction surface patches. wang et al. ( b) proposed a computational method learned by the expectation maximization (em) algorithm, insite, to search for motifs whose presence in a pair of interacting proteins determined which motif pairs have high affinity that would lead to an interaction between proteins. yan et al. ( ) also provided a two-stage method consisting of an svm and a bayesian classifier for predicting the surface residues of proteins that participate in protein-protein interaction. the method exploits the fact that interface residues tend to form clusters in the primary amino acid sequence. in addition, chou and cai ( ) provided a covariant discriminant algorithm to predict active sites of enzyme molecules. the high accuracy of prediction shows the effectiveness of the method. protein-dna interactions are critical for deciphering the mechanisms of gene regulation. yan et al. ( ) presented a supervised machine learning approach for the identification of amino acid residues involved in protein-dna binding sites. a naive bayesian classifier was trained for predicting whether a given amino acid residue is a dna-binding residue based on its identity and the identities of its sequence neighbors. mclaughlin and berman ( ) developed statistical models for discerning protein structures containing the dbhth motifs. the method uses a decision tree model to identify the key structural features required for dna binding. these features include a high average solvent-accessibility of residues within the recognition helix and a conserved hydrophobic interaction between the recognition helix and the second alpha helix preceding it. the adaboost algorithm was used to search the pdb with the aim of identifying the structure containing the motifs with high probability. metal ions are crucial in facilitating the function of a protein. identifying the features of metal binding sites provides crucial knowledge of the function performance of the local structures. because the residues that coordinate a metal often undergo conformational changes upon binding, the detection of binding sites based on simple geometric criteria in proteins without bound metal is difficult. however, aspects of the physicochemical environment around a metal-binding site are often conserved, even when this structural rearrangement occurs. ebert and altman ( ) developed a bayesian classifier using known zinc-binding sites as positive training examples and nonmetal-binding regions as negative training examples. babor et al. ( ) reported an approach that identifies transition metal-binding sites in proteins by combining the decision tree and svm. in the first step, the geometric search of structural rearrangements following metal binding was taken into account by a decision tree classifier. a second classifier based on svms was then used to identify the metal-binding sites. nayal and honig ( ) proposed a comprehensive method to identify drug-binding sites in which attributes were first computed for each cavity, and these were then used to distinguish drug-binding sites by the random forest classification scheme. the cavity properties cover eight broad categories, such as cavity size, cavity shape, hydrophobicity, electrostatics, hydrogen bonding, amino acid composition, secondary structure and rigidity. an interesting method to identify function motifs is based on the graph theory and the network concept. the main methods are listed in table . one subcategory of the method represents the protein structure as a complex network. a node represents a c a of the backbone, and an edge linking two nodes represents the physical distance or the functional relationship between the nodes. greene and higman ( ) viewed protein structures as network systems. the systems are identified to exhibit small-world, single-scale and, to some degree, scale-free properties. using the network model, amitai et al. ( ) identified active site residues. the method transforms a protein structure into a residue interaction graph, where graph nodes represent amino acid residues, and links represent their interactions. the active site, ligand-binding and evolutionary conserved residues are identified typically with a high closeness value, from which the functional residues are filtered out. del sol et al. ( ) also represented a protein liu et al. ( b) structure as a small-world network and searched the topological determinants related to functionally important residues. the method investigates the performance of residues in protein families. the results indicate that enzyme active sites are located in surface clefts, and hetero-atom binding residues have deep cavities, while protein-protein interactions involve a more planar configuration. wangikar et al. ( ) reported a method for detecting recurring side-chain patterns using an unbiased and automatic graph theoretic approach. the method first lists all structural patterns as subgraphs. the patterns are compared in a pairwise manner based on content and geometry criteria. the recurring pattern is then detected using an automatic search algorithm from the all-againstall pairwise comparison proteins. similarly, huan et al. ( ) defined a labeled graph representation of a protein structure in which edges connecting pairs of residues are labeled by the euclidian distance between the c a atoms of the two residues. based on this representation, a structural motif corresponding to a labeled clique occurs frequently among the graphical representation of the protein structures. the paper further presented an efficient mining algorithm aimed at discovering structure motifs in this setting. in studies on protein structure and function, identifying calcium-binding sites in proteins is one of the first steps towards predicting and understanding the role of calcium in biological systems. calcium-binding sites are often complex and irregular, and it is difficult to predict their location in protein structures. deng et al. ( ) reported a rapid and accurate method for detecting calcium-binding sites. this algorithm uses a graph theory algorithm to identify oxygen clusters of the protein and a geometric algorithm to identify the center of these clusters. a cluster of four or more oxygen atoms has a high potential for calcium binding. a potential calcium-binding position is a clique and can be detected by a clique-detecting algorithm. the high accuracy of prediction shows that the majority of calcium-binding sites in proteins are formed by four or more oxygen atoms in a sphere center with a calcium atom. the above network methods all focus on individual proteins and represent a protein structure a complex network. the specific topology features clearly imply a particular function module (zhang and grigorov ; zhang et al. ). recently, a novel category of networkbased analysis of the protein local structures at the macro level has been proposed (liu et al. ) . the similarity of the local structures, specifically the pockets on the protein surface, is mapped to constitute a similarity network. the nodes represent the pockets, and the edges represent the certain similarity relationships among the pockets. the properties of the pocket similarity network are like other complex networks (liu et al. ) . the similar pockets are identified by the clusters and community structures, and the special features of the network are helpful in clustering the pockets into similar groups (liu et al. b) , which may imply clusters of structure motifs and correspond to special functional implications (liu et al. ) . with the network concept, the pockets can also be used to characterize and predict protein functions by annotating the topology neighbors. in this way, the accuracy of the prediction is better than that with the global structural similarity approach (liu et al. a ). prediction of functions at the cellular level most of the methods used to annotate protein functions that are listed above are based on molecular function at the biological processing level. at the cellular component and location levels, the importance of protein local structure is also critical. in fact, information on the subcellular locations of proteins is important because it can provide useful insights into protein functions as well as how and in what kind of cellular environments they interact with each other and with other molecules. such information is also fundamental and indispensable to systems biology because a knowledge of the localization of proteins within cellular compartments can facilitate our understanding of the intricate pathways that regulate biological processes at the cellular level. from this perspective, the functions of proteins at different levels are strongly inter-related to each other. at the cellular component level, local structures are still crucial in determining the roles of proteins and specific functions. many methods for predicting the subcellular location of proteins have been proposed recently because the location of such proteins in the cell can provide useful insights or clues about their functions . one of the more powerful methods applied in location prediction is based on an important descriptor of the protein sample, i.e. the pseudo-amino acid (pseaa) composition (chou ) . this descriptor can be used to represent a protein sequence with a discrete model yet without completely losing the sequence-order information. since the concept of pseaa composition was introduced, various pseaa composition approaches have been developed, all with the aim of improving the prediction quality of protein attributes (gao et al. ; zhou et al. a, b; diao et al. ; fang et al. ; li and li ) . the pseaa method has been widely used and extended. a very flexible pseaa composition generator (pseaac) was established which enables users to generate different kinds of pseaa composition. a web server called cell-ploc has recently been developed that allows users to predict the subcellular locations of proteins in various different organisms. pseaa composition and pssm have also been combined in various algorithms to improve the prediction quality for membrane protein type (i.e. memtype- l: chou and shen a) , enzyme main-functional class and sub-functional class (i.e. ezypred: shen and chou a) and protein subnuclear localization (i.e. nuc-ploc: shen and chou b) . a comprehensive review published recently provides a summary of these topics. in addition to sequence information, local structural information is useful, interesting and important in protein localization function prediction. a quality assessment of the results is necessary at all three levels of function prediction. the predicted functions of proteins can be taken as indicators of the directions to be taken by researchers when carrying out experiments to validate the functions of proteins. many of the computational methods used to annotate protein functions as well as those used to predict functionally important local structures use cross-validation methods to assess the performance of a prediction; these include the independent dataset test, subsampling test and jackknife test (chou and zhang ) . however, as elucidated by chou and shen ( ) , of these cross-validation methods, the jackknife test is considered to be the most objective and has been increasingly used by investigators to examine the accuracy of various predictors (zhou ; zhou and assa-munt ; zhou and doctor ; xiao et al. ; zhou and cai ; chen et al. ; shi et al. ) . it is important to consider the relationship among the functional terms and the semantic similarity with the aim of avoiding biases in the assessment of functional similarity (liu et al. a ). the global structure similarity-based methods provide a straightforward approach to annotate protein functions. however, since the relationships between structures and functions are so complex, local structure-based methods can be used to predict protein function directly by identifying the local structures carrying out particular functions. laskowski et al. ( ) proposed a novel method of predicting protein function using local three-dimensional templates. the authors build a template database and use four types of templates-enzyme active sites, ligandbinding residues, dna-binding residues and reverse templates-to construct the relationship between templates and functions. ferre et al. ( ) described a method for the functionrelated annotation of protein structures based on the detection of local structural similarity with a library of annotated functional sites. an automatic procedure was used to annotate the function of the local surface regions, and then a sequence-independent algorithm was developed to compare exhaustively these functional patches with a larger collection of protein surface cavities. after tuning and validating the algorithm on a dataset of well-annotated structures, the results are able to provide functional clues to proteins that do not show any significant sequence or global structural similarity with proteins in the current databases. binkowski et al. ( ) provided similar methods to annotate protein functions from the protein surface similarity. pockets are identified by castp from several proteins. these pockets are queried in the pvsoar to locate similar pockets corresponding to annotated proteins. the conservation among the pockets can be detected by the sequence identities and other similarity metrics. tseng and liang ( ) developed a bayesian markov chain monte carlo method for rate estimation of the special substitution rates of the short sequence of local structure. moreover, a method for protein function prediction is presented by surface matching using scoring matrices derived from estimated substitution rates for residues located on the binding surfaces. the method is effective in identifying functionally related proteins that have overall low sequence identity. the method provided by pazos and sternberg ( ) first identifies functional sites in proteins by bridging the local structures and functions, then the functions of a target proteins can be inferred from the similarity of the functional sites in the position-specific scoring matrices. information on the functional importance of local structure can facilitate the annotation of protein function more precisely. george et al. ( ) proposed an effective method to annotate protein function through the use of functional clues of conservation among the catalytic residues. this method improves the precision of annotation significantly. the advantages of predicting protein functions from local structures are based on the fact that such methods can be implemented without any prior homology hypothesis. the methods can be used in proteins in midnight zone without sequence similarity, and local structures often provide concrete and specific functional annotations. to compare the precision and coverage of the global structural similarity and that of local structures, liu et al. ( a) proposed a novel method to predicted protein from the pockets on the protein's local surface region. the similarity of regional local surface pockets and the global similarity of proteins are all represented by networks. the prediction is based on the network topology. a comparison of the results show that the local-structure-based prediction is better than the global-structure-based prediction (liu et al. a ). in this paper, we have reviewed protein function prediction methods at different levels, i.e. sequence, structure, interaction and integration. we have mainly focused on the importance of local structures and the method used to predict functionally important local structures. in summary, we discuss possible future directions. the interaction between proteins provides high-level information on protein function, especially in various biological processes. although there are thousands of known interactions, a tiny fraction of these are available in precise molecular details. if we are able to examine structural details, systematic representation of the interaction would accurately reflect biological reality. for example, we can predict which part of the structures is most likely to be involved in interaction with other macromolecules, proteins, dna or rna by analyzing the properties of different local patches on the protein surface. the patch analysis, which considers properties of the surface such as flatness, hydrophobicity, charge and, in particular, residue conservation, is effective in identifying protein-protein interaction surfaces and has also been shown to successfully identify dna-binding sites (aloy and russell ) . structural systems biology is a very effective approach that combines protein interactions and protein three-dimensional structures. the mechanisms of protein and protein interaction lie in the local structures between the two protein surfaces. from this perspective, structural systems biology provides us with a new direction in the fields of structural biology and systems biology. it combines the key features of the two directions to provide more insight into linking the single protein and systematic interaction between proteins. the relationships between local structures and functions are expected to play important roles in structural systems biology. the computational methods used to bridge the relationship between local structures and functions can be further improved. the community of computational biology has a strong need for comprehensive feature selection in concise and effective ways. in addition, there is still much room for improvement in terms of the accuracy of the methods used to align the features between two local structures. the validation of the functions of structural motifs should also be conducted more carefully and by more reliable biological experiments. recent advances in the field inspired by developments in sequences and structures demonstrate the great potential of such research in protein science in elucidating essential functional roles of the local structures. in our opinion, research aimed at bridging the gaps between local structures and function is still in its infant stage, and further advances in such areas will greatly enhance our ability to study the fundamental properties of proteins at a system-wide level. in other words, we expect to gain deep insight into essential mechanisms of biological systems from both structural and functional perspectives. different methods based on the local similarity, global similarity and interaction require and use different information, and they have different aspects, intentions and advantages. to our knowledge, the function annotation problem is still in its developing period and needs more comprehensive or hybrid approaches. none of the existing methods are likely to be successful in all cases to annotate a protein with its functions correctly and comprehensively. one reason for this is that protein functions not only rely on the sequence and/or folding characteristics, but also on the cell environment, the cycle of the biological processes and other chemical compounds. there are still many difficultto-decipher proteins that researchers have been unable to annotate correctly by any existing method. hence, a sensible strategy is to use different methods to incorporate data from multiple sources and to extensively utilize existing function annotations. future directions include using combinations of different methods at different levels so as to efficiently explore the overall sequences, global structures and local structures and to obtain more information on interactions between the target proteins and others in the cellular context. although computational methods generally cannot directly validate protein functions, the predefined tentative annotations provide valuable information as a basis for further efficient validation experiments. network analysis of protein structures identifies 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using amino acid pair antigenicity scale revealing divergent evolution, identifying circular permutations and detecting active-sites by protein structure comparison improvement in protein functional site prediction by distinguishing structural and functional constraints on protein family evolution using computational design prediction of protein cellular attributes using pseudo amino acid composition (erratum: ibid structural bioinformatics and its impact to biomedical science a novel approach to predict active sites of enzyme molecules predicting protein-protein interactions from sequences in a hybridization space memtype- l: a web server for predicting membrane proteins and their types by incorporating evolution information through pse-pssm recent progresses in protein subcellular location prediction cell-ploc: a package of web-servers for predicting subcellular localization of proteins in various organisms prediction of protein structural classes binding mechanism of coronavirus 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protein universe an algorithm for predicting protein-protein interaction sites: abnormally exposed amino acid residues and secondary structure elements global mapping of the protein structure space and application in structure-based inference of protein function prediction of functional sites in proteins using conserved functional group analysis pdbsitescan: a program for searching for active, binding and posttranslational modification sites in the d structures of proteins pdbsite: a database of the d structure of protein functional sites a new bioinformatic approach to detect common d sites in protein structures principles of protein-protein interactions searching for functional sites in protein structures protein-rna interactions: a structural analysis using electrostatic potentials to predict dna-binding sites on dnabinding proteins quantitative assessment of relationship between sequence similarity and function similarity shape variation in protein binding pockets and their ligands 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molecular surfaces, cavities and intermolecular interactions protein clefts in molecular recognition and function from protein structure to biochemical function? protein function prediction using local d templates q-sitefinder: an energy-based method for the prediction of protein-ligand binding sites automatic multiple structure alignment and detection of a common substructural motif using pseudo amino acid composition to predict protein subnuclear location with improved hybrid approach protein-protein interactions: hot spots and structurally conserved residues often locate in complemented pockets that pre-organized in the unbound states: implications for docking webfeature: an interactive web tool for identifying and visualizing functional sites on macromolecular structures automatic construction of structural motifs for predicting functional sites on protein structures protein binding site prediction using an empirical scoring function evolutionary predictions of binding surfaces and interactions predicting gene ontology functions from protein's regional surface structures an approach for clustering protein pockets into similar groups analysis of protein surface patterns by pocket similarity network protein-dna interactions: amino acid conservation and the effects of mutations on binding specificity an overview of the structures of protein-dna complexes amino acidbase interactions: a three-dimensional analysis of protein-dna interactions at an atomic level protein-protein interaction: structurally conserved residues distinguish between binding sites and exposed protein surfaces a combined algorithm for genome-wide prediction of protein function statistical models for discerning protein structures containing the dna-binding helix-turn helix motif real spherical harmonic expansion coefficients as d shape descriptors for protein binding pocket and ligand comparisons scop: a structural classification of proteins database for the investigation of sequences and structures on the nature of cavities on protein surfaces: application to the identification of drug-binding sites promate: a structure based prediction program to identify the location of protein-protein binding sites predicted protein-protein interaction sites from local sequence information cath-a hierarchic classification of protein domain structures ssap: sequential structure alignment program for protein structure comparison from protein structure to function inference of protein function from protein structure prediction of functional sites by analysis of sequence and structure conservation identifying cysteines and histidines in transition-metal-binding sites using support vector machines and neural networks automatic prediction of protein function and detection of functional sites from structure the catalytic site atlas: a resource of catalytic sites and residues identified in enzymes using structural data molecular shape comparisons in searches for active sites and functional similarity twilight zone of protein sequence alignments the funcat, a functional annotation scheme for systematic classification of proteins from whole genomes detection of protein three-dimensional side-chain patterns: new examples of convergent evolution a structural perspective on protein-protein interactions computational methods of analysis of protein-protein interactions integrating structure, bioinformatics, and enzymology to discover function-bioh, a new carboxylesterase from escherichia coli a new method to detect related function among proteins independent of sequence and fold homology structure and mechanism of the m proton channel of influenza a virus predicting enzyme function from sequence: a systematic appraisal network-based prediction of protein function ezypred: a top-down approach for predicting enzyme functional classes and subclasses nuc-ploc: a new web-server for predicting protein subnuclear localization by fusing pseaa composition and psepssm pseaac: a flexible web-server for generating various kinds of protein pseudo amino acid composition using pseudo amino acid composition to predict protein subcellular location: approached with amino acid composition distribution protein structure alignment by incremental combinatorial extension (ce) of the optimal path siteengines: recognition and comparison of binding sites and protein-protein interfaces structural alignment of protein-dna interfaces: insights into the determinants of binding specificity hierarchical protein structure alignment using both secondary structure and atomic representations identifying structural motifs in proteins predicting metal-binding site residues in low-resolution structural models annotation in three dimensions. pints: patterns in non-homologous tertiary structures finding functional sites in structural genomics proteins a model for statistical significance of local similarities in structure annotating nucleic acid-binding function based on protein structure analysis and prediction of carbohydrate binding sites gene ontology: tool for the unification of biology displar: an accurate method for predicting dna-binding sites on protein surfaces using a library of structural templates to recognise catalytic sites and explore their evolution in homologous families estimation of amino acid residue substitution rates at local spatial regions and application in protein function inference: a bayesian monte carlo approach structure-based prediction of dna-binding sites on proteins using the empirical preference of electrostatic potential and the shape of molecular surfaces global protein function prediction from protein-protein interaction networks tess: a geometric hashing algorithm for deriving d coordinate templates for searching structural database. application to enzyme active sites virtual screening for finding natural inhibitor against cathepsin-l for sars therapy insite: a computational method for identifying protein-protein interaction binding sites on a proteome-wide scale functional sites in protein families uncovered via an objective and automatic graph theoretic approach predicting protein function from sequence and structural data prediction of protein function from protein sequence and structure assessing annotation transfer for genomics: quantifying the relations between protein sequence, structure and function throng traditional and probabilistic scores prediction of protein-protein interactions: the capri experiment, its evaluation and implications using complexity measure factor to predict protein subcellular location a two-stage classifier for identification of protein-protein interface residues predicting dna-binding sites of proteins from amino acid sequence an accurate, sensitive, and scalable method to identify functional sites in protein structures fatcat: a web server for flexible structure comparison and structure similarity searching lga-a method for finding d similarities in protein structures neural networks in optimization similarity networks of protein binding sites tm-align: a protein structure alignment algorithm based on tm-score discovering functions and revealing mechanisms at molecular level from biological networks prediction protein homo-oligomer types by pseudo amino acid composition: approached with an improved feature extraction and naive bayes feature fusion gene function prediction using labeled and unlabeled data protein domain annotation with integration of heterogeneous information sources an intriguing controversy over protein structural class prediction some insights into protein structural class prediction predicting protease types by hybridizing gene ontology and pseudo amino acid composition subcellular location prediction of apoptosis proteins interaction-site prediction for protein complexes: a critical assessment prediction of protein interaction sites from sequence profile and residue neighbor list improved prediction of subcellular location for apoptosis proteins by the dual-layer support vector machine using chou's amphiphilic pseudo-amino acid composition and support vector machine for prediction of enzyme subfamily classes fast: a novel protein structure alignment algorithm acknowledgments this work was supported by the national natural science foundation of china (nsfc) under grant no. and no. . lyw and xsz are also supported by the grant no. cb from the ministry of science and technology, china. the research was also supported by nsfc-jsps collaborative project no. . the authors are grateful to the anonymous referees as well as editors for comments and for helping to improve the earlier version. we recognize that this review is far from comprehensive, and we apologize for any papers related to the subject that were not mentioned. key: cord- -kvuti authors: benjin, xu; ling, liu title: developments, applications, and prospects of cryo‐electron microscopy date: - - journal: protein sci doi: . /pro. sha: doc_id: cord_uid: kvuti cryo‐electron microscopy (cryo‐em) is a structural biological method that is used to determine the d structures of biomacromolecules. after years of development, cryo‐em has made great achievements, which has led to a revolution in structural biology. in this article, the principle, characteristics, history, current situation, workflow, and common problems of cryo‐em are systematically reviewed. in addition, the new development direction of cryo‐em—cryo‐electron tomography (cryo‐et), is discussed in detail. also, cryo‐em is prospected from the following aspects: the structural analysis of small proteins, the improvement of resolution and efficiency, and the relationship between cryo‐em and drug development. this review is dedicated to giving readers a comprehensive understanding of the development and application of cryo‐em, and to bringing them new insights. for decades, electron microscopy has been used to study biomacromolecules and related complexes. with the accumulation of experience and the deepening of understanding, electron microscopy has been developed to study the structure of biomacromolecules. in recent years, the continuous development of cryogenics and rapid freezing technology has led to the birth of cryo-electron microscopy (cryo-em), that is, the micro technology of using transmission electron microscope to observe samples at low temperature. the basic principle of cryo-em is to image biological macromolecules frozen and fixed in glassy ice, thereby obtaining the projection of protein molecules in all directions. a computer is then used to process and calculate a large number of d (two-dimensional) images, and reconstruct the d (three-dimensional) structure of the biomacromolecule. d reconstruction is used to deduce d structure from d images. its theoretical basis is the central section theorem proposed by aaron klug in : that is, the fourier transform of a function projected along a certain direction is equal to the cross-section function of the function whose fourier transform passes through the origin and is perpendicular to the projection direction. in recent years, cryo-em has made great achievements in the determination of macromolecular structure, especially the structures of supramolecular systems. the breakthrough of this technology has led to a revolution in structural biology. in , the nobel prize in chemistry was awarded to three biophysicists, jacques dubochet, joachim frank, and richard henderson (figure ), for their pioneering contributions to the development of cryo-em. xu benjin and liu ling contributed equally to this work structural biology was born in the middle of the last century. it is a discipline that elucidates life phenomena by studying the structure and function of biological macromolecules. at present, there are three kinds of commonly used structural biology research methods, namely, x-ray crystallography, nuclear magnetic resonance (nmr), and cryo-em, which together constitute the basis of highresolution structural biology research. in the past half century, x-ray crystallography has been dominant in structural biology. as of october , , the crystal structures in the pdb database account for about % of the total, the nmr structures account for . %, and the em structures account for . % ( figure ). x-ray crystallography is mainly applicable to proteins with molecular weight of ~ kda. after decades of development and maturity, more than , protein structures have been resolved, which has become the main means to analyze the structure of biological macromolecules. nmr is often used to study proteins with molecular weight less than kda. the advantage of this method is that it can determine the structure of protein molecules in solution, and study the dynamic process and the corresponding energy change of biological samples. however, the application of nmr is limited due to the requirement of high sample concentration (usually at the mm level) and high stability. nmr structures are also at relatively low resolution. with the rapid development of cryo-em, the minimum molecular weight limit of sample particles has been pushed to kda, the resolution has also been continuously improved, and the freezing method has been more advanced. this has made cryo-em faster and more efficient, so that it can compete or even replace x-ray crystallography in many aspects. compared with traditional structural biology methods such as x-ray crystallography f i g u r e three nobel prize winners in chemistry in (https://www.nobelprize.org/prizes/chemistry/ ) f i g u r e structure proportion of the three structural biology methods. blue corresponds to structures determined by x-ray crystallography, dark orange corresponds to structures determined by nuclear magnetic resonance, and gray corresponds to structures determined by electron microscopy and nmr, cryo-em has the following advantages: (a) it does not need crystals; (b) it is suitable for proteins and their complexes of large molecular weight; (c) it reduces radiation damage and maintains the native activity and functional state of samples, including posttranslational modifications; (d) multiple different conformational states can be captured in one experiment; (e) it is suitable for the structural analysis of membrane proteins such as gpcr and their complexes; (f) when encountering some structures that cannot be resolved by conventional x-ray crystallography, cryo-em is still the mainstream. of course, cryo-em also has its own shortcomings or defects: (a) cryo-em can only be used for a large protein complex, which generally requires a minimum of kda or even more than kda, otherwise, the protein is too small to find under the electron microscope; (b) the requirement of sample homogeneity is very high, it will be difficult to get good results if the protein is flexible; (c) the current resolution of cryo-em is about Å, and is below Å only in a few cases, which is still a certain gap from the demand of pharmaceutical research and development ( Å). in , max knoll, a german physicist, and his student ernst ruska, invented the first transmission electron microscope, and in , they broke through the limit of the optical microscope for the first time, making it possible to see even smaller particles. electron microscopy imaging needs to be performed in a high vacuum, and the radiation damage of electrons to biological samples is very large. therefore, in the following decades, scientists could only image biological samples through heavy metal salt staining. in , de rosier and klug proposed and established the general concept and method of d reconstruction, and reported the d structure of the t phage tail, the first electron microscope structure. in order to reduce the radiation damage caused by high-energy electrons, taylor and glaeser in proposed the use of cryo-em. the wheel of historical development is rolling forward! in , jacques dubochet and his colleague alasdair mcdowall made a breakthrough in electron microscopy technology. they introduced the method of rapid freezing, , which quickly froze molecules in a single layer of vitreous state water. this solved the above two problems of high vacuum and radiation damage. the process of vitrification can not only keep the sample in its natural state, but also protects them from dehydration. however, there are still many challenges to achieve atomic resolution. for example, the contrast and signalto-noise ratio in electron microscope images is low. also one needs to transform d images into d structure. joachim frank and his colleague, marin van heel, took the lead in using computational image processing techniques [ ] [ ] [ ] to calculate and average multiple image copies of biomacromolecules to improve the signal-tonoise ratio. after obtaining d images with different angles, d reconstruction is carried out by computer software to analyze the d structure of biomolecules. by , the resolution of s large subunit of escherichia coli ribosome had been improved to . Å. after about , the number of electron microscope structures began to increase year by year, but the resolution was not high. however, after , the number of structures analyzed by electron microscopy began to improve rapidly and the resolution also increased to near atomic level. the structure of β-galactosidase reported in had a resolution of . Å. also, the resolution of the s proteasome and the s-ef-tu complex were . Å and . Å, respectively ( figure ). during the past few years, cryo-em has made a lot of progress in computing image processing, such as the development of user-friendly software, [ ] [ ] [ ] and the use of direct electronic detectors. in , the d structure of glutamate dehydrogenase ( kda) was reported, and the resolution even reached . Å. it is the development of these technologies that has made the application of structural analysis based on cryo-em more and more extensive (see timeline in figure ). by october , , the total number of electron microscope structures in the pdb database had reached , , and it is believed that the growth momentum will be more rapid in the future. the general process of structure analysis by cryo-em is as follows: (a) protein expression and purification: samples with high purity, homogeneity, and integrity need to be obtained, and the molecular sieve needs to show a single peak and symmetrical distribution; (b) negative staining: the sample molecules are embedded in a layer of heavy metal salt (commonly uranium acetate) solution, so that the heavy metal salt surrounds the molecules. with the yeast small subunit processome, for example, μl of ssu processome at an absorbance of . mau at nm can make a sample ; (c) data collection of negative staining sample (sometimes omitting this step according to the situation); (d) freezing sample: this is very important and usually includes two steps, first load the sample on the grid to form a thin water film, and then quickly freeze. in most cases, water can be made glassy by immersing the grid into liquid ethane quickly by manual operation, which has the advantage that the sample can be close to the "natural" state. because biochemical reactions, especially some enzymatic reactions, take place rapidly, another method named fast mixing/spraying microfluidic chips [ ] [ ] [ ] has been developed in order to obtain the structural information of the intermediate state of the reaction. that is, mixing the two molecular systems in milliseconds, and then freezing them quickly so that one can capture the intermediate steps in the biochemical reactions. this method can achieve time resolution of tens of milliseconds, which is very suitable for studying short-term biological events, such as ribosome recycling, translation initiation, and other processes. , generally speaking, the sample concentration of soluble protein is about mg/ml and membrane protein is about mg/ml. take eukaryotic ribosome for example, μl of nm can make a sample ; (e) load the sample in the cryo-em; (f) screen sample: it is necessary to screen the sample before data collection to check whether the water in the sample is in a glassy state and whether the thickness of ice layer is appropriate. if there is a problem, the sample needs to be prepared again; (g) data collection: select good particles to take photos, and the minimum exposure technology must be used in the photography because the high-energy electrons have radiation damage to the sample; (h) three-dimensional reconstruction. some problems may be encountered in the process of using cryo-em, mainly in the following aspects: (a) the sample is unstable, degraded, or aggregated; (b) some ligands with small molecular weight, may not be seen in the density map; (c) there may be organic substances such as sugar, dmso, or glycerin in the buffer, resulting in the decrease of sample contrast and resolution; (d) the purity of the sample may be good or even very good, but the homogeneity is poor, which greatly reduces the resolution; (e) the target area may have greater flexibility, after d or d averaging, the resolution of the target area becomes very poor; (f) samples may be destroyed during freezing; (g) in addition to the samples, there are many parameters to be optimized, such as sample concentration, block time, temperature, grid specifications, and so forth. therefore, cryo-em needs rich experience and sufficient machine time, and good experimental results really need the right time and the right place. during the last few years, a series of important breakthroughs have been made in the field of structural biology by the combination of cryo-em and d f i g u r e timeline of key events in the development of cryo-em reconstruction. the breakthrough of cryo-em mainly comes from the development of hardware and software. the hardware includes the electron microscope and the image recording device; the software includes d reconstruction algorithms and image data processing. the titan krios is the most advanced cryo-em at present, which has made great improvements in the following six aspects: (a) kv field emission low temperature transmission electron microscope, equipped with field emission electron gun and three-stage condenser system, which can realize parallel illumination within a certain illumination range; (b) an automatic sample injection system that can load and store frozen samples at one time, improving the sample efficiency; (c) an automatic liquid nitrogen filling system, which can automatically maintain the low temperature of the sample chamber and the lens cone for extended operation; (d) a maximum tilt angle of the sample table of , and a horizontal rotation of ; (e) a constant power mode magnetic lens system that ensures the high stability of imaging; (f) a point resolution that can reach . nm, and on information resolution limit which can reach . nm. the development of new detector hardware led to the resolution revolution of cryo-em. a series of new and attractive near atomic resolution structures have been obtained, which fully proves this point. for example, the complex structure of pathogen ribosome and drug molecules, the large subunit structure of mitochondrial ribosome, the structure of mammalian ribosome complex, and the structure of trpv ion channel. the new direct electronic detector is more sensitive and faster than the traditional medium (ccd camera or photographic film), and can record image data at the rate of multiple frames per second instead of single long-time exposure. the direct electronic detection camera not only has a significant improvement in the detection quantum efficiency, but also has a high frame rate, enables images to be acquired as "movies" made of stacked frames. in the future, desire for higher resolution will certainly promote the development of electron microscopy in many aspects: thinner detector chip and faster readout rate, so as to greatly improve the signal-to-noise ratio of resolution; improve the sample preparation method and use the holder that is not easy to move under electron bombardment; develop new image recording programs to reduce sample movement caused by light beams. low signal-to-noise ratio or poor image contrast is the main challenge of cryo-em, which is difficult to avoid as biological samples are sensitive to radiation. it can only be improved by reducing the electron dose, which in turn results in a very low signal-to-noise ratio. the development and application of new direct electronic detectors have greatly promoted the development of image processing and algorithms. , in order to extract valuable structural information from noisy images, researchers have developed an assembly based on the maximum possible and prior information of undetermined structure. this new software can work without user intervention to a large extent, and provides objective resolution standards, which quickly become the "gold standard" of cryo-em. the new standardized verification procedure makes the results (including particle orientation, absolute chirality, and contrast loss) more reliable, and can avoid over fitting noise to generate clearer images. the powerful classification method can separate different molecules or different structural states of the same molecule in the same field of vision. therefore, the development and progress of the new algorithm make people have a new understanding of sample processing: samples are purified in computers, rather than through arduous, time-consuming, and often destructive biochemical processes. | applications of cryo-em cryo-em is mainly used to determine the structure of biological macromolecules and macromolecular super complexes. the most typical examples are spliceosomes and chromosomes. the splicing of rna is performed by the spliceosome, a super protein enzyme complex, and abnormal splicing leads to many genetic diseases. yigong shi, a member of tsinghua university, reported the highresolution cryo-em structures of yeast and human spliceosomes in different catalytic states, - revealing the mechanism of spliceosome catalyzed mrna processing, providing an important basis for understanding the rna splicing process of higher organisms. in , song et al. successfully established a platform for in vitro reconstruction and structural analysis of chromatin, and took the lead in analyzing the highdefinition d structure of nm chromatin by using cryo-em, making an important breakthrough in the research of higher-order structure of nm chromatin, the carrier of "life information," which provides a structural basis for understanding the mechanism of epigenetic regulation of organisms. in , li et al. analyzed the cryo-em structure of the snf -nucleosome complex in different nucleotide states, answered the basic principle of dna translocation in the process of chromatin remodeling, and revealed the mechanism of chromatin remodeling. the most typical examples are the ribosome and the proteasome. the ribosome is a molecular machine for protein synthesis, the determination of its d structure is the basis for correct understanding of ribosome function. the high-resolution structures of prokaryotic and eukaryotic cytoplasmic ribosomes have been resolved by x-ray crystallography and cryo-em, but the high-resolution structures of eukaryotic mitochondrial ribosomes have not been resolved for a long time. the breakthrough of cryo-em and the development of data classification algorithm provide a powerful means for the determination of mitochondrial ribosome structure and for the investigation of internal rearrangements of the translational apparatus. v. ramakrishnan of mrc molecular biology laboratory analyzed a series of high-resolution structures of mitochondrial ribosomes by means of cryo-em. in , they reported the structures of human and yeast mitochondrial ribosome large subunits with resolutions of . Å and . Å respectively; they also resolved the structures of human mitochondrial ribosome and yeast mitochondrial ribosome, with resolutions of . Å and . Å, respectively. in addition, ban et al. analyzed the structure of mammalian mitochondrial ribosomal large subunits in , with resolutions of . Å and . Å, respectively. these results provide an important structural basis for understanding the molecular mechanism of protein synthesis in mitochondria. the proteasome is a giant protein complex that is common in eukaryotes and archaea, and also exists in some prokaryotes. in eukaryotes, proteasome is widely distributed in the cytoplasm and nucleus. its core function is to regulate the concentration of specific proteins and to remove misfolded proteins in cells. it is one of the indispensable large-scale all enzyme supramolecular complex machines in the cell, and also the largest protein degradation machine found so far. in , the low resolution cryo-em structures of human and yeast s proteasome were reported. in , huang et al. and schweitzer et al. reported the atomic structure of human s proteasome with resolution of . Å and . Å, respectively. in , dong et al. determined the high-resolution ( . ~ . Å) atomic structures of seven intermediate conformations of human s proteasome during the degradation of substrate by combined use of cryo-em and machine learning technology. this work provides an important structural basis for the activation mechanism of the proteasome core particle gated switch and the kinetic mechanism of substrate transport channels of regulatory particle subcomplex. these include various ion channels, transporters, and supermembrane protein complexes. they are very important in organisms that participate in many biological processes such as cell proliferation and differentiation, energy transfer, signal transduction, and material transport. it is estimated that about % of the drug targets are membrane proteins. the membrane proteins are difficult to purify and crystallize, and it is difficult to resolve their d structure by x-ray crystallography. therefore, the development and breakthrough of cryo-em bring new hope for the structural analysis of membrane proteins and their complexes. in , gong et al. first reported the . Å cryo-em structure of human cholesterol transporter npc ; wu et al. reported the first highresolution d structure of the eukaryotic voltage-gated ca + channel, which laid a foundation for understanding the working mechanisms of voltage-gated ca + and na + channels with important physiological and pathological functions. in addition, zhao et al. in reported the cryo-em structure of the mammalian voltage-gated ca + (cav) channel and ligand complex. this clarified the molecular basis for the recognition and regulation of cav channel by three ligands at the atomic level, and laid the foundation for the future drug research and development of cav channel disease. in the field of membrane complexes, wei et al. reported the . Å cryo-em structure of spinach photosystem ii-light-harvesting complex ii (psii-lhcii) supercomplex in . the results are of great significance to further understand the time dynamics and light protection mechanism of energy transfer in psii-lhcii supercomplex at the molecular level. in , su et al. reported the structure of pea psii-lhcii supercomplex, revealing the supramolecular basis of efficient light harvesting in plants under low light conditions. in , pi et al. reported the cryo-em structure of psii-fcpii supercomplex of chaetoceros gracilis, which provided an important basis for elucidating the unique mechanism of light energy capture, transfer and transformation and efficient light protection in psii-fcpii supercomplex of diatom. in addition, gu et al. first reported the cryo-em structure of the respiratory chain super complex, respirasome, which has the highest resolution so far in . the results of this study provide an important structural basis for further understanding of the assembly form, molecular mechanism of mammalian respiratory chain complexes and the treatment of cellular respiratory related diseases. viruses are pathogens that cause various diseases of human or animals. the determination of their d structures is not only helpful to elucidate the mechanism of virus self-assembly and infection, but also has guiding significance for drug design based on structure. the size of virus particles is larger than that of proteins, so it is more suitable to analyze their d structure with cryo-em. in , yuan et al. first reported the atomic resolution structure of the nucleocapsid of herpes simplex virus type (hsv- ) of the herpesvirus α family. they clarified the complex interaction mode and fine structural information of the nucleocapsid protein, and proposed the assembly mechanism of the nucleocapsid of the herpesvirus. in the same year, song et al. revealed the cryo-em structure of the spike glycoprotein of sars coronavirus and its receptor ace complex. yuan et al. revealed the structure and function of mers-cov and sars-cov spike proteins, providing a key d structure map for the follow-up design of broad-spectrum antibody and vaccine. in , liu et al. first reported the key mechanism of human herpesvirus genome packaging and the genome structure of the virus, which is helpful to prevent and control a variety of diseases caused by herpesvirus, and is expected to transform herpesvirus for targeted treatment. wang et al. determined the cryo-em structure of african swine fever (asf) virus capsid, with a resolution of . Å, which revealed the basis of capsid stability and assembly, and opened a new way for the development of asf vaccine. neurodegenerative disease results in dysfunction due to the gradual loss or even death of neuron structure or function, including amyotrophic lateral sclerosis, parkinson's disease, alzheimer's disease, and huntington's disease, among others. at present, the cause of this kind of disease is not clear and cannot be cured, which seriously threatens human health and daily life. in , yang et al. reported the cryo-em structure of human γ-secretase in complex with a notch fragment, which revealed the structural basis of notch recognition, and is of great significance for γ-secretase to recruit amyloid precursor protein. then, zhou et al. reported the cryo-em structure of human γ-secretase in complex with transmembrane app fragment, which provided important support for the design of substrate specific inhibitors and the understanding of the function of γ-secretase and the pathogenesis of ad. in the same year, gu et al. resolved the cryo-em structure of mammalian atpase tetramer. this explained the structural composition pattern and molecular mechanism of mammalian atpase, the synergistic relationship between respiratory chain complexes and the influence on the shape of mitochondrial cristae, which provided an important structural basis for the treatment of energy metabolism diseases and neurodegenerative diseases. the innate immune system forms an evolutionarily ancient defense line, which can resist invading pathogens and endogenous danger signals. in some innate immune cells, inflammasome can sense a series of stimuli, which can produce inflammatory response. the imbalance of inflammatory signals leads to a wide range of immune disorders leading to diseases such as gout, crohn's disease and sepsis. in , hu et al. reported the cryo-em structure of prgj-naip -nlrc Δcard complex (inflammasome) in mice, revealing the molecular mechanism of nlrc protein induced self-activation in naip-nlrc . zhang et al. reported the cryo-em structure of the activated naip -nlrc inflammasome and revealed its nucleated polymerization mode. in , sharif et al. analyzed the cryo-em structure of human nlrp -nek complex, and revealed the molecular mechanism of nek mediated activation of nlrp inflammasome through the multi-interface interaction with nlrp subunit. wang et al. , reported the assembly, structure, and function analysis of plant nlr protein complex. this work revealed the key molecular mechanism of nlr function, promoted people's understanding of plant immune mechanism, and was a milestone event in plant immune research. dong et al. reported the cryo-em structure of human tcr α/β in complex with cd hexamer complex. it revealed the structural basis of tcr-cd complex assembly, provided clues for tcr triggering, and laid a foundation for the rational design of immunotherapy for the complex. at present, most of the biomacromolecule complexes analyzed by cryo-em are assembled in vitro. only a few have been studied in vivo, as will be necessary to understand the essence of life phenomenon. the next development goal of structural biology will be "in situ structural biology", that is, to study biological macromolecules in physiological and pathological states directly from the cell in situ, and to unify structure and function in the cell. , cryo-electron tomography (cryo-et) can obtain the d projection of the sample in multiple directions by tilting the sample in the microscope, and then reconstructing the d structure by calculation. it combines the d imaging technology with the sample preparation method to maintain the complete cell structure, so as to achieve the purpose of studying the d structure of macromolecules in a more real state. it is very suitable to study the d structure of viruses, proteins, and organelles with heterogeneous structure on the nanometer scale. cryo-et can not only map the position and interaction of macromolecules in the whole cell environment, but also reveal their in situ high-resolution structures. the d reconstruction of cryo-et generally includes four steps: (a) data collection of d electronic projection images with different angles; (b) registration of a series of oblique projection images; (c) d reconstruction of tomograms by reverse projection; (d) denoising and distinguishing of reconstruction results. cryo-et is widely used. it can not only analyze the d structure of proteins and their complexes at the molecular level, but also study the organelles at the subcellular level and even the tissue structure at the cell level. in recent years, much new knowledge has been gained in the field of cell biology by means of cryo-et, including the ultrastructure of mitochondria, ribosomes, the endoplasmic reticulum and the golgi complex, the structure of spindles involved in cell division, the tissue structure of insect flying muscles, the d structure of the nuclear pore complex, and the structure of asymmetric and polymorphic viruses. compared with single particle technology, the sample resolution obtained by the cryo-et is not as high, but it is suitable to analyze the structure of some viruses and their complexes that are asymmetric, amorphous, and important in function. cryo-et can effectively make up for the shortcomings of other structural analysis techniques, and will be widely used in more laboratories. as predicted by timothy baker, a microscientist at purdue university, in the next years or so, cryo-et will show explosive development, and it will undoubtedly play a leading role in the field of cell biology. | prospects for cryo-em for a long time, the poor contrast transfer function (ctf) made it difficult for cryo-em to analyze the structure of small molecular complexes. therefore, cryo-em has been considered to be only useful for the structure determination of macromolecular complexes with molecular weight greater than kda. however, many drug targets are of low molecular weight. in recent years, cryo-em has developed rapidly, more and more high-resolution structures less than kda have been resolved. in , khoshouei et al. introduced the voltage phase plate (vpp) into cryo-em. by doing so, they analyzed the structure of kda human hemoglobin, and pushed the lowest molecular weight limit of the electron microscope sample to a new height. in , hongwei wang's group from tsinghua university reported the high-resolution structure of kda streptavidin with a resolution of . Å, which is the smallest protein resolved by cryo-em. although the phase plate technology needs to be further improved in imaging stability, it provides the possibility for cryo-em to break through the limit of small molecular scale. cryo-em has made a lot of progress in the past years, but there is still a lot of room for improvement. one of the keys is to further improve the resolution to about Å. the homogeneity of the sample and the flexibility of the particle interior are two limiting factors of the resolution. a nonhomogeneous sample will lead to difficulty in data processing, and may also damage the integrity of the structural information and reduce the resolution. at present, many cryo-em studies still use intuition to get structure through continuous multiple rounds of classification. we expect focused d alignment and classification methods to overcome this problem in the future. in addition, the resolution can also be improved by optimizing the grid preparation process, data acquisition, and data processing. moreover, the further improvement of direct electronic detectors and phase plates, as well as the comprehensive upgrades of software and hardware, including more advanced optical lens systems and better detectors and algorithms, will enable us to enter the golden age of cryo-em, by then, the resolution will reach a new height. some in industry point out, it may take another - years for cryo-em to become a conventional tool for new drug development. but for many pharmaceutical and biotech companies, now may be the best time to integrate this tool into the r&d system. in x-ray crystallography, data collection only takes a few minutes, and structure analysis is very fast, making it the main means of structure-based research. cryo-em samples, however, need to be optimized many times before data collection, and data collection takes hours, if not days. therefore, the throughput is much less than for crystallography. therefore, the automation and optimization of sample preparation is one of the most urgent needs of cryo-em development in the future. at present, the rapid, perfect, automatic, and easy to quantify crystal structure screening system is mature. it is hard to imagine what cryo-em will become in the next few decades. therefore, it is not impossible for cryo-em to replace crystallography for conventional drug discovery. however, the more likely outcome is that the two technologies will continue to be the mainstream means of drug discovery, complementing each other and developing together for a long time. with the continuous development of cryo-em, we firmly believe that our understanding of many molecular mechanisms, such as those that drive translation or transcription, dna repair, rna splicing, cytoskeletal transport, and channel gating mechanisms, will be more profound. structural biology revolution led by technical breakthroughs in cryo-electron microscopy reconstruction of three dimensional structures from electron micrographs single particle cryo-em reconstruction of kda streptavidin at . angstrom resolution cryo-em captures the dynamics of ion channel opening cryo-em in drug discovery: achievements, limitations and prospects electron diffraction of frozen, hydrated protein crystals vitrification of pure water for electron microscopy electron microscopy of frozen water and aqueous solutions use of multivariate statistics in analysing the images of biological macromolecules spider-a modular software system for electron image processing imagic: a fast, flexible and friendly image analysis software system the escherichia coli large ribosomal subunit at . Å resolution . Å resolution cryo-em structure of beta-galactosidase in complex with a cellpermeant inhibitor carragher b. . Å resolution reconstruction of the thermoplasma acidophilum s proteasome using cryo-electron microscopy structure of the e. coli ribosome-ef-tu complex at < Å resolution by cs-corrected cryo-em accelerated cryo-em structure determination with parallelisation using gpus in relion- high resolution single particle refinement in eman cryosparc: algorithms for rapid unsupervised cryo-em structure determination breaking cryo-em resolution barriers to facilitate drug discovery architecture of the yeast small subunit processome monolithic microfluidic mixing-spraying devices for time-resolved cryo-electron microscopy structural dynamics of ribosome subunit association studied by mixing-spraying timeresolved cryogenic electron microscopy key intermediates in ribosome recycling visualized by time-resolved cryoelectron microscopy late steps in bacterial translation initiation visualized using time-resolved cryo-em structure and assembly model for the trypanosoma cruzi s ribosomal subunit cryo-em structure of the plasmodium falciparum s ribosome bound to the antiprotozoan drug emetine structure of the yeast mitochondrial large ribosomal subunit structure of the mammalian ribosome-sec complex to . Å resolution structure of the trpv ion channel determined by electron cryo-microscopy single-particle cryo-em data acquisition by using direct electron detection camera. microscopy cryo-em enters a new era ribosome structures to near-atomic resolution from thirty thousand cryo-em particles electron counting and beaminduced motion correction enable near-atomic-resolution single-particle cryo-em a bayesian view on cryo-em structure determination optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy prevention of overfitting in cryo-em structure determination structure of a human catalytic step i spliceosome structure of the postcatalytic spliceosome from saccharomyces cerevisiae structure of a yeast step ii catalytically activated spliceosome structure of a yeast catalytic step i spliceosome at . Å resolution the . Å structure of the u /u . u tri-snrnp: insights into spliceosome assembly and catalysis cryo-em study of the chromatin fiber reveals a double helix twisted by tetranucleosomal units mechanism of dna translocation underlying chromatin remodelling by snf structure of the large ribosomal subunit from human mitochondria the structure of the human mitochondrial ribosome the structure of the yeast mitochondrial ribosome the complete structure of the large subunit of the mammalian mitochondrial ribosome architecture of the large subunit of the mammalian mitochondrial ribosome molecular model of the human s proteasome near-atomic resolution structural model of the yeast s proteasome an atomic structure of the human s proteasome structure of the human s proteasome at a resolution of . Å cryo-em structures and dynamics of substrate-engaged human s proteasome structural insights into the niemann-pick c (npc )-mediated cholesterol transfer and ebola infection structure of the voltage-gated calcium channel ca(v) . at . Å resolution molecular basis for ligand modulation of a mammalian voltage-gated ca + channel structure of spinach photosystem ii-lhcii supercomplex at . Å resolution structure and assembly mechanism of plant c s m -type psii-lhcii supercomplex the pigment-protein network of a diatom photosystem ii-light-harvesting antenna supercomplex the architecture of the mammalian respirasome cryo-em structure of a herpesvirus capsid at . Å. science cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains cryo-em structures of herpes simplex virus type portal vertex and packaged genome architecture of african swine fever virus and implications for viral assembly structural basis of notch recognition by human gamma-secretase recognition of the amyloid precursor protein by human γ-secretase cryo-em structure of the mammalian structural and mechanistic elucidation of inflammasome signaling by cryo-em structural and biochemical basis for induced self-propagation of nlrc cryo-em structure of the activated naip -nlrc inflammasome reveals nucleated polymerization structural mechanism for nek -licensed activation of nlrp inflammasome ligand-triggered allosteric adp release primes a plant nlr complex reconstitution and structure of a plant nlr resistosome conferring immunity structural basis of assembly of the human t cell receptor-cd complex single-particle cryo-em-how did it get here and where will it go cryo-electron tomography and applications cryo-electron tomography and its application to biological research cryo-electron tomography-the cell biology that came in from the cold structural studies by electron tomography: from cells to molecules a new window on the cell's inner workings reaching the information limit in cryo-em of biological macromolecules: experimental aspects cryo-em structure of haemoglobin at . Å determined with the volta phase plate how to cite this article: benjin x, ling l. developments, applications, and prospects of cryoelectron microscopy the authors declare no potential conflict of interest.orcid xu benjin https://orcid.org/ - - - key: cord- - bik authors: hillisch, alexander; pineda, luis felipe; hilgenfeld, rolf title: utility of homology models in the drug discovery process date: - - journal: drug discovery today doi: . /s - ( ) - sha: doc_id: cord_uid: bik abstract advances in bioinformatics and protein modeling algorithms, in addition to the enormous increase in experimental protein structure information, have aided in the generation of databases that comprise homology models of a significant portion of known genomic protein sequences. currently, d structure information can be generated for up to % of all known proteins. however, there is considerable controversy concerning the real value of homology models for drug design. this review provides an overview of the latest developments in this area and includes selected examples of successful applications of the homology modeling technique to pharmaceutically relevant questions. in addition, the strengths and limitations of the application of homology models during all phases of the drug discovery process are discussed. - / /$ -see front matter © elsevier ltd. all rights reserved. pii: s - ( ) - the majority of drugs available today were discovered either from chance observations or from the screening of synthetic or natural product libraries. the chemical modification of lead compounds, on a trial-and-error basis, typically led to compounds with improved potency, selectivity and bioavailability and reduced toxicity. however, this approach is labor-and time-intensive and researchers in the pharmaceutical industry are constantly developing methods with a view to increasing the efficiency of the drug discovery process [ ] . two directions have evolved from these efforts. the 'random' approach involves the development of hts assays and the testing of a large number of compounds. combinatorial chemistry is used to satisfy the need for extensive compound libraries. the 'rational', protein structure-based approach relies on an iterative procedure of the initial determination of the structure of the target protein, followed by the prediction of hypothetical ligands for the target protein from molecular modeling and the subsequent chemical synthesis and biological testing of specific compounds (the structure-based drug design cycle). the rational approach is severely limited to target proteins that are amenable to structure determination. although the protein data bank (pdb; http://www.rcsb.org/pdb) is growing rapidly (~ new entries daily), the d structure of only - % of all known proteins has as yet been experimentally characterized. however, advances in sequence comparison, fold recognition and protein-modeling algorithms have enabled the partial closure of the so-called 'sequence-structure gap' and the extension of experimental protein structure information to homologous proteins. the quality of these homology models, and thus their applicability to, for example, drug discovery, predominantly depends on the sequence similarity between the protein of known structure (template) and the protein to be modeled (target). despite the numerous uncertainties that are associated with homology modeling, recent research has shown that this approach can be used to significant advantage in the identification and validation of drug targets, as well as for the identification and optimization of lead compounds. in this review, we will focus on the application of homology models to the drug discovery process. homology, or comparative, modeling uses experimentally determined protein structures to predict the conformation of another protein that has a similar amino acid sequence. the method relies on the observation that in nature the structural conformation of a protein is more highly conserved than its amino acid sequence and that small or medium changes in sequence typically result in only small changes in the d structure [ ] . generally, the process of homology modeling involves four steps -fold assignment, sequence alignment, model building and model refinement ( figure ). the fold assignment process identifies proteins of known d structure (template structures) that are related to the polypeptide sequence of unknown structure (the target sequence; this is not to be mistaken with drug target). next, a sequence database of proteins with known structures (e.g. the pdb-sequence database) is searched with the target sequence using sequence similarity search algorithms or threading techniques [ ] . following identification of a distinct correlation between the target protein and a protein of known d structure, the two protein sequences are aligned to identify the optimum correlation between the residues in the template and target sequences. the next stage in the homology modeling process is the model-building phase. here, a model of the target protein is constructed from the substitution of amino acids in the d structure of the template protein and the insertion and/or deletion of amino acids according to the sequence alignment. finally, the constructed model is checked with regard to conformational aspects and is corrected or energy minimized using force-field approaches. several improvements and modifications of this general homology modeling strategy have been developed and applied to the prediction of protein structures. to subject the available structure prediction methods to a blind test, community-wide experiments on the critical assessment of techniques for protein structure prediction (casp - ) have been performed and their results presented and published. as a result, the current state-of-the-art in protein structure prediction has been established, the progress made has been documented and the areas where future efforts might be most productively concentrated have been highlighted [ , ] . homology modeling techniques are dependent on the availability of high-resolution experimental protein structure data. the development of effective protein expression systems and major technological advances in the instrumentation used for structure determination (x-ray crystallography and nmr spectroscopy) has contributed to an exponential growth in the number of experimental protein d structures. by may , the pdb contained ~ , experimental protein structures for ~ different proteins (proteins with less than % sequence identity). a recent analysis of all protein chains in the pdb shows that these proteins can be grouped into protein families ddt vol. , no. august reviews research focus www.drugdiscoverytoday.com figure . the steps involved in the prediction of protein structure by homology modeling. structure modeling of the bacterial transcriptional repressor copr is shown [ ] . although the model is based on a low-sequence identity of only . % between copr and the p c repressor, several experimental methods support this homology model. reproduced, with permission, from ref. [ ] . abbreviation: copr, plasmid copy control protein. target sequence: homolog , no d structure: homolog , no d structure: template , d structure: template , d structure: comprising unique protein folds [ ] (updates can be found at http://scop.mrc-lmb.cam.ac.uk). the majority of the structures in the pdb ( %) were determined by x-ray crystallography, with % of the structures being characterized by nmr spectroscopy. the pdb database encompasses experimental information on an extensive array of ligands (small organic molecules and ions) bound to more than , different binding sites that can be analyzed using programs including relibase (http://relibase.ebi.ac.uk) [ ] , ligbase (http://alto.compbio.ucsf.edu/ligbase) [ ] and pdbsum (http://www.biochem.ucl.ac.uk/bsm/pdbsum) [ ] . although the experimental structure database is growing rapidly, there is still a substantial gap between the number of known annotated sequences [ , , unique sequences in swiss-prot-trembl (http://www.expasy.org/ sprot) as of august ] and known protein d structures ( , ). if only significantly different proteins are considered (~ ), which omits muteins, artificial proteins and multiple structure determinations of the same proteins (e.g. hiv-protease and carbonic anhydrase ii), then less than % of the d structures of known protein sequences have been elucidated. this sequence-structure gap can partly be filled with homology models. for example, the queryable database modbase (http://alto.compbio. ucsf.edu/modbase-cgi/index.cgi) provides access to an enormous number of annotated comparative protein structure models [ ] . the program psi-blast was used to assign protein folds to all , , unique sequence entries in swiss-prot-trembl. for % of these sequences, comparative models with an average model size of amino acids could be built using the program modeller [ ] . thus, by august , , d structure models of proteins were accessible via the internet. the models are predicted to have at least % of their c α atoms superimposed within . Å of their correct positions. information on binding sites and ligands can be retrieved from this database using ligbase [ ] . however, the majority of the models are built on a low sequence identity and it should be realized that this level of accuracy is, in most cases, not sufficient for a detailed structure-based ligand design. the swiss-model repository (http://swissmodel.expasy. org/repository) [ ] is also a database of annotated comparative protein d structure models, which have been generated using the fully automated homology-modeling pipeline swiss-model. as of august , this database contained models for , different protein sequence entries ( %) from the swiss-prot-trembl databases ( , , sequences), with an average model size of ~ amino acids. researchers from eidogen (http://www.eidogen.com) have created a database system called target informatics platform™ [ ] that currently includes homology models for , proteins. homology modeling of , human protein sequences resulted in the construction of , models for , different sequences ( %). thus, putative and known ligand binding pockets can be detected, analyzed and compared and the resulting data used to support target prioritization and lead discovery and/or optimization procedures. accelrys (http://www.accelrys.com) produces discovery studio (ds) atlasstore™ as a complete oracle ® -based protein and ligand structural data management solution. currently, ds atlasstore™ contains , , homology models that have been automatically generated from the sequences of , proteins from different genomes. in conjunction with homology models, cengent therapeutics (http://www.cengent.com) offers dynamic structural information generated from molecular dynamics simulations for human drug target proteins. this structural information can be used for target prioritization and virtual screening. the quality of the homology models is dependent on the level of sequence identity between the protein of known structure and the protein to be modeled [ ] . for a sequence identity that is greater than %, homology can be assumed; the two proteins probably have a common ancestor and are, therefore, evolutionarily related and are likely to share a common d structure. in this case, pairwise and multiple sequence alignment algorithms are reliable and can be used for the generation of homology models ( figure ). if the sequence identity is below %, structure modeling becomes speculative, which could lead to misleading conclusions. when the sequence identity is between % and %, conventional alignment methods are not sufficiently reliable and only sophisticated, profile-based methods are capable of recognizing homology and predicting fold. for regions of low sequence identity, threading methods [ ] are often applied. protein models that are built on such low sequence identities can be used for the assignment of protein function and for the direction of mutagenesis experiments ( figure ). models that have a sequence identity between ~ % and % could facilitate the structure-based prediction of target drugability, the design of mutagenesis experiments and the design of in vitro test assays ( figure ). if sequence identity is greater than ~ %, the resulting models are frequently of sufficient quality to be used in the prediction of detailed protein-ligand interactions, such as structure-based drug design and prediction of the preferred sites of metabolism of small molecules ( figure ). there are numerous applications for protein structure information and, hence, homology models at various stages of the drug discovery process [ ] . the most spectacular successes are clearly those where protein structural information has helped to identify or to optimize compounds that were subsequently progressed to clinical trials or to the drug market [ ] . the applications of homology models that had an impact on target identification and/or validation, lead identification and lead optimization are reviewed here ( figure ). it is clear that only a minute fraction of the entire proteome can be affected by drug-like (preferentially orally bioavailable) small molecules. based on the total numbers of known genes, disease-modifying genes and drugable proteins, the number of drug target proteins, for humans, has been estimated at - [ ] . for small molecules, sets of properties have been established that differentiate drugs from other compounds [ , ] ; these properties can be used to identify compounds with, for example, poor oral absorption properties [ ] . drug molecules and their corresponding target proteins are highly complementary, which suggests that some rules that distinguish good target proteins from others should be deducible [ ] . deep lipophilic pockets that comprise distinct polar interaction sites are clearly superior to shallow highly charged protein surface regions. the inhibition of protein-protein interfaces as a valuable therapeutic principle has recently been shown with inhibitors of the p -murine double minute clone (mdm ) interaction [ , ] . the binding site for these inhibitors is a distinct lipophilic pocket that normally interacts with the α-helical surface patches of the p tumor suppressor transactivation domain. advances in the rapid detection, description and analysis of ligand-binding pockets [ ] [ ] [ ] , together with the availability of more than . million homology models, will open new possibilities for the prioritization of proteins with regards to drugability. in the pharmaceutical industry, structural aspects are being increasingly implemented as additional decision criteria on the drugability of potential drug targets. companies such as inpharmatica (http:// www.inpharmatica.com) have developed an integrated suite of informatics-based discovery technologies that contain software tools for the structure-based assessment of target drugability. the design of site-directed mutant proteins is one further important option for the application of homology models to target validation. introducing point mutations and subsequently studying the effects in vitro or in vivo is a common approach in molecular biology. this strategy enables the identification of amino acids that are functionally or relationship between target and template sequence identity and the information content of resulting homology models. arrows indicate the methods that can be used to detect sequence similarity between target and template sequences. applications of the homology models in drug discovery are listed to the right. the higher the sequence identity, the more accurate the resulting structure information. homology models that are built on sequence identities above ~ % can frequently be used for drug design purposes. superimpositions of x-ray crystal structures of the ligand-binding domains of members of the nuclear receptor family are shown to the left. these x-ray structures illustrate the increase in structure deviation with a decreased sequence identity. the pr is red, the gr is green, the erα is blue and the trβ is cyan. sequence identities: pr:gr, %; pr:erα, %; and pr:trβ, %. abbreviations: erα, estrogen receptor α; gr, glucocorticoid receptor; pr, progesterone receptor; trβ, thyroid receptor β. structurally important in the protein under investigation, which ultimately contributes to biological knowledge on, for example, potential target proteins. typically, the amino acids that are to be modified in these studies are selected on the basis of sequence alignments by focusing on conserved residues. however, if at least some structure information is available, the selection of the amino acids that are to be mutated can be much more precise and successful [ ] . this approach is even more powerful when applied in conjunction with pharmacologically active compounds. site-directed mutants of the target protein can be made to render that target sensitive to an existing pharmacological agent. based on homology models, some members of the mitogen-activated protein (map) kinase family were mutated to make them sensitive to a kinase inhibitor from the pyridinyl imidazole class [ ] . this enabled the use of the compound for broader target validation studies. one of the most attractive ways to validate a target protein is to administer a pharmacologically active compound that selectively acts on that protein and to study the effects in a relevant animal model. similar strategies have been described under the term 'chemogenomics' [ ] . it has recently been shown that it is possible to design small molecules based on homology models and then to use these compounds as tools to study the physiological role of the respective target protein of that particular drug [ ] . eight years after the discovery of estrogen receptor β (erβ), the distinct roles of the two er isotypes, erα and erβ, in mediating the physiological responses to estrogens are not completely understood. although knockout animal experiments have provided an insight into estrogen signaling, additional information on the function of erα and erβ was imparted by the application of isotype selective er agonists. based on the crystal structure of the erαligand-binding domain (lbd) and a homology model of the erβ-lbd ( % sequence identity to erα), hillisch et al. [ ] designed steroidal ligands that exploit the differences in size and flexibility of the two ligand-binding cavities ( figure ). compounds that were predicted to bind preferentially to either erα or erβ were synthesized and tested in vitro. this approach led directly to highly er isotype-selective ( - -fold) ligands that were also highly potent. to unravel the physiological roles of each of the two receptors, in vivo experiments with rats were conducted using the erα-and erβ-selective agonists in parallel with the natural ligand of er, β-estradiol. the erα agonist was shown to be responsible for most of the known estrogenic effects (e.g. induction of uterine growth and bone-protection), in addition to pituitary (e.g. reduction of luteinizing hormone plasma levels) and liver (e.g. increase in angiotensin i plasma levels) effects [ ] . however, the erβ agonist had distinct effects on the ovary, for example, the stimulation of early folliculogenesis [ ] , which possibly presents clinicians with a new option for tailoring classical ovarian stimulation protocols. a comparison of the homology model with the x-ray crystal structure of the erβ-lbd complexed with genistein [ ] revealed that the homology model had a root-mean-square deviation (rmsd) of the backbone atoms (not considering helix ) of . Å. the x-ray crystal structure confirmed the presence of essential interactions between the ligand and the erβ and did not reveal, at least in this case, any new aspects for the design of erβ agonists that were not covered by the homology model. these studies show that it is possible to design highly selective compounds, if structure information on all of the relevant homologs of the target is available, and ddt vol. , no. august reviews research focus www.drugdiscoverytoday.com figure . applications of homology models in the drug discovery process. the enormous amount of protein structure information currently available could not only support lead compound identification and optimization, but could also contribute to target identification and validation. reproduced, with permission, from [ ] . there are numerous examples where protein homology models have supported the discovery and the optimization of lead compounds with respect to potency and selectivity. currently, the structures of of the known different human protein kinases have been characterized by x-ray crystallography [ ] . homology model-based drug design has been applied to epidermal growth factor-receptor tyrosine kinase protein [ , ] , bruton's tyrosine kinase [ ] , janus kinase [ ] and human aurora and kinases [ ] . using the x-ray crystal structure of cyclin-dependent kinase (cdk ), honma et al. [ ] generated a homology model of cdk . this model guided the design of highly potent and selective cdk inhibitors that were targeted towards the atp binding pocket. the diarylurea class of compounds were subsequently synthesized and tested. in an in vitro inhibition assay, the most potent compound had an ic of nm. the predicted binding mode of the lead compound was verified by co-crystallization with cdk [ ] . vangrevelinghe et al. [ ] identified a cdk inhibitor using a homology model of the protein and highthroughput docking. siedlecki et al. [ ] have demonstrated the utility of homology modeling in the prediction of pharmacologically active compounds. alterations in dna methylation patterns play an important role in tumorigenesis; therefore, inhibitors of dna methyltransferase (dnmt ), which is the protein that represents the major dna methyltransferase activity in human cells, are desired. known inhibitors from the -azacytidine class were docked into the active site of a dnmt homology model, which led to the design of n -fluoroacetyl- -azacytidine derivatives that acted as highly potent inhibitors of dna methylation in vitro. thrombin-activatable fibrinolysis inhibitor (tafi) is an important regulator of fibrinolysis, and inhibitors of this enzyme have potential use in antithrombotic and thrombolytic therapy. based on a homology model of tafi (~ % sequence identity to carboxypeptidases a and b), appropriately substituted imidazole acetic acids were designed and were subsequently found to be potent and selective inhibitors of activated tafi [ ] . homology models of the voltage-gated k + -channel k v . and the ca + -activated channel ik ca were used to design selective ik ca inhibitors that were based on the polypeptide toxin charybdotoxin. comparison of the two models revealed a unique cluster of negatively charged residues in the turret of k v . that were not present in ik ca . to exploit this difference, the homology model was used to design novel analogs, which were then synthesized and tested. research demonstrated that the novel compounds blocked ik ca activity with ~ -fold higher affinity than k v . [ ] . . (a,b) comparison of the two isotypes of the estrogen receptor. in the homology model, erα (blue) and erβ (green) ligand-binding pockets are shown in complex with the natural ligand of the er, β-estradiol. the binding of β-ve , a highly potent and selective erβ agonist, modeled into the erβ ligand-binding niche is depicted to the right. reproduced, with permission, from ref. [ ] . (c,d) a model of the antiprogestin ru (mifepristone) bound to hpr. a single amino acid mutation renders this compound inactive at the cpr and hamster pr. steric clashes between ru and cpr are shown on the right side. abbreviations: er, estrogen receptor; her, human estrogen receptor; cpr, chicken progesterone receptor; hpr, human progesterone receptor; pr, progesterone receptor; rba, relative binding affinity; β-ve , β-vinylestra- , , ( )-triene- , β-diol. the key proteinase (m pro , or cl pro ) of the new coronavirus (cov) that caused the severe acute respiratory syndrome (sars) outbreak of (sars-cov) is another example of successful homology model building; in this case, success is defined as the ability to use the model to propose an inhibitor that has significant affinity for the target enzyme. x-ray crystal structures for the m pro s of transmissible gastroenteritis virus (tgev, a porcine coronavirus) and of human coronavirus e [ , ] have been characterized. these proteinases have and % sequence identity, respectively, with the key proteinase of sars-cov. following publication of the genome sequence of the new virus, first on the internet and a few weeks later in print [ , ] , the level of sequence identity between the proteinases enabled anand et al. [ ] to construct a d homology model for the m pro of human cov. however, the d homology model generated was insufficient for the design of inhibitors with reasonable confidence. to establish the structural basis of the interaction with the polypeptide substrate of the m pro , anand and co-workers [ ] synthesized a substrate-analogous hexapeptidyl chloromethylketone inhibitor that was complexed with tgev m pro . the x-ray crystal structure of the complex was then determined, which revealed that, as expected, the chloromethylketone moiety had covalently reacted with the active-site cysteine residue of the proteinase. the p , p , and p side chains of the inhibitor had bound to, and thereby defined, the specificity binding sites of the target enzyme. the experimentally determined structure of the inhibitor-tgev m pro complex was then compared with all inhibitor complexes of cysteine proteinases in the pdb, which revealed a surprisingly similar inhibitor binding mode in the complex of human rhinovirus type (hrv ) c proteinase with ag ( figure ) [ ] . at that time, ag was in late phase ii clinical trials as a drug for the treatment of the strain of the common cold that is caused by human rhinovirus. the comparison of the crystal structures of hrv in complex with ag and tgev m pro in complex with the hexapeptidyl chloromethylketone inhibitor revealed little similarity between the two target enzymes, except in the immediate neighborhood of the catalytic cysteine residue, but an almost perfect match of the inhibitors. to investigate these findings further, ag was docked into the substrate-binding site of the sars-cov m pro model without much difficulty, although it was noted that there could potentially be steric problems with the p-fluorobenzyl group in the s pocket, and also with the ethylester moiety in s ′. therefore, it was proposed that, although ag was not an ideal inhibitor, this compound should be a good starting point for the design of anti-sars drugs. indeed, only a few days after the on-line publication of these results in sciencexpress [ ] , it was confirmed that ag had anti-sars activity in vitro. derivatives of ag with modified p residues have since been shown to have k i values in the lower µmolar range (rao et al., pers. commun.) . the crystal structure of the authentic sars-cov key proteinase was determined a few months later [ ] . although the dimeric structure showed the expected similarity to the homologous enzymes of tgev and human cov e, there were interesting differences in detail. in particular, one of the monomers in the dimer was observed to be in an inactive conformation, which was thought to be the result of the low ph of crystallization. the overall rmsd for the entire dimer from the homology model of anand et al. [ ] was > . Å (i.e. no residues excluded from the comparison), which dropped to . Å when a few outliers at the carboxy terminus were excluded from the comparison, and to < . Å for each of the three individual domains of the enzyme. other homology models were generated (d. debe, unpublished and [ ] ) and virtual screening has been performed using a sars-cov m pro model [ ] . taken together, these findings confirm that homology modeling is often inadequate for the prediction of the mutual orientation of domains in multidomain proteins. however, the homology model generated by anand et al. [ ] also shows that a reasonable model of a substrate-binding site can serve to develop useful ideas for inhibitor design that can inspire medicinal chemists to start a synthesis program long before the d structure of the target enzyme is experimentally determined. in the case of g-protein-coupled receptors (gpcr), homology-modeling approaches are limited by the lack of experimentally determined structures and the low sequence similarity of those structures that have been characterized with respect to pharmacologically important target proteins. the x-ray crystal structure of only one gpcr, bovine rhodopsin, has been determined [ ] . this structure is complemented by bacteriorhodopsin, which is a transmembrane protein that comprises seven helices and is also of relevance for modeling approaches, even though this protein is not a gpcr. some examples of homology models for gpcrs and their utility have recently been reviewed [ ] . high-throughput docking has been applied to verify the ability of homology models to identify agonists (glucocorticoid receptor agonists) [ ] , antagonists of retinoic acid receptor α [ ] , d -dopamine-, m -muscarinic acetylcholine-and v a -vasopressin-receptors [ ] and inhibitors of thrombin [ ] . in the identification of thrombin inhibitors, homology models of thrombin were built retrospectively and were based on homologous serine proteases ( %- % sequence identity); the best docking solutions were yielded with those models that were derived from proteins of higher sequence identity. recently, the performance of docking studies into protein active sites that had been constructed from homology models was assessed using experimental screening datasets of cdk and factor viia [ ] . when the sequence identity between the model and the template near the binding site was greater than ~ %, there was an approximate fivefold increase in the number of active compounds identified than would have been be detected randomly. this performance is comparable to docking to crystal structures. a further application of homology models is the design of test assays for the in vitro pharmacological characterization of compounds or hts. based on the structure of the coiledcoil domain of c-jun, models for α-helical proteins were designed such that they can be used as affinity-tagged proteins that incorporate protease cleavage sites [ ] . the resulting . kda recombinant proteins were synthesized and used as molecularly defined and uniform substrates for in vitro detection of hiv- and iga endoprotease activity, which enabled the surface plasmon resonance-based screening of inhibitors. the enormous volume of structure information on entire target protein families that is available might also have an impact on screening cascades. many drug discovery projects endeavor to identify ligands that are highly selective for particular drug targets. selective compounds are supposed to be superior because such compounds typically lead to fewer adverse side effects (e.g. cox- inhibitors). however, the most important homologs that should not be targeted by the desired drug, with respect to the actual target, are not always clear, particularly within the larger target protein families. the sequence similarity of the full-length proteins or entire domains might not always be representative of the target protein when considering the conservation of the ligand-binding pockets. comparison of the shape and features of the binding pockets within a protein family could indicate which homologs should be included in the screening cascade for so-called 'counter screening'. the structure information that is currently available on entire protein families (e.g. proteases, kinases and nuclear receptors) could contribute to the design of selective compounds or better screening cascades, both of which could potentially advance the design of drugs that have fewer side effects. a detailed structural knowledge of the ligand-binding sites of target proteins was also shown to facilitate the selection of animal models for ex vivo or in vivo experiments. the proposal is that animals having target proteins with significantly different binding sites compared with human orthologs should be excluded as pharmacological models. many promising compounds showing high-potency in human in vitro assays have not reached clinical trials because efficacy could not be demonstrated in animal models. single amino acid differences between humans and animals might, in some cases, be sufficient to cause such effects. the er selectivity of ligands described by hillisch et al. [ ] was shown by homology models and in vitro assays to be crucially dependent on the interaction of ligand substituents with one particular amino acid that differs between erα and erβ (figure a ) [ ] . to ensure that this important interaction is present in estrogen receptors of all animal models that are used to characterize compounds [ ] , homology models of murine, rat and bovine erβ were built and compared with the binding pocket of human erβ (herβ). a complete conservation of amino acids within the binding pockets of human, murine and rat erβ was observed. however, bovine erβ showed one amino acid difference at the exact position that was determined to be crucial for erβ ligand selectivity. the prediction that the herβ selective compounds should not bind to bovine erβ was later verified using transactivation experiments (unpublished results). thus, the implementation of uninterpretable experiments could be avoided at an early stage and the otherwise attractive bovine tissues (later available in larger amounts) could be excluded from ex vivo investigations. similarly, information on the structure of progesterone receptors (pr) can be used to explain the abolished binding of the progesterone antagonist mifepristone (ru ) to chicken pr and hamster pr [ ] . a single point mutation (human pr gly to chicken pr cys ) prevents antiprogestins containing β-aryl substituents (e.g. ru ) from binding to chicken (and hamster) pr (figure c) , which therefore excludes hamsters, for example, from pharmacological studies with antiprogestins [ ] . in the future, such effects could be predicted and particular species could then be excluded from pharmacological studies at an early stage, which would ultimately reduce attrition rates in the drug discovery process. one of the challenges in lead optimization is to identify compounds that not only show a high potency at the desired target protein but also have adequate physical properties to reach systemic circulation, to resist metabolic inactivation for a specific time period and to avoid undesired pharmacological effects. knowledge of the structure of the proteins that are involved in these processes, such as drugmetabolizing enzymes, transcription factors or transporters, could help to design molecules that do not interact with these 'non-target' proteins. the cytochrome p s (cyp) are an extremely important class of enzymes that are involved in phase i oxidative metabolism of structurally diverse chemicals. only ~ hepatic cyps are responsible for the metabolism of % of known drugs. recently, the x-ray crystal structures of three mammalian cyps, cyp c [ ] , cyp c [ ] and cyp c [ ] , have been solved and represent a solid basis for the homology modeling of this entire superfamily. models of cyp a , cyp a , cyp b , cyp c , cyp c , cyp c , cyp d , cyp e , cyp a and cyp a have been generated using different structure templates. these models have been used to explain and to predict the probable sites of metabolic attack in a variety of cyp substrates [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, the large lipophilic and highly flexible character of some cyp binding cavities renders pure in silico approaches towards the prediction of the occurrence and site of small molecule metabolism extremely difficult. if protein structure information is combined with pharmacophoric patterns and quantum mechanical calculations, some predictions concerning the preferred sites of metabolism within small molecules are possible [ ] . regarding this aspect of homology modeling, cyp d is a particularly interesting cyp because - % of the caucasian population does not produce this polymorphic member of the cyp superfamily. the resulting deficiencies in drug oxidation can lead to severe side effects in these individuals. predictions on whether or not a lead compound could act as a cyp d substrate could help to identify problematic cases early in drug discovery. combined homology modeling and quantitative sar approaches are able to predict such cyp inhibitors [ ] . thus, in the future, protein structure information in conjunction with high-throughput docking and pharmacophore-based methods could be used to decide which compounds have the potential to inhibit particular cyps. this approach could facilitate the detection of potential drug-drug interactions early in the drug discovery process and measures could then be taken to avoid such interactions [ ] . cyp substrates and inhibitors are not the only compounds to have been studied using homology models. these approaches have been used recently to investigate cyp inducers. the induction of cyps is primarily mediated via the activation of ligand-dependent transcription factors, such as the aryl hydrocarbon receptor (ahr) for the cyp a family, the constitutive androstane receptor (car) for the cyp d family and the pregnane x receptor (pxr), glucocorticoid receptor (gr) and vitamin d receptor (vdr) for the cyp a family [ ] . in principle, the in silico prediction of drug-metabolizing enzyme induction could be reduced to predicting the binding and activation of transcription factors (e.g. ahr and car). however, recent xray structure analyses of pxr have shown that the lbd of this nuclear receptor contains a large lipophilic and flexible binding pocket [ ] . this renders pure in silico structure-based predictions concerning whether or not a small molecule will activate pxr difficult. the homology modeling of car [ , ] and other members of the nuclear receptor family involved in cyp induction [ ] have recently been described. these models predict reasonably shaped potential ligand binding pockets. however, further results on the utility of these models are needed. with respect to the structure-based prediction of adverse health effects, progress has been described with the human ether-a-go-go-related gene (herg). this tetrameric potassium channel contributes to phase three repolarization of heart muscle cells by opposing the depolarizing ca + influx during the plateau phase. inhibition of this protein results in cardiovascular toxicity (qt-prolongation) and has caused several drugs to be withdrawn from the market. therefore, in silico predictions on the probability of the formation of an interaction between a drug and herg have gained enormous attention and have recently been reviewed [ ] . homology models of herg, which are based on the x-ray crystal structures of the bacterial kcsa [ ] and mthk channels [ ] , have already shed light on some details of the molecular interactions that initiate herg inhibition. however, the complexity of this potassium channel signifies that detailed x-ray structure analyses of the protein in the open-and closed-state are required before these molecular interactions can be fully understood and predicted, which has implications for the prediction of cardiotoxicity. numerous examples for the successful application of homology modeling in drug discovery are described here. in the absence of experimental structures of drug target proteins, homology models have supported the design of several potent pharmacological agents. one of the advantages of homology models is that these models can be generated ddt vol. , no. august reviews research focus relatively easily and quickly. furthermore, such models could support the hypotheses of medicinal chemists on how to generate biologically active compounds in the important early conceptual phase of a drug discovery project. the design of compounds that are selectively directed at particular drug target proteins is one of the strengths of this technique. such selective compounds can even be applied to gain insights into the physiological role of novel drug targets. the in silico protein structure-based prediction of metabolism and toxicity of small molecules, particularly cyp inhibition and induction and herg inhibition, is currently in its infancy and predictive capabilities could be limited to classification only. however, while complete experimental structures of pharmacologically important proteins are missing, the homology modeling technique provides one approach to bridge the gap until this information becomes available. modern methods of drug discovery: an introduction the response of protein structures to amino-acid sequence changes fold recognition methods progress in protein structure prediction assessment of homology-based predictions in casp scop: a structural classification of proteins database for the investigation of sequences and structures databases for protein-ligand complexes ligbase: a database of families of aligned ligand binding sites in known protein sequences and structures pdbsum: summaries and analyses of pdb structures modbase, a database of annotated comparative protein structure models, and associated resources comparative protein modelling by satisfaction of spatial restraints the swiss-model repository of annotated threedimensional protein structure homology models supporting your pipeline with structural knowledge the relation between the divergence of sequence and structure in proteins casp assessment of fold recognition target predictions the role of protein d structures in the drug discovery process the impact of structure-guided drug design on clinical agents the druggable genome prediction of 'drug-likeness' a scoring scheme for discriminating between drugs and nondrugs drug-like properties and the causes of poor solubility and poor permeability structural bioinformatics in drug discovery inhibition of the p -hdm interaction with low molecular weight compounds inhibition of the p -mdm interaction: targeting a protein−protein interface a new method to detect related function among proteins independent of sequence and fold homology inferring functional relationships of proteins from local sequence and spatial surface patterns structural classification of protein kinases using d molecular interaction field analysis of their ligand binding sites: target family landscapes transcriptional repressor copr: structure model-based localization of the deoxyribonucleic acid binding motif use of a drug-resistant mutant of stress-activated protein kinase a/p to validate the in vivo specificity of sb chemogenomics: an emerging strategy for rapid target and drug discovery dissecting physiological roles of estrogen receptor alpha and beta with potent selective ligands from structurebased design impact of isotype-selective estrogen receptor agonists on ovarian function structure of the ligand-binding domain of oestrogen receptor beta in the presence of a partial agonist and a full antagonist the protein kinase complement of the human genome design and synthesis of novel tyrosine kinase inhibitors using a pharmacophore model of the atp-binding site of the egf-r rational design of potent and selective egfr tyrosine kinase inhibitors as anticancer agents rational design and synthesis of a novel antileukemic agent targeting bruton's tyrosine kinase (btk), lfm-a structure-based design of specific inhibitors of janus kinase as apoptosis-inducing antileukemic agents targeting aurora kinase in oncogenesis: a structural bioinformatics approach to target validation and rational drug design structure-based generation of a new class of potent cdk inhibitors: new de novo design strategy and library design discovery of a potent and selective protein kinase ck inhibitor by high-throughput docking establishment and functional validation of a structural homology model for human dna methyltransferase synthesis and evaluation of imidazole acetic acid inhibitors of activated thrombin-activatable fibrinolysis inhibitor as novel antithrombotics structure-guided transformation of charybdotoxin yields an analog that selectively targets ca + -activated over voltage-gated k + channels structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain coronavirus main proteinase ( clpro) structure: basis for design of anti-sars drugs characterization of a novel coronavirus associated with severe acute respiratory syndrome structure-assisted design of mechanismbased irreversible inhibitors of human rhinovirus c protease with potent antiviral activity against multiple rhinovirus serotypes the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor evaluation of homology modeling of the severe acute respiratory syndrome (sars) coronavirus main protease for structure-based drug design a d model of sars cov cl proteinase and its inhibitors design by virtual screening crystal structure of rhodopsin: a g-proteincoupled receptor modeling the d structure of gpcrs: advances and application to drug discovery nuclear hormone receptor targeted virtual screening rational discovery of novel nuclear hormone receptor antagonists protein-based virtual screening of chemical databases. ii. are homology models of g-protein-coupled receptors suitable targets? docking ligands onto binding site representations derived from proteins built by homology modelling performance of d database molecular docking studies into homology models design of helical proteins for real-time endoprotease assays a single amino acid that determines the sensitivity of progesterone receptors to ru ru is not an antiprogestin in the hamster mammalian microsomal cytochrome p monooxygenase: structural adaptations for membrane binding and functional diversity structure of human microsomal cytochrome p c . evidence for a peripheral fatty acid binding site crystal structure of human cytochrome p c with bound warfarin molecular modeling of human cytochrome p -substrate interactions modelling human cytochromes p involved in drug metabolism from the cyp c crystallographic template homology modelling of human cyp a based on the cyp c crystallographic template structure homology modelling of cyp a based on the cyp c crystallographic template: enzyme-substrate interactions and qsars for binding affinity and inhibition molecular modelling of cyp b based on homology with the cyp c crystal structure: analysis of enzyme-substrate interactions a molecular model of cyp d constructed by homology with the cyp c crystallographic template: investigation of enzyme-substrate interactions investigation of enzyme selectivity in the human cyp c subfamily: homology modelling of cyp c , cyp c and cyp c from the cyp c crystallographic template prediction of drug metabolism: the case of cytochrome p d a novel approach to predicting p mediated drug metabolism. cyp d catalyzed n-dealkylation reactions and qualitative metabolite predictions using a combined protein and pharmacophore model for cyp d competitive cyp c inhibitors: enzyme inhibition studies, protein homology modeling, and three-dimensional quantitative structure-activity relationship analysis molecular basis of p inhibition and activation: implications for drug development and drug therapy prediction of human drug metabolizing enzyme induction coactivator binding promotes the specific interaction between ligand and the pregnane x receptor a structural model of the constitutive androstane receptor defines novel interactions that mediate ligandindependent activity insights from a three-dimensional model into ligand binding to constitutive active receptor molecular modelling of the human glucocorticoid receptor (hgr) ligand-binding domain (lbd) by homology with the human estrogen receptor alpha (heralpha) lbd: quantitative structure-activity relationships within a series of cyp a inducers where induction is mediated via hgr involvement predicting undesirable drug interactions with promiscuous proteins in silico a structural basis for drug-induced long qt syndrome characterization of herg potassium channel inhibition using comsia d qsar and homology modeling approaches modern methods of drug discovery we gratefully acknowledge fruitful discussions with mario lobell (bayer healthcare; http://www.bayerhealthcare.com), derek debe, sean mullen (eidogen) and sunil patel (accelrys). key: cord- - cwp xbw authors: yamasaki, satoshi; amemiya, takayuki; yabuki, yukimitsu; horimoto, katsuhisa; fukui, kazuhiko title: togo-wf: prediction of rna tertiary structures and rna–rna/protein interactions using the knime workflow date: - - journal: j comput aided mol des doi: . /s - - -y sha: doc_id: cord_uid: cwp xbw recent progress in molecular biology has revealed that many non-coding rnas regulate gene expression or catalyze biochemical reactions in tumors, viruses and several other diseases. the tertiary structure of rna molecules and rna–rna/protein interaction sites are of increasing importance as potential targets for new medicines that treat a broad array of human diseases. current rna drugs are split into two groups: antisense rna molecules and aptamers. in this report, we present a novel workflow to predict rna tertiary structures and rna–rna/protein interactions using the knime environment, which enabled us to assemble a combination of rna-related analytical tools and databases. in this study, three analytical workflows for comprehensive structural analysis of rna are introduced: ( ) prediction of the tertiary structure of rna; ( ) prediction of the structure of rna–rna complexes and analysis of their interactions; and ( ) prediction of the structure of rna–protein complexes and analysis of their interactions. in an rna–protein case study, we modeled the tertiary structure of pegaptanib, an aptamer drug, and performed docking calculations of the pegaptanib-vascular endothelial growth factor complex using a fragment of the interaction site of the aptamer. we also present molecular dynamics simulations of the rna–protein complex to evaluate the affinity of the complex by mutating bases at the interaction interface. the results provide valuable information for designing novel features of aptamer-protein complexes. electronic supplementary material: the online version of this article ( . /s - - -y) contains supplementary material, which is available to authorized users. recent advances in micro-array and sequencing technologies have revealed that large amounts of non-coding rnas exist in cells. for example, in human cells, tens of thousands of long non-coding rnas have been identified [ ] . the functions of non-coding rnas have been studied extensively, and relationships between mutations in non-coding rnas and diseases have been investigated [ ] [ ] [ ] . these non-coding rnas have various functions, including regulation of gene expression and catalysis of biochemical reactions inside cells. protein-rna and rna-rna interactions are key elements in such functional processes. the rna-induced silencing complex (risc), small nuclear ribonucleoproteins (snrnps) and small nucleolar rna-protein complexes (snornps) are protein-rna complexes that regulate gene expression. additionally, rna interference can be used readily as a mechanism to decompose target mrnas [ ] [ ] [ ] . several types of non-coding rnas are associated with serious diseases. in cancer cells, many types of micro-rna (mirna) are expressed and affect tumor progression or metastasis by interacting with various tumor factors [ ] [ ] [ ] [ ] [ ] . interaction between cellular human mirna and viral rna is important for infection or defense processes of pathogenic viruses, including hiv, influenza virus, sars coronavirus and ebola virus [ ] [ ] [ ] [ ] [ ] . moreover, there is a general acceptance that many kinds of structured non-coding rnas exist in nature and play important roles in living cells; however, details of their functional mechanisms remain largely unknown [ ] [ ] [ ] . non-coding rnas have therapeutic value as potential drugs and drug targets [ ] . there is high expectation that nucleic acid medicines will alleviate or cure disease processes, because specifically designed nucleic acids (oligonucleotides) will directly target dna, rna, or proteins that cause diseases. the depletion of drug discovery targets in low molecular weight drug discovery programs, the problems associated with mass production of antibody drugs that show high specificity, and issues of high cost to sales ratios will possibly be solved by nucleic acid medicine. consequently, the development of nucleic acid medicinal approaches is attracting significant attention. currently, five nucleic acid-based drugs have been approved and are on the market worldwide. several companies are considering commercialization based on industry-academia co-operations, and a dramatic increase in this market is anticipated in the near future [ , ] . nucleic acids generally play important roles in preserving genetic information and protein synthesis, and these biomolecules form various three-dimensional structures. in an effort to exploit their potential benefit to human health, it is important to carry out detailed analyses of protein-rna or rna-rna interactions. thus, solving the tertiary structures of rna molecules, as seen with proteins, should aid nucleic acid drug development. therefore, a large body of research investigating or predicting the tertiary structures of rna has been performed [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . previously-developed software for predicting the secondary (rnafold [ ] , mfold [ ] , centroidfold [ ] ) and tertiary structures (farna [ ] , mc-fold and mc-sym [ ] , rna d d [ ] , simrna [ ] , spqr [ ] ) of rna molecules from their nucleotide sequences are available. a recent computational challenge is the prediction of tertiary structures of more difficult targets such as rna-rna and rna-protein complexes. the current difficulties associated with using available tools and software are the requirement of several complex interactive manipulations and a user that has a strong understanding of rna tertiary structures. to simplify the task of computational rna analysis, we provide a novel workflow, which facilitates the study of rna molecules from their sequence to their tertiary structure, and their interactions with various biomolecules. by using this workflow, it is possible to use various analysis tools, software and databases developed for rna analytics in a single platform. for example, in the case of tertiary structure prediction, the input sequence selected from a database passes to secondary structure prediction software and the calculated secondary structure is used for tertiary structure calculations in a one-stop platform. the intermediate results of each computational step can be visualized. workflow systems with a graphical user interface are useful tools for addressing how to treat large-scale data and to efficiently integrate analytical resources [ ] . we introduce the togo-wf workflow as a bioinformatic analytical resource, which is capable of analyzing, modeling and visualizing biological data by using the knime (konstanz information miner) platform [ ] . the knime workflow platform is freely available via http://www.knme.org. over the last few years, we have released several bioinformatic nodes, mainly developed by bioinformatics groups at aist, and have deposited them in the node repository using the knime environment. on this platform, users can combine independent analytical resources, called nodes in knime, from the repository of bioinformatics tools developed at aist and external tools/databases by selecting them in an easy drag-and-drop manner. in this study, we have developed a workflow that combines rna-related analytical nodes to predict rna tertiary structures and rna-rna/protein interactions that provide insights and a starting point for tertiary structural analysis by rna molecular simulations. using the structure of an rna-protein complex obtained from this workflow, we have performed molecular dynamics simulations of this rna-protein complex to analyze the structural stability and affinity of the interaction. we have created several novel rna analytical nodes for predicting rna tertiary structures and rna-rna/protein interactions. the knime environment, including the rna nodes, can be downloaded from the togo-wf website (http://togo.medal s.jp/activ e_local _rna_predi ction .eng.html) for microsoft windows, linux and mac osx operating systems. we have also posted installation instructions and user manuals for the rna workflow. after installation, three workflow lines are released by selecting rna structure prediction in the knime explorer, as shown in fig. . nodes in the workflow that were developed by aist include sparql, centroidfold, ipknot, rna dchecker, viewer, fragment-selector, intminmm, ractip, rassie and rascal. prediction of rna secondary structures is performed using the programs centroidfold [ ] , ipknot and ractip [ ] , and tertiary structure prediction is achieved using the programs rassie [ ] and rascal [ ] . the rna dchecker node is used to judge the difficulty of the tertiary structure prediction based on the secondary structure prediction results and from this analysis either rassie or rascal is selected. the details of rassie and rascal based on fragment-based methods are described in references [ , ] . in brief, prediction using rassie is performed by assembling small tertiary structural elements based on secondary structures (stem(duplex), hairpin-loop, internal-loop, single-strandloop). those small structural elements were derived from rna structures found in the pdb. targets with long loop structures that are not found in the pdb or targets with high complexity (e.g., targets with too many structural elements) are switched to the rascal node by rna dchecker. rascal is based on a fragment-assembly method, which uses dihedral angles of single strand three base fragments and randomly replaces them during - thousands of steps of metropolis monte carlo simulation. rascal can predict tertiary structures with any secondary structures because the single stranded three-base fragments derived from rna structures found in the pdb are not based on their secondary structure like rassie. rascal can handle single and double stranded structures as input with a secondary structure and joint secondary structures of rna-rna interactions. rascal also has the feature of predicting tertiary structures by adjusting base-pair potentials without the secondary structure. rassie predicts relatively short chain rnas with simple secondary structure elements in rapid computational time. long-chain rnas with complex secondary structures can be predicted by rascal; however, more computational time is required to obtain good prediction results. in the workflow for determining rna-protein complexes, fpockts [ ] and autodockvina [ ] are used as the analytical resources for rna-protein docking. the node of the fragment selector for rna can create the fragment structure the bottom workflow presents the structure prediction of the rnaprotein complex of the given rna and the rna fragment is used for docking calculations with the target protein whose active sites are specified by the fpockts node. after docking, the rna fragment-protein complex structures are obtained using the merge target and ligand node. the original input rna structure is reconstructed by the rebuild node based on the docking position of the rna fragment, and the dockinganalyzer node is used to cluster the complex structures. by selecting one of the complex structures, the structure is energetically minimized by molecular mechanics, initminmm. in the knime environment, users can configure each node by clicking the right mouse button and can view a simple annotation in the node description by clicking the left mouse button. the nodes developed by aist allow users to execute jobs on our aist remote computer (not on the user's local computer). the nodes, rassie, rascal, autodockvine and initminmm demand high amounts of computing power and memory. these nodes are in our cloud-based workflow system and jobs with these nodes are submitted to the message passing interface (mpi) parallel jobs system of the aist cluster machine. the manuals on how to install and use togo-wf for rna can be downloaded from our website (https ://togo.medal s.jp/activ e_local _rna_predi ction .eng.html). we constructed a database by searching the literature for nucleic acid-based drugs and web services. rna-based drugs can be classified by the mechanism of activity, and include inhibitors of mrna translation (antisense) and rnas that bind proteins and other molecular ligands (aptamers) [ ] . information about the authorization status of nucleic acid drugs, primary structure (sequence information) and previous researchwas primarily collected from the eu clinical trials register (https ://www.clini caltr ialsr egist er.eu), u.s. food and drug administration (https ://www. fda.gov/) and ionis pharmaceuticals, inc. (http://www.ionis pharm a.com/pipel ine/). the developed database for medals nucleic acid drug database (mnadd) provides "atc classification", "product name", "synonymous", "phase", "description" and "target protein id". in the workflow, the sparql node (fig. ) is used to select a sequence from the mnadd database, which contains approved rna drugs. the database also has short non-coding rna entries less than nucleotides obtained from frnadb [ ] , which is accessible at the national bioscience database center (https ://dbarc hive.biosc ience dbc.jp/en/frnad b/desc.html). molecular dynamics (md) simulations in explicit solvent water were performed to relax the structures derived from either the rassie or rascal module of the workflow. all initial structures were placed at the center of the water box and water molecules were modeled using the tip p -point charge model potential [ ] . k + and cl − ions were added using the leap module in the amber package to neutralize the net charge of the system and to give the system an ionic concentration of . m. these systems were energyminimized. all calculations were performed using amber [ ] with the protein.ff sb (for protein), rna.ol (for rna) and tip p (for solvent) force fields [ ] [ ] [ ] [ ] with a time step of integration set to fs. all bond lengths involving hydrogen atoms were constrained to their respective equilibrium values by the shake method [ ] . electrostatic interactions were treated with the particle mesh ewald method with a cut-off of Å [ ] . each system was gradually heated to k during the first ps at a heating rate of k/ps in the nvt ensemble. following a ps heating and equilibrating nvt simulation, for each system, a ns dynamic calculation was performed in the ntp ensemble, with pressure constant at atm and temperature constant at k using the berendsen's thermostat [ ] . molecular mechanics-generalized born surface area (mm-gbsa) calculations were performed to evaluate the stability of the docked complex structure. theoretical free energies were calculated using the mm-gbsa approach [ ] . the md simulations of docked structures were performed using the same method described above. snapshot structures were sampled every ps (from . to . ns of the md trajectory) and used in estimating the binding free energies. in the analysis of the binding free energies, water molecules were replaced with the implicit solvent gb model. this evaluation of free energies using the mm-gbsa approach was performed for complex structures, which were selected from using the rebuild node to confirm that the molecules do not overlap following the rebuilding of the fragments after the docking calculation. the structure of the complex that gave the lower binding free energy with adequate distance was used as the template model for building and carrying out simulations of the rna mutants. the workflow, rna structure prediction, at the top of fig. models the tertiary structure of the rna target according to secondary structure information, which is based on the calculated results from centroidfold [ ] . the rna sequence input is provided by the user or using the sparql node, which is connected to our mnadd database. figure a, b show the predicted secondary and tertiary structures for rna aptamer [ ] calculated from the centroidfold and ras-sie nodes. the input sequence was obtained from a human igg-aptamer complex (pdb id: agv). in the workflow, rna dchecker automatically selects rassie or rascal, depending on the complexity of the secondary structure. for the short sequence, rassie was selected and succeeded in constructing the tertiary structure based on the secondary structure. figure c , d show the predicted secondary and tertiary structures for the non-coding rna (fr of frnadb) calculated by the centroidfold and rascal nodes. the input sequence was selected from mnadd using the sparql node in the workflow. the tertiary structure of such a long hairpin-loop was predicted using rascal. the input sequence and the predicted secondary structure in fig. c were more complicated than those in fig. a . thus, rna- dchecker automatically selected rascal for further tertiary structure calculations in the workflow. to evaluate the performance and capabilities of togo-wf, the predicted structures obtained from the workflow were compared with secondary structures predicted by mcfold, and tertiary structures predicted by mc-sym [ ] and simrna [ ] . the detailed comparison is given in supplementary material . the workflow predicted the rna secondary and tertiary structures to a higher accuracy than mc-fold/mc-sym and simrna. a reason for this higher accuracy is that the fragment-based method with high-resolution potential energy functions in rassie and rascal enables highly accurate tertiary structure predictions. togo-wf uses a platform that aims to provide one-stop rna analysis that can switch the workflow depending on the difficulty of the problem. additionally, by linking the analysis workflow and database with one platform, large-scale analysis is possible. the predicted rna structures can be used easily as initial structures for md simulations to obtain detailed tertiary structure analysis of conformational changes and the stability of potential chemical modifications. the calculations introduced here for prediction of tertiary structures of rnas are described in sect. of the online manual (https ://togo. medal s.jp/downl oad/rna_userm anual _e_v . .pdf). rna-rna interactions are important for many biological functions and considered to be potential targets of new medicines. the kissing hairpin loop of the dimerization initiation site (dis) of hiv- is a typical example of such interactions. in the lifecycle of hiv- , the dimerization of genomic rna is crucial for viral replication [ ] . formation of a kissing loop hairpin structure in the dis region is the primary event of dimerization. therefore, inhibiting formation of the kissing loop is a potential target for treating hiv. the workflow, rna-rna structure prediction, in the middle of fig. predicts the tertiary structure of rna-rna interacting complexes with rascal using the results of ractip, which provides rna two-dimensional complex structures for rna-rna interactions with kissing hairpins. we have previously predicted the tertiary structures of many kissing loops and duplex targets successfully from nucleotide sequences by using the ractip-rascal pipeline [ ] . recently, patisiran (onpattro), which is the first sirna therapeutic agent, was approved by the fda for treating the rare disease hereditary transthyretin amyloidosis [ ] . using the workflow, we have modeled the tertiary structure of the double-stranded sirna, patisiran (fig. ) . this workflow can model tertiary structures of rna-rna interactions .(((....) ))))))), was calculated by the centroidfold node in the workflow. gamma was set to ^ using the configuration from centroidfold. b one of the predicted tertiary structures derived with the rassie node. the predicted and native (pdb id: agv) rna structures are shown in red and blue, respectively. the rmsd (root-mean-squared deviation) between the predicted and native structure ( agv) is . Å. c the input sequence of the putative conserved noncoding region for rascal is tgt tga tcc ggc gtg taa tga ggt gtt cct gga atc aata, which was selected from the mnadd database using the sparql node (fr ). the secondary structure, ((( ((((((((.................) )))).)))))), was calculated by the centroidfold node in the workflow. d one of the predicted tertiary structures derived with the rascal node and double-stranded rna structures. the obtained tertiary structures can be used for further molecular simulations to compute the dimerization process from the kissing structure and the interaction of rna-protein complexes. the results may provide new suggestions or data that cannot be explained without an understanding of the tertiary structures of the complexes. by collecting information about the authorization status of nucleic acid drugs, primary structures and observations from previous research, we found drugs that have gone through clinical trials. they are classified into two major types of nucleic acid-based drugs: one type targets nucleic acids and represents antisense drugs, whereas the other type targets proteins and are termed aptamers. five of the drugs tested have been approved. eteplirsen (exondys ), vitravene (fomivirsen), kynamro (mipomersen) and nusinersen (spinraza) are antisense drugs that bind exon of dystrophin pre-messenger ribonucleic acid (pre-mrna), target mrna of cytomegalovirus, degrade apob- mrna expressed in the liver and target survival motor neuron (smn ) pre-mrna to inhibit factor suppressing splicing of exon from binding to pre-mrna, respectively. the other, pegaptanib (macugen), is an aptamer that binds to vascular endothelial growth factor (vegf). pegaptanib is the first approved aptamer of a new therapy to slow vision loss in people with the eye disease neovascular (wet) age-related macular degeneration (amd) [ ] [ ] [ ] [ ] . antisense drugs represent the majority of nucleic acids in clinical trials with most targeting cancer and viral infections. consequently, there is increasing interest in antisense technology. ions pharma is currently conducting ongoing clinical trials of other antisense drugs for the treatment of common medical conditions, including rheumatoid arthritis, cancers and crohn's disease, a serious intestinal illness. figure shows major adaptation diseases that are current targets of nucleic acid medicines. we summarize the information of nucleic acid drugs in terms of "approval status of nucleic acid drugs", "target proteins and number of its nucleic acid drugs" and "number of nucleic acid drugs participating in pathway", which are provided at the website (https ://medal s.jp/enucl eicac id.html). the database is readily accessible to the corresponding server's url (https :// medal s.jp/edrug nainf o.html) and can be downloaded from https ://dbarc hive.biosc ience dbc.jp/en/nucle ic-acid-drug/ downl oad.html. in the workflow, rna based drugs and non-coding rnas less than nucleotides obtained from frnadb are stored in the rdf-formatted database. users can access the database with the sparql node and use the environment in which database and analysis tools are connected seamlessly in the workflow. the rna-protein workflow is presented at the bottom of fig. and is used to predict the structure of a nucleic acid drug-target protein complex by molecular simulations. we applied our workflow to the complex formed between the antagonistic aptamer pegaptanib and vegf. figure shows the secondary structure of pegaptanib, (((.. ..(((....)) ).......))). when we used the workflow for simple prediction of the secondary structure (fig. , top workflow of the centroidfold with default parameters), the predicted structure is ((((........ ...........) ))). the secondary structure predicted is very different from the experimentally obtained secondary structure in fig. . it is difficult to estimate the tertiary structure using an inaccurate secondary structure as input. thus, in this the input sequences of rna and rna are gua acc aag agu auu cca u (red) and aug gaa uac ucu ugg uua cuu (blue), respectively. the predicted tertiary structure is derived with the rascal node using the result obtained with the ractip node case, the tertiary structure is derived using rascal, which can model structures with internal loops without the need of a secondary structure as a starting point. in obtaining the candidate tertiary structures by rascal, the potential function was set as a penalty if base pairs were formed between a -c and u -u . a matrix file of dimensions (number of bases × number of bases) defining the weight of base-pair potentials is used as input together with the sequence. a detail explanation of how to use the workflow to obtain ten candidate structures of pegaptanib is given in supplementary material . from the ten candidate structures derived from the workflow for tertiary structure determination of rna by the rascal module, two candidate structures shown in figure s ) . nmr experiments have shown [ ] that u interacts with the side chain of cys of vegf. u faces outward in the two selected structures. to relax these structures in a water environment, ns md simulations of each structure immersed in tip p water + kcl ( . m) were performed.one of the rna structures that has the u base facing outward was selected as the tertiary structure in the rna-protein docking process. the relaxed rna structure and protein structures in pdb format were given as input for the complex structure workflow analysis. using the fragmentselector node in the workflow, a three-base fragment a -u -g from the structure was extracted for docking. coordinates of residues in the protein that are in the vicinity of the three-base fragment can be easily specified using the fpocket node. the coordinates near cys in the protein were used as input, and then docking calculations of the pegaptanib fragment and vegf were performed. the docking result is given in supplementary material .the structures of the complex were rebuilt based on the docking poses of the rna fragment. by confirming that the protein and rna structures do not sterically clash with each other in the process of rebuilding the full-length rna, we selected three structures of the complex that are reasonable for carrying out md calculations. figure shows the selected structures of the complex after the docking and rebuilding processes. md simulations and mm-gbsa calculations were performed after energy minimization with the initminmm node to evaluate the stability and binding free energy of the docked structures. for the three docked structures (fig. ), . ns md simulations in tip p water + kcl ( . m) were performed, and snapshot structures every ..) )).......))),which is an antagonistic aptamer that targets vegf ps were taken during the . - . ns simulation period and used for mm-gbsa calculations. the distance between the centroid of base heavy atoms of u and the centroid of the cys -cys disulfide bond was also assessed to evaluate the protein-rna distance (fig. ) . previous experiments reported that cys displayed the largest chemical shift change upon aptamer binding in nmr spectra and the mechanism for the formation of a photo-crosslink between u and the disulfide bond, cys :s-cys :s [ ] . among the c -c structures, the simulation of c showed the closest distance between the aptamer and protein. table shows the binding free energy, ΔΔg, derived from the mm-gbsa calculations for the three complexes. although the calculations showed that the c structure had the lowest ΔΔg, indicating the highest affinity, the distance is Å. this distance is too large to reconcile the experimental observation of a photo-cross link. thus, the c structure is considered to be consistent with the experimental results and the probable complex between the aptamer pegaptanib and vegf. furthermore, the structure was used for simulations of the mutants, which investigated the stability and affinity of the complex. three simulations were performed, c _a, c _g and c _c, where u in the c structure was mutated to a, g and c nucleotides, respectively. the simulations were performed using the same conditions as those used for the native sequence. table shows the results of these simulations. the native c structure showed the shortest distance and the c _g had the highest binding affinity. this indicates that the native and the mutated c _g structures form the probable complex, and these c structures support the experimentally obtained results. in aptamers that recognize proteins by a three-dimensional structure, there are cases where the base moiety is modified to gain diversity of the three-dimensional structure or enhance affinity with the protein. development of an artificial base pair for aptamers has also been attempted [ ] . our workflows that integrate rna analytical tools can easily provide complex structures that may provide new suggestions for the design of aptamers by interpreting the recognition mechanism of rna-protein complexes. the current cloud-based workflow on our website integrates analytical resources for rna tertiary structure determination into knime workflow systems. we have developed three analytical workflows for comprehensive structural analysis of rna: ( ) prediction of the tertiary structures of rna molecules; ( ) prediction of the structures of rna-rna complexes, including analysis of the interface; and ( ) prediction of the structures of rna-protein complexes, including analysis of the interface. taking advantage of the graphical tools offered by knime communities, users visually interpret the structural results, reproduce the calculated data and generate data suitable for fast and reliable preparation of md simulations. we have also introduced database-linking tools to offer seamless connection between tools and the rna database in the workflow. using the workflow, we have evaluated the rna-protein complex formed between the rna aptamer pegaptanib and vegf. molecular simulations were used to calculate changes in the affinity of complexes where key bases in the aptamer involved in complex formation were mutated. the results provide valuable information and insights that could aid the design of rna aptamer-protein complexes with enhanced affinity, which affords an efficacy at a lower concentration and circumvents toxicity issues. our workflows for the comprehensive structural analysis of rna based on knime offer a powerful tool for structural analysis and design of nucleic acid drugs, and these structural analyses can be directly inputted into md simulations. towards a complete map of the human long noncoding rna transcriptome long noncoding rnas and the genetics of cancer rna and disease role of non-coding sequence variants in cancer rnai therapeutics: principles, prospects and challenges potent and specific genetic interference by double-stranded rna in caenorhabditis elegans rna interference the micrornas mir- and mir- c promote tumour invasion and metastasis microrna expression profiles classify human cancers a microrna regulon that mediates endothelial recruitment and metastasis by cancer cells -with comments micro-rna- targets tumor suppressor genes in invasion and metastasis identification of ebola virus micrornas and their putative pathological function micrornome analysis unravels the molecular basis of sars infection in bronchoalveolar stem cells viruses, micrornas and cancer cellular micror-nas inhibit replication of the h n influenza a virus in infected cells suppression of microrna-silencing pathway by hiv- during virus replication the amazing world of bacterial structured rnas cooperative tertiary interaction network guides rna folding direct-coupling analysis of nucleotide coevolution facilitates rna secondary and tertiary structure prediction non-coding rnas: therapeutic strategies and delivery systems micrornas in cancer: biomarkers, functions and therapy automated de novo prediction of native-like rna tertiary structures the mc-fold and mc-sym pipeline infers rna structure from sequence data rna-puzzles: a casp-like evaluation of rna three-dimensional structure prediction polymorphism of bulged-out residues in hiv- rna dis kissing complex and structure comparison with solution studies rna d d: a program for generating, viewing, and comparing -dimensional models of rna towards d structure prediction of large rna molecules: an integer programming framework to insert local d motifs in rna secondary structure bridging the gap in rna structure prediction prospects for tertiary structure prediction of rna based on secondary structure information tertiary structure prediction of rna-rna complexes using a secondary structure and fragment-based method simrna: a coarsegrained method for rna folding simulations and d structure prediction a nucleobase-centered coarse-grained representation for structure prediction of rna motifs the viennarna web services mfold web server for nucleic acid folding and hybridization prediction cen-troidfold: a web server for rna secondary structure prediction scientific workflow systems: pipeline pilot and knime knime: the konstanz information miner ractip: fast and accurate prediction of rna-rna interaction using integer programming mdpocket: open-source cavity detection and characterization on molecular dynamics trajectories software news and update autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading rna-based therapeutics: current progress and future prospects the functional rna database . : databases to support mining and annotation of functional rnas comparison of simple potential functions for simulating liquid water refinement of the cornell et al. nucleic acids force field based on reference quantum chemical calculations of glycosidic torsion profiles rna structural dynamics as captured by molecular simulations: a comprehensive overview simmerling c ( ) ff sb: improving the accuracy of protein side chain and backbone parameters from ff sb refinenement of the amber force field for nucleic acids: improving the description of alpha/gamma conformers numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes particle mesh ewald: an n 	extperiodcentered log(n) method for ewald sums in large systems molecular dynamics with coupling to an external bath generalized born models of macromolecular solvation effects matsumura h ( ) conformational plasticity of rna for target recognition as revealed by the . a crystal structure of a human igg-aptamer complex a therapeutic aptamer inhibits angiogenesis by specifically targeting the heparin binding domain of vegf( ) pegaptanib, a targeted anti-vegf aptamer for ocular vascular disease imino proton exchange rates imply an induced-fit binding mechanism for the vegf( )-targeting aptamer nucleic acid aptamers: clinical applications and promising new horizons unnatural base pair systems toward the expansion of the genetic alphabet in the central dogma publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -h n ywbd authors: roy, urmi title: insight into the structures of interleukin- systems date: - - journal: comput biol chem doi: . /j.compbiolchem. . sha: doc_id: cord_uid: h n ywbd structure-based molecular designs play a critical role in the context of next generation drug development. besides their fundamental scientific aspects, the findings established in this approach have significant implications in the expansions of target-based therapies and vaccines. interleukin- (il- ), also known as interferon gamma (ifn-γ) inducing factor, is a pro-inflammatory cytokine. the il- binds first to the il- α receptor and forms a lower affinity complex. upon binding with il- β a hetero-trimeric complex with higher affinity is formed that initiates the signal transduction process. the present study, including structural and molecular dynamics simulations, takes a close look at the structural stabilities of il- and il- receptor-bound ligand structures as functions of time. the results help to identify the conformational changes of the ligand due to receptor binding, as well as the structural orders of the apo and holo il- protein complexes. immunologically relevant proteins are critical for the functioning of cellular pathways. therefore, in the general contexts of target-based therapies and drug designs, it is necessary to fully understand how these proteins and their variants participate in the progressions of inflammatory, autoimmune and infectious human diseases (kabat ; voit ; taylor et al. ; gupta et al. ; huang et al. ; jang et al. ) . the task of gathering detailed knowledge about this topic can be aided to a large extent with residue-level structural analyses of immunologically significant proteins and their interaction with receptors (moczydlowski ; xiao et al. ; yao et al. ) . this investigative approach assists in the proper identifications of therapeutic-targets, their structural-assemblies and consistent ligand-receptor interfacial interactions, and thus, plays a vital role in the development of new, innovative medicines (freudenberg et al. ; asbury et al. ; haydarlou et al. ; mariethoz et al. ; janwa et al. ) . the present study of molecular dynamics (md) simulation (ho and hamelberg ; jin et al. ; weako et al. ) , focusing on interleukin- (il- ), falls in this category of computational structural immunology. the main goal of this study is to explore the essential structural and conformational aspects of il- ligand and ligand bound receptor systems. il- , also known as interferon gamma (ifn-) inducing factor, is a pro-inflammatory cytokine. il- induces t helper (th) cells and responses, and il- signal transduction j o u r n a l p r e -p r o o f cascade can also activate nuclear factor kappa beta (nf) (mak and saunders ) . il- , a member of interleukin- (il- ) family, is similar to interleukin- , and is important for supporting the host defense (dinarello ; gracie et al. ) . il- bridges the gap between innate and adaptive immune responses and is responsible for various inflammatory, autoimmune and physiological conditions, such as inflammatory bowel disease, psoriasis, sepsis, myocardial infarction, crohn's disease, arthritis and cancer. the il- first binds to the il-  receptor (il- r and forms a lower affinity complex. upon binding with il-  receptor (il- ra hetero-trimeric complex with higher affinity is formed, which initiates the signal transduction process and, triggers the nf-κβ, leading to a downstream transcription through mitogen-activated protein kinase (mapk) signaling cascades (kato et al. ; ohnishi et al. ; tsutsumi et al. ) . as reported by previous authors, il- rrp, the main il- r, binds to il- , but not to il- β (nakamura et al. ) . kato et al. have described the first solution structure of il- ligand (kato et al. ) . this apo version of il- is further modified by tsutsumi et al. (tsutsumi et al. ) . the ligand bound holo forms of il- /il- r are also elucidated by binding protein (il- bp) is a cell surface receptor that binds to and eventually leads to il- neutralization (dinarello et al. ) . a recent study by krumm et al. has identified the conserved il- bp interfacial binding-region on human il- (krumm et al. ). in recent years, inhibitor based therapies have shown great potentials for further developments in the pharma and biotech sectors (vogt and hofmann ; boulaki et al. ; zamiri et al. ; gonzalez et al. ; lin et al. ; kwak et al. ) . the treatments of il- related diseases may involve the use of selective or potent il- ligand/-receptor inhibitors that would interfere with, or effectively block the targeted functions (hamasaki et al. ; j o u r n a l p r e -p r o o f tsutsumi et al. ) . in our earlier papers we have described the structures and structure-related functional changes of several physiologically relevant proteins (roy and luck ; roy a roy , b roy , roy , a roy , b roy , . the present simulation study examines the structural stabilities of the ligand-protein, il- and il- receptor (il- r) bound ligand structures as functions of time. we analyze here the conformational changes within the ligand-protein, due to receptor binding and, we also identify the possible structurally ordered and disordered region within the apo and holo (ligand-bound) protein complexes. all protein structures were collected from the protein databank (pdb) website (berman et al. ) in the form of standard pdb files. for structural studies, we have used nuclear magnetic resonance (nmr) and x-ray crystallographic structures of the human il- ligand-protein j s.pdb (kato et al. ) and wo .pdb, (tsutsumi et al. ) . additionally wo .pdb and wo .pdb, the x-ray crystallographic structures of il-  receptor bound il- and the ternary signaling complex of il- have been utilized (tsutsumi et al. ). the f .pdb, an il- complex with il- bp of ectromelia virus has also been briefly mentioned (krumm et al. ). the resolution values for the x-ray structures wo , wo , wo and f are . Å, . Å, . Å and . Å respectively. nanoscale molecular dynamics (namd) and visual molecular dynamics (vmd) programs were used for time-based simulations (humphrey et al. ; phillips et al. ) , and the j o u r n a l p r e -p r o o f aligned sequences were studied with molecular evolutionary genetics analysis (mega ) (kumar et al. ) . il- interactome analyses were performed using the cytoscope package (shannon et al. ) , and d protein graphics were developed with biovia discovery studio visualizer, v . . . (dassault systèmes biovia discovery studio modeling environment ) . the plots were constructed with origin (originlab corporation ). the experimental simulation setup for il- and il- /il- r were similar to those reported previously (roy a (roy , b (roy , (roy , a (roy , b (roy , . based on the native/original protein, the protein structure file (psf) along with a corresponding pdb structure was generated. the namd software required both the psf and the modified pdb files as simulation inputs, and these files were created using the automatic psf builder (autopsf) graphical user interface (gui) of vmd. the topology file, psf had all the structural information while the modified pdb file contained the predicted hydrogen atoms coordinates. the solvated and neutralized protein structures were obtained using solvate and ionize gui of vmd, where a . mol/l concentration of nacl was used for system neutralization. energy minimization for the system was performed in , steps using the npt ensemble. the final production run was carried out for ns at k using the nvt ensemble and employing the charmm force field. a combined version of charm /charmm /cmap force field was used for this purpose. charm and charmm provided the necessary parameter files for the proteins as well as the lipids. the proteins were subjected to phi, psi cross term map (cmap) corrections (a built-in feature of namd plugin gui); these force field parameter files were necessary to calculate the energies. the periodic boundary condition with "langevin on" control was applied in each case, with an "active" setting of particle mesh ewald (pme). the langevin damping was set to . the time steps were set to femtosecond (fs), with steps per cycle selected. for the basic dynamics, the dielectric value was set to . . the periodic cell basis and periodic cell center were selected according to the pdb coordinates. the time frames of each system were saved as "dcd" files. the output parameters, like dcd/xst output frequency, energy output frequency and restart file frequency, were chosen according to system specifications. most of these parameters were kept at their default values incorporated in the namd gui. this simulation approach is based on the conformational sampling of the predominant (especially early-stage) structural changes of the four systems studied here (chhatbar et al. ; dimić et al. ). the computational protocol was designed to obtain the basic frameworks for the il- ligand and the ligand bound holo structures. additional computational details of the present work have been described elsewhere (roy a (roy , b (roy , (roy , a (roy , b (roy , . table s has been included in the supplementary data (sd) to list the abbreviations of fig. . the ppi is one of the most valuable tools for identifying the ligand-receptor interactions and signaling cascades (jiang et al. ; wang et al. ). the results have been generated by cytoscape, on the basis of experimental data, as well as manually curated results obtained using the mentha database (calderone et al. ) . mentha compiles the proteins' key interaction data j o u r n a l p r e -p r o o f collected by international molecular exchange (imex) databases (orchard et al. ) and analyzes the experimental data with a thorough annotation. the interactions considered here are dominated by physical association (as those studied in affinity chromatography, two hybrid array and two hybrid prey pooling), direct interactions (pull down, cross-linking/molecular sieving/xray crystallography/enzymatic study and two hybrid), physical interactions (biochemical) and antitag co-immunoprecipitation. the confidence scores are based on a scale of to where these values reflect the number of functional ppis curated by experimental procedures as well as based on data from scientific literature. nonetheless there is always a possibility that a few interactions may be absent. the score between il- and il- r interaction is . , which is slightly higher than the usual upper bound of a "medium" range. the score near the range of denotes many ppis as published in scientific literature whereas lower range represents fewer experimental processes. the ppis identified between il- and il- r are primarily of physical/biochemical origins. the high affinity signaling complex il- /il- r initially activates toll like receptor (tlr) domains, which, via the recruitment of certain adaptor and recruiter molecules, ultimately trigger the nf and mapk dependent pathways. binding of il- bp to il- leads to neutralization, and the il- /il- r signaling cascades are no longer induced (kimura et al. ; krumm et al. ; dinarello et al. ) . the biological sequence alignment between il- /il-  and il- r /il- r type and are (kato et al. ; yamamoto et al. ; tsutsumi et al. ). the alignment schemes presented here (figs. s and s ) follow the format used by them. these multiple sequence alignments are generated using the clustal omega (clustalw) algorithm (sievers et al. ) of mega software, (kumar et al. ) along with the incorporation of the blocks substitution matrix (blosum) (henikoff and henikoff (yamamoto et al. ). in il-  the s-glutathionylation of a highly conserved cys residue is considered as a therapeutic target (zhang et al. ) . at this time, the possibility of utilizing the conserved il- or il- r cys residues as potential drug targets for various infectious or chronic diseases remains a subject of future investigations. ligand-protein (kato et al. ) . this is a single subunit protein that mostly contains sheets. the undefined loop structure in j s consists of residues - . fig. a (kato et al. ). these residues have been highlighted in a "green stick" mode within the primary interactions site of il- and the il- bound receptor structures. wo is the x ray crystal structure of wildtype (wt) il- ligand-protein (tsutsumi et al. sd table s . the x ray crystal structure of the il- signaling complex, wo , is displayed in fig. d (tsutsumi et al. ). this core signaling complex consists of a ternary structure of il- d . these primary site iii residues have also been highlighted accordingly in sd table s . f , an il- complex with the il- binding protein (il- bp) of ectromelia virus is displayed in fig. f . this structure of f has been described by krumm et al. (krumm et al. ). part of the il- r resembles il- bp and thus, the binding of il- bp with il- blocks the interactions between il- and il- r (krumm et al. buried cys residues play a leading role in determining the protein's structural stability, since buried cys residues are usually embedded within a protein's hydrophobic core. being surrounded by various hydrophobic and aromatic residues they form hydrophobic and other nonbonded interactions and thus make the protein structure more compact and stable. in wo / wo , most of the cys residues and disulfide bridges of il- and il- r receptor chains are buried, and hence, are subject to the aforesaid stability criterion. in the wo : il- rsubunit, however, the cys residues and some of the disulfide bridges are mostly surfaceexposed. in our previous paper, we have addressed the cys residue stability resulting from metal binding or structural effects (roy and luck ) . here, our goal is to examine how disulfide connectivity increases the overall stability in a protein complex. previous authors explored the possibility of mutating cys (sh) to increase the overall stability of a protein (yamamoto et al. ). to check the overall efficacy of the d models, the initial protein structures were assessed. ramachandran plots for the initial structures used in md simulation are depicted in sd fig s . these plots illustrate the favored, allowed and disfavored distributions of phi (φ)/psi (ψ) dihedral angles. the structural evaluations of these figures ensure that most residues are within the j o u r n a l p r e -p r o o f favored and allowed region (lovell et al. ) . overall, these plots indicate the effectiveness of the d structures; further details are explained in the sd. greater ( . Å) than wo ( . Å). the first . ns of the simulation was skipped for averaging as initial sampling indicated a steep rise owing to the process of protein-unfolding. these observations gathered from plot a lead us to conclude that, apo ligand wo is more stable than j s. the undefined loop structure and cys (-sh) residues in j s make this structure less stable. four cys (-sh) residues also exist in wo , where the cys and cys species support sulfur-pi interactions with tyr and phe , respectively. these sulfur-pi interactions are absent in j s. though the cys -phe pair involves pi-alkyl interactions in both cases. although some minor variations are observed in wo loop, overall it maintains a stable nature. the partial conversion of turns into -helices in wo strengthens and stabilizes this structure. the undefined loop region in j s is relatively less stable. for the ligand bound ternary structure wo , the  receptor exhibit strong rmsd variations compared to those of the ligand and receptor structures (data not shown). the larger interfacial regions of the  receptor (both with ligand and the  receptor) most likely boost the latter's stability, because the stronger inter-subunit interactions operate in these regions. the existence of three cys residues in the  receptor could be attributed to the latter's unstable nature. the presence of surface exposed cys in the il- r receptor chain may also be accountable for the -chain's higher instability. it is possible that, these fluctuations in the  j o u r n a l p r e -p r o o f receptor (fig. b ) cause the overall instability of the ligand bound receptor's trimeric structure (fig. a) . sd fig. s shows the superposed cys and the disulfides within the il- systems. a change from - helices to turn is observed around residues - , within the ligand bound form of the wo ligand. like the case of wo , in the wo ligand, the residues around - also persistently change from - helical structure to turn. aside from this, no other major changes are observed in this case, and overall the ligand in wo seems slightly more stable than that of wo . the receptor residues - and - in the linker-turn region of wo show a few changes from turn to coils and - helices respectively. nevertheless, within the receptor structure, the prominent loop region - seems rather stable. it is likely that, the protein-protein inter-subunit interactions act to stabilize this loop. from the structure of wo it is also clear that the il- fits very well within the typical ligand binding pocket by surrounding receptor domains. certain accommodative arrangements taking place between the ligand and the receptor's three different domains may make the corresponding region further stable. it is also evident from fig. that, the ligand is a bit more stable in the receptor bound form of wo , as compared to the corresponding case of the apo structure wo . the secondary structure of wo ligand has an approximately comparable or marginally higher stability with respect to that of the wo ligand. however, the wo ligand residues - show some additional variability from -sheets to turns. nevertheless, the changes that are observed around residue in wo ligand are absent here. the secondary structural changes of the ligand within the ligand bound receptors are plotted in sd fig. s . from figs. and s , it is evident that, the ligand is more stable within the receptor-bound forms than in its apo form, and that correspondingly, fewer conformational changes occur within the ligand of wo upon receptor binding. the il- in wo is somewhat more stable than wo , even though the overall stability in wo is higher than that of wo ; the relatively higher interfacial intersubunit interactions present in wo ligand may be responsible for this higher stability. the maximum changeability in the secondary structure is observable in the c subunit furthermore, along with several other cytokines, il- can be used as adjuvants with hiv and several other vaccines (bradney et al. ) . in recent years, due to outbreak of sars-cov- pandemic, considerable attentions have been directed towards the immunity development against viral pathogens. the present paper demonstrates that, il- itself is a structurally steady protein and that it can retain this stability over a relatively extended time-scale. this observation points at the potential use of il- as an adjuvant for antibody generation and immune enhancement, which, in turn could facilitate future strategies to develop novel treatments for communicable viral diseases. the computational results presented in this work describe a set of residue-level analyses of the il- systems based on their published structures. this investigation also explores the ppis between the il- /il- r and the il- /il- bp systems, and notes the relevance of the findings. the time-based structural stabilities of il- , in both its apo and ligand bound holo forms have been measured using md simulation. the results show that, the ligand is somewhat more stable in its receptor bound structures. the ligand bound receptor monomer wo has been found to be somewhat more stable than the ligand bound receptor dimer wo . the immunological significance of il- signaling is rooted in the fact that, il- is related to numerous autoimmune and inflammatory diseases, and that the interruption of il- /il- r signaling cascades impact the development of anti-inflammatory drugs/drug-targets. additionally, il- upregulates fas ligand and interferon gamma (ifn-) leading to the process j o u r n a l p r e -p r o o f of apoptosis or cell death. il- biologics may be relevant also in the contexts of immunity enhancement approaches. thus the detailed identification of the ligand/receptor interfaces of this protein, and subsequent identification of targeted-therapy based on computer modeling efforts, as those discussed in this paper, could be helpful in the development of predictive immunotherapy and precision bio-therapeutics. the author has attached all high resolution figures (figs to and graphical abstract) as zip files. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. the author declares no conflict of interest. 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covid- pandemic has led to an unprecedented push across the whole of the scientific community to develop a potent antiviral drug and vaccine as soon as possible. existing academic, governmental and industrial institutions and companies have engaged in large-scale screening of existing drugs, in vitro, in vivo and in silico. here, we are using in silico modelling of sars-cov- drug targets, i.e. sars-cov- protein structures as deposited on the protein databank (pdb). we study their flexibility, rigidity and mobility, an important first step in trying to ascertain their dynamics for further drug-related docking studies. we are using a recent protein flexibility modelling approach, combining protein structural rigidity with possible motion consistent with chemical bonds and sterics. for example, for the sars-cov- spike protein in the open configuration, our method identifies a possible further opening and closing of the s subunit through movement of sb domain. with full structural information of this process available, docking studies with possible drug structures are then possible in silico. in our study, we present full results for the more than thus far published sars-cov- -related protein structures in the pdb. at the end of a cluster of pneumonia cases was discovered in wuhan city in china, which turned out to be caused by a novel coronavirus, sars-cov- . since then the virus has spread around the world and currently has caused over million infections with more than , deaths worldwide (july st). sars-cov- is the seventh coronavirus identified that causes human disease. four of these viruses cause infections similar to the common cold and three, sars-cov, mers-cov and sars-cov- cause infections with high mortality rates. as well as affecting humans, coronaviruses also cause a number of infections in farm animals and pets, and the risk of future cross-species transmission, especially from bats, has the potential to cause future pandemics. thus, there is an urgent need to develop drugs to treat infections and a vaccine to prevent this disease. some success has already been achieved for sars-cov- with dexamethasone reducing mortality in hospitalised patients and a number of vaccine trials are currently ongoing. the viral spike protein is of particular interest from a drug-and vaccine-development perspective due to its involvement in recognition and fusion of the virus with its host cell. the spike protein is a heavily glycosylated homotrimer, anchored in the viral membrane. it projects from the membrane giving the virus its characteristic crown-like shape. the ectodomain of each monomer consists of an n-terminal subunit, s , comprising two domains, s a and s b , followed by an s subunit forming a stalk-like structure. each monomer has a single membrane-spanning segment and a short c-terminal cytoplasmic tail. the s is involved in recognition of the human receptor, ace . this subunit has a closed or down configuration where all the domains pack together with their partners from adjacent polypeptides. , however, in order for recognition and binding to ace to take place, one of the three s b domains dissociates from its partners and tilts upwards into the open or up configuration. , binding of ace to the open conformation leads to proteolytic cleavage of the spike polypeptide between s and s . s then promotes fusion with the host cell membrane leading to viral entry. drugs that target the spike protein thus have the potential to prevent infection of host cells. the main protease of the virus (m pro ) is another important drug target. m pro is responsible for much of the proteolytic cleavage required to obtain the functional proteins needed for viral replication and transcription. these proteins are synthesised in the form of polyproteins which are cleaved to obtain the mature proteins. m pro is active in its dimeric form but the sars-cov m pro is found as a mixture of monomer and dimers in solution. sars-cov m pro has been crystallised in different, ph-dependent conformations suggesting flexibility, particularly around the active site. md simulations support this flexibility. the protease is highly conserved in all coronaviruses so targeting either dimerization or enzymatic activity may give rise to drugs that can target multiple coronaviruses, known and yet unknown. , since the discovery of sars-cov- , a plethora of structures have been determined including m pro and the ectodomain of the spike protein , as well as other potential drug and vaccine targets. these structures provide the opportunity for rational drug design using computational biology to identify candidates and optimise lead compounds. however, crystal structures only provide a static picture of proteins, whereas proteins are dynamic and this property is often important in drug development. for example, agonists and antagonists often bind different conformations of g coupled-protein receptors. . flexibility also affects thermodynamic properties of drug binding, , yet the ability to assess flexibility is often hampered by the long computational times needed for md simulations. we use a recent protein flexibility modeling approach, combining methods for deconstructing a protein structure into a network of rigid and flexible units (first) with a method that explores the elastic modes of motion of this network, [ ] [ ] [ ] [ ] [ ] and a geometric modeling of flexible motion (froda). , the usefulness of this approach has recently been shown in a variety of systems. [ ] [ ] [ ] [ ] [ ] methods similar in spirit, exploring flexible motion using geometric simulations biased along "easy" directions, have also been implemented using frodan and nmsim. we have performed our analysis through multiple conformational steps starting from the crystal structures of sars-cov- -related proteins as currently deposited in the pdb. this results in a comprehensive overview of rigidity and the possible flexible motion trajectories of each protein. we emphasize that these trajectories do not represent actual stochastic motion in a thermal bath as a function of time, but rather the possibility of motion along a set of most relevant low-frequency elastic modes. each trajectory leads to a gradual shift of the protein from the starting structure and this shift may reach an asymptote, where no further motion is possible along the initial vector, as a result of steric constraints. energies associated with such a trajectory for bonds, angles, electrostatics, and so forth, have be estimated in previous studies of other proteins and shown to be consistent and physically plausible. computing times for the method vary with the size of the proteins, but range from minutes to a few hours and are certainly much faster than thermodynamically equilibrated md simulations. the approach hence offers to possibility of large-scale screening of protein mobilities. we have downloaded protein structure files as deposited on the protein data bank, including all pdb codes that came up when using "sars-cov- " and "covid- " as search terms, as well as minor variations in spelling. in our first search of april th, , this gave protein structures. a second and third search on may th and th, , respectively, resulted in a total of structures. in addition we included a few protein structures from links provided in selected publications to give a grand total of protein structure included in this study. many of the structures found, as outlined above, have been deposited on the pdb in dimer or trimer forms. hence one has the choice to study the rigidity and motion of the monomer or the dimer/trimer. clearly, the computational effort for a dimer/trimer is much larger than for a monomer since in addition to the intra-monomer bond network, also inter-monomer bond need to be taken into account. furthermore, it is not necessarily clear whether the possible motion of a monomer or dimer should be computed to have the most biological relevance. we have computed the motion of full dimer and trimer structures only for a few selected and biologically most relevant such structures while the default results concentrated on the monomers. nevertheless, we wish to emphasize that when results for a certain monomer exist, it should nearly always be possible to also obtain their motion in dimer/trimer configuration. for some protein structures, we have found that steric clashes were present in the pdb structures that made a flexibility and sometimes even just the rigidity analysis impossible. usually, this is due to a low crystal resolution. a list of all current protein pdb ids included in this work is given in table s . in figs. and we show examples of different rigidity patterns that emerge from the first analysis. in line with previous studies comparing these rigidity patterns across various protein families, we find that they can be classified into about four types. in fig. (a) we see that for the crystal structure of sars-cov- nucleocapsid protein n-terminal rna binding domain (pdb: m m), the largest rigid cluster in the pristine structure, i.e. at e cut = , largely remains rigid through the dilution process of consecutively lowering e cut values. when bonds are opened the rigid cluster shrinks but the newly independent parts are flexible and not part of any new large rigid cluster themselves. only very small parts of the protein chain break to form their own independent rigid structures before at a certain e cut the whole protein is essentially flexible. we shall denote such a behaviour as brick-like. in contrast, in fig. (b) we observe that for chain a of the co-factor complex of nsp and the c-terminal domain of nsp , which forms part of the rna synthesis machinery from sars cov- (pdb: wiq), already the crystal structure has fallen into three independent rigid structures. opening bonds, we find that the largest rigid cluster breaks into rigid structures. gives chain a of the co-factor complex of nsp and the c-terminal domain of nsp from sars cov- (pdb: wiq). different rigid clusters of the polypeptide chain appear as identically coloured blocks along the protein chain with each c α labelled from its n-terminal at to its c-terminal. when the energy cutoff e cut decreases (left-most column, downward direction towards larger, negative e cut magnitudes), rigid clusters break up and more of the chain becomes flexible. the colour coding shows which atoms belong to which rigid cluster. flexible regions appear as black thin lines. the second column on the left indicates the mean number r of bonded neighbours per atom as e cut changes. we note that the e cut scale is not linear since new rows are only added when the rigidity of a structure has changed. then these now four domains retain their character upon opening more and more bonds until they simply dissolve into full flexibility. while brick-and domain-like behaviour is found only occasionally, more prevalent are two further types of rigidity dissolution. in fig. (a) we see that for, e.g. the monomer of crystal structure of covid- main protease (m pro ) in apo form (pdb: m ), the rigid cluster dominating the crystal structure quickly falls apart upon change in e cut with five newly formed independent rigid clusters emerging towards to n-terminal of the protein chain. these new clusters remain stable to the opening of further bonds, even when the remnants of the original rigid cluster has become fully flexible. such a behaviour relies on a certain critical e cut value to dominate the rigidity dissolution and is reminiscent of so-called firstorder phase transitions in statistical physics. hence this rigidity-type is usually denoted as st order. the behaviour seen in (b) for the crystal structure of the complex resulting from the reaction between the sars-cov main protease and tert-butyl here, there are many values of e cut where large parts of the original rigid structure break off one after another so that towards the end of the bond-opening process, the original cluster is still present, but no longer dominates the rigidity pattern. this behaviour is called nd order. in table s , we have indicated the classification for each protein structure into the four classes. obviously, this classification is not perfect and there are also sometimes intermediate rigidity patterns. nevertheless, this rigidity classification already provides a first insight into the possible flexibility and range of motion for each structure. it should be clear, that a brick-like rigidity should show the least flexibility until it dissolves completely. on the other hand, for domain-like structures, one can expect possible intra-domain motion while inter-domain motion might be harder to spot. similarly, for a st-order rigidity, one would expect little dynamic mobility until the "transition" value for e cut has been reached, although high levels of flexibility should be possible afterwards. last, a protein with nd-order rigidity should have the most complex behaviour in terms of flexibility since new possible mobility can be expected throughout the range of e cut values. for each value of e cut , the first analysis has provided us with a map of rigid and flexible regions in a given crystal structure. we can now translate this into propensity of motion by allowing the flexible parts to move perturbatively, subject to full bonding and steric constraints (see methods section). moving along directions proposed by an elastic normal model analysis of the crystal structure, we can therefore construct possible motion trajectories that are fully consistent with the bond network and steric constraints. each trajectory corresponds to one such normal mode, denoted m up to m for the first low-frequency non-trivial such modes, as well as the chosen e cut value. generally, a larger value of |e cut | implies less rigidity and results in larger scale flexible motion. in fig. we give examples of such motion trajectories. fig. (a) shows a monomer of the sars-cov- spike glycoprotein (closed state) (pdb code: vxx ). we can see that there is a good range of motion from the crystal structure when following the normal mode modes into either positive or negative changes along the mode vector. fig. (b) shows the motion for the dimer structure of the sars-cov- main protease (pdb code: lu , in complex with inhibitor n ) . again, one can see considerable motion, although due to the complexity of the structure, it is difficult to distinguish individual movement patterns from such a frozen image. much better insight can be gained when watching for full range of motion as a movie. we have therefore included movies for all figures as supplemental materials. we also have made a dedicated web download page where movies for the complete set of pdb codes as given in table s are publicly available. the movies are being offered for various modes m to m as well as e cut values, at least containing results for e cut = kcal/mol, kcal/mol and kcal/mol, respectively. in addition, the site includes the rigidity resolutions discussed above and all intermediate structures needed to make the movies. this allows for the calculation of relative distances and other such structural measures along each motion trajectory as desired. for a detailed analysis of the sars-cov- main protease with similar methods, we refer the reader to ref. . as discussed above, the trans-membrane spike glycoprotein mediates entry into host cells. , as such, it is a "main target for neutralizing antibodies upon infection and the focus of therapeutic and vaccine design". it forms homotrimers protruding from the viral surface. up to the end of may , structures of the trans-membrane spike glycoprotein have been deposited in the pdb. these transmission electron cryomicroscopy (cryo-em) studies have led to structures with pdb codes vsb , vxx and vyb now being available. with rms resolution of . Å, vsb has a slightly lower resolution than vxx at . Å and byv at . Å. in the following, we shall discuss the resulting rigidity and flexibility properties of these three structures in their full trimer form. results for individual monomer rigidities, as given in fig. (a) , are also available at ref. . the rigidity pattern of the homotrimer for this structure (pdb: vsb, . kda, atoms, residues) is dominated by a large rigid cluster encompassing most of the trimer structure except for a region from roughly residue to residue . this indeed corresponds to the s b domain of s in each monomer. in the trimer configuration, it is known that one of the s b domains can change from the closed to an open configuration at |e cut | = . kcal/mol, the vsb structure of the trimer breaks into many different rigid parts, but the original large cluster remains a dominating feature across the rigidity dilution plot. a motion analysis analysis does not compute but breaks with bad sterics, apparently due to the comparably low resolution of the crystal structure. for the closed state structure ( vxx, . kda, atom count: atoms, residues) of the sars-cov- spike glycoprotein, the rigidity pattern is again "brick"-like and the whole of the crystal structure is part of a single cluster. at |e cut | = . kcal/mol, there is a first order break of the large rigid cluster into dozens of smaller rigid units. nevertheless, the original cluster retains a good presence in each chain of the trimer. in terms of motion, we are now able to produce motion studies for the full set of modes m to m and various e cut 's. in fig. we show motion results, using again the structures for the extreme ranges of the motion as in fig. . in addition, we are showing side, c.p. fig. (a) and top, c.p. fig. (b) , views similar to ref. . looking at the whole range of modes and e cut 's computed, we find that motion is very reminiscent of the vibrational excitations of a rigid cone or cylinder. there is twist motion around the central axis, bending of the trimer along the central axis, relative twist motion of chains relative to the remaining chain (c.p. fig. (b) ), etc. already at m (with |e cut | = kcal/mol), large scale motion has stopped and one only observes smaller scale motion is flexible parts of the trimer chains. overall, this behaviour is very consistent with the elastic behaviour of a "closed" structure similar to a cone or cylinder. we now turn to the last sars-cov- spike ecto domain structure ( vyb, . kda, atoms, residues). this structure is in the open configuration similar to the conformation seen in vsb. the structure is shown in fig. , again in side and top view. from the rigidity plots we find, in addition to the largest rigid cluster, already for the crystal structure at |e cut | = kcal/mol a second rigid cluster, also roughly spanning from residue to . again, this region identifies the s b domain as in the vsb structure. compared to the closed state ( vxx), we see that this cluster has more internal structure, i.e. consists of more flexible parts in the s b region from to and also seems to fall apart upon further changing e cut . this suggests that it is more flexible as a whole. upon motion simulation, we observe a very high mobility in that prominent s b subdomain. starting from the crystal structure, we find for |e cut | = kcal/mol a clear further opening towards a negative froda mode, m , while in the other, positive direction of m , the structure can again close the trimer. the distance range of the motion can be expressed as follow: the distance from residue in the middle of the central β -sheet of the s b to the most opened conformation is Å while distance to the same residue in the most closed conformation is Å. the distance from open to closed is Å. hence the motion simulation adds additional insight into the distinction between the open ( vyb) and the closed ( vxx) structures while also showing that a transition from open to closed is indeed possible. as stated in ref. , this interplay of closing and opening is expected to be central to the viral entry into the human cell. sars-cov- infectivity is dependent on binding of the spike protein to ace . this binding is only possible when the spike protein is in the open conformation. structures of both the open and closed conformation have already been determined and the flexibility of the s b domain inferred from these static structures , . the spike trimer consists of almost amino acids and hence is not an easy target for dynamics simulations due to its size. in the open structures ( vsb and vyb) the s b domain is clearly identifiable in the rigidity analysis as a separate cluster to the rest of the trimer. this shows that this domain has increased flexibility. we can clearly observe the hinge movement of the s b domain in the open configuration ( vyb) with the s b domain moving back into the closed configuration. the range of movement from the most opened to the most closed conformation can be measured to be quite large and the flexibility within the s b domain itself during the hinge movement is also seen to be considerable. all these findings suggest that the s b domain of the spike protein has the necessary flexibility to attach itself readily to ace . however, when starting from the closed structure ( vxx), we do not see an opening. this suggests possibly stronger bonds and steric constraints which need to be overcome before the structure is able to open up. nevertheless, to our knowledge this is the first time the hinge motion of the s b domain has been predicted solely based on the dynamics of a structure. in principle, the full structural information provided in our download site we start the rigidity, flexibility and mobility modelling in each case with a given protein crystal structure file in pdb .pdb format. hydrogen atoms absent from the pdb x-ray crystal structures are added using the software reduce . alternate conformations that might be present in the protein structure file are removed if needed and the hydrogen atoms renumbered in pymol. we find that for some protein structures the addition of hydrogen atoms is not possible without steric clashes. consequently, identification of a viable structure and its continued analysis is not possible. these proteins are labelled in table s . for the remaining proteins we produce the 'rigidity dilution' or rigid cluster decomposition (rcd) plot using first. the plots show the dependence of the protein rigidity on an energy cutoff parameter, e cut < . it parametrizes a bonding range cutoff based on a mayo potential , such that larger (negative) values of e cut correspond to more open bonds, i.e. a smaller set of hydrogen bonds to be included in the rigidity analysis. elastic network modes we obtain the normal modes of motion using elastic network modelling (enm) implemented in the elnemo software. , this generates a set of elastic eigenmodes and associated eigenfrequencies for each protein. the low-frequency modes are expected to have the largest motion amplitudes and thus be most significant for large conformational changes. the six lowest-frequency modes (modes - ) are just trivial combinations of rigid-body translations and rotations of / the entire protein. here we consider the six lowest-frequency non-trivial modes, that is, modes - for each protein. we will denote these modes as m , m , . . ., m . the modes are next used as starting direction for a geometric simulation, implemented in the froda module within first. this explores the flexible motion available to a protein within a given pattern of rigidity and flexibility. froda then reapplies bonding and steric constraints to produce an acceptable new conformation. since the displacement from one conformation to the next is typically small, we record only every th conformation. the computation continues for typically several thousand conformations. a mode run is considered complete when no further projection along the mode eigenvector is possible (due to steric clashes or bonding constraints). this manifests itself in slow generation of new conformations. we have performed froda mobility simulation for each protein at several selected values of e cut . this allows us to study each protein at different stages of its bond network, roughly corresponding to different environmental conditions, such as different temperatures as well as different solution environments. in a previous publication, we discussed the criteria for a robust selection of e cut . ideally, for each protein structure, a bespoke set of e cut values should be found, with the rcd plots providing good guidance on which e cut values to select. clearly, for a large-scale study as presented here, this is not readily possible due to time constraints. instead, we have chosen e cut = − kcal/mol, − kcal/mol and − kcal/mol for each protein. these values have been used before in a multi-domain protein with kdalton and shown to reproduce well the behaviour of (i) a mostly rigid protein at e cut = − kcal/mol, (ii) a protein with large flexible substructures/domains at e cut = − kcal/mol and (iii) a protein with mostly flexible parts connecting smaller sized rigid subunits at e cut = − kcal/mol. , in addition, we have also performed the analysis at other values of e cut when upon inspection of the rcd plots it was seen that the standard values (i) -(iii) would not be sufficient. the exact values used are given in table s . we emphasize that these trajectories do not represent actual stochastic motion in a thermal bath as a function of time, but rather the possibility of motion along the most relevant elastic modes. each trajectory leads to a gradual shift of the protein from the starting structure. this shift eventually reaches an asymptote, where no further motion is possible along the initial vector, as a result of steric constraints. energies associated with such a trajectory for bonds, angles, electrostatics, and so forth, can be estimated and shown to be consistent and physically plausible. a familial cluster of 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sars-cov main proteinase (mpro) dimer: molecular dynamics simulations and multiple x-ray structure analyses structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design targeting the dimerization of the main protease of coronaviruses: a potential broad-spectrum therapeutic strategy structure of m pro from covid- virus and discovery of its inhibitors importance of protein dynamics in the structure-based drug discovery of class a g protein-coupled receptors (gpcrs protein conformational flexibility modulates kinetics and thermodynamics of drug binding rapid simulation of protein motion: merging flexibility, rigidity and normal mode analyses protein flexibility and dynamics using constraint theory normal mode analysis of macromolecular motions in a database framework: developing mode concentration as a useful classifying statistic conformational change of proteins arising from normal mode calculations on the potential of normal mode analysis for solving difficult molecular replacement problems elnemo: a normal mode web server for protein movement analysis and the generation of templates for molecular replacement normal mode analysis and applications in biological physics constrained geometric simulation of diffusive motion in proteins docking of photosystem i subunit c using a constrained geometric simulation protein flexibility is key to cisplatin crosslinking in calmodulin inhibition of hiv- protease: the rigidity perspective structure and function in homodimeric enzymes: simulations of cooperative and independent functional motions the flexibility and dynamics of protein disulfide isomerase something in the way she moves': the functional significance of flexibility in the multiple roles of protein disulfide isomerase (pdi) generating stereochemically acceptable protein pathways multiscale modeling of macromolecular conformational changes combining concepts from rigidity and elastic network theory the protein data bank flex-covid data repository asparagine and glutamine: using hydrogen atom contacts in the choice of side-chain amide orientation the pymol molecular graphics visualization programme automated design of the surface positions of protein helices comparative analysis of rigidity across protein families amplitude elastic motions in proteins from a single-parameter, atomic analysis this work received funding by the cy initiative of excellence (grant "investissements d'avenir" anr- -idex- ) and developed during r.a.r.'s stay at the cy advanced studies, whose support is gratefully acknowledged. we thank warwick's scientific computing research technology platform for computing time and support. special thanks to overleaf.com for free premium access during covid- lockdown. uk research data statement: data accompanying this publication are available for download. rar conceived the study, nsr and rar assembled table s and identified e cut values, rar performed the computations and curated the data, akw identified biologically most relevant structures. all authors wrote and reviewed the manuscript. the authors declare no competing interests. is chain a of complex resulting from the reaction between the sars-cov main protease and a carbamate (pdb: y m). different rigid clusters of the polypeptide chain appear as identically coloured blocks along the protein chain with each c α labelled from its n-terminal at to its c-terminal. when the energy cutoff e cut decreases (left-most column, downward direction towards larger, more negative e cut magnitudes), rigid clusters break up and more of the chain becomes flexible. the colour coding shows which atoms belong to which rigid cluster. flexible regions appear as black thin lines. the second column on the left indicates the mean number r of bonded neighbours per atom as e cut changes. . colors are chosen identical to fig. . as in fig. , the arrows in each panel show the range of motion for chain b (blue shades) in (a+b) and also for chain c (reds) in (b). key: cord- -s tdllby authors: burton, aaron s.; di stefano, marco; lehman, niles; orland, henri; micheletti, cristian title: the elusive quest for rna knots date: - - journal: rna biol doi: . / . . sha: doc_id: cord_uid: s tdllby physical entanglement, and particularly knots arise spontaneously in equilibrated polymers that are sufficiently long and densely packed. biopolymers are no exceptions: knots have long been known to occur in proteins as well as in encapsidated viral dna. the rapidly growing number of rna structures has recently made it possible to investigate the incidence of physical knots in this type of biomolecule, too. strikingly, no knots have been found to date in the known rna structures. in this point of view article we discuss the absence of knots in currently available rnas and consider the reasons why knots in rna have not yet been found, despite the expectation that they should exist in nature. we conclude by singling out a number of rna sequences that, based on the properties of their predicted secondary structures, are good candidates for knotted rnas. the systematic characterization and classification of knots has engaged scientists since the introduction of tait's knots table, whose first entries are shown in fig. a . it is readily seen that the different types of entanglement are permanently trapped in the closed curves. in fact, any two curves cannot be continuously deformed into one another unless they are cut open, rearranged and resealed. in our everyday life, the knots that typically surround us differ from these well-defined mathematical ones because they are tied in open chains (fig. b) . these knots are clearly not permanently trapped and hence are termed "physical knots". many of the physical knots that we encounter have been purposely introduced in, for example, the yarn of sewn clothes or in ropes secured to anchoring points. not infrequently, however, we have to deal with their unwanted spontaneous occurrence, as in the earphone cords pulled out of our pocket. these spontaneous forms of entanglement arise by statistical necessity in microscopic filaments too. as a matter of fact, equilibrated polymers cannot escape the formation of knots, if they are sufficiently long, especially if they are densely packed. , it is therefore a remarkable fact that the statistical incidence of topological entanglement in biomolecules such as proteins and dna filaments, which are both long and compact, is substantially smaller than expected for general models of equilibrated polymers with equivalent length and packing conditions. [ ] [ ] [ ] the observed limited incidence of knots probably results from evolutionary pressure to limit the likely detrimental implications on the biological viability and functionality of these molecules. for instance, if a protein was as prone as a generic flexible polymer to the stochastic formation of knots, then its folding route would be riddled with kinetic traps and dead ends. it is accordingly plausible, as remarked by levinthal in more general contexts, , that protein sequences have evolved to encode not only the thermodynamically-stable native fold, but also the sequence of steps leading to the native structure formation so to minimize the incidence of misfolded states, including knotted ones which would be very challenging to backtrack. arguably, the most vivid illustration of the seemingly general aversion to entanglement of biomolecular systems is provided by dna molecules in higher eukaryotes, where they are hierarchically organized in chromatin fibers and form very long chromosomes. it has long been a puzzle how the chromosomes could dynamically reconfigure through the decondensed interphase configurations and the rod-like mitotic ones without being hindered by the cisand trans-chromosome entanglement expected to arise from their dense packing in the nucleus. the complementary advancements in imaging techniques , and computer modeling , have nowadays clarified that the incidence of intra-and inter-chromosome entanglement is kept at a minimum by the fact that the cell cycle lasts only a tiny fraction of the time needed to equilibrate decondensed chromosomes. consequently, interphase chromosomes are quenched in a state that retains memory of the disentangled mitotic state and are thus efficiently primed for the subsequent recondensation step. these considerations support the long-held notion that biomolecules should typically be free of significant forms of self-and mutual entanglement. yet, although expectedly disfavoured, knots do occur both in proteins and in dna. for instance, it is known that about ¡ % of the proteins in the protein data bank (pdb) contain physical knots. [ ] [ ] [ ] furthermore, electrophoretic experiments have shown that viral dsdna, once circularised either in bulk or inside viral capsids, is substantially knotted. [ ] [ ] [ ] in particular, the incidence of knots in p dna, which is kb-long, is at least %. the incidence of knots in longer dna packed inside viral capsids is expected to be even higher, though at the moment this has not been ascertained yet due to limitations of current topological profiling techniques. motivated by these considerations, last year some of us undertook a survey of the occurrence of knots in the rna structures deposited in the pdb as of june . this was motivated by the fact that, since the early study of ref. , no systematic profiling of rnas topology had apparently been carried out, despite the rapid increase of the database of publicly available structures. at that time, the pdb contained several thousand rna entries, spanning a total of , chains. assuming a knot incidence comparable to that found in proteins, » %, tens of knotted entries could have been expected. strikingly, no genuinely-knotted rna chain was identified in the survey. for this point of view article, we considered the reasons why knots in rna have not yet been found, despite the expectation that they should exist in nature. to initiate this query, we first updated our survey by profiling the rna structures deposited in the pdb as of august ( fig. and supplemental material). again, despite the appreciable increase in the number of distinct chains over one year, from , to , , no genuinely-knotted rna structure was found. to be precise, the two surveys did return three knotted entries, which are listed in table , but, as already noted in ref. they are most likely artifactual results of cryo-em reconstructions. though an equal footing comparison with proteins is difficult for the different typical length (number of monomers) of the two types of molecules and the pdb entry redundancy, the total absence of knots in the > , available rnas structures is notable. assuming that this dataset provides an unbiased representation for naturally-occurring rnas, it is appealing to speculate on the biological rationale behind the apparent aversion of rna to knots. a first general consideration regards rna folding kinetics, particularly its possible co-transcriptional component. , it appears plausible that newly transcribed nucleotides in a nascent rna chain could anneal with other nearby nucleotides establishing ephemeral local secondary structures. while these temporary motifs may be traded off for longer range ones at later folding stages, their presence would make it more difficult to develop knots by increasing the effective thickness and local rigidity of the chain. the possible outof-equilibrium accretion of the rna chain growing sequentially in layers around the already-transcribed, compact, portion would clearly also push toward entanglement-free structures, too. it is worth noting that the sequential modular growth of rna chains could be also relevant from an evolutionary point of view. in fact, it has been recently suggested that rna ribosomal complexes have reached their current large and articulated conformations by the sequential addition of small modular structures. similarly to the kinetic ones, such evolutionary mechanisms should limit the incidence of selfentanglement too. it also appears plausible that rna knots, and other forms of entanglement, are not favored thermodynamically. in fact, independent of kinetic considerations, naturally occurring rnas appear to be primed for acquiring native structures with rather low geometrical complexity. this observation is prompted by the analysis of secondary-structure predictions of algorithms that are exclusively based on the minimization of the (model) free energy of rna sequences. these phenomenological approaches clearly do not account for any kinetic effects and yet their returned structures are geometrically much simpler than randomly reshuffled variants of the sequences. for instance, even when using structure prediction algorithms based on planar graph representations one observes that viral rna sequences have a significantly smaller graph diameter, a proxy for three-dimensional size, than their reshuffled versions. , this point is reinforced by the analysis of the minimum energy structures returned by mcgenus. , specifically, it is found that the predicted non-planar graphs of the reshuffled variants have a topological complexity, as measured by the graph genus, that is more than an order of magnitude larger than for the wild-type counterparts. although rna knots may be elusive today, it is conceivable that they were important in earlier evolutionary stages, even during the origins of life. the rna world concept posits a period of time when rna, or something chemically similar, was responsible for all living processes including heredity and metabolism. rna-rna interactions have often been proposed to be critical to maintaining the physical integrity of networks of distinct sequences. [ ] [ ] [ ] typically these interactions have focused solely on secondary structure interactions, but many environments on an early earth could have experienced rather extreme temperature fluctuations that could disrupt weak basepairing. more severe entanglements such as knotsbut without covalent bondingwould have been a means to preserve spatial sympatry of rna network individuals in harsh environments. the observation that many catalytic rna species have structures that are pseudoknotted seems to hint at closer interand intra-molecule associations as one goes back in time. it is, again, important to recall that these conclusions and considerations are based on the set of currently known rna structures which, though not negligible, is inevitably still limited. there is also almost certainly a selection effect among the solved rna structures for molecules that adopt a single stable conformation, meaning that if rna adopts a knotted conformation as one of multiple conformations or as a misfolding event, then it is less likely to be detected. in this regard, a possible parallel can be drawn with proteins, where the discovery of knotted structures was inextricably tied with the growth of the pdb size. in fact, while the occurrence of a knot in transcarbamylase was reported as early as , its genuine character was disputed for its proximity to one of the termini. it was only after the protein database had grown enough that later surveys of mansfield and taylor established beyond doubt that genuine, deeply-tied knots were present in naturally occurring proteins. , it is therefore appealing to speculate that a similar pathway may follow for rna too. with the number of established structures rapidly increasing, it is possible that genuine knotted rnas can be found. the plausibility of knotting in biological rnas is illustrated strongly by two considerations based on the seminal work of n. seeman and coworkers. in a remarkable set of experiments, they designed synthetic » -nt long rna sequences that could spontaneously fold in circularised knotted structures and be unknotted by the top bacterial dna. more recently, the same assay has been used to demonstrate that the human top b enzyme also has rna topoisomerase activity. two important facts emerge from these experiments. first, they vividly demonstrate that even short rna molecules can be knotted. secondly, that the molecular machinery of the cell is already predisposed to deal with rna knots and simplify them. from the principle of biological parsimony, such simplifying action would hardly be expected if complex rna topologies were not present in vivo, particularly when one considers the exquisite specificity of enzymes that act on nucleic acids for rna or dna, but almost never both. in view of these considerations, the occurrence of knots in naturally occurring rna appears at least possible, and could be very likely. they would arise from a balance between evolutionary pressures to avoid detrimental entanglement and the functional advantage that knotted rnas could have in specific, and arguably rare cases. for instance, physical entanglement might allow rnas to modulate gene expression or to enhance their resilience to in vivo degradation. the latter aspect might be relevant for the recently discovered circrnas - which thanks to their circularised form, could trap long-lived knots, as suggested by frank-kamenetskii. the knotted forms of any rna would be expected to be only a fraction of total folding space, such that any given genotype could have multiple phenotypes. these could arise from the delicate interplay of folding kinetics and thermodynamics or from the interaction with partner molecules. perhaps only - % of a "knottable" rna is actually knotted in vivo. this could help explain why they have eluded detection so far. to guide the search for knotted rna, we carried out specifically for this point of view article a bioinformatics survey aimed at identifying candidate rna sequences that could be susceptible to knotting. as the seeman laboratory elegantly demonstrated, nucleic acid sequences designed to contain two self-complementary -nucleotide sequences (x, y, x', y' in which x and x' pair and y and y' pair) can spontaneously form knots. we have diagrammed this scenario in an example rna, and show pathways through which knotted structures could form, at least in some sub-population of folded structures (fig. ) . we note that in addition to forming physical knots, these molecules are also pseudoknotted, in the sense that the strands involved in the two helices are interleaved. thus, pseudoknotted rna sequences with relatively long base-pairing table . knotted rnas in the survey data set. the knots are likely artifactual results of cryo-em reconstruction. the entries are the same as those shown in table of ref. , though some of the pdb codes differ because the pdb introduced a new archiving system of large structures (with no change of atomic coordinates). the overall knot type was established by using the minimally-invasive closing procedure of ref. . the # sign in the knot label of the last entry denotes the composition (concatenation) of various prime knots. stretches appear to be the most likely candidates to form knots. the minimal number of basepairs needed to form a knot can be estimated from a theoretical standpoint, based on the requirement of a minimum of three strand-crossings for the simplest knot. because » basepairs are necessary for two strand crossings (a full helical turn), pseudoknotted structures containing multiple helices of at least ten basepairs would be prime candidates for knotting. a review of the rna pseudoknot database reveals two candidates that approach or meet these criteria (table ) . specifically, the plasmid colib-p mrna (with helices of and basepairs), and homo sapiens ccr prf (with helices of and basepairs) and telomerase rna (helices of and basepairs) appear very likely to have at least a slip-knotted structure (in which a free-end has not been completely threaded through a loop). incidentally we note that such structures have also been observed in proteins. [ ] [ ] [ ] these candidate sequences are found within larger rna mole-cules, which might seem to preclude the threading events necessary for knotting. in proteins, however, it has been demonstrated in vitro that addition of large structured domains to the n-and c-termini of a knotted protein does not prevent it from adopting its native, knotted conformation so it should not be assumed a priori that longer rnas cannot form knots. in addition, we have also identified a number of pseudoknotted rnas with one helix of or more basepairs and a second helix of seven or more basepairs. although the lowest energy conformations of these molecules would likely be only pseudoknotted, the potential would seemingly exist for knotting to occur to at least a small extent as an alternative or misfolded conformation ( table ) . as a matter of fact, their secondary structures, as predicted by mcgenus, , are not a priori incompatible with proper knots. the table of rna sequences we have identified, while not exhausting the repertoire of possible knotted candidates, should nevertheless provide a starting point for a targeted search for knotted rnas. previous biochemical analyses raised the possibility that two naturally-occurring rna intron sequences were capable of adopting knotted conformations, but the presence of a physical knot could not be confirmed irrefutably with the available data. a significant challenge for the unambiguous confirmation of true knots in rna is the difficulty associated with solving rna structures; this challenge could be overcome by the development of an appropriate high-throughput assay to detect knotted rnas, especially when present as isoforms coexisting with other stable unknotted conformations. the lack of such systematic assays is, at present, the main obstacle toward advancing our understanding of the incidence of physical knots in rnas. in conclusion, the lack of physical knots in presently available rna structures is particularly intriguing and also unexpected by comparison with other biopolymers, such as proteins and encapsidated viral dna where knots are known to occur. it is possible that the sequence of naturally-occurring rnas, some of which need to be efficiently translocated through biological pores, have evolved to harness folding kinetics and thermodynamics so as to minimize the incidence of various forms of selfentanglement, including knots, in their native structures. at the same time, the fact that knotted rnas with as few as » nucleotides have been successfully designed and synthesized, suggests that there is no fundamental reason why relatively short knotted rnas should not exist in nature, possibly in circularised form where knots could be essential to enhance their mechanical stability. according to this standpoint, the current situation would parallel the historic route taken for proteins, where the occurrence of knots was once deemed implausible, if not impossible, due to the inevitable initial limitations in size and representation bias of the set of available protein structures. to aid the ongoing efforts to detect knotted rnas we have presented a list of sequences whose predicted native structures are expectedly susceptible to the formation of knots, possibly as isoform competing with other unknotted ones. we hope that the list of selected candidates will serve as a useful starting point toward the systematic search of rna knots. no potential conflicts of interest were disclosed. knots in self-avoiding walks polymers with spatial or topological constraints: theoretical and computational results statistics of knots, geometry of conformations, and evolution of proteins crumpled globule model of dna packing in chromosomes: from predictions to open questions the fractal globule as a model of chromatin architecture in the cell how to fold graciously from levinthal to pathways to funnels kinetics of chromosome condensation in the presence of topoisomerases: a phantom chain model cell biology: chromosome territories chromosome territories, nuclear architecture and gene regulation in mammalian cells structure and dynamics of interphase chromosomes structures and folding pathways of topologically knotted proteins knotted vs. unknotted proteins: evidence of knot-promoting loops intricate knots in proteins: function and evolution protein knots and fold complexity: some new twists probability of dna knotting and the effective diameter of the dna double helix dna knots reveal a chiral organization of dna in phage capsids knotting of a dna chain during ring closure absence of knots in known rna structures to knot or not to knot? examination of s ribosomal rna models sequential folding of transfer rna: a nuclear magnetic resonance study of successively longer trna fragments with a common end sequential folding of a messenger rna molecule evolution of the ribosome at atomic resolution predicting the sizes of large rna molecules synonymous mutations reduce genome compactness in icosahedral ssrna viruses tt ne: a novel algorithm to predict rna secondary structures with pseudoknots mcgenus: a monte carlo algorithm to predict rna secondary structures with pseudoknots frequency of rna-rna interaction in a model of the rna world the dawn of the rna world: toward functional complexity through ligation of random rna oligomers lehman n. spontaneous network formation among cooperative rna replicators beta-sheet topology and the relatedness of proteins are there knots in proteins? a deeply knotted protein structure and how it might fold an rna topoisomerase top b is an rna topoisomerase that works with fragile x syndrome protein to promote synapse formation circular rnas are abundant, conserved, and associated with alu repeats circular rnas are a large class of animal rnas with regulatory potency circular rnas are the predominant transcript isoform from hundreds of human genes in diverse cell types identification of rare slipknots in proteins and their implications for stability and folding conservation of complex knotting and slipknotting patterns in proteins knotprot: a database of proteins with knots and slipknots knotted fusion proteins reveal unexpected possibilities in protein folding characterization of novel functions and topologies in rna probing the entanglement and locating knots in ring polymers: a comparative study of different arc closure schemes we thank sandro bottaro and giovanni bussi for useful discussions. we acknowledge financial support from the italian ministry of education, grant prin hxaw key: cord- - l jznkj authors: holbrook, stephen r title: rna structure: the long and the short of it date: - - journal: curr opin struct biol doi: . /j.sbi. . . sha: doc_id: cord_uid: l jznkj the database of rna structure has grown tremendously since the crystal structure analyses of ribosomal subunits in – . during the past year, the trend toward determining the structure of large, complex biological rnas has accelerated, with the analysis of three intact group i introns, a- and b-type ribonuclease p rnas, a riboswitch–substrate complex and other structures. the growing database of rna structures, coupled with efforts directed at the standardization of nomenclature and classification of motifs, has resulted in the identification and characterization of numerous rna secondary and tertiary structure motifs. because a large proportion of rna structure can now be shown to be composed of these recurring structural motifs, a view of rna as a modular structure built from a combination of these building blocks and tertiary linkers is beginning to emerge. at the same time, however, more detailed analysis of water, metal, ligand and protein binding to rna is revealing the effect of these moieties on folding and structure formation. the balance between the views of rna structure either as strictly a construct of preformed building blocks linked in a limited number of ways or as a flexible polymer assuming a global fold influenced by its environment will be the focus of current and future rna structural biology. stephen r holbrook the database of rna structure has grown tremendously since the crystal structure analyses of ribosomal subunits in [ ] [ ] . during the past year, the trend toward determining the structure of large, complex biological rnas has accelerated, with the analysis of three intact group i introns, a-and b-type ribonuclease p rnas, a riboswitch-substrate complex and other structures. the growing database of rna structures, coupled with efforts directed at the standardization of nomenclature and classification of motifs, has resulted in the identification and characterization of numerous rna secondary and tertiary structure motifs. because a large proportion of rna structure can now be shown to be composed of these recurring structural motifs, a view of rna as a modular structure built from a combination of these building blocks and tertiary linkers is beginning to emerge. at the same time, however, more detailed analysis of water, metal, ligand and protein binding to rna is revealing the effect of these moieties on folding and structure formation. the balance between the views of rna structure either as strictly a construct of preformed building blocks linked in a limited number of ways or as a flexible polymer assuming a global fold influenced by its environment will be the focus of current and future rna structural biology. recent trends in rna structure determination and analysis have accelerated in two seemingly disparate directions: the determination of larger, more biologically relevant rna structures by both x-ray crystallography and nmr methods; and the identification, classification and characterization of the small, ubiquitous structural motifs found in these rna structures. surprisingly, these two directions are related and are mutually reinfor-cing the advancement of the field of rna structural biology. continuing improvements in methods for the synthesis, purification, crystallization and derivatization of large rna molecules, together with technical advances at and increased availability of synchrotron x-ray beamlines, and the development of advanced structure solution software, are allowing more and larger rna structures to be determined [ ] . the parallel development of high-resolution nmr methods has increased the feasible size of rna structures that can be determined, as evidenced by the structure of the -nucleotide rna responsible for signaling core encapsidation in moloney murine leukemia virus (mmlv) [ ] . figure shows the average size of rna structures determined by year, as catalogued in the nucleic acid database (ndb) [ ] . the solution of the first ribosomal subunit structures in - demonstrated that methods for the determination of large rna structures were available. these methods are now being widely applied to a variety of biologically important rna structure analyses. structural studies and comparative sequence analyses have suggested that biological rnas are largely modular in nature, composed primarily of conserved structural building blocks or motifs [ ] of secondary (helices, and internal, external and junction loops) and tertiary (coaxial stacks, kissing hairpin loops, ribose zippers, etc.) structure. although many secondary and tertiary structure motifs have been identified and characterized in terms of sequence preference, structural constraints, energetics and dynamics, it is an open question as to whether we have observed a large or small fraction of the universe of motifs. immediately after the structure determination of the large [ , ] and small [ ] ribosomal subunits, inspection of the rrna structures suggested that they consisted mainly of known rna structural elements (i.e. few, if any, novel motifs were observed). more detailed studies are showing, however, that there is an abundance of information still to be extracted from these structures and correlated with rna structural features observed in other biomolecules. the determination of large rna structures in many cases has benefited from the design of more crystallizable sequences, based on an understanding of rna structural motifs. reciprocally, the identification and characterization of known and novel rna motifs are greatly advanced by the structure determination of new types of large biological rnas. in this review, recent structure determinations of several biological rnas are presented and described. the secondary structure and tertiary interaction motifs of these rnas are analyzed in terms of our current knowledge of rna motifs and the novel motifs observed in these structures. the ability to partition large rna structures into well-characterized subunits, or motifs, is an important advance in understanding the relationship between structure and function, and in rna structure prediction and design. in addition to the updated ribosomal subunit models, with better resolution, and the refinement and analysis of rrna interactions with protein and metal [ , ] , many structures of the ribosomal subunits bound to antibiotics [ ] and other ligands [ , ] have been deposited in the protein data bank (pdb). other biological rna structures that have recently been determined are summarized in table . three group i introns are among recently solved, new, high-impact, biological rna structures: an intact, selfsplicing group i intron with both its and exons from the purple bacterium azoarcus sp [ , ] , a group i ribozyme-product complex from phage twort [ ] and a group i intron ribozyme from tetrahymena [ ] . other structures include the specificity domains of both a- [ ] and b-type ribonuclease p [ ] ; rnas corresponding to a guanine-responsive riboswitch (xpt) complexed with guanine [ ] or hypoxanthine [ ] , and an adenosine-responsive riboswitch (add) complexed with adenosine [ ] ; a highly conserved stem-loop motif found at the end of the genome of sars (severe acute respiratory syndrome) virus and other coronaviruses [ ] ; the core encapsidation signal of mmlv [ ] ; and complexes between a high-affinity rna aptamer and the nf-kb p homodimer [ ] , and between the archaeal rnabinding protein l ae and an rna k-turn derived from a h/aca small rna [ ] . in addition, a series of crystal structures of the hepatitis virus ribozyme in its precleaved state [ ] showed that significant conformational changes take place and a metal ion is lost in comparison to the product form of the ribozyme [ ] . finally, structures of a high-salt left-handed rna duplex [ ] and a mirror image (or l-configuration) [ ] rna duplex show variations on the canonical 'standard' rna double helix. these structures demonstrate not only the prevalence of well-known rna structural motifs, but also the presence of numerous novel structural elements that potentially may serve as common modules in biological rna. conversely, the structure of an alternative trna conformation, designated as lambda form, has been found in complex with the trna modification enzyme archaeosine trna-guanine transglycosylase [ , ] . in this complex, the tertiary interactions between the d-loop and tloop are disrupted, leading to an alternative base pairing pattern. this suggests that rna structure in general, and motif structure specifically, are subject to change through interaction with environmental agents such as proteins, other rnas, metals or other ligands. number of rna structures deposited in the ndb (http://ndbserver.rutgers.edu/) (dark red) and the average number of nucleotides per structure (yellow) given by year. although the number of structure determinations has grown only slowly, the average structure size has dramatically increased since . the availability of numerous diverse, large rna structures has made possible the identification and classification of an increasing number of rna secondary and tertiary structure motifs. both manual and automated classification procedures are being used to identify and characterize motifs [ ] . a database of non-canonical base pairs in rna structures [ ] , coupled with tools for the automatic identification and classification of rna base pairs [ ] , provides an initial description of rna secondary structure. this description can be coupled with automated motif searches [ ] [ ] [ ] , identifying both previously known and new rna motifs. the scor (structural classification of rna) database is a comprehensive, manually curated resource of rna structural motifs that utilizes automated tools and literature descriptions to assist in the classification of rna secondary and tertiary structure motifs [ , ] . characterization of these motifs or rna building blocks in turn is enabling rna design [ , ] , rna structure prediction, rna modeling and rna gene finding [ ] . recently determined crystal structures of rna and rna-protein complexes (in addition to rrna structures) are revealing many new structural elements, in addition to previously characterized motifs (as summarized in table ). as additional rna structures are determined, sequences and topology the occurrence of these elements may indicate that they are common structural motifs, and thus enable their characterization by sequence, structure and function. presented below are examples of recently determined rna structures that have both known rna motifs and novel structural features that may correspond to new structural motifs. these motifs are likely to be crucial to both biological function and three-dimensional structure. the end of the genome of sars virus and related coronaviruses and astroviruses contains a highly conserved sequence called the s m or stem-loop ii element. as shown in table , the crystal structure of a -nucleotide rna corresponding to this element has been solved at . Å resolution [ ] . in addition to previously characterized rna structural motifs, such as a gnra-like pentaloop and a dinucleotide platform base triple [ ] , novel structural features were observed. a unique base quartet is formed between watson-crick gc pairs at the junction of two helices, as shown in figure . another novel rna structural feature is formed by a three-purine bulge that excludes an adenosine from the central stack and forms tertiary interactions with residues from an asymmetric internal loop, forming a 'tunnel' that serves as a binding site for two magnesium ions [ ] . the interaction between two hairpin loops in the guanineresponsive riboswitch is required for its biological function. crystal structures of the riboswitch-hypoxanthine complex [ ] and the riboswitch-guanine complex [ ] show the detail of this tertiary interaction between these seven-nucleotide loops, as shown in figure . these quartets are similar to those found in the sars virus s m element (see above) in that they include watson-crick pairs (one base from each loop), but in this case they interact with non-canonical pairs (formed between loops) in their minor groove. this tertiary interaction pulls the loops together along with their attached stems. the moloney murine leukemia virus core encapsidation signal specificity of packaging of retroviral genomes is a result of interaction between nucleocapsid domains of the gag polyproteins and the c-site of the viral genome. conserved rna secondary structure elements within the csite have been identified for mmlv and other retroviruses. in mmlv, such a region, which includes three stem-loop structures, is highly conserved. the sequences of the three stem-loops (tetraloops) in this region were changed to prevent intermolecular interaction and enhance crystallization, while retaining function. the structure of this engineered, functional rna, which corresponds to the core encapsidation signal, has been determined by nmr methods [ ] . as seen in figure , two of the stem-loops form a coaxial stack connected to the third stem-loop by a flexible linker. in addition to several familiar structural motifs, a new element, termed an 'a-minor kink turn', was observed. this structural feature, with similarities to both the a-minor motif and a kink turn, is formed by the interaction of an extruded uridine and a ggaa bulge. in this element, two unpaired adenosines pack in the minor groove of a neighboring stem, inducing a kink in the helix. more structural examples are needed to determine whether this is a recurring rna structural motif or unique to this structure. considering the large fraction of rna that can be attributed to secondary and tertiary structure motifs, the prediction of these motifs from sequence would be a large step toward the prediction of rna three-dimensional structure. currently, the most successful approach to predicting rna motifs begins by predicting the secondary structure by means of either energy minimization using nearest neighbor thermodynamic rules, as implemented in mfold [ ] and the vienna rna package [ ] , or covariation analysis, or a combination of both [ ] . next, potential motifs are identified in the predicted rna structure holbrook a watson-crick base quartet found in a highly conserved region of the sars virus genome. the quartet is shown in orange, a gnra-like pentaloop with a closing base pair is in green, a base pair from a second helix at a sharp angle to the tetraloop is shown in magenta and a dinucleotide linker between bases of the quartet is in yellow. secondary structure loop regions using sizes and sequences known to be compatible with the motifs. the sequence profiles of homologous aligned rnas are then checked against these potential motifs to confirm compatibility. this approach was used successfully to identify sarcin-ricin loops and loop e motifs in rrna [ ] . the ribose zipper tertiary structure motif has been characterized [ ] in terms of sequence and secondary structure based on instances observed in rrna. the most common, or canonical, type of ribose zipper shows a strong sequence bias, and is found to link stem or stem-like regions with loop regions. the residues involved in ribose zippers were also found to be phylogenetically conserved and to demonstrate covariation patterns. thus, the application of sequence and structure conservation analysis, as used for the prediction of secondary structure motifs, is also a powerful tool for predicting this tertiary interaction motif. further studies of the application of this approach to the prediction of other secondary and tertiary structure motifs are necessary to confirm and make this approach standard. the focus of rna structure determination has switched from small model structures and molecular fragments to large, naturally occurring biological rnas, some in protein complexes and some engineered to enhance crystallization. these larger, more biological structures provide a diverse picture, and represent a more uniform sampling of the secondary and tertiary structure motifs available to rna. detailed examination of recently determined biological rnas has revealed many new structural elements, in addition to finding repeated examples of previously characterized motifs. in fact, by far the greatest proportion of rna structure can be described as consisting of combinations of well-characterized structural modules: double helices, motifs in hairpin loops and internal loops, and tertiary interaction motifs. although we can identify many motifs in rna structures, we are far from having a complete library of rna structural motifs, and even farther from understanding the context of these motifs and their effect on the surrounding structure, and the ability to predict them directly from sequence. because the potential applications of being able to predict, design [ ] and engineer [ , ] rna structure through an understanding of its building blocks are so great, directed effort in this area is warranted. although we will eventually obtain this knowledge through research on individual biological rnas, we should consider a structural genomics type project to map the universe of rna motifs, and a parallel computational effort to predict motifs from sequence and determine how they interact combinatorially to build up an overall rna structure. an emerging trend in rna structural biology is the association of rna secondary structure and tertiary interaction motifs with human disease. three examples have recently appeared in the literature recognizing these relationships and correlating them with specific structures. the most recent of these is a proposed rna target, sequences and topology interacting hairpin loops from the guanine-responsive riboswitch. one loop is in cyan and the other is in magenta, with stacked quartets in the loops colored yellow and orange. helical stems of the hairpin loops are colored blue. mmlv core encapsidation signal. green, gnra tetraloop; salmon, disordered tetraloop; violet, watson-crick double helix; blue, non-canonical base pairs; cyan, base triple; orange, a-minor k-turn; yellow, linker region and bulge base. formed by kissing hairpin loop tertiary interaction motifs, for the binding of the kh domain of the fragile-x mental retardation protein (fmrp) [ ] . this complex interaction competes with the binding of fmrp to brain polyribosomes and is associated with a crucial known mutation. another example is a proposed explanation for lead toxicity by hydrolysis of mrnas containing the leadzyme motif [ ] . finally, models of the role of mrna secondary structure in a variety of human neurodegenerative diseases (trinucleotide repeat expansion diseases) caused by the expansion of cag repeats in the open reading frames of certain genes have been proposed and are under active study [ , ] . crystallization of rna and rna-protein complexes nmr structure of the -nucleotide core encapsidation signal of the moloney murine leukemia virus the rna structure of the core recognition signal for encapsidation of the positive strand genome of mmlv is reported. this is the largest nmr structure of an rna determined to date the nucleic acid database: a comprehensive relational database of three-dimensional structures of nucleic acids analysis of rna motifs the complete atomic structure of the large ribosomal subunit at . Å resolution high resolution structure of the large ribosomal subunit from a mesophilic eubacterium crystal structure of the s ribosomal subunit from thermus thermophilus: purification, crystallization and structure determination the roles of ribosomal proteins in the structure assembly, and evolution of the large ribosomal subunit the contribution of metal ions to the structural stability of the large ribosomal subunit functional insights from the structure of the s ribosomal subunit and its interactions with antibiotics atomic structures of the s subunit and its complexes with ligands and antibiotics crystal structure of an initiation factor bound to the s ribosomal subunit crystal structure of a group i intron splicing intermediate crystal structure of a self-splicing group i intron with both exons crystal structure of a phage twort group i ribozyme-product complex structure of the tetrahymena ribozyme: base triple sandwich and metal ion at the active site crystal structure of the specificity domain of ribonuclease p basis for structural diversity in homologous rnas the crystal structure of the specificity domain of a-type ribonuclease p rna from thermus thermophilus was determined. comparison of the aand b-type ribonuclease p structures shows that, although the secondary and tertiary structures differ structural basis for discriminative regulation of gene expression by adenine-and guanine-sensing mrnas crystal structures of guanine-and adenine-responsive riboswitches are determined. these structures illustrate how the rnas discriminate between adenine and guanine, and how they perform their regulatory function structure of a natural guanine-responsive riboswitch complexed with the metabolite hypoxanthine the structure of a rigorously conserved rna element within the sars virus genome the crystal structure of a highly conserved fragment of the sars virus genome was determined. the structure shows novel rna secondary and tertiary structure elements crystal structure of nf-kappab (p ) complexed to a high-affinity rna aptamer structure of protein l ae bound to a k-turn derived from an archaeal box h/aca srna at . Å resolution a conformational switch controls hepatitis delta virus ribozyme catalysis crystal structure of a hepatitis delta virus ribozyme high salt solution structure of a left-handed rna double helix betzel c: first look at rna in l-configuration alternative tertiary structure of trna for recognition by a posttranscriptional modification enzyme trna structure goes from l to lambda ncir: a database of non-canonical interactions in known rna structures tools for the automatic identification and classification of rna base pairs rna structure comparison, motif search and discovery using a reduced representation of rna conformational space the identification of novel rna structural motifs using compadres: an automated approach to structural discovery motif prediction in ribosomal rnas. lessons and prospects for automated motif prediction in homologous rna molecules scor: structural classification of rna, version . three-dimensional motifs from the scor, structural classification of rna database: extruded strands, base triples, tetraloops and u-turns ) is a compilation and classification of rna secondary and tertiary motifs. several new motifs are identified in the database and described in this article tectorna: modular assembly units for the construction of rna nano-objects building programmable jigsaw puzzles with rna self-assembling rna building blocks were used to generate defined structures termed tectosquares. this illustrates the modularity of rna, the ability to use known structural motifs to design building blocks and the potential of rna in nanotechnology a computational approach to identify genes for functional rnas in genomic sequences mfold web server for nucleic acid folding and hybridization prediction vienna rna secondary structure server secondary structure prediction for aligned rna sequences sequence and structural conservation in rna ribose zippers paradigms for computational nucleic acid design kissing complex rnas mediate interaction between the fragile-x mental retardation protein kh domain and brain polyribosomes lead toxicity through the leadzyme rna structure of trinucleotide repeats associated with human diseases molecular architecture of cag repeats in human disease related transcripts the author acknowledges jasmin yang of the ndb, and donna hendrix and elizabeth holbrook of uc berkeley for assistance in the preparation of this manuscript. srh was supported by national institutes of health grants r gm and r hg . key: cord- -p iazl z authors: ziółkowska, natasza e.; o'keefe, barry r.; mori, toshiyuki; zhu, charles; giomarelli, barbara; vojdani, fakhrieh; palmer, kenneth e.; mcmahon, james b.; wlodawer, alexander title: domain-swapped structure of the potent antiviral protein griffithsin and its mode of carbohydrate binding date: - - journal: structure doi: . /j.str. . . sha: doc_id: cord_uid: p iazl z the crystal structure of griffithsin, an antiviral lectin from the red alga griffithsia sp., was solved and refined at . Å resolution for the free protein and . Å for a complex with mannose. griffithsin molecules form a domain-swapped dimer, in which two β strands of one molecule complete a β prism consisting of three four-stranded sheets, with an approximate -fold axis, of another molecule. the structure of each monomer bears close resemblance to jacalin-related lectins, but its dimeric structure is unique. the structures of complexes of griffithsin with mannose and n-acetylglucosamine defined the locations of three almost identical carbohydrate binding sites on each monomer. we have also shown that griffithsin is a potent inhibitor of the coronavirus responsible for severe acute respiratory syndrome (sars). antiviral potency of griffithsin is likely due to the presence of multiple, similar sugar binding sites that provide redundant attachment points for complex carbohydrate molecules present on viral envelopes. a number of lectins, particularly those that bind highmannose sugars, have been shown to exhibit significant activity against human immunodeficiency virus (hiv), as well as against a number of other viruses (botos and wlodawer, ) . for these reasons, several such proteins, among them cyanovirin-n and scytovirin (bokesch et al., ) , have been under active development as topical antiviral therapeutics. most recently, another potent antiviral lectin, griffithsin, was isolated from the red alga griffithsia sp., collected from the waters off new zealand (mori et al., ) . griffithsin was shown to inhibit the cytopathic effects of different isolates of hiv- at concentrations as low as pm (mori et al., ) , and its binding to viral envelope glycoproteins was shown to be directly dependent on their glycosylation states. these properties make griffithsin a promising potential candidate for development as a future pharmaceutical agent. knowledge of the three-dimensional structure of putative therapeutic proteins is very helpful in their development, yet the amino acid sequence of griffithsin was initially reported to be unique, giving no clues to its structure (mori et al., ; giomarelli et al., ) . more careful analysis of the sequence of griffithsin indicates that this protein belongs to a diverse family of b-prism-i (or jacalin-related) lectins (raval et al., ) and shares structural characteristics with proteins such as jacalin (aucouturier et al., ) , artocarpin (jeyaprakash et al., ) , and heltuba (bourne et al., ) ( figure a ). however, sequence identity between griffithsin and any of these other proteins is less than % (mori et al., ) (see also figure a ). thus, structural details, particularly of the carbohydrate binding site(s), cannot be readily modeled, necessitating solution of an experimental structure. we report here the structures of griffithsin in three crystal forms, with and without bound carbohydrate molecules. since the antiviral activity of griffithsin against hiv- has already been proven (mori et al., ) , we have investigated other viral targets of this lectin. we have now shown that griffithsin is a potent inhibitor of the life cycle of the coronavirus that causes severe acute respiratory syndrome (sars), a life-threatening disease that first manifested itself only a few years ago. such broad antiviral activity makes this newly characterized lectin a particularly promising target for the development as a novel antiviral agent. the structure of griffithsin was solved by single-wavelength anomalous diffraction (sad) with selenomethionine (semet)-labeled protein produced in e. coli and was refined at high resolution with material grown in plants. three different crystal forms of griffithsin were studied (table ). the fold of griffithsin corresponds to the well-known b-prism motif (chothia and murzin, ) , found mainly in a variety of lectins but also in some other proteins (shimizu and morikawa, ) . the motif consists of three repeats of an antiparallel four-stranded b sheet that form a triangular prism. however, unlike other lectins whose sequences are compared in figure a , griffithsin forms an intimate dimer ( figure ) in which the first two b strands of one chain (consisting of residues located at its n terminus) are associated with ten strands of the other chain (and vice versa). that arrangement is unique among the family members that have been described so far and is seen in all crystal forms of griffithsin, despite the presence of a significant nonnative n-terminal extension in one of them. thus, griffithsin can be described as a domainswapped dimer, although it is not clear whether a corresponding monomeric forms does (or even could) exist. a considerable distance between the residues a and b ( . Å between their ca atoms) is much larger than a corresponding distance in the domain-swapped proteins that have been shown to exist in both mono-meric and dimeric forms, such as, for example, another antiviral lectin, cyanovirn-n (yang et al., ) . in our previous study that utilized size-exclusion chromatography, griffithsin was shown to exist as a homodimer in solution (giomarelli et al., ) . griffithsin contains three strictly conserved repeats of a sequence ggsgg ( figure b ). these repeats were residues that are strictly conserved among all nine proteins are shown on red background, and those of a similar character are highlighted in yellow. residues that are strictly conserved among the other lectins but not in griffithsin are highlighted in green in the former and magenta in the latter. (b) structure-based sequence alignment of the three b sheets (blades) in griffithsin. conserved residues that interact directly with the bound carbohydrates are highlighted in red, whereas conserved residues that do not make such contacts are shown on magenta background. structure predicted to represent boundaries between subdomains (mori et al., ) , since similar sequences are often reported for typical flexible linkers. however, in griffithsin, these sequences are found in loops connecting the first and fourth strand of each sheet rather than in interdomain linkers and are very well ordered. as discussed below, the main chain amide of the last residue of each of these sequences participates in creation of a ligand binding site, and the strict conservation of this sequence may be related to the presence of three monosaccharide binding sites on each molecule of griffithsin. the structure of griffithsin was compared to the structures of a number of proteins that display a similar overall fold, including mannose binding lectins. for the purpose of such comparisons, we utilized a compact domain of griffithsin derived from the coordinates of the high-resolution, orthorhombic form of the carbohydrate-free protein. the domain used for the comparison consisted of residues a -a and b -b . for other proteins used in these comparisons for which multiple structures exist, we preferentially chose the structures of complexes with carbohydrates and, in case of multiple molecules in the asymmetric unit, arbitrarily chose the first molecule present in a file if bound to a carbohydrate. we found artocarpin (pdb code vbo) (jeyaprakash et al., ) to be the closest homolog of griffithsin, with an rms deviation of only . Å between pairs of ca atoms. the deviations for the closely related lectins including banana lectin ( bmz) (meagher et al., ) , heltuba ( c m) (bourne et al., ) , artocarpus hirsuta lectin ( toq) (rao et al., ) , maclura pomifera agglutinin ( jot) (lee et al., ) , and jacalin ( ugw) (jeyaprakash et al., ) were only slightly larger, at . Å ( pairs), . Å ( pairs), . Å ( pairs), . Å ( pairs), and . Å ( pairs), respectively. the deviation from molecule d of calsepa (pdb code ouw; that molecule chosen due to the presence of bound mannose) is . Å for pairs, whereas the deviation for the first of the three covalently linked domains of parkia lectin was . Å for pairs. by comparison, the deviation from the coordinates of vitelline membrane outer layer protein i (pdb code vmo) (shimizu and morikawa, ) , a protein that may exhibit enzymatic rather than strictly carbohydrate binding properties, was . Å for pairs of ca atoms. the first of the repeated broad ggsgg loops in griffithsin (residues - ) is located between strands and that are swapped between the two molecules forming the dimer. this region is close to the principal carbohydrate binding sites in this family of lectins, yet it shows only partial structural conservation, limited to glycines and . jacalin, maclura pomifera agglutinin, and artocarpus hirsuta lectin contain two separate chains, with a break between chains b and a falling in exactly this area. the trace of that chain in other lectins, on the other hand, is very similar to that in griffithsin (figure ) , although the amino acid sequence is not very highly conserved between them ( figure a) . a loop connecting the two inner strands of the same sheet (gly -tyr in griffithsin), on the other hand, follows an almost identical path in all the compared structures. this structural element interacts closely with the carbohydrate molecules (or with a sulfate in the structure of griffithsin solved in the absence of a sugar ligand), and it is not surprising that its sequence is also very similar. griffthsin differs very considerably from all other compared lectins in the conformation of the adjacent a compact domain of griffithsin was defined as residues a -a and b -b . the following colors were used: griffithsin, red; artocarpin ( vbo), blue; heltuba ( c m), magenta; parkia lectin ( zgs), yellow; calsepa ( ouw), pink; banana lectin ( bmz), black; jacalin ( ugw), violet; ahl ( toq), brown; mpa ( jot), green. b-hairpin, consisting of gly and asp . this loop is much shorter than in any other lectins, leaving this part of the structure much more open. with the exception of artocarpin, in which this loop is even longer, all other lectins are similar in this area. another conserved broad loop with the ggsgg sequence, - , is located near the secondary carbohydrate binding site in calsepa and follows a course quite different than in banana and parkia lectins, yet similar to the path in the other proteins. however, since that site is created in calsepa also by the previously described loop that is significantly shorter in griffithsin, it is unlikely that an analogous site could exist in the latter. another short inner loop formed by residues - in griffithsin bears considerable similarity to the equivalent loops in banana and parkia lectins as well as artocarpin but is quite different in the remaining proteins. that loop, together with the last broad ggsgg loop ( - ) creates a second carbohydrate binding site in banana lectin. the latter loop differs very significantly between most of these proteins, with only its path in banana lectin having any resemblance to its trace in griffithsin. only residues are strictly conserved in all the compared structures ( figure a ). it appears that almost all, if not all of them, are crucial in establishing the b-prism-i fold. as mentioned above, gly and gly are part of a broad loop in the first sheet and are found in the vicinity of the principal carbohydrate binding site. interestingly, even though these residues are conserved also in the two-chain lectins such as jacalin, they are located on two separate chains that are not connected directly. glu , phe , thr , and asn are all part of a partially buried cluster that fills the space between sheets and and maintains their spacing. the side chains of glu and thr make a strong ( . Å ) hydrogen bond, and this feature is also strictly conserved. gly , pro , and gly are part of an outer strand in sheet and are located in a region that shows surprising conservation, including the cis conformation of the gly-pro peptide bond. gly and gly are found in a very highly conserved loop connecting the inner strands of sheet , whereas the nearby asp may be the only side chain of a residue directly involved in carbohydrate binding to be found in all the structures. six residues that are conserved in all other structures are not preserved in griffithsin ( figure ). four of them (arg , phe , ile , and tyr ) are part of a cluster between sheets and . the strand containing the latter two residues is shifted considerably in griffithsin compared to all other structures and thus may need differently sized residues to maintain its location. ile (phe in all other structures) is deeply buried and its nature depends on the other neighbors found in its vicinity. the side chain of tyr points at the unique carbohydrate binding site of griffithsin (see below), and this residue may be partially responsible for the properties of this lectin. in addition to these residues, a deletion in the aligned sequences, located between residues and of griffithsin, corresponds to an isoleucine in all other structures. deletion of this residue may be related to the domain-swapped nature of griffithsin that differentiates this protein from all the other related lectins. it should be noted that the site of domain swapping that follows the second b strand is topologically different than the location of the separation between chains a and b of jacalin, the latter following the first b strand. the structure presented here does not shed much new light on the nature and/or importance of residue , which was reported not to be among the standard amino acids (mori et al., ). an alanine inserted at that position in the artificial genes used for the work described here is buried, but the side chain of his at which it is pointing (at a distance of . Å to ce ) could easily move if a larger residue were to substitute for ala . although this residue follows asp , the side chain of which interacts directly with a bound sugar (see below), it does not appear itself to be implicated in carbohydrate binding since its side chain points into a direction opposite to that of asp . the residues that follow the other two aspartic acids involved in carbohydrate binding are asparagine and serine; thus the identity of these residues does not appear to be an important contributor to the carbohydrate binding properties of griffithsin. cocrystallization of griffithsin with mannose yielded a new, monoclinic crystal form with excellent diffraction properties, containing a griffithsin dimer in the asymmetric unit. a comparison of the coordinates resulting from the two high-resolution structures of the unliganded and mannose bound griffithsin yielded rms difference of . Å . the largest differences were observed for sera , glyb , and glyb , as well as in the orientation of the side chains of tyr , tyr , asp , and tyr of both molecules. native griffithsin incorporated so ions in places corresponding to the localization of man , man , man , and man in the structure of the complex with mannose and a molecule of ethylene glycol in place of man . seven mannose molecules were found to be bound to each dimer of griffithsin in the . Å structure but only six in the atomic-resolution structure. the latter carbohydrates are very well ordered in both structures, whereas the seventh molecule in the lower-resolution structure, bound in a site created through intermolecular interactions in the crystal, shows some indications of disorder. it is likely that low-occupancy mannose might also be present in this site in the atomic-resolution structure, but such variability suggests that ligand binding in this site is not very specific, and thus site will not be considered further. however, the six principal sites are arranged in two groups of three, one on molecule a and the other on molecule b, and are very similar in both structures. the only significant difference is that whereas the electron density in the lower-resolution structure (figure ) could be interpreted as resulting from the presence of exclusively a mannose, clear indications of the presence of an admixture of b mannose are seen in the atomic-resolution structure. similar presence of both anomers of mannose was also indicated in the ralstonia solanacearum lectin, solved at atomic resolution (pdb code vyy; no other reference available). this minor component will not be considered in the further discussion. the interactions between man , man , and man with molecule a are virtually identical to the interactions of man , man , and man with molecule b. man (and man ) are found in a site that corresponds to the major carbohydrate binding site described for a variety of lectins from this family. due to domain swapping that exists in griffithsin, however, this site is formed by the residues from both molecules a and b. the protein side chain most responsible for the specific interactions with the mannose is aspa , which interacts through its carboxylate with the hydroxyl oxygens o and o of the mannose. oxygen o also accepts a hydrogen bond from the main chain amide of tyra , whereas o is a recipient of a bond from the n of aspa . hydroxyl o accepts a proton from the main chain amide of glyb , while o and o interact only with solvent (water or glycol). the interactions for man are the same with the opposite chains. this pattern of interactions is very similar for the other mannose residues. man (and man ) bind in an area anchored by aspa and b , respectively, with the hydrogen bonds to the main chain amides of residues , , and . similarly, man (and man ) interact primarily with asp , with the other hydrogen bonds supplied by the main chain amides of residues , , and ( figure ) . the presence of three carbohydrate binding sites that form an almost perfect equilateral triangle on the edge of the lectin (figure ) is unprecedented for this family, although seen in lectins with b-prism-ii fold (hester et al., ; wright et al., ) . however, the distances between the sites in griffithsin are w Å , whereas corresponding distances in galanthus nivalis agglutinin are w Å . while the presence of site was reported for all b-prism-i lectins, site has so far only been seen in banana lectin (meagher et al., ) . as seen in figure , asp is strictly conserved in all lectins that were compared, explaining the conservation of this site. not surprisingly, the only other lectin that has an equivalent of asp is banana lectin since that residue is crucial for creation of site . site , however, appears to be unique to griffithsin, and asp is not conserved at all in any other b-prism-i lectin. the three carbohydrate binding sites of griffithsin are formed from the parts of the structure with extensive sequence conservation. a tyrosine precedes the aspartic acid by two positions, and a glycine by four. either a leucine or isoleucine is immediately preceding the aspartate, and position is occupied by an aspartic acid or a serine. with the sequence and the structure of this region highly conserved, it is not surprising that the main chain amides of these residues can make very similar interactions with the sugars in all three sites. the sequence of site is conserved among other lectins. gly is strictly conserved, and the positions corresponding to tyr and leu in griffithsin are occupied by similar residues in all b-prism-i lectins compared in figure a . sequences corresponding to site are not well conserved, except for banana lectin where a glycine precedes by four positions the aspartic acid involved in carbohydrate binding. sequences corresponding to site show also limited conservation. for example, gly is conserved in all other lectins except for banana lectin. a position corresponding to tyr in griffithsin is occupied by valine, and the position of ile is occupied by similar residues in the other lectins. the final interactions in the three sites are provided by the main chain amides of gly , gly , and gly , respectively. all these glycines are the c-terminal residues of the strictly conserved sequence ggsgg, and this observation may indeed explain the unprecedented conservation of this feature of the primary structure of griffithsin. gly , together with gly , is strictly conserved in all compared lectins. the sequence ggsgg is also present in banana lectin in site . gly is strictly conserved, and gly is present in all lectins except for heltuba. while the carbohydrate molecules observed in the cocrystal structure are remarkably clear, we have also observed limited binding of monosaccharides in the orthorhombic crystals soaked in either mannose or nacetylglucosamine, although in none of these structures the omitmap f o f c electron density map was calculated at . Å resolution, based on the final coordinates of the structure of crystal refined after the removal of the mannoses, and it was contoured at the . s level. were all six principal sites occupied. site is clearly occupied by n-acetylglucosamine in a conformation similar to that of man in the cocrystal structure, although other sites are occupied by other ligands present in the mother liquor. ethylene glycol is found in site , sulfate molecules are found in sites and , site is occupied by only water molecules, whereas site is involved in intermolecular contacts in this crystal form and is thus not accessible to ligands. with the carbohydrate binding sites at a variable distance from the symmetry-related protein molecules, it is not certain whether the preferential binding of a carbohydrate molecule in site indicates higher affinity or simply easier accessibility. the latter explanation seems to be more probable in view of the similarity of the sites as shown in the cocrystal structure. to further test the antiviral activity of griffithsin, the protein was submitted for testing against the coronavirus that was responsible for sars outbreak in . it had been reported previously that the coronovirus bored a surface ''spike'' protein that was heavily glycosylated and was able to bind to the monosaccharidespecific human lectin dc sign (yang et al., ) . based on the demonstrated promiscuity of griffithsin binding to oligosaccharides, it was hypothesized that griffithsin should be capable of binding to the spike protein of the sars virus and inhibiting subsequent infection. the results of three different antiviral assays with the sars virus showed that griffithsin could indeed inhibit both viral replication and the cytopathicity induced by the virus (table ) . though griffithsin was active against the virus at nanomolar concentrations, it was not as active against the sars virus as it is against hiv (table ) . although both the his-tagged and the plant-expressed griffithsin crystallized easily, crystals grown from the plant-produced material (resembling more closely the authentic protein) diffracted significantly better. crystals of the his-tagged griffithsin contained only a single molecule in the asymmetric unit, whereas both crystal forms grown from the plant-expressed material contained two. however, only the latter crystals diffracted to atomic (or at least near-atomic) resolution, both in the presence and absence of bound carbohydrate. in all crystals that did not contain bound carbohydrate, the binding sites exhibited considerable disorder, with even the side chains of the strictly conserved tyrosines , , and showing significant flexibility. the presence of carbohydrates, whether soaked in or cocrystallized, stabilized the binding sites and significantly lowered the temperature factors of the residues involved in their binding. griffithsin is an unusual protein in many ways, and consequently, the determination of its three-dimensional structure is illuminating on many levels. these include its unusual domain-swapped dimeric structure, its three repeated domains, and its six independent binding sites for monosaccharides. the antiviral lectin cyanovirin-n has previously been reported to form domain-swapped dimers but they differed from the griffithsin dimers in that half of the each molecule was involved in swapping, rather than two b strands out of . both monomers and dimers of cyanovirin-n could be isolated, whereas we have seen no indications of the existence of griffithsin in a monomeric form. in addition, only four carbohydrate binding sites are present in each dimer of cyanovirin-n, rather than six in griffithsin. the presence of three almost identical domains in each molecule of griffithsin is a unique adaptation of the b-prism-i family and an advance in our knowledge of the structural complexity of this group of lectins. the other structurally closely related lectins ( figures a and ) contain either a single carbohydrate binding site, or at most two such sites, in each molecule. although griffithsin shares structural homology with many mannoseor n-acetylglucosamine-specific lectins, its reported biological activity against hiv was > -fold better than those previously reported for several monosaccharidespecific lectins (charan et al., ) . of those lectins most closely related to griffithsin, only jacalin has been previously reported to exhibit anti-hiv activity (corbeau et al., ) . jacalin was, however, found to be relatively weak in its anti-hiv activity, not reaching an ec level at a high dose of mg/ml ( nm) in the same whole-cell assay system in which griffithsin displayed an ec = pm (mori et al., ) . the source of this enhanced potency and, presumably, higher affinity for hiv envelope glycoproteins, is of considerable interest. an explanation is suggested from the structure of griffithsin, which offers no less than six separate binding sites for mannose in a compact domain-swapped dimer. the enhanced binding potential for the high-mannose oligosaccharides present on the hiv envelope glycoprotein gp to be gained from this multivalency is significant. the sites on each monomer are much closer than in the lectins that belong to the b-prism-ii family, presumably allowing tighter binding of complex carbohydrates. similar enhancement was seen in previous studies on the anti-hiv protein cyanovirin-n, which detailed the increased relative binding affinity, due to multivalent interactions, of this domain-swapped dimer for high mannose oligosaccharides (shenoy et al., ) . initial attempts at the crystallization of griffithsin with larger oligosaccharides have shown signs that similar multivalent binding to these carbohydrate structures occurs (charan et al., ) urtica diocia agglutinin a (charan et al., ) p. tetragonolobus lectin a (charan et al., ) n. pseudonarcissus lectin a (charan et al., ) myrianthus holstii lectin a (charan et al., ) scytovirin (bokesch et al., ) . cyanovirin-n . griffithsin a (mori et al., ) . a monosaccharide-specific lectins. with griffithsin, leading to rapid precipitation of the complexes (data not shown). the specificity of griffithsin for monosaccharides was at first thought to be a disadvantage for this protein's potential as a prophylactic or therapeutic agent. however, in some ways, the specificity displayed by griffithsin may be an advantage. it was previously speculated that griffithsin, due to its more promiscuous carbohydrate binding, might be active against viruses such as the coronavirus responsible for the recent sars outbreak (mori et al., ) . this prediction was based on the ability of griffithsin to bind a broader spectrum of oligosaccharide structures than other antiviral lectins such as cyanovirin-n (bolmstedt et al., ) and scytovirin (adams et al., ) . this prediction has proved to be true as recent data from anti-sars assays has shown that griffithsin is indeed active against this virus at nanomolar concentrations, while both cyanovirin-n and scytovirin were inactive (data not shown). whether or not this protein will prove to have any clinical utility as a prophylactic or therapeutic agent for the treatment or prevention of viral infections remains to be answered, but its unique structure and interactions with carbohydrates, in addition to its unusual potency against hiv, provide ample justification for continued study. cloning and bacterial expression of griffithsin his-tagged griffithsin was expressed and purified as previously reported (giomarelli et al., ) . protein labeled with semet (calbiochem, san diego, ca) was expressed in a similar manner. briefly, his-griffithsin expressing strain bl (de ) cells were grown overnight at ºc on an lb agar plate containing kanamycin at a concentration of mg/ml. a single colony was picked from the plate and inoculated in a ml culture shake flask containing ml luria-bertani (lb) medium (difco, gaithersburg, md). the overnight culture was grown at ºc with rpm shaking for hr. the total of liters of overnight express autoinduction systems medium (novagen, madison, wi) was equally divided into five liters baffled culture shake flasks. kanamycin was added to this medium to a final concentration of mg/ml. each flask then received ml of the original seed culture and was grown at ºc with rpm shaking. after hr of growth, cells were collected for extraction and purification of semet-labeled his-griffithsin as previously reported (giomarelli et al., ) . his-tagged protein produced in e. coli was used for all of the in vitro studies on the antiviral activity of griffithsin against the sars virus. nicothiana benthamiana plants ( days postsowing) were grown in a controlled environment (shivprasad et al., ) and inoculated with infectious recombinant tobamovirus vectors that carried a synthetic cdna of griffithsin. twelve days post inoculation (dpi), infected leaf and stem material was harvested at one leaf above the inoculation leaves, and tissue was homogenized in mm acetate buffer (ph . ) containing mm nacl, mm ascorbic acid, and . mm sodium meta-bisulfite at tissue-to-buffer ratio of : . w/v. green juice was recovered by passing the homogenate through four layers of cheesecloth. recovered green juice was adjusted to ph . and then clarified by centrifugation at g for min (s fraction). s fraction was dialyzed overnight at ºc against phosphate-buffered saline (pbs) (ph . ) with mwco tubing and centrifuged at , g for min to remove insoluble proteins and particles. prior to column chromatography, the ph of the s fraction was adjusted to . by using hcl and filtered through a . mm filter. clarified s was loaded on a sp sepharose fast flow (ge healthcare, piscataway, nj) column equilibrated with mm acetate buffer (ph . ) (solution a). after washing the unbound proteins from the column, griffithsin was eluted with a linear nacl gradient from to mm in solution a, with subsequent ph adjustment of eluted protein fractions to ph . by using m phosphate buffer (ph . ) to a final concentration of mm. fractions were analyzed by sds-page with % tris glycine sds-page gels and by matrix-assisted laser desorption-ionization time of flight (maldi-tof) mass spectrometry with a perseptive biosystem voyager-de str (applied biosystems, foster city, ca) to verify fractions for pooling. protein was then loaded on a source rpc column (ge healthcare, piscataway, nj) equilibrated with mm phosphate (ph . ) (solution b) and eluted with a . % n-propanol gradient, followed by a % linear n-propanol gradient in solution b. fractions containing > . % pure grft were pooled and dialyzed against pbs (ph . ) by using mwco dialysis tubing. the plant-expressed griffithsin did not bear a his-tag, but was acetylated on its n terminus. this material, like the e. coli produced griffithsin, contained an alanine residue at position to replace the unidentified amino acid reported in the original publication (mori et al., ) . three types of assays were used to assess the activity of griffithsin against the severe acute respiratory syndrome (sars) coronavirus. griffithsin was tested for its ability to inhibit the cytopathic effect (cpe) of the sars virus (strain ). for the cpe assay, four log dilutions of griffithsin (e.g., , , , mg/ml) were added to triplicate wells of a -well flat-bottomed microplate containing the vero cell monolayer; within min, the sars virus was added, the plate was sealed and incubated at ºc, and cpe was read microscopically when untreated infected controls developed a to + cpe (approximately to hr). a known positive control drug (interferon a-n ) was evaluated in parallel with griffithsin. the data were expressed as % effective concentrations (ec ). this test was run to validate the cpe inhibition seen in the initial test and utilizes the same -well microplates after the cpe has been read. neutral red (nr) was added to the medium, and the resulting dye uptake by the vero cells was read on a computerized microplate reader as previously published (mcmanus, ). an ec was also determined from this dye uptake. decrease in virus yield assay following positive cpe inhibition and nr dye uptake results, griffithsin was retested both by cpe inhibition and, using the same plate, for effect on reduction of virus yield assay. frozen and thawed eluates from each cup were assayed for virus titer by serial dilution onto fresh monolayers of vero cells. development of cpe in these cells is an indication of presence of infectious virus. as in the initial tests, interferon a-n was run in parallel as a positive control. the % effective concentration (ec ), which is the test drug concentration that inhibits virus yield by log , was determined from these data. plant-expressed griffithsin and an e. coli-expressed semet derivative were purified as reported earlier (mori et al., ; giomarelli et al., ) . the protein samples were concentrated to about mg/ml. crystallization was carried out by the hanging drop vapor diffusion method (wlodawer and hodgson, ) . the plant-expressed protein and semet derivative crystallized under identical conditions ( . m ammonium sulfate and % peg at low ph), with the largest crystals reaching the size of . . . mm. before flash freezing, the crystals were transferred into a cryoprotectant solution containing % ethylene glycol. x-ray data were collected at the ser-cat beamline -id, the advanced photon source (aps), argonne, illinois, on a mar ccd detector. all data were processed with the hkl package (otwinowski and minor, ) (table ) . we have obtained two different crystal forms of unliganded griffithsin. the protein expressed in e. coli crystallized in the trigonal space group p , with a single molecule in the asymmetric unit. this protein construct was prepared with an n-terminal -his affinity tag followed by a putative thrombin cleavage site, extending the sequence by amino acids (mori et al., ; giomarelli et al., ) . however, enzymatic removal of the tag proved to be impractical and thus the crystallized protein consisted of amino acids, rather than the that are present in plant-expressed griffithsin. isomorphous crystals were grown for both the plant-expressed protein as defined above and for a similarly produced semet derivative. the structure of griffithsin in the trigonal crystal form was solved by single-wavelength anomalous diffraction (sad), initially with hkl (minor et al., ) , followed by density modification with resolve (terwilliger, ) and by automated tracing with arp/ warp (perrakis et al., ) . the latter program built out of residues expected in this construct, with only a single one-residue break at asn . that residue as well as one residue each on the n and c termini were added with the program o (jones and kjeldgaard, ) during subsequent refinement and rebuilding. the structure was refined with refmac (murshudov et al., ) utilizing data extending to . Å resolution ( table ). the resulting model contains all residues expected in the structure of authentic griffithsin, as well as additional five residues derived from the n-terminal extension. the rest of the n-terminal extension, including the his-tag, was found to be disordered. the second crystal form, grown from material expressed in plants and containing only residues in a protein chain, was orthorhombic (p ) and contained two griffithsin molecules in the asymmetric unit. these crystals diffracted better than the trigonal ones, and data were collected to near-atomic resolution. the structure was solved by molecular replacement with phaser (storoni et al., ) and was refined with refmac (murshudov et al., ) at . Å resolution. both chains could be traced end-to-end, and even their terminal residues, including acetylated n-terminal serines, were clearly observed in the electron density. a complex of griffithsin with n-acetylglucosamine was prepared by soaking the orthorhombic crystals described above in a reservoir solution supplemented with mm n-acetylglucosamine. x-ray data were collected on a mar image plate detector mounted on a rigaku rotating anode x-ray source. cuka radiation was focused by an msc/osmic mirror system. data were processed with the hkl package (otwinowski and minor, ) (table ). the structure of the complex was refined with refmac (murshudov et al., ) at . Å resolution, with the structure of unliganded griffithsin in the orthorhombic crystal form as the initial model. a complex of griffithsin with mannose was prepared by soaking in a similar manner and data were collected, but that structure was not refined further when cocrystals became available. a distinct crystal form of a complex of griffithsin with mannose was obtained by cocrystallization by the hanging drop vapor diffusion method. the crystals were grown in . m mgso , . m mes (ph . ) with : molar ratio of griffithsin monomers to mannose. crystals belonged to space group p (table ) and diffracted to . Å resolution on the home source described above and to . Å on a synchrotron. full data sets were collected on two crystals of the complex. a complete data set extending to . Å resolution was collected on the home source from crystal . high-resolution data were collected at the synchrotron to a nominal resolution of . Å from crystal , but since that crystal suffered from the presence of considerable ice rings, these data were merged with another lowresolution data set collected on crystal , this time on the synchrotron. data from these two crystals were scaled together with hkl package (otwinowski and minor, ) , retaining reflections extending only to . Å . the structure that utilized home data was solved with phaser (storoni et al., ) by using the orthorhombic structure of unliganded griffithsin and was refined with refmac (murshudov et al., ) . the synchrotron structure was refined starting directly from that model (table ) . oligosaccharide and glycoprotein microarrays as tools in hiv glycobiology; glycan-dependent gp / protein interactions characterization of jacalin, the human iga and igd binding lectin from jackfruit a potent novel anti-hiv protein from the cultured cyanobacterium scytonema varium cyanovirin-n defines a new class of antiviral agent targeting n-linked, high-mannose glycans in an oligosaccharide-specific manner proteins that bind high-mannose sugars of the hiv envelope helianthus tuberosus lectin reveals a widespread scaffold for mannose-binding lectins discovery of cyanovirin-n, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp : potential applications to microbicide development the free r value: a novel statistical quantity for assessing the accuracy of crystal structures isolation and characterization of myrianthus holstii lectin, a potent hiv- inhibitory protein from the plant myrianthus holstii new folds for all-b proteins jacalin, a lectin interacting with o-linked sugars and mediating protection of cd + cells against hiv- , binds to the external envelope glycoprotein gp recombinant production of anti-hiv protein, griffithsin, by auto-induction in a fermentor culture structure of mannose-specific snowdrop (galanthus nivalis) lectin is representative of a new plant lectin family structural basis for the carbohydrate specificities of artocarpin: variation in the length of a loop as a strategy for generating ligand specificity electron-density map interpretation structure of the complex of maclura pomifera agglutinin and the t-antigen disaccharide, galb , galnac microtiter assay for interferon: microspectrophotometric quantitation of cytopathic effect crystal structure of banana lectin reveals a novel second sugar binding site hkl- : the integration of data reduction and structure solution -from diffraction images to an initial model in minutes isolation and characterization of griffithsin, a novel hiv-inactivating protein, from the red alga griffithsia sp refinement of macromolecular structures by the maximum-likelihood method isolation and characterization of niphatevirin, a human-immunodeficiency-virusinhibitory glycoprotein from the marine sponge niphates erecta processing of x-ray diffraction data collected in oscillation mode automated protein model building combined with iterative structure refinement two orthorhombic crystal structures of a galactose-specific lectin from artocarpus hirsuta in complex with methyl-a-d-galactose a database analysis of jacalin-like lectins: sequence-structure-function relationships multisite and multivalent binding between cyanovirin-n and branched oligomannosides: calorimetric and nmr characterization the b-prism: a new folding motif heterologous sequences greatly affect foreign gene expression in tobacco mosaic virus-based vectors likelihood-enhanced fast rotation functions solve and resolve: automated structure solution and density modification crystallization and crystal data of monellin structural characterisation of the native fetuin-binding protein scilla campanulata agglutinin: a novel two-domain lectin crystal structure of cyanovirin-n, a potent hiv-inactivating protein, shows unexpected domain swapping ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign accession numbers coordinates and structure factors have been deposited in the pdb with accession codes gux (semet structure of the e. coli-expressed griffithsin), gty (plant-expressed, unliganded griffithsin), guc (a complex with mannose at . Å resolution), gud (mannose complex at . Å resolution we would like to thank dr. w. minor key: cord- -ihh q f authors: nan title: posters p - p date: - - journal: eur biophys j doi: . /s - - -x sha: doc_id: cord_uid: ihh q f nan in order to understand mrna stability, we proceed to the studie of life time model system composed of the regb ribonuclease and the ribosomal protein s . the t bacteriophage life cycle is modulated by regb which mediate speci c mrna degradations. regb cleaved the mrna at the middle of the translation initiation region (shine-dalgarno sd). studies had shown that regb activity is enhanced with addition of s which is normaly required for the translation of mrna in case of unusual sd regions. s is considered as a key factor of translation's inititiation. composed of six similar domains (f -f ), it interacts with the ribosome by his f -f domains and with the mrna by f -f . we had shown that the f fragments got the same properties than the whole protein. many global architectures had been published (linear or globular conformation). then we try to understand what is the catalysis way of s in the regb activation and in rna recognition. we study the global conformation of f fragment. for that, we use nmr to determinate the domains interfaces. at rst we made the nmr backbone assignement of n/ c/ h labeled fragments (f -f ). then we analysed hsqc overlapping of each fragments in order to identify interfaces residues. the residues identi cation on s homologous model allowed us to determinate interaction region between domains. currently we study of s -rna interactions to determinate if the ligand conformation could change the interaction region and involve conformationals changes. translocation of amino acids from the membrane interface to the interior: theory and experimiment c. aisenbrey , e. goormaghtigh , j.-m. ruysshaert , b. bechinger ulp/cnrs, chemistry, rue blaise pascal, strasbourg, université libre, campus plaine cp / , brussels the interactions of a series of histidine-containing peptides with biological model membranes have been investigated by attenuated total re ection fourier transform infra red (atr-ftir) and oriented solid-state nmr spectroscopies. related peptides have previously been shown to exhibit antibiotic and dna transfection activities. the -residue lah x and lah x peptides were designed in such a manner to form amphipathic helical structures in membrane environments. four histidines and four/six variable amino acids x constitute one face of the helix whereas leucines and alanines characterize the opposite hydrophobic surface. the dichroic ratio of atr-ftir spectra, or the orientation-dependent n solid-state nmr chemical shift have been used to follow the ph-dependent transition from in-plane to transmembrane alignments upon increase in ph. a theoretical model of the topological modulations is presented and the experimental transition curves analysed in order to reveal the gibbs free energy of transition. the novel concept provides access to the free energy changes associated with the amino acids x incorporated into an extended -helix and in the context of phospholipid bilayers. for the peptides of the lah x series the gibbs free energies associated with the transition from the membrane interface to the bilayer interior follow the sequence of amino acids: l < a i < s f < t g < v w << y. here we present a novel solid-state nmr approach which allows for the accurate determination of the tilt and rotational pitch angles of peptides reconstituted into uniaxially oriented membranes. the method works with transmembrane or in-plane oriented peptides that have been labelled with , , - h -alanine and n-leucine at two selected sites. proton-decoupled n and h solid-state nmr spectroscopy at sample orientations of the membrane normal parallel to the magnetic eld direction have been used to characterize the tilt and rotational pitch angle of these peptides in considerable detail. the same samples when inserted into the magnetic eld at degrees tilted alignments provide valuable information on the rotational diffusion constants in membranes and thereby of the association and size of peptide complexes within the membrane environments. whereas monomeric transmembrane peptides exhibit spectral averaging and well-de ned resonances, larger complexes are characterized by broad spectral line shapes. in particular the deuterium line shape is sensitive to association of a few transmembrane helices. in contrast, the formation of much larger complexes affects the n chemical shift spectrum. aisenbrey ch. & bechinger, b. biochemistry , - ( ) a meccano set approach of joining trpzip a water soluble -hairpin peptide with a didehydrophenylalanine containing hydrophobic helical peptide p. chetal, v. chauhan, d. sahal international centre for genetic engineering & biotechnology,new delhi ,india a residues long, water soluble, monomeric -hairpin peptide "trpzip" [cochran et al ( ) pnas , - ], stabilized by tryptophan zipper has been linked via a tetraglycyl linker to a hydrophobic didehydrophenylalnine ( f) containing helical octapeptide. circular dichroism studies of this residues long peptide, "trpzipalpha" (ac-gewtwddatktwtwte-gggg-fal fal fa-nh ) in water have revealed the presence of both the -hairpin and the helical conformations. this is the rst instance where a f containing peptide has been found to display a helical fold in water. the uorescence emission wavelengths of tryptophan in ac-g-w-g-nh , trpzip and trpzipalpha were . , . and . nm respectively. the uorescence quantum yield of trpzip was . fold higher than trpzipalpha suggesting that proximal interactions between the -hairpin and the helix caused the quenching of tryptophan uorescence in the former by the fs in the latter. the molar ellipticity of the far uv couplet characteristic of trpzip was reduced in trpzipalpha and the cd based thermal melting temperatures at nm were c (trpzip) and c (trpzipalpha). a concentration dependent variable temperature cd study in water showed that in trpzipalpha, increasing temperature is detrimental to the -hairpin, but it augments the helical motif by intermolecular oligomerization. our results show that in water, trpzipalpha exhibits long-range interactions between two different secondary structures. structural-based differential stability in the yoeb-yefm toxin-antitoxin module i. cherny, e. gazit department of molecular microbiology and biotechnology, faculty of life sciences, tel aviv university, tel aviv , israel the speci c physiological role of natively unfolded proteins is only recently beginning to be explored. a notable case in which natively unfolded state appears to have physiological signi cance is the e. coli yoeb-yefm toxin-antitoxin (ta) module. a crucial element in proper functioning of ta systems requires physiological instability of the antitoxin in contrast to the stable pro le of its toxin partner. we have shown that yefm antitoxin is a natively unfolded protein, lacking secondary structure even at low temperatures. in contrast, its toxin partner has a well-folded conformation at physiological temperatures. we suggest that the structural-based differential thermodynamic stability between the two components is the cause for their differential physiological stability, since structural instability of the antitoxin exposes it to cellular quality-control machinery. we further revealed that yefm and yoeb interact and form tight complex and determined it stoichiometry. a potential use of ta systems is as novel antibacterial targets. indeed, we identi ed homologous yefm-yoeb systems in a large number of bacteria including major pathogens. we aim to design peptides capable of interfering with the yefm-yoeb interaction, thus releasing the toxin to execute its detrimental function. for this purpose, we identi ed a short linear determinant within yefm that is involved in yoeb interaction. this peptide motif will be optimized for development of antibacterial lead molecules. a. chatterjee, a. prabhu, a. ghosh-roy, r. v. hosur tata institute of fundamental research, mumbai, india- dynein light chain (ddlc ), a member of the cytoplasmic motor assembly exists as a monomer or a dimer (functional form) under different experimental conditions. here we report the unfolding characteristics of the monomeric ddlc at ph , due to urea and guanidine hydrochloride, by various biophysical techniques. it is observed that the unfolding pathways due to the two denaturants have many differences. urea unfolding seems to be two state, while guanidine unfolding is more complex. the nmr experiments carried out at low denaturant concentrations have enabled detailed characterization of the structure and dynamics of the near native excited states of the protein. these are similar to the native state in structure, except for the small extensions of the helices in the nterminal half of the protein. however, the local stabilities of the and -strands are perturbed and this occurs differently in the two denaturants. in the guanidine case the entire multi-stranded -sheet in the c-terminal half is destabilized. in either case the motional characteristics, seem to suggest the presence of a nite population of the dimer in the excited state ensemble. these states are suggested to be likely intermediates in the momoner-dimer transition, and their characterization here thus provides clues to the molecular mechanism of the transition. it is also envisaged that the near native excited states could play regulatory roles in the functioning of the protein. kinetic bottlenecks identi cation in different folding models f. cecconi , c. guardiani , r. livi infm -istituto di sistemi complessi -cnr, italy, centro interdipartimentale per lo studio delle dinamich complesse, università di firenze, italy, dipartimento di sica, università di firenze, italy the ww domains are a family of fast folding, compact, modular domains featuring a triple-stranded, antiparallel beta-sheet. the ww domain of the pin protein, due to the availability of a complete picture of the residues involved in thermodynamic stability and in the formation of the transition state, in particular, represents an excellent benchmark to test computational methods. the objective of the present work is to identify the kinetic bottlenecks in the folding process through md unfolding simulations at increasing temperatures. the kinetic bottlenecks are related to the establishment of contacts requiring the overcoming of a large entropy barrier and acting as a nucleus for the creation of further contacts. the key sites are therefore those involved in contacts showing a dramatic decrease in fractional occupation near the speci c heat peak. the technique was applied to the go model and to a model based on the knowledge of secondary structure, providing in both cases a picture of the folding process consistent with the experimental data. evidence is also shown that while the go model allows a more accurate prediction of the native structure, the folding pathway is better described by the other model. the protein talin plays a key role in coupling the integrin cell adhesion molecules to the actin cytoskeleton and in integrin activation. the globular head of talin, which binds -integrins, is linked to a rod containing an actin-binding site and binding sites for the protein vinculin, which regulates the dynamic properties of cell-matrix junctions. we have determined the structure of three domains which contain vinculin binding sites (vbss) and shown that each of these are made up of helical bundles. the structures of complexes between vinculin and peptides corresponding to the vbss show that the residues which interact with vinculin are buried in the hydrophobic core of the helical bundles of the talin domains. nmr studies of the interaction of one of these domains with vinculin shows that it involves a major structural change in the talin fragment, including unfolding of one of its four helices, to make the vbs accessible. while the observation of folding of unstructured regions of a protein on interaction with a 'partner' is quite common, this kind of major unfolding to permit a protein-protein interaction is much less common. ways in which it may be regulated will be discussed. conformational changes of eye lens proteins studied by combined saxs and high pressure s. finet , f. skouri-panet , a. tardieu european synchrotron radiation facility, rue horowitz, bp , grenoble france, institut de minéralogie et de physique des milieux condensés, rue de lourmel, paris france, protéines: biochimie structurale et fonctionnelle, case , quai st-bernard, paris france -, -and -crystallins are the main components of vertebrate eye lenses, with exceptional structural and associative properties. the crystallins are known to be exceptionally stable in vivo since they have to last the lifetime of the organism. they therefore represent an extreme case of stability versus unfolding and aggregation. these proteins are mainly beta strands. -crystallins are kda monomers (from to % sequence identity), and -crystallins are large hetero-oligomers of about kda. -crystallins are molecular chaperone; they belong to the ubiquitous superfamily of small heat shock proteins, shsps. here, the conformation and the stability of -and -crystallins were investigated by small angle x-ray scattering (saxs) and high pressure, depending upon temperature and ph. at room temperature, -crystallins have shown a partially reversible change in size from to kb, and this effect was enhanced by the combination of temperature and pressure. in the case of -crystallins and in the pressure range up to kb, the pressure was combined with temperature and ph. the results depend upon the different itself. structural studies on pore-forming peptides s. m. ennaceur , d. bown , j. m. sanderson chemistry department, university of durham, biology department, university of durham the resistance of pathological microorganisms to conventional antibiotic drugs has created a need for new antibacterial agents. biologically active antimicrobial peptides that act as primary defense agents in a large variety of species are thought to have the potential as precursors for a new range of drugs from antibiotics to cancer treatments. this study has attempted to analyse the structural properties of membrane peptides and proteins through the use of model systems that have been designed to mimic their natural counterparts: we have successfully synthesised model membrane peptides with a beta-sheet structural motif and have used a wide range of techniques to analyse their interactions with phospholipid (pc) membranes. the synthetic peptides were very hydrophobic and only soluble in uorinated alcohols such as hfip and to a lesser extent tfe. we found hfip to have a very strong af nity for pc membranes and carried out a series of experiments to investigate this af nity. binding of hfip to pc membranes was found to be reversible and we exploited this property in d crystal trials of our synthetic peptides. we over expressed the c-terminal domain of brka, a gram negative autotransporter protein, which forms a beta-barrel channel in the outer membrane (omp), for comparison with our model peptides. we performed d crystal trials on the omp and imaged the resulting protein arrays by stem and afm. a protein spontaneously folds into a unique native structure in physiological conditions. this process accompanies a huge loss of the conformational entropy (ce). our major concern is to specify the factor that can compete with the ce loss. the previous discussions concerning protein folding have been focused on contributions to the free energy of folding from the interaction potentials in a system. a view lacking in earlier studies is that the folding is critically in uenced by the translational movement of water molecules. when solute particles contact each other in a solvent, the excluded volumes for the solvent molecules overlap, and the total volume available to their translational movement increases. this leads to a gain in the translational entropy (te) of the solvent. this type of te effect should be much stronger in protein folding where the tight packing of the side chains occurs. an elaborate statistical-mechanical theory is employed to analyze the te of water in which a peptide or a protein molecule is immersed. it is shown that the te gain upon folding is large enough to compete with the ce loss. when water is replaced by another solvent whose molecular size is larger, the te gain decreases to a remarkable extent. we suggest that the entropic loss accompanying the self-assembly and the formation of ordered structures in a living system is compensated mostly by the te gain of water, highlighting an aspect of the crucial importance of water in sustaining life. domain ii of ribosome recycling factor is required for disassembly of the post-termination complex p. guo, l. zhang, y. feng, g. jing institute of biophysics, chinese academy of sciences, national laboratory of biomacromolecules, beijing, china ribosome recycling factor (rrf) consists of two domains and, in concert with elongation factor ef-g, triggers dissociation of the post-termination ribosomal complex. however, the exact function of the individual domains of rrf remains unclear. to clarify this, two rrf chimeras, ecodi/ttedii and ttedi/ecodii, were created by exchanging the rrf domains between the proteins from escherichia coli and thermoanaerobacter tengcongensis. the ribosome recycling activity of the rrf chimeras was compared with their wild-type rrfs by using in vivo and in vitro activity assays. the experiments show that like wild-type tterrf, the ecodi/ttedii chimera fails to complement the rrf ts phenotype of e.coli lj (frr ts ) strain and has no polysome breakdown activity. however, under the same conditions, the ttedi/ecodii chimera complements the rrf ts phenotype and has polysome breakdown activity equivalent to that of wild-type ecorrf. the results indicate that domain ii of rrf is the functional domain that is mainly responsible for the disassembly of the post-termination ribosomal complex, and the speci c interaction between rrf and ef-g on the ribosomes mainly depends on the interaction between domain ii of rrf and ef-g; while domain i of rrf is the main contributor for binding ribosomes and maintaining the stability of the rrf molecule. this study provides direct genetic and biochemical evidence for the assignment of individual functions of rrf domains. self-assembly of natural somatostatin into liquid crystalline nano brils w the natural neuropeptide somatostatin- is a cyclic tetradecapeptide hormone, with broad inhibitory effects on both endocrine and exocrine secretions. we report the self-assembly of somatostatin in solution, into stable liquid crystalline nano brils, based on the neuropeptide bioactive backbone conformation. the system was studied as a function of peptide concentration, milieu composition and time, using optical and electron microscopy, x-ray scattering, vibrational spectroscopy and sec/rp-hplc. in pure water, the formation of twisted nano brils (around nm wide and a few microns long) was characterized. their structure relies on the native somatostatin -hairpin and on intermolecular antiparallel -sheets networks. the nano brils were observed to laterally associate further with increased concentration and time, as well as to generate hexagonal phases. increase in ionic strength (sodium chloride, phosphate) was found to signi cantly favor the self-association process. the soft conditions of formation of the somatostatin nano brils support biological relevance, for instance to the biological mechanism of storage of the neuropeptide hormone. unraveling the physical origin of the structure of fully denatured ubiquitin s. golic grdadolnik, f. avbelj national institute of chemistry, hajdrihova , ljubljana, slovenia the structure and dynamics characterization of non-native states of proteins is crucial for understanding the mechanism of protein folding. recently many experimental studies have shown variations of conformational propensity and exibility along the backbone chain of fully denatured proteins. it has been supposed that areas of residual structure may serve as initiation sites of protein folding. however, the physical origin of these variations is still unclear. we analyze the structure of fully urea-denatured ubiquitin. the experimental veri cation of conformational propensities of protein backbone is obtained through structurally dependent nmr parameters. although the secondary structure of ubiquitin under strong denatured conditions is not detectable and no correlation with the native overall topology is found, the variations of nmr parameters along the backbone follow the secondary structure elements of its native state. we show that these variations are in accord with the recently developed electrostatic screening model of denatured proteins ( ). in this model, the backbone conformations of residues in unfolded protein are determined by local backbone electrostatic interactions and their screening by backbone solvation. many virulence factors from gram-negative bacteria are autotransporter proteins. the nal step of autotransporter secretion is c nterminal threading through the outer membrane (om), followed by folding. this process requires neither atp hydrolysis nor a proton gradient. pertactin, an autotransporter from bordetella pertussis and the largest -helix structure solved to date, folds much more slowly than expected based on size and native state topology, yet folding intermediates are not aggregation-prone. equilibrium denaturation results in the formation of a partially folded structure, a stable core comprising the c-terminal half of the protein. examination of the pertactin crystal structure does not reveal the origin of the enhanced c-terminal stability. yet sequence analysis reveals that, despite size and sequence diversity, all autotransporters are predicted to fold into parallel -helices, suggesting this structure may be important for secretion. for example, slow folding in vivo could prevent premature folding of in the periplasm prior to the assembly of the om porin. moreover, extra stability in the c-terminal rungs of the -helix may serve as a template for the formation of the native protein during secretion, and formation of the growing template may contribute to the energy-independent translocation mechanism. coupled with the sequence analysis, these results suggest a general mechanism for autotransporter secretion. thermal and functional properties of e.coli outer membrane protein-receptor fhua fhua is e.coli outer membrane protein, which transports iron into the bacterial cell and also serves as receptor for several phages. in order to get more deep information about structural properties of fhua, we've studied its thermal properties by means of calorimetry. we've also investigated the interaction between t , ira phages and directly fhua by means of viscometry. the calorimetric result of heat denaturation of membrane protein fhua and next deconvolution of the recorded calorimetric curve with two transitions has shown that in a chosen conditions the structure of fhua consists of domains. though both t and ira phages grow on the common bacterial strain (e.coli k ho ), expressing fhua the results of viscometric investigation show that under direct interaction of phages with fhua the receptor activity of protein revealed only for t phage. therefore, we conclude that other than fhua protein serves as receptor for ira phage. it should be mentioned that the phage dna ejection process induced by receptor was observed for the rst time by us in an incessant regime. electron spray ionization mass spectroscopy (esi-ms) is a powerful tool for the investigation of the protein folding or proteins non-covalent interactions in solution since charge state distributions (csds) in esi-ms are affected by the conformational state and mass relates on the association state. we used this tecnique to inquire at different ph and different conditions the dimerization process of the porcine and bovine -lactoglobulins that share a high sequences similarity and close d structures. dimerization oflactoglobulins is reversible and involves both electrostatic and hydrophobic interactions. it was possible to detect simultaneously both the monomeric and dimeric form of the proteins in solution, pointing out the different dimerization behaviour of the two isoforms. we assessed the maximal stability of the dimeric structure at ph for the porcine protein and ph for the bovine one. moreover we showed that bovine lactoglobulin has a stronger dissociation costant than the porcine protein. further we showed that it is possible to modulate the dimerization equilibrium of the bovine isoform at ph both increasing temperature and adding methanol without inducing denaturation of the protein. a possible novel method of protein structure prediction; a. ikehata division of structural bioinformatics science of biological spramolecular systems graduate school of integrated sciences mechanism of protein folding has been mysterious since an nsen's dogma was sugested.here i would like to propose a possible novel method of protein structure prediction; origami method. the method comes from a protein backbone property of uctuation and the residue hydrophobicity. l. marsagishvili , m. shpagina , z. podlubnaya institute of theoretical and experimental biophysics ras, pushchino state university amyloid brils are formed by proteins or their peptides in the result of a conformational transition from alpha helix into beta-sheet structure. despite the different nature of proteins-precursors their amyloids have common properties: beta-pleated sheet structure with individual beta-sheets oriented parallel to the main axis; insolubility in vivo; speci c binding to congo red and thyo avin-t. amyloid deposits are observed in different diseases such as myositis, myocarditis, cardiomyopathies and others. we showed that sarcomeric cytoskeletal proteins of titin family (x-, c-, h-proteins) of rabbit skeletal muscles are capable to form amyloid brils in vitro. these proteins already contain % of beta-sheet structure necessary for formation of amyloids. the amyloid nature of their brils was conrmed by electron, polarization and uorescence microscopy. as x-, c-, h-proteins form amyloid brils easily in vitro, there is a danger of fast growth their amyloid deposits in vivo. taking into account common properties of amyloids formed by different proteins, our results clear the ways for conducting by amyloidogenesis in human organs and tissues. work is supported by rfbr grants - - , "universities of russia" . . and program of the presidium ras "fundamental sciences for medicine". probing the folding capacity and residual structures in -and -residue fragments of staphylococcal nuclease d. liu, x. wang, y. feng, l. shan, j. wang national laboratory of biomacromolecules, institute of biophysics, chinese academy of sciences n-terminal fragments of staphylococcal nuclease (snase) with different chain lengths were used as a model system in the folding study. the detailed characterization of conformational states of - and - residues snase fragments (snase and snase ) and their v w and g w mutants can provide valuable information on the development of conformations in the folding of snase fragments of increasing chain lengths in vitro. in this study, the presence of retained capacity for folding and residual structures in snase and snase is detected by cd, uorescence, ftir, and nmr spectroscopy. snase is represented as an ensemble of interconverting conformations. the uctuating nascent helix-and âsheet-like structures, localized in regions of a -a and t -v , respectively are transiently populated in snase . the native-like tertiary conformations are obtained for g w and v w and for snase in the presence of . m tmao. analysis of the results of such studies indicate that folding of snase fragments is dominated by developing the local and non-local nucleation sites from native-like secondary structures and by intensifying the longrange interactions of residues at nucleation sites with residues further removed in sequence. thermal disruption of a spanning network of hydration water and conformational changes of elastin a. krukau , i. brovchenko , a. oleinikova , a. geiger international max-planck research school in chemical biology, otto-hahn str. , d- , germany, physical chemistry, university of dortmund, otto-hahn str. , d- , germany hydration water strongly in uences the structural and dynamical properties of biomolecules. the existence of a spanning hydrogenbonded network formed by the hydration water enables the function of biomolecules at low hydration levels. we can expect that the formation/disruption of the spanning network formed by the hydration water in solution also affects crucially the protein properties. we present the rst computer simulation study of the thermal disruption of a spanning network formed by the hydration water of a biomolecule (elastin-like peptide). this process obeys the laws of d percolation transition, similarly to the formation of a spanning water network with increasing hydration level [ ]. the spanning water network transforms into an ensemble of small water clusters with increasing temperature: it is still permanent at k and exists with probability % at k. in the same temperature interval, the conformation of the peptide changes noticeably: its radius of gyration increases sharply (by %) at about k. these two phenomena may be related to the "inverse temperature transition" at about k, where an elastin solution separates into two phases. in our simulations, the displacement of hydration water by the addition of a denaturant (urea) or by other peptide molecules causes an even stronger increase of the radius of gyration (up to % lipidic cubic phases formed by distinct water and lipid volumes provide bicontinuous d bilayer matrices that have speci c and controllable water channel sizes and large surface areas. these systems have proven to be also valuable as membrane mimetic structures, as promising matrices for controlled-release and delivery of proteins, vitamins and small drugs in pharmacological applications, and they offer a d lipid matrix for successful crystallization of membrane proteins which do not easily crystallize in bulk solution. the present study is directed towards a better understanding of the interplay between curved cubic lipid phases and the protein entrapped within their aqueous channel structures. as model systems, we have chosen a cubic ia d phase formed by an uncharged lipid, monoolein, and incorporated different proteins, such as cytochrome c, -chymotrypsin and insulin, in its narrow water channels. we show that the protein secondary structure and unfolding behaviour may be in uenced by the con nement and, vice versa, the topology of the lipid matrix may change as a function of protein size and concentration. in fact, even new cubic lipid structures may be formed that are not known in pure lipid systems. furthermore, we compare the aggregation scenario of insulin in bulk solution and in the narrow water channels of the cubic lipid matrix and discuss the differences found in terms of the geometrical limitations imposed by the con nement. after endocytosis, i.e. at acidic ph, the t domain inserts in the membrane of the target cell and helps the translocation of the catalytic domain into the cytoplasm. therefore, the t domain has a key role in the strategy of internalization of the toxin. the study of the interaction of the t domain with membranes and its ph dependence is important for a better understanding of the diphtheria toxin translocation mechanism. at least, two steps can be distinguished during the membrane insertion of the t domain. the rst step involves hydrophobic interactions with the membrane and is related to the ph-induced stabilization in a molten-globule state. in the second step, electrostatic interactions are preponderant and the ph-sensitivity comes from changes of the balance between repulsive and attractive electrostatic interactions. the role of the n-terminal part of the t domain in the second step has been investigated by studying peptides corresponding to the amphiphilic helices found in this part of the domain. the results are correlated with those obtained with a single trp mutant probing the n-terminal region of the whole domain. the translocation mechanism will be discussed in view of the physico-chemical properties of the peptides. in complex systems with many degrees of freedom such as peptides and proteins there exist a huge number of local-minimumenergy state. one way to overcome this multiple-minima problem is to perform a simulation in a generalized ensemble where each state is weighted by a non-boltzmann probability weight factor so that a random walk in potential energy space may be realized (for reviews, see refs. [ ] [ ] [ ] xas spectroscopy results show that there are two different structures of the metal binding site in the a peptide according to whether they are complexed with cu or zn ions. while the geometry around copper is suggestive of an intra-peptide binding with three histidine residues bound to the metal, the zinc site geometry is compatible with an inter-peptide aggregation mode. this result reinforces the hypothesis that assigns opposite physiological roles to the two metals, with zinc favouring and copper blocking peptides aggregation and consequent plaque formation. effect of pressure on the conformation of proteins. a molecular dynamics simulation of lysozyme a. n. mccarthy, r. grigera instituto de física de líquidos y sistemas biológicos (iflysib), conicet-unlp-cic, university of la plata, argentina the effect of pressure on the structure and dynamics of lisozyme was studied by md computer simulation at bar ( pa) and kbar using gromacs package. all-atoms (ff gmx) force eld were used for the minimization process and for all the md simulation and kept all protein bond lengths constrained (lincs algorithm). water molecules (spc/e model) were constrained using the settle algorithm. for the electrostatic forces we applied the reaction field method. lennard-jones interactions were calculated within a cut-off radius of . nm. the results have good agreement with the available experimental data, allowing the analysis of other features of the effect of pressure on the protein solution. the studies of mobility show that although the general mobility is restricted under pressure this is not true for some particular residues. from the analysis of secondary structures along the trajectories it is observed that the conformation under pressure is more stable, suggesting that pressure acts as a 'conformer selector' on the protein. the difference in solvent accessed surface (sas) with pressure shows a clear inversion of the hydrophilic/hydrophobic sas ratio, which consequently shows that the hydrophobic interaction is considerably weaker under high hydrostatic pressure conditions. direct observation of mini-protein folding using uorescence correlation spectroscopy h. neuweiler, s. doose, m. sauer applied laser physics & laser spectroscopy, university of bielefeld, bielefeld, germany the "trp-cage" motif represents the smallest and one of the fastest folding mini-proteins known to date. the globular fold is characterized by a hydrophobic core burying a single tryptophan (trp) residue. here, we report on the direct observation of trp-cage folding kinetics using uorescence correlation spectroscopy (fcs). our method is based on the selective uorescence quenching of oxazine dyes by trp which becomes ef cient only upon contact formation between the dye and the indole moiety of trp. by sitespeci cally labeling the dye to trp-cage, temporal uorescence uctuations of the dye-peptide conjugate, caused by intramolecular contact formation between dye and trp, directly report on conformational dynamics and folding transitions of the peptide chain. in order to measure uorescence uctuations directly in solution we used fcs on a confocal uorescence microscope setup. fcs allows us to reveal conformational dynamics with nanosecond timeresolution, under thermodynamic equilibrium conditions, and in highly dilute solutions (i.e. at nano-molar sample concentrations). our method con rms microsecond folding kinetics of the trp-cage motif, previously estimated with non-equilibrium temperature-jump techniques. we further investigated stability and folding rates under denaturing conditions and at various temperatures, giving further insight into structural transitions during the folding process. identi cation and mutagenesis of a region of tnt required for the stability of tnt-tni coiled-coil evolutionarily conserved heptad hydrophobic repeat (hr) domains present in troponin subunits tnt and tni are involved in alpha helical coiled-coil formation. using recombinant peptides from fast-skeletal tnt and tni, we examined the contributions of amino acid residues within these hr domains as well as anking these domains, to the stability of the coiled-coil interaction. a series of tnt fragments were tested for their ability to form coiledcoil with tni hr domain. we show that the tnt region - , although remains outside of the coiled-coil domain, is absolutely required for the stability of the coiled-coil. interestingly, the region tnt - contains few absolutely conserved residues that are potential candidate for ionic interaction, as predicted by molecular modeling. using single point mutants we show that among all the conserved residues, residue lysine is most important in stabilizing the coiled-coil interaction, whereas others play accessory role. we propose that the lysine initiates the stabilization of the coiled-coil interaction and then the other residues acts in a zipper like fashion. magnesium promotes conformational switching of ca sensor s. mukherjee, k. v. r. chary tata institute of fundamental research the importance of mg , one of the most abundant metal ion in the cell cytoplasm, relating to the calcium sensor mechanism is demonstrated. in this study it has been shown that the amino acid long calcium binding protein from entamoeba histolytica (ehcabp) can exist in three different forms namely, the calcium-free (apo) form, the magnesium bound (apo-mg) form and the calcium bound (holo) form. these three forms have been characterized using chromatographic, calorimetric as well as various spectroscopic techniques. there is a radical difference in the stability between the calcium free and ca bound forms. the calcium free form has molten globule like characteristics. mg stabilizes the closed conformation of the apo form, where the hydrophobic core remains buried. the presence of mg signi cantly alters the calcium binding cooperativity thereby increasing the cooperativity of the conformational switching between the open and closed conformation which is an important aspect of such regulatory proteins. a structural model for the molten-globular form of apo-ehcabp and its equilibrium folding towards completely folded holo state in presence and absence of mg will be presented. intermediate states of formin binding protein ww domain: explored by replica-exchange simulation y. mu school of biological science, nanyang technological university, singapore ww domain of formin-binding protein (fbp) is a model system for beta strand folding study. although it is small, only amino residues in total, the folding kinetics of fbp ww domain proved to be biphasic. an extensive molecular dynamics-monte carlo hybrid method, called replica-exchange simulation strategy is employed to study the folding/unfolding of the fbp ww domain in explicit water model. begin with randomly chosen conformations from high temperature unfolding trajectory distributed in replicas ( different temperatures covering from k to k), the simulation lasts nanoseconds in each replica. in the end an interesting distribution of conformations shapes up. we nd that there are three distinctive subgroups, one being the unfolded conformations with rmsd averaging around Å , one being native conformations with rmsd . Å, more interestingly, the third group having rmsd Å, an intermediate folding ensemble. by checking the intermediate ensemble in detail, we nd that it is quite heterogeneous and the heterogeneity mainly comes from the exibility of the c-terminal loop region. our ndings provide a microscopic picture of folding kinetics of the ww domain: the stable intermediate states with mis-registered hydrogen bonds on the c-terminal beta strand make this peptide folding as a three-state folding model rather than a usual two-state model. h. rezaei, f. eghiaian, j. perez, y. quenet, y. choiset, t. haertle, j. grosclaude institut national de la recherche agronomique, france in pathologies due to protein misassembly, low oligomeric states of the misfolded proteins rather than large aggregates play an important biological role. to get better insight into the molecular mechanisms of prpc/prpsc conversion, we studied the kinetic pathway of heat induced amyloidogenesis of the full length recombinant ovine prp (arq) at ph . . according to the size exclusion chromatography experiments, three sets of oligomers were generated from the partial unfolding of the monomer. the effect of concentration on the oligomerization kinetics was different for the three species obsreved suggesting that they are generated from distinct kinetic pathways. limited proteolysis and peptide analysis of the two best separated peaks showed a difference in the accessibility of the c-terminal domain of these two oligomers, and allowed the identi cation of regions undergoing a structural change during the conversion process. the analysis of correlations between oligomer populations as well as numerical kinetic modeling led us to propose a multi-step kinetic pathway describing the evolution of each species as a function of time. the existence of at least three distinct oligomerization pathways on one hand and the differences in the accessibility of the two puri ed oligomers on the other hand re ect the structural plasticity of the prp protein. studying the mechanism of retention of ovine prion protein in soils will tackle the environmental aspect of potential dissemination of scrapie infectious agent. the conformational transition from the monomeric cellular prion protein prp c in -helical structure into the aggregated -sheet-rich multimer prp sc is supposed to be responsible for the so-called prion diseases. it is commonly admitted that the recombinant prp could serve as model of conversion of the normal prion protein prp c . fundamentally, the interaction of proteins with surfaces either uid or solid involves both protein binding and unfolding. our goal in studying protein adsorption is to determine the nature and the amplitude of the structural changes occurring during non-speci c adsorption. the protein-clay interaction depends on several parameters such as protein hydration, net charge, charge distribution on the protein surface. ftir spectroscopy is well-suited to probe structural changes of proteins at a molecular level at aqueous/solid interfaces. the conformational states of the full-length ovine prp adsorbed on the electronegative clay surface are compared to its solvated state in deuterated buffer in the pd range . - , using ftir spectroscopy. during the intoxication of a cell, the diphtheria toxin binds to a cell surface receptor, is internalized and reaches the endosome. the translocation (t) domain from the toxin interacts with the membrane of the endosome in response to the acidic ph found in this compartment. this process drives then the passage of the catalytic domain of the toxin through the membrane into the cytoplasm. the interaction of the t domain with the membrane involves at least two steps. in the rst step occurring at ph , t adopts a molten globule conformation, which is able to bind super cially to the membrane through hydrophobic interactions by the c-terminal region (helices th and th ). in a second step occurring between ph and , penetration into the membrane involves electrostatic interactions. this step leads to a functional inserted state. trp was mutated to phe in order to use trp located in helix th as a probe of its behavior during the interaction with the membrane as a function of ph. we found that the second step is correlated with the reorganization of the n terminal region in the membrane and is controlled by electrostatic interactions. peptide conformational search using generalized simulated annealing method p. g. pascutti , f. p. agostini , c. osthoff , k. c. mundim , m. a. moret ibccf -ufrj -brazil, lncc -brazil, iq-unb -brazil, ceppev -fundação visconde de cairú / df-uefs -brazil the three-dimensional structure of proteins is mainly determined by the sequence of amino acids, making possible the development of ab initio methods for peptides and protein structure prediction. we proposed a stochastic method based on a classical force field and the generalized simulated annealing (gsa), which utilizes tsallis generalization of boltzmann-gibbs statistics. we have applied this method for peptide conformational search and as a complement for comparative modeling of proteins, searching the conformation for loops and mismatched sequence alignments. the gsa ef ciency depends on the right choice of the parameters involved in the conformational calculation: the qv parameter which de nes a function for visiting the molecular energy surface; and the qa parameter de ning an acceptance probability, both according with tsallis statistics. to avoid the conformational trapping in local energy minima we introduced a new parameter in this work, qt, to control the temperature decreasing. to investigate the qv, qa and qt best parameters set, we used the -alanine and -alanine peptides, which have a known alpha-helical structure in low dielectric environment. the global minimum energy occurs for the alpha-helix folded structure, and was found for qv ranging from . to . , qa from . to . , and qt from . to . . we observed also that convergence values for qv decrease while for qa and qt increase for folded structures. p. s. santiago, É. v. de almeida, m. tabak instituto de quimica de são carlos-usp cp são carlos sp brasil extracellular hbgp is a giant hemoglobin, similar to other annelid hemoglobins, having a molecular weigth of . mda. the effect of ctac in the oligomeric protein structure was assessed by optical absorption and emission spectroscopies. optical absorption spectra of hbgp . mg/ml as a function of ctac concentration from up to . mm evidentiate changes both in soret band at nm, and at nm associated to light scattering which appears at low ctac concentration. below mm of surfactant extensive light scattering occurs together with signi cant shifts of max of soret band from nm to around nm, which is probably due to oxidation of the original oxyhbgp. light scattering reaches a maximum value disappearing for higher ctac concentrations. fluorescence spectra show a signi cant increase in intensity ( fold) upon titration with ctac. this is consistent with the dissociation of the oligomer with signi cant reduction of intrinsic quenching of tryptophan uorescence due to the heme groups. similar data were obtained at protein concentration of mg/ml. in this case a signi cant increase of light scattering is observed with protein precipitation at a narrow ctac range followed by re-disolution at higher ctac concentration. differently from anionic sds surfactant, cationic one induces protein aggregation. support: cnpq and fapesp. in situ formation of silk protein nano-particles studied by small angle x-ray scattering m. w. roessle , c. riekel , d. i. svergun european molecular biology laboratory; outstation hamburg/germany, european synchrotron radiation facility; grenoble/france silk threads from the mulberry silkworm bombyx mori are used for the production of textiles since centuries. in modern applications silk proteins are chosen because of their good tensile strength, their high biocompatibility as well as of the resorbability for the human body. however, the processing of silk by the silkworm is barely understood. the silk proteins are stored as a solution in the glands of the silkworm and processed during the spinning into a co-block polymer like ber. in a rst step the random coil silk proteins are transformed into molecules with beta-sheet subdomains, which provide a protein-protein interface for the ber assembly. this transformation can be mimicked by a rheometer applying a shear force to the silk protein solution. applying combined small/wide angle x-ray scattering (saxs/waxs) transient formed silk nanoparticles upon increasing shear force were found. further details of these macromolecules were derived by xing the transient state with chemicals such as polyethylene oxide (peo). the resulting data can be analyzed in detail by saxs data evaluation software and low resolution models of the found nanoparticles were derived. moreover, the internal structure of the particles was explored as well as suggestions for the silk processing of the silkworm could be made. on the three-dimensional information of a protein sequence v. a. risso, l. g. gebhard, r. g. ferreyra, j. santos, m. noguera, m. r. ermacora universidad nacional de quilmes conicet in this work, lactamase of b. licheniformis (es l) was used as an experimental model to (a) study the conversion of sequential information into d structure and (b) to investigate the distribution of conformational information in the polypeptide chain. by a novel approach, over thirty connectivity variants of the polypeptide chain were prepared; witch were also nterminally truncated to a variable degree. the variants produced in e. clil were puri ed to homogeneity, refolded, and its structure content analyzed by circular dichroism, hydrodynamic behaviour and aggregation state. several variants were dimeric in solution, suggesting a possible general inespeci c stabilization mechanism. most variants were compact and had different degrees of secondary and tertiary structure. a strikingly large number of variants showed native like spectroscopic signatures and signi cant enzymatic activity, which means that the very elaborate active site of beta lactamase is formed, at least in fractions of the molecules, despite the absence of long stretches of sequence. these ndings are discussed in the light of the current knowledge of the protein folding process. var and lgg contributed equally to this work. on the three-dimensional information of a protein sequence v. a. risso, l. g. gebhard, r. g. ferreyra, j. santos, m. e. noguera, m. r. ermacora universidad nacional de quilmes conicet in this work, lactamase of b. licheniformis (es l) was used as an experimental model to (a) study the conversion of sequential information into d structure and (b) to investigate the distribution of conformational information in the polypeptide chain. by a novel approach, over thirty connectivity variants of the polypeptide chain were prepared; witch were also nterminally truncated to a variable degree. the variants produced in e. clil were puri ed to homogeneity, refolded, and its structure content analyzed by circular dichroism, hydrodynamic behaviour and aggregation state. several variants were dimeric in solution, suggesting a possible general inespeci c stabilization mechanism. most variants were compact and had different degrees of secondary and tertiary structure. a strikingly large number of variants showed native like spectroscopic signatures and signi cant enzymatic activity, which means that the very elaborate active site of beta lactamase is formed, at least in fractions of the molecules, despite the absence of long stretches of sequence. these ndings are discussed in the light of the current knowledge of the protein folding process. var and lgg contributed equally to this work. unfolding of the extrinsic proteins of photosystem ii ( kda, kda and kda) induced by pressure unfolding of the three extrinsic proteins of spinach photosystem ii induced by pressure has been systematically investigated. thermodynamic equilibrium studies indicated that these proteins are very sensitive to pressure. at o c all the proteins show a reversible unfolding transition by about mpa for kda, mpa for kda and mpa for kda. the ph and temperature dependence of pressure unfolding of these proteins were explored. the stabilization effect of reagents sucrose etc on the proteins was found to associate not only with the increase in the unfolding free energy, but also with the reduction of the absolute value of v u . pressure-jump studies of unfolding of kda protein revealed a negative activation volume for unfolding and a positive activation volume for refolding, indicating that in terms of system volume, the transition state lies between the folded and unfolded states. comparison of temperature dependence of v u # , v f # and v u indicated that the thermal expansivities of the transition state and the unfolded state are similar and larger than that of the folded state. aggregation-prone intermediate protein structures on the refolding pathway l. smeller , j. fidy , k. heremans semmelweis university dept. biophysics and radiation biology, budapest, hungary, department of chemistry, katholieke universiteit leuven, belgium the folding of the polypeptide chain into a native conformation can be studied by experimental systems, where the environmental parameters causing the denatured state can be easily and fast eliminated. one of such parameters is the high hydrostatic pressure. refolding of the unfolded protein can be studied after decompression. the refolding pathway can contain several intermediate states. on the other hand, the destabilization of the native proteins can populate conformations, where the polypeptide chain is not completely folded. these metastable conformations can easily aggregate. deposition of insoluble protein aggregates plays a crucial role in the conformational diseases (parkinson, alzheimer's disease, amyloidozis, etc.) stability and conformation of the above mentioned metastable conformations were investigated in case of myoglobin, apo-horseradish peroxidase, lipoxygenase. fluorescence and infrared spectroscopy and light scattering experiments were used to explore not only the structure but also the aggregation af nity of the intermediates in case of all the above mentioned proteins a well de ned temperature range could be determined, where the metastable not completely folded structures were populated considerably during the refolding process. these intermediate conformations were significantly more aggregation prone, than the native conformers existing in the same temperature range. aggregation processes in beta-amyloid peptides: effects of molecular chaperons a. sgarbossa, d. buselli, f. lenci cnr istituto bio sica, pisa, italy several neurodegenerative pathologies, like parkinson's, hungtington's and alzheimer's diseases, are related to the formation of small peptides aggregates, which amyloid brils originate from. understanding the molecular mechanisms responsible for these processes can, therefore, contribute to clarify the origin and, hopefully, to control the development of the afore mentioned diseases. here we report the results of an in vitro study aiming to affect the aggregation kinetic of - and - beta-amyloid peptides by means of an endogenous chaperone-like protein (alpha-crystallin) and an exogenous polycyclic aromatic pigment (hypericin) that can perturb the aggregation process through stacking interactions with the peptides aromatic residues. because of the well known problems in getting reproducible and reliable results, particular attention has been devoted to carefully check the preparation procedures of the samples. the effects of both alpha-crystallin and hypericin on the self-assembly process have been examined at different times of the aggregation kinetics. the results are discussed in relation with the involvement of different molecular structures in the amyloid brillation phenomenon. remodelling the folding of thioredoxin by removal of the c-terminal helix j. santos, j. m. del no iquifib and departamento de química biológica, facultad de farmacia y bioquímica, uba, junín , c aad buenos aires, argentina e. coli thioredoxin (trx) is a monomeric / protein of amino acids with a fold characterized by a central beta sheet surrounded by alpha helices. two subdomains are topologically noticeable, but it is unclear whether their folding occurs in a concerted fashion. subdomain trx - has been extensively studied as a model of a partially folded, with no tertiary or persistent secondary structure. this work describes the expression and characterization -by circular dichroism (cd), uorescence emission, size exclusion chromatography, chemical cross-linking and light scattering-of a novel engineered fragment (trx - ) lacking the last stretch of amino acids. after refolding from inclusion bodies, trx - shows a strong propensity to form soluble oligomers endowed with distinctive optical properties unlike those observed for the full protein. although trx - also shows signi cant changes in secondary structure, trp residues appear to occupy rigid and apolar environments. these ndings support the existence of an alternatively folded form for trx - . in addition, the secondary structure content of chemically synthesized peptide trx - and its ability to complement fragment trx - upon refolding were also evaluated by cd. taken together, the data herein presented shed light upon issues such as the distribution of information content relevant for folding along the polypeptide chain in regard to conformational stability. with grants from anpcyt, ubacyt and conicet. thermal aggregation of two "beta-protein" models at different ph values v. vetri, f. librizzi, v. militello, m. leone physical and astronomical sciences, univ. of palermo, italy & infm the structural stability of proteins strongly depends on the environment and the lost of this stability may trigger a partial unfolding, leading in turn to the formation of aggregates. such processes have been extensively studied also in view of their biotechnological and medical implications. in fact a large number of diseases is associated with protein misfolding and aggregation. conformational changes play a keyrole in the aggregation processes and have their onset under particular external conditions. the aggregation pathways and the topology of the obtained supramolecular structures sizeably depend on the details of the involved conformational changes, which are determined by the details of the external conditions. here we present an experimental study on thermal aggregation processes of two model proteins mainly composed from structures: -lactoglobulin and concanavalin a, at different ph values. the conformational changes of the proteins (whose association state depends by ph) and the aggregation pathways were monitored by intrinsic and suitable external dyes uorescence. at the same time, the growth of supramolecular structures was followed by measuring the rayleigh scattering of the excitation light. secondary structure changes were followed by circular dichroism measurements. the results show that at different ph values the aggregation processes of both proteins follow different pathways determined by the variations in the native structure and by the details of the involved conformational changes. a. verma , t. herges , w. wenzel iwr, forschungszentrum karlsruhe, po box , , karlsruhe, germany, int, forschungszentrum karlsruhe, po box , , karlsruhe, germany ab-initio protein structure prediction and the elucidation of the mechanism of the folding process are among the most important problems of biophysical chemistry. investigations of the protein landscape may offer insights into the folding funnel and help elucidate folding mechanism and kinetics. we investigate the landscape of the internal free-energy of the amino acid villin headpiece with a modi ed basin hopping method in the all atom force eld pff , which was previously used to fold several helical proteins with atomic resolution. we identify near native conformations of the protein as the global optimum of the force eld. more than half of the twenty best simulations started from random initial conditions converge to the folding funnel of the native conformation, but several competing low-energy metastable conformations were observed. from , independently generated conformations we derived a decoy tree which illustrates the topological structure of the entire low-energy part of the free energy landscape and characterizes the ensemble of metastable conformations. these emerge as similar in secondary structure content, but differ in the tertiary arrangement. exploring the free energy landscape of a folded protein by means of afm stretching experiments m. vassalli , b. tiribilli , l. casetti , a. torcini , a. pacini , a. toscano isc -cnr, florence -italy, csdc, univ. of florence -italy, anatomy dept, univ. florence -italy the aim of our work is to study the mechanically induced folding and refolding of single proteins by means of an atomic force microscope (afm). the resulting data will be analyzed with theoretical methods, both to determine the folding pathway and to gain information on the energy landscape of real systems. recently, experiments employing atomic force microscopy have shown that mechanical and thermal unfolding share several common features. we are using an afm to perform mechanical stretching experiments on single biomolecules. the experiments that can be performed are particularly well-suited to reconstruct the folding-unfolding pathways as well as the free energy landscape of the examined protein. in particular we are interested in the free energy pro le associated to titin and elastin, by considering a periodic loading of the afm cantilever, instead of the usual linear ramp, and measuring the force as a function of displacement. these experiments will be complemented by theoretical and numerical studies. molecular dynamics simulation of simple models but including the experimental geometry, will allow to examine in detail the effect of different experimental procedures (periodic loading versus linear ramp) proposed to reproduce equilibrium energy landscapes. moreover, we will investigate the limit of applicability of the jarzynski's equality which has been claimed to be able to be used to extract equilibrium results from non equilibrium measurements. association of subunits is a prerequisite for formation of the native structure of the dimeric ipmdh a. varga, É. gráczer, i. hajdú, p. závodszky, m. vas institute of enzymology, biological research center, hungarian academy of sciences, budapest, hungary to answer the question whether subunits are autonomous folding units, or their association at an early stage of folding is required for formation of the native protein structure, denaturation-renaturation experiments were carried out with the dimeric isopropylmalatedehydrogenase (ipmdh). denaturation was induced by guanidine hydrochloride, renaturation was initiated by dilution and followed by activity measurements and uorimetry. reactivation is a complex process with an initial lag phase, indicating the presence of an inactive intermediate. the kinetics of the process is independent of protein concentration, suggesting that association of the two polypeptide chains takes place much faster than the rate limiting rst order isomerisation step(s). restoration of protein uorescence during renaturation is also protein concentration independent, biphasic process, however the initial lag phase is replaced by an even faster burst increase of uorescence. the rst step leads to formation of an intermediate with a native-like uorescence spectrum. based on our experiments the following mechanism is proposed for refolding of ipmdh: d+d i i n , where d is denaturated monomer, i and i are inactive dimer intermediates, n is native dimer, that means initial association of the polypeptide chains during refolding is a prerequisite for formation of the native dimensional structure of ipmdh. we describe a new beamline for optical biophysic in construction on the synchrotron soleil. the high briliance of the synchrotron beam, associated with its tunability on a broad part of the electromagnetic spectrum make it an excitation source of choice for several biophysical optical techniques. the disco beamline we present will consist of three endstations : . the circular dichroism (cd) endstation will bene t from the inclusion of the energies accessible in the vuv wavelength range ( - nm) and from the natural polarization of the synchrotron beam. cd spectra of proteins covering a broad range of wavelenghts will enable better and ner structural analysis. moreover, new biological chromophores such as sugars which absorb in the deep uv will be accessible in cd. . the mass spectrometry endstation,will bene t from an ionisation beam with even greater energies (down to nm) comprising nebulisation at atmospheric pressure. photoionisation of macromolecular bio-structures without any solvent restriction will produce perfect analytes for mass spectrometry, . the multiparametric imaging endstation, build on a confocal microscope, will use the great tunability of the synchrotron radiation ( - nm) to excite samples at many wavelenghts simultaneously. the temporal component of the beam will allow natural lifetime imaging by phase modulation -demodulation. predictive all-atom protein folding with stochastic optimization methods the prediction of protein tertiary structure, in particular based on sequence information alone, remains one of the outstanding problems in biophysical chemistry. according to the thermodynamic hypothesis, the native conformation of a protein can be predicted as the global optimum of its free energy surface with stochastic optimization methods orders of magnitude faster than by direct simulation of the folding process. we have recently developed an all-atom free energy force eld (pff ) which implements a minimal thermodynamic model based on physical interactions and an implict solvent model. we demonstrated that pff stabilizes the native conformation of several helical proteins as the global optimum of its free energy surface. in addition we were able to reproducibly fold several helical proteins (the amino acid (aa) trp-cage protein, the villin headpiece ( aa), the conserved headpiece of the hiv accessory protein ( aa), the headpiece of protein a ( aa) and the -helix bacterial ribolsomal protein l ( aa), as well as several beta-sheet peptides. we used several stochastic optimization methods: the stochastic tunneling method, an adapted version of parallel tempering, basin hopping techniques and distributed evolutionary optimization strategies. we discuss advantages and limitations of this approach to de-novo allatom protein structure prediction. the independent thermal unfolding simulations of gb have been performed. physical property parameters of protein structure were chosen to construct a -dimension physical property space. then the -dimension property space was reduced to dimensions principle component property space. under the property space, the unfolding pathway ensemble of gb was obtained. the pathway ensemble likes a funnel that was gradually emanative from the native state ensemble to the unfolded state ensemble. the unfolding trajectories have the similar variable trend during the native state and the transition state ensemble. during the unfolded state, the unfolding trajectohries were divided into two types that one includes only one trajectory and the other include trajectories. the rst type of unfolded state was a discontinuity step distribution model, which is not random distribution. the second type of unfolded state was a near ellipsoid distribution model and a near random. there were substantial overlaps of unfolded state, indicating that thermal unfolded state consists of a con ned set of property values that makes the number of unfolded state of protein to be much smaller than that was believed before. the protein circular dichroism data bank (pcddb): a bioinformatics and spectroscopic resource b. a. wallace , l. whitmore , r. w. janes dept. of crystallography, birkbeck college, university of london, school of biological sciences, queen mary, university of london we describe the development and creation of the protein circular dichroism data bank (pcddb), a deposition data bank for validated circular dichroism spectra of biomacromolecules. its aim is to provide a resource for the biological and bioinformatics communities, by providing open access and archiving facilities for circular dichroism spectra. it is named by parallel with the protein data bank (pdb), a long-existing valuable reference data bank for protein crystal structures. it will permits spectral deposition via userfriendly web forms and will include automatic reading of a range of data formats and data mining from le headers to facilitate the process. it will be linked, in the case of proteins whose crystal structures and sequences are known, to the appropriate pdb and sequence data band les, respectively. a series of validation tools that will provide reports on data quality are included (and are accessible as stand-alone software). it is anticipated that this data bank will provide readily-accessible biophysical catalogue of information on folded proteins that may be of value in structural genomics programs, for quality assurance and archiving in industrial and academic labs, as a resource for programs developing spectroscopic structural analysis methods, and in bioinformatics studies. the relation of n-terminal residues and structural stability of l-chain apoferritin k. yoshizawa , k. iwahori , y. mishima , i. yamashita crest/jst, atrl-matsushita, nara institute of science and technology the denaturation of apoferritin by acidic solution was studied. ferritin, the ubiquitous iron storage protein, represents a well known polymeric assembly that is highly resistant to chemical and physical denaturants. it is a cage-shaped protein which is composed of subunits. natural vertebrate ferritins are copolymers of two different subunits, l-and h-chains. in the recombinant h-chain apoferritin (rhf), the structural stability is decreased by deletion of the n-terminal residues. we studied the effect of n-terminal residues of recombinant l-chain apoferritin (rlf) on the acidic ph denaturation and re-assembly. we constructed rlf and mutant rlfs which are lack of (fer ) or (fer ) amino acid residues from the n-terminus and investigated their stability by cd spectra. among three, fer has the least endurance against ph decrease. in the case of fer , the re-assembly of subunits into apoferritin can be performed by increasing solution ph without causing the by-product while huge aggregations are caused in fer and fer . the structural comparison of three mutants indicates that the hydrogen bonds of inter-and intra-subunits decrease by the loss of the n-terminal residues. therefore, it is elucidated that the hydrogen bonds of inter-and intra-subunits from n-terminal residues affect the molecule stability and re-assembly of l-chain apoferritin. pressure perturbation calorimetic studies of solvation, unfolding and aggregation of proteins r. winter university of dortmund, physical chemistry, otto-hahn-str. , d- dortmund pressure perturbation calorimetry (ppc) was used to study the solvation and volumetric properties of various proteins in their native and unfolded state. in ppc, the coef cient of thermal expansion of the partial speci c volume of the protein is deduced from the heat consumed or produced after small isothermal pressure jumps, which strongly depends on the interaction of the protein with the solvent or cosolvent at the protein-solvent interface. the effects of ph and various chaotropic and kosmotropic cosolvents (glycerol, sucrose, urea, guhcl, salts, etc.) on the solvation and unfolding behavior of the proteins was also investigated, and the observed volume and expansivity changes are correlated with further thermodynamic and spectroscopic properties of the systems. depending on the type of cosolvent and its concentration, speci c differences are found for the solvation properties of the proteins, and the volume change upon unfolding may even change sign. taken together, the data obtained lead to a deeper understanding of the solvation process of proteins in different cosolvents in their native and unfolded states. in addition, the effects of con nement and crowding on the solvational properties of the proteins were studied. finally, the use of ppc for studying intermolecular interactions and aggregation (amyloidogenesis) phenomena of proteins (e.g., insulin, prp) will be discussed. the in uence of semisynthetic derivatives of phenolic lipids on activity of yeast abc pumps phenolic lipids are the natural amphiphilic long-chain homologues of orcinol ( , -dihydroksy- -methylbenzene). they occur in numerous plants and microorganisms. resorcinolic lipids exhibit high af nity for lipid bilayer and biological membranes and are able to modify the activity of membrane enzymes (e.g. pla , ache). the in uence of semisynthetic derivatives of phenolic lipids on yeast pdr protein activity was studied by spectro uorimetric method using the potentiometric uorescence probe dis-c ( ). the probe is expelled from s. cerevisiae by abc pumps and can conveniently be used for studying their performance. two pump-competent s. cerevisiae strains and different pump-free mutant strains were used to experiment to check the effect of the semisynthetic derivatives of phenolic lipids on activity of abc transporters. two of these derivatives, named . and . , seem to affect the activity of pdr pumps. their in uence on activity of yeast plasma membrane multidrug resistance abc pumps is concentration-dependent. in uence of lipid membrane composition on pglycoprotein activity k. bucher, s. d. krämer, h. wunderli-allenspach department chemistry and applied biosciencies, eth zurich, switzerland p-glycoprotein (p-gp), a membrane atpase expelling many structurally unrelated compounds, is one of the major contributors to multidrug resistance. it is proposed that substrates bind to it within the membrane and are exported from there out of the cell. p-gp substrates are generally hydrophobic and their binding to the transporter is governed by their ability to partition into the membrane. the intimate association of both p-gp and its substrates with the membrane suggests that p-gp function may be regulated by the composition of the lipid bilayer. as detergents in uence the membrane properties and have been shown to affect p-gp atpase activity, we developed virtually detergent-free proteoliposomes to investigate the in uence of the membrane environment on the atpase activity of p-gp. the basal and substrate induced atpase activity was dependent on the cholesterol level of egg phosphatidylcholine (pc) membranes. the compound concentration at half maximal activation of p-gp (k m ) in proteoliposomes correlated with the af nity of the respective compound to liposomes consisting of the same lipids as the proteoliposomes tested. in conclusion, the basal and drug-induced atpase activity of p-gp is strongly dependent on the cholesterol content in detergent-free p-gp/egg pc/cholesterol proteoliposomes. m. berchel , j. jeftic , t. benvegnu , j.-y. thepot , d. plusquellec enscr umr cnrs , campus de beaulieu, rennes, université de rennes , umr cnrs , rennes bipolar lipids found in archaebacterial membranes, generally termed bolaamphiphiles, induce increased stability in membranes exposed to environments such as acidic conditions, high temperatures, high salt concentrations and/or absence of oxygen. we have synthesized a spin labeled unsymmetrical bolaamphiphile that selforganises in water solutions in multilamellar vesicles and shows slow ip-op phenomenon in comparison to conventional liposomes. generally, the ip-op from the exovesicular to the endovesicular membrane surface is a relatively slow process, which is due to the high energy barrier in transferring the polar amphiphilic heads through the lipophilic membrane. it can be involved in membrane transport mechanisms and in facilitating the transport, cells have evolved to use various supramolecular strategies. the half-life of the ip-op is estimated to more than twelve hours. we are now modulating the ip-op rate by incorporating chemical modi cations such as addition of cyclopentanes, double or triple bonds into the bridging chain of the molecule, in order to control the membrane transport via the ip-op mechanism. transport activity of the monocarboxylate transporter is increased by carbonic anhydrase h. m. becker, j. w. deitmer abt. allgem. zoologie, tu kaiserslautern, kaiserslautern the enzyme carbonic anhydrase (ca), which catalyses the conversion of co and h o to bicarbonate and protons, is present in nearly all animal cells, and is highly expressed in astrocytes. it is known that ca can bind to several membrane transporters, forming a transport metabolon and thereby enhancing the transport activity of the protein. in this project we have studied the functional interaction of the enzyme with the monocarboxylate transporter (mct ), which transports lactate and other monocarboxylates together with protons and is believed to play a pivotal role in the metabolite shuttling between astrocytes and neurons. therefore we expressed mct and then injected ca into xenopus oocytes. our results indicate a direct binding of ca to the mct , leading to a ca-induced increase in acid/base ux mediated by the transporter. interestingly, the effect was insensitive to the ca inhibitor ethoxyzolamide and to the nominal absence of co /hco , but disappeared when binding of ca to the mct was hindered. it seems, that ca, bound to the mct , mediates local buffer capacity by removing protons transported into the cell via the mct . this helps to stabilise the proton gradient close to the cell membrane, and thereby enhances the transport activity of the mct . these ndings suggest that ca can enhance metabolite-acid/base transport, by forming a transport metabolon with the mct . melibiose permease of e.coli (mel b) is a membrane bound ioncoupled sugar symporter that uses the favorable na , li , or h electrochemical potential gradient to drive cell accumulation ofor -galactosides. cysteine scanning mutagenesis, electrophysiological (ssm -solid supported membranes) and uorometric measurements, were used in order to better understand the role of speci c parts of the protein in the function of this symporter. the ssm technique combines a rapid solution exchange with the high sensitivity of planar lipid membranes. it employs a solid supported membrane as a capacitive electrode and allows the time resolved investigation of charge translocation during the catalytic cycle of such transporters as na /solute symporters. in order to obtain some more precise information about the function of mel b symporter, starting from the c-less melb, the mutant g c was constructed and from electrical, spectro uorimetric and fret measurements carried out on this mutant, in the absence and in the presence of speci c inhibitors, conclusions were drawn about the possible role of the helix iv in the function of the symporter. two-dimensional crystallization of co-reconstituted ca +-atpase, phospholamban and sarcolipin the sarcoplasmic reticulum ca -atpase and its regulators phospholamban (plb) and sarcolipin (sln) form a primary control mechanism in the recovery of resting state calcium levels in the myocardium. defects in the regulation of ca -atpase by plb and sln are central determinants in cardiac contractility and disease states such as cardiomyopathy. given the signi cance of these proteins, the structural details of their regulatory mechanisms remain an important future goal for the clinical improvement of heart disease. using co-reconstitution into proteoliposomes at low lipidto-protein ratios, we have examined the effects of mutation on the functional properties of plb and sln, revealing novel insights into calcium pump regulation. in addition, these same co-reconstituted proteoliposomes have been used for structural studies by electron cryo-microscopy. in an attempt to better de ne the structural interactions between plb, sln and ca -atpase, we have sought methods for the production of large two-dimensional crystals suitable for high resolution electron crystallography. we previously utilized the co-reconstituted proteoliposomes to produce long, tubular crystals suitable for helical reconstruction. our new procedure comprises three steps -co-reconstitution, membrane fusion, and crystallization -producing large two-dimensional crystals suitable for high resolution structural studies. herein, we will present our latest results characterizing the structural interaction between plb, sln and ca -atpase. t. v. demina , n. s. melik -nubarov , h. frey , e. e. pohl state university, chemistry department, moscow, russia, institute of organic chemistry, johannes-gutenberg-university, mainz, germany, humboldt university, charite, institute of celland neurobiology, berlin, germany multidrug resistance (mdr) of tumours is associated with overexpression of the p-glycoprotein responsible for an active drug ef ux from cells. block copolymers of ethylene oxide and propylene oxide (pluronics) are known to cause a pronounced chemosensitization of tumour cells. the effect may be due either to speci c polymer -protein interactions or to unspeci c lipid bilayer disturbance. we have shown recently that amphiphilic copolymers with various hydrophilic and hydrophobic blocks can disturb lipid bilayers. importantly, that block copolymers of propylene oxide and glycerol (ppo-pg) with hyperbranched "corona" induced larger effects then pluronics with linear polyethylene oxide chains. in the present work we have shown that ppo-pg copolymers increase dox cytotoxicity towards human erythroleukemia (k is , k /dox) and breast carcinoma (mcf /dox) resistant cell lines. using confocal and two-photon microscopy, we demonstrated that these copolymers accelerated dox penetration into resistant cells, inhibited efux and caused drug redistribution into nuclei. a clear correlation between the ability of the polymers to disturb lipid bilayers and favour drug accumulation in mdr cells was disclosed. this nding points to an unspeci c mode of the copolymers' chemosensitizing activity. voltage dependence of processes related to electrogenic membrane transporters electrogenic membrane transporters, such as the sodiumbicarbonate cotransporter (nbce ), may induce dependence on membrane potential upon processes which are innately voltageindependent. we tested this hypothesis by heterologously coexpressing electrogenic nbce from human kidney in oocytes of the frog xenopus laevis with the electroneutral rat monocarboxylate transporter mct . the apparent intracellular buffer capacity was increased by nbce expression and became voltage-dependent by mm/ mv membrane depolarisation. lactate transport via the mct not only became enhanced after co-expression with nbce , but also dependent upon membrane potential. injection of carbonic anhydrase caii from bovine erythrocytes into oocytes enhanced the ef cacy of nbce activity, identifying an additional, ca-sensitive, membrane current via nbce . our results show that nbce adds voltage-dependent buffer capacity to the cytosol; this is suggested to be the prime cause for enhancing acid/base-coupled transport and conferring membrane potential dependence on transporters which are stoichiometrically electroneutral. these interactions may have functional consequences for cells and tissues, where electrogenic and electroneutral processes interact, such as in brain, heart and muscle. . by using carboxy-snarf- as ph-sensitive uoroprobe and microspectro uorimetry, we now show that nhe activation is due to jun kinase (jnk) activation, resulting from reactive oxygen species (ros) produced during metabolism of b(a)p and might involve lipid raft. when analysing b(a)p-induced apoptosis, we have found that cariporide signi cantly reduces both nuclear fragmentation and caspase- like activity. we further show that nhe activation and/or alkalinization affects the mitochondrial ros production detected during the apoptotic cascade, likely via an effect on the complex iii of the electron transport chain. altogether, our results suggest that apoptotic xenobiotics, such as benzo[a]pyrene, induce an early activation of nhe that might play a signi cant role in the subsequent mitochondria-dependent apoptosis. conformational dynamics of a lactose proton symporter j. c. holyoake, m. s. sansom biochemistry department, university of oxford, south parks road, oxford, ox qu, u.k. transporter proteins are an essential class of proteins, catalysing the transfer of molecules across membranes. increasing numbers of transporter structures are becoming available, opening the way to study their dynamic properties using computational techniques. we present a study on the conformational dynamics of the lactose proton symporter lactose permease using molecular dynamics simulations. these simulations exhibit large scale conformational changes from the initial intracellularly open conformation to a more closed conformation that may be signi cant to the transport mechanism. the conformational change is analysed to identify the contributing motions. effects of nortriptyline and chlorpromazine on anthroylouabain-labeled na,k-atpase e. a. guevara , m. l. barriviera , a. hassón-voloch , s. r. louro departamento de física, puc-rio, rio de janeiro, brazil, instituto de biofísica carlos chagas filho, ufrj, rio de janeiro, brazil the effects of nortriptyline and chlorpromazine (cpz) on the uorescence properties of anthroylouabain (ao)-treated na ,k -atpase of electrocyte membranes from e. electricus are studied. na ,k -atpase oscillates between two major conformations e and e during ion transport cycle. the cardiotonic steroid ouabain speci cally inhibits this enzyme binding to the e conformation. the uorescent label ao presents increased uorescence when binding to the ouabain site of na ,k -atpase. tricyclic drugs such as the antipsychotic cpz and the antidepressant nortriptyline inhibit na ,k -atpase activity in the micromolar range. for the e enzyme, but not e , nortriptyline was found to increase the uorescence in a concentration dependent manner, suggesting a further stabilization of e . for both conformations, cpz induces negligible uorescence change up to µm. the uorescence of atpasebound ao, however, strongly increases upon ultraviolet exposure after cpz treatment at concentrations around µm. fluorescent products of cpz-photodegradation were studied in pure buffer and in the presence of membranes. the results suggest that cpz binds to na ,k -atpase and photolabels amino-acid residues near the ouabain binding site. how important is protein exibility for transport through ion channels? a. a. gray-weale university of sydney certain ion channels are selective for k+ over other ions, but the geometry of the pore does not explain selectivity because thermal uctuations are too large. i extend the usual treatment of ion channels with molecular dynamics simulation by calculating the static and dynamic pair correlations between monovalent ions and ion channels (gramicidin-a and kcsa), and also between certain small, complex cations and the gramicidin channel. this means not only the radial-distribution functions or the density pro les, but also correlations between the ion and the mass and charge densities of various regions of the protein. the advantage of this approach is that it systematically identi es the elements of the protein and modes of motion that contribute to selectivity, and illustrates the decay of correlations. recently, noskov et al. [ ] showed that thermal uctuations protect selectivity. my results on the interaction of ions with carbonyl groups agree with theirs, but take the analysis further to higher correlations. the key new element is the study of the time-correlation functions that describe the motion of the ions through the channel, borrowing methods originally developed for the study of dense or even supercooled liquids. the surface-enhanced infrared absorption spectroscopy (seiras) is used for the investigation of two membrane proteins, the cytochrome c oxidase (cco) and the bacteriorhodopsin (br). of central interest are the transport-mechanisms of electrons (cco) and ions (br). the main parts of the setup are an infrared light source, a hemispherical si-crystal, in which the beam is internal re ected and a plexiglas cell with buffer solution (see schematic scetch). the beam is totally re ected on the inner at surface of the crystal, but the evanescent wave excites surface plasmons in a chemically adsorbed gold-layer on the crystal (attenuated total re ection spectroscopy-atr). the protein to be analysed is attached at the gold surface and can absorb certain wavelengths. the gold is need for the surfaceenhancing effect. cco plays a major role in the respiratory chain, the retinal protein br is a photosynthetic protein. the cco is immobilized on the gold surface via the af nity of its histidine-tag to a nickel-chelating nitrilo-triacetic acid (nta) surface. for the br we incorporate the protein in a lipid membrane, which is attached on the gold surface by the sulfuric bindings of , -di-o-phytanyl-sn-glycerol- -tetraethylene glycol-d,l-lipoic acid ester lipid (dptl). the sensitivity of this method is further enhanced by modulation of an external parameter, like the electric potential. effects of copper ions on the escherichia coli growth and proton-potassium exchange copper ions are required for the function of many important enzymes in escherichia coli but can cause a number of toxic cellular effects also. it's interesting to reveal the in uence of copper ions on the growth of bacteria and proton-coupled membrane systems. upon transition of e. coli mc wild-type culture to stationary growth phase a decrease in redox potential (e h ) from the positive values ( + mv) to the negative ones (of - to - mv), resulting h production by formate hydrogenlyase (fhl) have been studied. copper increased a latent growth phase duration as well as delayed a logarithmic growth phase in concentration-dependent manner. during the anaerobic growth, the production of h was strongly inhibited in the presence of cucl ( mm). . mm cucl was inhibited h production under experimental conditions with glucose. the inhibitory effect of copper ions ( . mm) on n',n'dicyclohexylcarbodiimide (dcc)-sensitive h /k exchange was also observed: k uptake was decreased and the stoichiometry of dcc-inhibited ion uxes varied. interestingly, these effects on h and k uxes were absent for the mutant hd (hyc-operon for hydrogenase was deleted). we suggest that copper ions, inhibiting the activity of fhl, have an effect on h /k -exchanging mechanism which is the proton f f -atpase associated with k uptake trk system. this effect may be due to the relationship of fhl with the ion-exchanging mechanism above under fermentation at alkaline ph. lateral diffusion in tethered bilayer membranes m. jung, v. atanasov, w. knoll, i. koeper max planck institute for polymer research, mainz, germany tethered bilayer membranes (t'blm) provide a useful platform for the investigation of bilayer membranes as well as embedded membrane proteins. we have developed a modular system, which is suitable for surfaces providing gold and oxide surface coatings. these systems serve as a quasi natural environment for the study of membrane proteins, being functionally incorporated into a lipid bilayer, which is covalently bound (tethered) to the substrates. functionality could be shown using electrochemical methods. here we present a study of these systems, basically the membrane itself, using uorescence recovery after photobleaching (frap) studies in order to investigate lateral motion in the lipid bilayers. lateral mobility is essential for successful incorporation of large membrane protein complexes. we will present rst results of experiments that try to differentiate motions hindered due to the tethering from diffusion in free oating or suspended bilayers. the information gained in this study will serve for improvements in the chemical structure of the tethered molecules. we will develop the system as a basis for bio sensing applications, where embedded proteins will serve as actual sensing elements. a. d. ivetac, j. campbell, m. s. p. sansom biochemistry department, university of oxford, south parks road, oxford, ox qu, u.k. atp-binding cassette (abc) transporters form an important superfamily of membrane proteins which couple atp hydrolysis to the active transport of diverse compounds across the cell membrane. their biomedical relevance is highlighted in examples such as multidrug resistance to antibacterial and anticancer agents, and cystic brosis. the availability of crystal structures of three complete bacterial abc transporters provides an opportunity to study structurefunction relationships at the atomic level. in this work, we carry out multi-nanosecond molecular dynamics simulations of the vitamin b importer from e. coli (btucd), with both the complete multimeric transporter embedded in a phospholipid bilayer and the soluble subunits in a membrane-free environment, in an attempt to elucidate some of the conformational changes which arise during the transport event. atp-bound and atp-free structures are used to investigate the effect of nucleotide on the system. a range of analytical techniques have been applied to assess the dynamic behaviour of the protein during the simulations, which includes measurements of: conformational drift, residue exibility, transmembrane domain (tmd) movement, concerted protein motions, nucleotide-binding and translocation pathway changes. an in-vitro method was designed to measure transmembrane transport rates. liposomes were prepared by extrusion with dipalmitoyl phosphatidylcholine (dppc) and optionally cholesterol, and loaded with a peptide (zinc-insulin tagged with a uorescent group or bsa). after removal of the non-encapsulated peptide from the liposome solution by gel ltration, the release of the peptide from the liposomes was monitored by uorescence as a function of time at various temperatures. the transport was greatly accelerated by the presence of a speci c proprietary excipient molecule (cyclopentadecanolide -cpe ™), which effectively triggered the release of the peptide. a mathematical model was developed to quantify these results. a semi-empirical nonlinear equation involving four parameters ts the protein release pro les. then a neural network predictions model was used to correlate the different release condition parameters and the four semi-empirical tting parameters based on the experimental data sets. most release data t well with the mathematical model, further supporting our theory of a two step release mechanism. phenyltin the well known group of organotin compounds that exhibit toxic properties in relation to the biological systems are phenyltins. no studies have been performed as yet to establish directly whether organotins such as diphenyltin dichloride ( dpht) and triphenyltin chloride (tpht) cross the lipid bilayer. we have performed experiments that showed transfer of those compounds across the lipid bilayer using the stopped-ow technique and desorption of those compounds from a monolayer using the langmuire technique. obtained results demonstrate that dpht and tpht rst adsorb onto the lipid bilayer surface, in diffusion controlled manner, within a very short time ( . s), whereas the membrane passing was observed in a minute's time range. the long time kinetics show a complex dependence on the kind of compound, its concentration and the presence of cholesterol in the membrane. the desorption of both compounds from the monolayer to water subphase occurs in a minute's time range. these observations may explain the known fact, that the in uence of organic, amphiphilic tin (and also lead) compounds is more toxic than that of inorganic ones.the phenyltins much easier (compared with tin or lead ions ) penetrate e.g. blood -brain barrier. [ under the resting conditions ca concentration in agonistsensitive ca stores re ects a balance between active uptake mediated by a ca -atpase (serca) and passive ef ux of ca . this ca leak appears to be a common property of ca -storing organelles, but the nature of the leak in submandibular acinar cells remains unclear. we have studied the ca leak pathways in the endoplasmic reticulum (er) of acinar cells of rat submandibular salivary gland by directly measuring concentration ca in the er ([ca ] er ) in mag-fura /am preloaded cells while [ca ] i was clamped at a resting level with a egta/ca mixture. we have shown that thapsigargin (tg) or ca -free buffer treatment completely blocked ca uptake by serca after the rst minute of superfusion and caused a ca leak represented by continuous decline in [ca ] er . this ca leak from the er was not sensitive to tg, heparin and ruthenium red and therefore appears to be independent of the serca, the insp receptor and the ryanodine receptors. however, treatment with puromycin ( . - mm) to remove nascent polypeptides from er-ribosome translocon pores increased ca leak from the er by a mechanism independent of the serca, insp or ryanodine receptors. thus we conclude that basal ca leak from the er of submandibular acinar cells occurs through translocon pores in the er membrane. r. b. kishore, j. reiner, e. edgu-fry, a. jofre, k. helmerson physics laboratory, national institute of standards and technology we have developed a procedure to make lipid and polymer nanotubes of up to one cm long and nm in diameter, from the surface of giant liposomes and polymersomes, using micro uidics and optical tweezers. the liposomes and polymersomes were formed, using elcetroformation method, from phospholipids and amphiphilic diblock copolymers, respectively. the polymer tubes were made extremely robust by cross-linking them using chemical reactions. we are currently studying the transport of molecules in the crosslinked nanotubes for use in nano uidic networks. p-glycoprotein (p-gp) is an active membrane transporter capable of expelling out of the cell a large number of potentially cytotoxic amphiphilic molecules with unrelated chemical structures. as a consequence, p-gp may be responsible for multidrug resistance of tumors against chemotherapy (mdr); it also plays a key role in absorption, biodisposition and elimination of many pharmaceuticals. to understand the molecular mechanisms of the transmembrane drug transport mediated by pgp, it is highly desirable to design a convenient assay for measuring both p-gp atpase activity and p-gp transport function. to do so, we used inside-out native membrane vesicles, prepared from mdr cells and containing high amounts of p-gp. we took advantage of the speci c property of a uorescent dye, the carbocyanin jc- , known to be expelled out of mdr cells: above a critical concentration (the "cjc"), this dye forms j-aggregates which emit a uorescence at a wavelength very different from that emitted by the monomer. in the presence of mgatp, the p-gp-containing vesicles accumulated jc- , which exceeded locally the cjc and thus formed intraluminal j-aggregates; these aggregates allowed accumulated jc- both to be sequestered inside the vesicles, by dramatically slowing down its passive backdiffusion, and to be speci cally detected. kinetic characterization of this transport suggests that jc- is rst translocated to the exoplasmic lea et of the vesicle membrane before its internalization into the aqueous phase of the vesicle lumen. interaction between the energy metabolism and externally applied electric elds in yeast cells electric elds are often used for biophysical or biomedical treatment of biological cells, e.g. cell fusion or killing of cells. however, only a few data about the possible mechanisms of electrosensitivity of biological cells are available. since electrostimulation always induces depolarization of biomembranes, an impact of the energy metabolism is obvious due to the regeneration of electrochemical gradients by the expenditure of cellular energy. our aim is to investigate the interactions between externally applied electric elds and the aerobic/anaerobic energy metabolism of yeast cells. for this, we have constructed a new electrical interface for local stimulation of biological cells with variable duration and amplitude. when applying short lasting electrical pulses to yeast cells, we nd a direct response of the energy metabolism (measured by nadh-uorescence) to these pulses. a sudden and fast decrease in nadh is followed by a slower recovery of the uorescence signal. these nadh-signals are abolished in the presence of antimycin a or kcn, demonstrating the importance of mitochondrial energy production for this phenomenon. we attribute these changes to the immediate break down of atp as a consequence of the regeneration of the membrane potential (atpases) and the slower regeneration of atp by mitochondrial respiration. new insights of hypericin blood transport and its incorporation into the plasma membrane b. m. macri , g. stoian , m. l. flonta department of animal physiology and biophysics, faculty of biology, university of bucharest, department of biochemistry, faculty of biology, university of bucharest, bucharest, romania hypericin (hyp) is one of the active compounds from hypericum perforatum (an herb usually prescribed as antidepressant). the bioavailability of this hydrophobic molecule is a very important issue for medical applications. the goal of our work was the study of hyp blood transport mechanisms. techniques of absorbtion spectroscopy, electrophoresis and uorescence microscopy were used in order to de ne the properties of hyp-albumin and hyp-lipoproteins complexes and to explain the action of hyp at the plasma membrane level. hyp bind to several electrophoretic (sds-page) bands evidenced by mice plasma migration. both albumin and lipoproteins bind to hyp, forming complexes, during the blood transport process. different types of lipoproteins from males and females plasma mice were evidenced by gradient electrophoresis to bind hyp. hyp-albumin complex was also identi ed by absorbtion spectra, and the ratio a /a has a ph-dependence. hyp interaction with plasma membranes was also examined on cell culture by uorescence microscopy, and hyp plasma incorporation is a dose-and incubating time-dependent process. our results partially elucidate the plasma fractions that bind hyp, contributing to its blood transport. this study proposes a new mechanism of hyp cellular insertion, discussing its plasmatic membrane penetration due to its high hydrophobicity. the overexpression of p-glyoprotein (p-gp) is one of the major causes of multidrug resistance (mdr) in cancer chemotherapies. many p-gp inhibitors have been designed to reverse the mdr effect, but the structure-activity relationships of the p-gp inhibitors and substrates still remain largely unknown. until now, it is still very challenging to obtain the high-resolution p-gp structures and currently only low-resolution electron microscopy structure is available. this has caused the structure-based design of "p-gp-ignoring" therapeutic agents a remote goal. however, the recent determination of x-ray crystal structures of bacterial lipid a transporter, msba, has provided eligible structure templates for homology modeling of p-gp. we have therefore conducted explicit solvent molecular dynamics simulations of the fulllength ef ux pump, human p-gp, in an excessively hydrated popc bilayer to re ne the homology model. both free and atp-bound forms of p-gp have been simulated. the entire system consists of more than , atoms. our molecular dynamics simulations have shown that the overall architecture of p-gp remained very stable for tens of nanoseconds, while the observed membrane undulation was rather large. the simulation results have allowed us to investigate the conformational changes of p-gp upon atp binding in the ef ux process and to predict the possible binding site of various known substrates and inhibitors. the re ned structure models of p-gp by our simulations could be used as the basis for further drug design. electroporation (ep) is a phenomenon where increased permeability of cells exposed to an external electric eld is observed. the induced transmembrane voltage presumably leads to the formation of aqueous pores in the phospholipid bilayer, which increases permeability of the membrane for molecules and ions. ep is currently used in many biomedical applications including transfer of genes and electrochemotherapy of tumors. still, the molecular mechanisms of the process are not fully explained. recently it was proposed that ep could be monitored in real-time by measuring electric conductivity of tissue. so far the studies focused mostly on a single pulse, however in biomedical applications usually several pulses are used. in our study we used a train of electric pulses to analyse the relationship between electric conductivity and cell permeabilization. current-voltage measurements during and after pulse application were performed in dense suspension of cells. conductivity changes were analysed numerically using nite elements method and compared with the percentage of permeabilized cells. we obtained a transient increase in conductivity above a certain voltage with complete relaxation in < s. substantial changes in conductivity are also due to the diffusion of ions through membrane pores and osmotic swelling. we further show that relation between conductivity and permeabilization level is indirect. interaction of quinolones with bacterial porin ompf: uorescence quenching studies quinolones are widely used antibiotics witch develop their antibacterial action by inhibition of important bacterial enzymes. consequence of the internal location of their target of action, the translocation of this drugs trough the outer membrane is an essential step for their antibacterial action. in vivo studies have been showing that ompf is important for the entrance of some of these antibiotics, but the exact degree of involvement of this protein in the transport of the different members of this group of antibiotics, remains unknown. in this study, the quenching of the intrinsic tryptophan uorescence of ompf, in presence and in the absence of the drugs and by two distinct quenchers, was used as a rst approach, to elucidate ligandinduced structural changes and consequently prove the differential involvement of ompf in the entrance of these antibiotics in the bacterial cell. the results obtained reveal that the degree of interaction with the protein is related with the hydrophobicity of the different antibiotics. this kind of evidence suggests that the entry by the porin channel is not the only path used by these antibiotics and that it is more important for the latest generations of this group because of their increased hydrophilic characteristics. inhibition of multidrug resistance-associated protein mrp and kv channels by natural polyphenols k. michalak, a. teisseyre, b. ania-pietrzak department of biophysics, wroclaw medical university, poland resistance to cytotoxic agents remains a major obstacle to successful chemotherapy in cancer. best-characterized form of drug resistance is caused by the overexpression of genes encoding membrane drug pumps, like p-gp or mrp . in present study, activity of several plant polyphenols ( avonoids and stilbene) against mrp has been studied using functional assay based on ef ux of mrp uorescent substrate. very recently, a role of kv . potassium channels in proliferation of various cancer cells was suggested. in our study the effect of the plant polyphenols on voltage-gated potassium channels kv . was investigated by patch-clamp electrophysiological method. some of studied compounds were found to be active inhibitors of multidrug resistance-associated protein mrp and voltage-gated potassium channels, and their properties are promising for further research in the eld of anticancer activity of natural products. the transport mechanism of melibiose permease: a study using electrical measurements and uorescence techniques k. meyer-lipp , c. ganea , t. pourcher , g. leblanc , k. fendler max planck institut für biophysik, frankfurt, germany, c. davila medical university, bucharest, romania, cea, université de nice so a antipolis, nice, france the melibiose permease (melb) of escherichia coli is a membrane bound carrier that uses the favorable na+, li+, or h+ electrochemical potential gradient to drive cell accumulation of alfa-galactosides (melibiose, raf nose) or beta-galactosides (methyl- -thio-beta-dgalactopyranoside). electrophysiological techniques have proved to be extremely useful tools to investigate the mechanism of ion transfer across the membrane by ion-coupled transporters. using a solid supported membrane (ssm) as a capacitive electrode a rapid solution exchange can be combined with the high sensitivity of planar lipid membranes and allows time resolved investigation of the charge translocation during the catalytic cycle of na+/solute symporters. this technique has been combined with uorescence measurements, which report on structural changes during the substrate transport process of the carrier. we have used time resolved tryptophane uorescence, uorescence energy transfer with a uorescent sugar substrate and site speci c uorescence of a label attached to a cysteine residue on the protein. this allowed us to identify conformational transitions during the reaction cycle of the melibiose permease. we could assess their electrogenicity and determine rate constants. a kinetic model for na+ and melibiose binding and transport is presented. electrophysiological characterization of the vast number of annotated channel and transport proteins in the postgenomic era would be greatly facilitated by the introduction of rapid and robust methods for the functional incorporation of membrane proteins into dened lipid bilayers. we present an automated method for reconstitution of membrane proteins into lipid bilayer membranes, that substantially reduces both the reconstitution time and the amount of protein required. we have applied this well-de ned system to the characterization of a novel mitochondrial uncoupling protein, ucp and demonstrated that ucp exhibits protonophoric function exclusively in the presence of fatty acids, similar to that previously shown for its homologue ucp . the membrane conductance was proportional to the concentration of the reconstituted ucp in presence of oleic acid or eicosatrienoic acid, and was inhibited by atp. amphipols are amphipathic polymers designed to replace or supplement detergents in membrane protein solution studies. for the study of the ca -atpase from sarcoplasmic reticulum, previous experiments have revealed both advantages and disadvantages to the use of a polyacrylate-based amphipol, a - . these issues have been reinvestigated using four different amphipols. size exclusion chromatography showed that, although a - aggregates in the presence of millimolar concentrations of calcium -an effect that probably accounts for most of the aggregation of atpase/a - complexes observed in our previous work-, aggregation can be avoided by resorting to a sulfonated version of a - . we also found that all amphipols tested slowed down the rate of calcium dissociation from its binding sites and reduced atpase activity, while protecting the solubilized protein against denaturation. this suggests that association with the polymer may damp the protein's dynamics, perhaps due to the multipoint attachment of the polymer to its hydrophobic transmembrane surface. such a "gulliver" effect could contribute both to the protection of membrane proteins against denaturation and to the reversible inhibition of serca a. clc proteins are found from prokaryotes to mammals. they function as plasma membrane chloride channels or provide neutralizing anion currents for v-type h -atpases that acidify compartments of the endosomal/lysosomal pathway. vesicular clcs have been thought to be cl -channels, in particular because clc- and clc- mediate plasma membrane cl -currents upon heterologous expression. we have shown, however, that these two mainly endosomal clc proteins rather function as electrogenic cl /h exchangers, resembling the transport activity of the bacterial clc-e that has been crystallized. neutralization of a critical glutamate residue not only abolished the steep voltage-dependence of transport, but also eliminated the coupling of anion ux to proton counter-transport. clc- and clc- may still compensate the charge accumulation by endosomal proton pumps, but are expected to tightly couple vesicular ph-gradients to cl -gradients. calorimetry and mechanics of ca transporting systems in rat myocardial bigeminies a series of new n-oxides of tertiary amines (nta) was checked for its biological activity. individual compounds differed in the length of substituted alkyl chain. the primary goal was to nd if they can be used as effective antioxidants and, to what degree they modify used model (liposomes) and biological (erythrocytes, algae and cucumber) membranes. various methods were used in order to do that. a mechanism of the interaction between nta and membranes was studied by measuring their potency to hemolyse erythrocytes, to in uence a phase transition temperature in dppc liposomes, to change a membrane potential of algae cells. the measure of the interaction of nta with cucumber cells were potassium leakage, chlorophyll content and inhibition of growth of hypocotyls. antioxidative abilities of nta were determined by measuring their ef ciency to protect erythrocytes against membrane lipid oxidation induced by uv irradiation and by comparising their antioxidative ef ciencies with that of trolox (vitamin e analogue) in chromogen experiments. the mostly widely employed mechanism of drug extrusion in bacteria is via membrane transport proteins called ef ux pumps. in gram-negative bacteria, multidrug resistance is conferred by tripartite complexes, rather than by a single transport protein. through these systems, a wide range of substrates is expelled from the cytoplasm, through the periplasmic region, to the exterior of the cell. among these complexes, the acrab/tolc system in escherichia coli is formed by an inner membrane ef ux pump, acrb, an outer membrane protein, tolc, and a periplasmic protein known as an adaptor, acra. the components of this complex are studied, in order to provide insights into drug transport in bacteria. here we present a dynamics study on mexa, homologue of acra from pseudomonas aeruginosa. the protein has been studied by molecular dynamics simulations in bulk water. a structural adjustment by the periplasmic protein is required in order to engage both the bottom part of the om protein and the top region of im protein. the dynamics on mexa reveals a exible behaviour of the protein in water. the major concerted motions observed are the hinge-bending of the two domains, and the rotation of the -barrel domain. these can be related to the adaptation of mexa (and acra) to the om and im proteins during the process of assembly in forming the complex, and during the opening of the channels. electrophysiologic study of ap in chara corallina -indication of its biochemical nature the speci c conductance's of aqueous solution of electrolytes (viz.naf, nacl, nano , na so , kf, kcl, kno , k so , mgcl , cacl , fecl , mncl ,crcl ,cucl , cocl , )have been measured across peritoneum at temperatures between( - ) c.conductance attains a maximum limiting value at higher concentrations for each electrolyte due to a progressive accumulation of ionic species within the transmembrane region. the membrane becomes more and more conductive to incoming ions and attaining a limiting value due to the fact that an electrically neutral pore, which is speci c for a particular ion, is unlikely to contain more than one type of ion. consequently, at high electrolyte concentration, the pore saturates and the conductance's approaches a limiting value. the values of speci c conductance measured follow the sequence for anions; so > cl > no > f . whereas for the cations the sequence is k > na ; ca > mn > co > cu > mg ; cr > fe . the energy of activation for the cations as well as for the anions follows the sequence (for cations): the low temperature ( k) chlorophyll uorescence, photochemical activity, oxygen ash yield and oxygen burst decay of thylakoid membranes with different organization of the light-harvesting chlorophyll a/b complex of photosysytem ii (lhcii) were investigated after freeze-thaw cycle in criotoxic and cryoprotective medium. the increase of lhcii oligomerization, which is associate with signi cant reduction of the surface charge density of the thylakoid membrane, correlates with lower extent of freezing damage of the photosynthetic apparatus, when the procedure is carried out in cryotoxic medium (nacl). in the presence of the cryoprotective compound (sucrose) freezing damage is less pronounced and is not affected by the degree of the lhcii oligomerization. the mechanisms of damage and protection of photosynthetic apparatus in the process of freeze-thaw treatment are discussed. spectral and redox characterization of the novel heme ci in the cytochrome b f complex . this is an indication of different spin delocalization in the primary donor, for the mutant being typical of a monomeric oxidized bchl. considering the fact that the properties of both isolated and membrane-associated mutant rcs were similar, we conclude that missing bchl molecule from the mutant rc was the result of the introduced mutation but not of the protein puri cation procedure. authors acknowledge the support by the russian brf. we created two site-directed mutants, a s and l i in the d protein of photosystem ii in thermosynechococcus elongatus. both mutations are within the binding pocket of the primary quinone acceptor (q a ). we investigated the effects of the mutations in vivo and in isolated psii. while the l i mutant exhibits characteristics similar to the wild type, the a s mutation effects q a charge recombination measured by thermoluminescence and uorescencedecay. these results strongly indicate that the a s mutation induce a shift in the redox potential of q a . the a s accelerates the rate of photoinhibition, an effect consistent with the negative shift in the redox potential. epr was used to measure the temperature dependence of the electron transfer from q a to q b in the a s mutant. it was found to be indistinguishable from the wild type despite the difference in the midpoint potential of q a . this is taken as an indication as a gating mechanism on the acceptor side of psii similar to that in bacterial reaction centers. protochlorophyllide oxidoreductase takes an abnormal reaction pathway below the glass transition g. durin , d. j. heyes , c. n. hunter , d. bourgeois ibs and esrf, grenoble, france, krebs institute and r hill institute for photosynthesis, shef eld university, shef eld, uk motions through the energy landscape of proteins lead to biological function. at temperatures below a dynamical transition ( - k), the activity of some proteins cease. in this work, we describe an enzyme that, instead, engages into a non-productive pathway below k. protochlorophyllide oxidoreductase (por) catalyzes the reduction of protochlorophyllide (pchlide) into chlorophyllide (chlide), a key step in chlorophyll biosynthesis. por is one of the two enzymes known to require light for catalysis. when illuminated with gentle light at k, the complex of t. elongatus por with pchlide and nadph transforms into a nonuorescent intermediate. upon warming, several uorescent intermediates develop, and at k chlide is released. when illuminated at temperatures below k, por behaves differently. if gentle light is used, the reaction can not start. instead, if a blue laser source is used, the initial complex disappears, like at k. however, upon warming, a new intermediate develops that uoresces at nm and leads to a dead-end product. by using uorescence microspectrophotometry, we have measured the solvent glass transition temperature of the system to be k. the solvent glass transition, possibly controlling a por dynamical transition, may be the determinant that switches the enzyme reaction pathway from a non productive to a productive one. the nonproductive pathway results from a two-photons absorption mechanism, whereas the productive pathway is a one-photon mechanism. sensory rhodopsin ii from n. pharaonis (npsrii) forms a complex with its cognate transducer nphtrii in a : stoichiometry . light activation of npsrii leads to a movement of helix f which triggers a rotation of tm in nphtrii . the mechanism of signal transduction through the hamp region to the cytoplasmic domain of the transducer is still unknown. structural information exists for the transmembrane and cytoplasmic regions, however the hamp domain is not yet characterized. in order to obtain structural information on this domain, twenty-four residues in the membrane adjacent region ( - ), and six residues in the following region were spin labeled and investigated by cw and pulsed x-band epr. to analyze the overall architecture of the complex, doubly spin labeled variants between the transducer and the receptor were also engineered. depending on their function, the absorption spectra of rhodopsins can be tuned by the protein over a wide range. a major determinant for spectral shifts between different rhodopsins are electrostatic interactions between the chromophore retinal and the protein. we compute and compare the classical electrostatic potential at the retinal of three archaeal rhodopsins: bacteriorhodopsin (br), halorhodopsin (hr), and sensory rhodopsin ii (srii). these proteins are an excellent test case for understanding the spectral tuning of retinal. the absorption maxima of br and hr are very similar, while the spectrum of srii is considerably blue shifted. we nd that the electrostatic potential is similar in br and hr, but differs signi cantly in srii. a quantum mechanical model of a particle in a box with a step potential can qualitatively relate the differences between the electrostatic potentials of the proteins to the relative shifts of their absorption maxima. by decomposing the electrostatic potential into contributions of individual residues, we could identify six residues that are responsible for the differences in electrostatic potential between the proteins. three of these residues are close to the retinal, while the other three residues are more then angstroem away from the retinal. the counterion of the schiff base, which is frequently discussed to be involved in the spectral tuning, does not contribute to the dissimilarities between the electrostatic potentials. effect of uv-a radiation on thylakoid membranes with different organization p. ivanova, a. dobrikova, t. markova, s. taneva, e. apostolova institute of biophysics, bulgarian academy of sciences, so a, bulgaria the effect of uv-a ( - nm) radiation on the energy transfer and the photosynthetic oxygen evolution of thylakoid membranes from pea mutants was investigated. the membranes have different pigment composition, stoichiometry and organization of pigment-protein complexes. the aim of our work was to nd out whether uv-a induced damage is affected by the altered content and/or oligomerization of the main light-harvesting chlorophyllprotein complex (lhcii) in thylakoid membranes. the data for the effect of uv-a radiation on the oxygen evolution demonstrate that: (i) the inhibition of photosystem ii (psii)-mediated electron transport and ash-induced oxygen yields strongly depend on the amount of lhcii; (ii) the increase of the s o populations of psii centers in darkness is more pronounced in thylakoid membranes with smaller amount of lhcii; (iii) the inhibition of the oxygen evolution is related to the reduced number of the functionally active psii centres; (iv) the degree of impairing of active psii centres depend on the amount and oligomerization of lhcii. the results also show that the altered content and organization of lhcii in uence the uv-a light-induced changes in the energy transfer between psii and psi and within the supramolecular lhcii-psii complex. the effects of uv-a radiation on leaves and isolated thylakoid membranes are compared. sudden polarisation -a large change in the electric dipole moment between the excited and the ground state -is a well-known phenomenon for retinal chromophore. some early models of the energy transduction mechanism in bacteriorhodopsin (br) even attribute a primary functional role of that. however, it was apparently unrecognized that the maxwell theory intuitively predicts the appearance of an ultrafast transient electromagnetic radiation due to this dipole moment change. here we show that the existence of this type of radiation can be derived from semiclassical quantum electrodynamics as a second order phenomenon. in optical terms it corresponds to the previously unstudied resonant case of optical recti cation. recently we experimentally observed a major component in the fs coherent infrared emission of oriented purple membranes of br corresponding well to this effect (groma et. al, proc. natl. acad. sci. , , ). our theory predicts that such a signal holds detailed information on the dynamics of excited state polarization, opening a new branch of impulsive spectroscopy on asymmetric systems. beyond optical recti cation we found a complex phase a coherent oscillation living for a few ps, i.e. much longer than the excited state of br. fitting analysis resulted in at least seven vibrating modes in the - cm region, while windowed fourier transform indicated time-dependent frequency distribution. a. ghignoli, g. cercignani, s. lucia, g. colombetti istituto di bio sica, cnr -pisa, dipartimento di fisiologia e biochimica, università di pisa, italy the life cycle of ophryoglena ava, a histophagous ciliate dwelling in fresh waters, reportedly includes several stages that feature morphology changes and different phototactic responses. previous studies on the phototactic responses in o. ava during its phase of maximal positive phototaxis led to an action spectrum with two main peaks at and nm, and a minor peak at nm. starting from those results, we analyzed the phototactic response at various cell ages, using three broad-band interferential lters (fwhm = nm) centred respectively at , and nm, and constructed dose-effect curves for each band. a higher photosensitivity at nm, and lower photosensitivies with the other two lters ( and nm) have been observed at any cell age. however, the photosensitivities in the blue and orange regions show a different time course vs. cell age with respect to the photosensitivity in the green region. measures were also carried out on cells whose feeding cycle was altered by a -day starvation (a double time with respect to the standard protocol) before being fed at t = . the maximal photoresponse values reached by starved cells are lower than the highest values reached with standard cultures; in other words, a general reduction of the phototactic response is observed. these results suggest that, while feeding optimally induces cell division, it does not generally reset all cellular functions. a. quaranta , f. lachaud , y. pellegrin , p. dorlet , m.-f. charlot , s. un , a. aukauloo , w. leibl service de bioénergétique, cea-saclay, bât. , gif-sur-yvette cedex (france), laboratoire de chimie inorganique, bât. , université paris-sud, orsay (france) coordination complexes based on a photoactive rutheniumpolypyridyl moiety linked to simple, rigid ligands with binding sites for transition metals, are developed to mimic the light induced charge separation and water oxidation processes taking place in the photosynthetic apparatus. inspired by the structure around the donor side of photosystem ii a family of phenanthroline based ligands holding an imidazole, a phenol or an indole unit simulating the amino acids histidine, tyrosine and tryptophan in the oxygen evolving complex, were developed as models for proton-coupled electron transfer. in some of the molecules investigated the hydrogen bonding interaction present in the natural system is reproduced. combined data from photophysical, spectroelectrochemical studies and dft calculations evidenced the photogeneration of a phenoxyl or a tryptophan radical upon excitation of the chromophore in presence of an external electron acceptor, therefore mimicking the electron trade between p and tyrz-hist . finite element model to predict the electric potential distribution in ps i containing vesicles c. p. a. pennisi , e. chemineau , e. greenbaum , k. yoshida center for sensory motor interaction, aalborg university, denmark, chemical sciences division, oak ridge national laboratories, usa, facultad de ingeniería, uner, argentina photosynthetic reaction centers (rc) are integral membrane proteins and molecular photovoltaic structures. recently, it was suggested their use as triggers of voltage-gated ion channels in excitable cells, where a certain voltage threshold has to be reached to evoke a response (kuritz et al., ieee trans. nanobiosci. in press ). experimental studies with rc's reconstituted in lipid vesicles have shown different values of transmembrane voltage, depending on parameters like light intensity, rc concentration and membrane passive properties. ultimately, the purpose of this work is to have a tool to estimate the proximity, number and density of rc's required near a voltage-gated channel to activate an excitable cell. as a starting point, we aim to predict the spatial distribution of the membrane potential in vesicles. a nite element model was realized using a commercial package (femlab, comsol a/s). the three-dimensional distribution of the electrical potential near a single rc in the surface of a spherical vesicle was calculated. in terms of density, in conditions of saturating light, a minimum of , e rcs/cm is needed to develop a potential of mv, capable to activate voltage-gated sodium channels. microsecond time-resolved x-ray diffraction study of purple membrane t. oka , k. inoue , m. kataoka , n. yagi faculty of science and technology, keio university, japan, japan synchrotron radiation research institute (jasri), japan, nara institute of science and technology, japan the structural changes in the photoreaction cycle of bacteriorhodopsin, a light-driven proton pump, was investigated at a resolution of Å by time-resolved x-ray diffraction experiment utilizing synchrotron x-rays from an undulator of spring- . the xray diffraction measurement system, used in coupling with a pulsed yag laser, enabled to record diffraction pattern from purple membrane lm at a time-resolution of µsec over the time domain of µsec to msec. the low temperature ( k) chlorophyll uorescence, photochemical activity, oxygen ash yield and oxygen burst decay of thylakoid membranes with different organization of the light-harvesting chlorophyll a/b complex of photosystem ii (lhcii) were investigated after freeze-thaw cycle in cryotoxic and cryoprotective medium. the increase of lhcii oligomerization, which is associate with signi cant reduction of the surface charge density of the thylakoid membrane, correlates with lower extent of freezing damage of the photosynthetic apparatus, when the procedure is carried out in a cryotoxic medium (nacl). in the presence of a cryoprotective compound (sucrose) freezing damage is less pronounced and independent of the degree of the lhcii oligomerization. the mechanisms of damage and protection of photosynthetic apparatus during the freeze-thaw process are discussed. we have studied the effect of a cytokinin meta-topolin (mt, m) on senescence-induced changes in the photosynthetic apparatus of detached primary leaves of wheat (triticum aestivum l. cv. hereward). the senescing leaves were kept under continuous light conditions. mt signi cantly slowed down the senescenceinduced decrease in chlorophyll content and markedly stimulated violaxanthin zeaxanthin (z) conversion. the high z content was maintained even after an hour in darkness. mt treatment caused also the appearance of an emission band f peaking at - nm. this emission band is attributed to aggregates of lightharvesting chlorophyll a/b-binding proteins (lhc), the production of which is associated with a higher z content. the presence of lhc aggregates in mt treated leaves was documented also by electron microscopy imagines. besides the lhc aggregation, mt induced also a decrease in photosystem i content which was documented by electrophoresis and k-uorescent spectra. supported by grants frvs / and msm . recently high resolution images of bacterial photosynthetic membranes have revealed the organization of membrane proteins in these native membranes. the organization revealed is remarkable, and all the more so when we realize that these specialized, protein rich, membranes differentiate from the cytoplasmic membrane which has a more complex composition and is richer in lipids. analysis of the protein organization in these specialized membranes from several different bacteria suggest that the organization results from a phase separation of several different contiguous phases. in order to better understand our observations we have undertaken an examination of the different phase behaviors that are possible for membrane proteins considered as a two dimensional colloid. monte-carlo modeling of the phase diagram of this system shows the importance of interaction distance in the determination of system behavior. transcription of our observations on the model systems to the photosynthetic membranes suggests that electrostatic and elastic forces in the membrane are of particular importance in determining the high level order of membrane proteins. the recent crystal structure of photosystem i (psi) from synechococcus elongatus shows two quasi-symmetric branches of potential electron transfer cofactors including primary donor (dimer of chlorophylls p ), monomeric chlorophylls a and a and quinone a , bound to the psaa/psab heterodimer. so far, it is not clear if both potential electron transfer pathways are active in this process or only one of them. to solve this issue, we studied a set of mutants with methionine coordinating the primary electron acceptor, a , replaced by histidine, leucine, or serine in either of two branches. our results obtained with a technique of femtosecond transient absorption spectroscopy show that both branches are equally active in electron transfer. mutation in either branch slows the forward electron transfer between a and a from ps in wilde type psi to - ns in all these mutants. this strong effect is explained by signi cant change in the redox midpoint potential and change in the position of a by the mutations. i. s. zaharieva department of biophysics and radiobiology, faculty of biology, university of so a an approach to the investigation of structural and functional properties of the photosystem ii supramolecular complex in native photosynthesizing objects based on the registration of delayed chlorophyll a uorescence is developed. using a disc phosphoroscope, we register simultaneously: i) changes of the intensity of millisecond delayed uorescence (decayed in . - . ms time range) during the transition of the photosynthetic apparatus from dark to lightadapted state; ii) changes of the intensity of delayed uorescence decaying in different subintervals of the investigated time range; iii) dark relaxation curves at different moments of the transition; iv) changes of the intensity of prompt chlorophyll a uorescence. the analysis of these data allows the correlation of the delayed uorescence characteristics to particular processes occurring in the photosystem ii complex -proton or electrical gradient accumulation, changes in the redox state of quinone acceptors, changes in the pigment-protein complexes caused by different stress factors, for example temperature. three-dimensional structure of major light-harvesting antenna of photosystem ii from cucumber h. yan , z. liu , k. wang , t. kuang , j. zhang , l. gui , x. an , w. chang national lab of biomacromolecules, institute of biophysics, chinese academy of sciences(cas), datun road, chaoyang distr., beijing , china, lab of photosynthesis and environmental molecular physiology, institute of botany, cas, nanxincun, xiangshan, beijing , china the major light-harvesting antenna complex of photosystem ii (lhc-ii), the most abundant integral membrane protein, functions in light capture, energy transfer/distribution and photoprotection. lhc-ii from different species or conditions shows different spectral properties and variation in polypeptide and pigment components. this indicates some speci c function-related alterations in the organization of lhc-ii. here we report a . -Å crystal structure of cucumber homo-trimeric lhc-ii, organized in a perfect virus-like icosahedral particle. the electron-density map shows the reasonable existence of a chlorophyll (chl) a/b mixed binding site in the complex. the occurrence and locus of lactucaxanthin (lac) was seen directly for the rst time. based on the credible structure information, a mechanism of the energy transfer, regulation and excess excited energy dissipation under high light condition was proposed. coherent anti-stokes raman scattering microscopy (cars) is a new approach for chemical imaging of molecular systems within cells and tissues, with high sensitivity, high spatial resolution, and three dimensional sectioning capabilities, without using uorophores that are prone to photobleaching. this technique permits to map selectively molecular species, by using vibrational properties of their chemical bounds. the epi detected (e-cars) and forward detected (f-cars) intensities depends on the shape, the size of the sample, as well as the index of the solvent. in this presentation, after introducing the cars microscopy technique, we show the rst cars studies of the refractive effect of the sample, comparing the e-cars and f-cars signals for different diameters of polystyrene beads, in different refractive index solvents. we present several simulations, comparing forward-detected and backward-detected signals in different sized polystyrene beads, embedded in different index solvents, and we show that, the backwardre ected f-cars dominates the experimentally epi-detected signals. furthermore, we demonstrate experimentally and theoretically that the maxima of forward and epi-detected signals are generated at different positions along the z axis in the sample. we nally discuss how index mismatch in cells can alter cars images. the effects of static magnetic elds on humans have been the subject of continuous investigations. since one of the major static magnetic eld sources is nuclear magnetic resonance imaging (mri), the present study aimed to investigate the effects of . t magnetic eld that is produced by mri on humans. the study is carried out with voluntary and healthy young men from to years of age. the subjects informed about the purpose of the study at the beginning. the subjects exposed to minutes of . t static magnetic eld by means of putting the subjects into the magnetic resonance unit. ml blood was taken from each subject one minute before and one minute after exposure. t and t relaxation times and trace elements were measured in of pre and post exposure plasma of the subjects. the obtained post exposure values were compared with pre-exposure values of the subjects. pre and post exposure results were analyzed by means of student t-test. evaluation of tumor response of breast cancer patients by diffusion weighted mri k. a. danishad , v. seenu , u. sharma , p. k. julka , g. k. rath , n. r. jagannathan department of nmr, department of surgery, department of radiotherapy, all india institute of medical sciences, new delhi, india diffusion weighted mr imaging (dwi) measures the diffusion of water molecules in tissues and is quanti ed by apparent diffusion coef cient (adc). dwi can be used to differentiate tumors from normal tissue and also can be used to monitor the response of tumor to chemotherapy. thirteen healthy volunteers and twelve patients were recruited for the study. dw images were obtained prior to therapy (n= ) and after three cycles of therapy (n= ). the mean adc value of tumors ( . x . mm /s) was signi cantly less (p < . ) compared to the normal tissue ( . x . mm /s). decrease in adc in tumor is due to an increase in the cellularity which restricts the diffusion of water molecules. in patients receiving neo-adjuvant chemotherapy, the adc values were higher ( . x . mm /s) and were closer to that of the normal tissue (p < . ), indicating response of the tumor to chemotherapy. the post-therapy increase in adc is due to the cell damage caused by the therapeutic agents which increases the fractional volume of the interstitial space, causing an increase in the mobility of water. the study showed that dwi can be used non-invasively to assess the response of breast cancer patients to neo-adjuvant chemotherapy. quanti cation by optical imaging of gene electrotransfer in mouse muscle and knee optical imaging was evaluated for monitoring and quanti cation of the mouse knee joint and tibial cranial muscle electrotransfer (et) of a luciferase encoding plasmid. the substrate of luciferase (luciferin) was injected i.p or locally in the muscle or the knee joint. luminescence resulting from the luciferase-luciferin reaction was measured with a cooled ccd camera. luminescence of the knee joint and muscle were higher after local than after i.p injection of luciferin, but both measurements were highly correlated. local injection procedure was adopted. a signi cant correlation was observed between measurements in vivo and in vitro on the same muscle. reproducibility of individual luminescence measurements was also veri ed, and the luminescence levels were clearly dependant of the amount of plasmid injected. in vivo luciferase in the electrotransfered knee joint was detected for two weeks. intramuscular electrotransfer of . or µg of plasmid led to stable luciferase expression for days, whereas injecting µg plasmid resulted in a luminescence fall two weeks after electrotransfer. these decreases were, at least partly, related to the production of antibodies against luciferase. thus, optical imaging was shown to be a relevant technique to quantify variations of luciferase activity in vivo in one given tissue. furthermore, evaluating the effective amount of luciferase in tissues from in vivo luminescence levels requires calibration since it relies on conditions of the enzymatic reaction and light absorption. acute effect of corticosterone on nmda receptormediated ca + elevation in mouse hippocampal slices m. saito , s. sato , h. osanai , a. hirano , y. komatsuzaki , s. kawato department of physics and applied physics, college of humanities and sciences, nihon university, department of biophysics and life sciences, graduate school of arts and sciences, university of tokyo corticosterone (cort) is a principal glucocorticoid synthesized in the rodent adrenal cortex and secreted in response to stress. we examined the rapid effects of cort on n-methyl-d-aspartate (nmda) receptor-mediated ca signals in adult mouse hippocampal slices by using ca imaging technique. application of nmda caused a transient elevation of intracellular ca concentration followed by a decay to a plateau within sec. the min preincubation of cort induced a signi cant decrease of the peak amplitude of nmda-induced ca elevation in the ca region. the rapid effect of cort was induced at a stress-induced level ( . - µm). because the membrane non-permeable bovine serum albuminconjugated cort also induced a similar rapid effect, the rapid effect of cort might be induced via putative surface cort receptors. in contrast, cort induced no signi cant effects on nmdainduced ca elevation in the dentate gyrus. in the ca region, cort effects were not evaluated, because the marked elevation of nmda-induced ca signals was not observed there. in vivo subcellular structures recognized with phase k. nagayama , r. danev , n. usuda , y. kaneko , k. nitta , a. nakazawa , k. atsuzawa , m. tanaka , m. setou okazaki institute for integrative bioscience, okazaki, japan, fujita health university, school of medicine, toyoake, aichi, japan, saitama university, saitama, japan, tokyo metropolitan institute of gerontology, itabashi-ku, tokyo, japan phase contrast transmission electron microscopy has been developed to enable a high contrast and a high resolution observation for unstained ice-embedded samples. to enhance the image contrast, two methodologies have already been developed; i) scattering contrast for stained samples with small aperture diaphrams and ii) defocus contrast for unstained or stained samples with deep defocusing. the former prevails in histochemical sciences and the latter is popular in electron crystallography. both methods, however, have a common drawback that the contrast is only improved by impairing the image quality. this drawback can be removed with use of the phase contrast method using phase plates, which has traditionally been used in visible light microscopy. due to the severe obstacle of the charging of phase plates, however, the idea has not yet been materialized. we have solved the phase-plate charging problem. an experiment kv with tem for a whole cell from cyanobacterium unstained and ice-embed ful lled the expectation. only weak and vague contrast was obtained for the conventional image of the cell even with a very deep defocus. contralily a high-contrasted image has appeared for phase contrast images, where various ne structures are clearly recognized. this may be a rst example to observe nanometer scale structures in details in the intact cell. other examples including intact state intravesicular structures will be shown. j. lichtenberger, p. fromherz max-planck-institute for biochemistry we cultured bovine chromaf n cells on an array of electrolyteoxide-silicon eld-effect transistors (eos fet) and monitored granule secretion. by stimulation with barium chloride, vesicles are released into the narrow sheet of electrolyte between the chip surface and the plasma membrane. the interaction of released protons with the silicon dioxide surface of the chip alters the threshold voltage of the transistor and gives rise to a measurable signal. simultaneously performed measurements with a carbon bre showed a correlation of the transistor signals and amperometric current traces. we conclude that the transistors are able to monitor exocytosis on a single vesicle level. to elucidate the role of protons, we destroyed the proton gradient across the vesicle membrane by nigericin and valinomycin. as a result a massive reduction of the transistor signals was induced, whereas there was only little change of the amperometric records. we conclude that released protons are responsible for the detection of vesicles with transistors. the individual transistor records of vesicle exocytosis can be explained by combining the dynamics of the exocytotic event with the diffusion in the cell-chip junction. transistor recording of exocytosis does not depend on the electrochemistry of transmitters. as many kinds of exocytotic vesicles contain a large amount of buffered protons it can be applied to numerous kinds of exocytotic events, independent on the nature of the transmitter. we tried various solvents for the solubility of the uorescent product, and found that the product was insoluble in water and most organic solvents. a quite bright uorescence emitted by the particles was observed by uorescent microscope when emitted by uv nm. sem indicated that the size of the particles was µm µm, depending on the reaction time and phospholipid concentration in hexane solution. endothelial cells from human vein grew better on the surface prepared from the particles than the culture plate, implies a possible application as a new type of biomaterial as a coating material for medical devices, and as a uorescent tracer for human bodies. confocal microscopy of the phototactic ciliate f. salina. fabrea salina is a marine heterotrich ciliate, which dwells in salt ponds. in previous works we have described the phototaxis and the uorescence properties of a hypericin like endogenous pigment in an albino strain. we have recently obtained a heavily pigmented strain from the saline of torre colimena (taranto, italy). we have used confocal microscopy to characterize the uorescent properties of this strain and to compare them with those of the albino strain. the results obtained by one and two photon confocal microscopy show that, as in the albino strain, the uorescence intensity of the pigment is higher in dead cells than in the living cells. the excitation and emission spectra are quite similar in the two strains and this is also true for the uorescence lifetime, which is about ns. all together, these measurements indicate that the pigment of the new strain belongs to the family of hypericin-like chromophores. the analysis of different confocal planes shows that the pigment is localized not only in granules under the somatic membrane in the cellular body, as currently thought, but also in the cilia. some experiments of fotobleaching "in situ" con rm this result, that might have important implications in the understanding of the mechanisms of the photomotile responses of f. salina and probably of other heterotrich ciliates. there is no doubt that modern physician should have the knowledge of basic sciences as physics, chemistry and biology. furthermore, the biophysics is incorporated into the curriculum of most european medical schools. at medical school in zagreb, the course of physics and biophysics is positioned in rst and forth year of study. the students learn basic physics phenomena of structure of matter, mechanics, thermodynamics, electromagnetism, optics and acoustics applied on the human body. additionally, the interactions of the body with the surrounding are thoroughly discussed as the basis for different diagnostic methods. the arguing at our school is still going on where to include the content of this course. should it be the autonomous course or the part of physiology and radiology courses in the problem based learning approach?! so far at zagreb medical school the biophysical courses are autonomous structured according to the biophysics programs at other european universities. the highlighting is on seminar work and lab, encouraging the students for individual learning. the seminars are made more vivid and instructive for students by inclusion of different model devices constructed in our department learning on science is also learning how scienti c knowledge is produced. in this sense, issues related to the dynamics of science should be brought into focus in science education. the idea has support in the "declaration on science and the use of scienti c knowledge", particularly in the statement that "science curricula should include science ethics, as well as training in the history and philosophy of science and its cultural impact". thinking on rst year undergraduate students, we are interested in an integrated approach of that kind of themes. this presentation describes a practical teaching module included in the basic biophysics course for biochemistry majors. organized in case studies, it deals with stories of biophysics (and biochemistry), and addresses the role of the biophysical approach in the progress of the life sciences. the module follows the whole course, and consists in small exercises on the way a given understanding has been constructed explored within the practice trend of contemporary science studies. examples of the chosen stories -anchored in the subjects covered in the lectures of the course -relate to the search for the mechanism of energy production through atp-synthase, the development of radioactive labelling techniques, and the discovery of protein water channels. beyond their value as cultural legacy and as motivating tools, the insight they might provide is vast. axiomatic theory of biophysics q. zhao china rehabilitation research centre, beijing, china. until now, all approaches to interpret biology by applying principles of physics have been announced failure. the achievement of biophysics is limited in area to provide essential tools for biological research and biophysics is far from to be the basic theory of biology. to resolve it requires the fundamental research about the logic features of biophysical processes of biology. we promoted system logic and then protein thermodynamics structure theory. based upon it, the axiomatic theory of biophysics and biology could be developed. our result shows that the real understanding of biology or biophysics must be constructed based upon new thinking methods. a successful ligand-receptor docking methodology depends strongly in the ef ciency of the global optimization algorithm used to explore the ligand conformational space. in this work we have implemented and analyzed the performance of a new exible ligand-receptor docking methodology. this methodology uses as optimization method a multisolution version of the generalized simulated annealing algorithm adapted to problems with box constraints. a grid-based methodology, considering the receptor rigid, and the gromos classical force eld are used to evaluate the ligand-receptor scoring function. the methodology was tested in redocking (ligand within it own protein conformation) and cross-docking (ligand within another protein conformation) experiments for ve hiv protease-ligand complexes with known threedimensional structures. all ligands tested are highly exible, having to conformational degrees of freedom. the implemented docking methodology was able to redock successfully all exible ligands with a success ratio % and a mean rmsd lower than . Å with respect to the corresponding experimental structures. in the cross-docking experiments we observed a strong dependence of the mean success ratio with respect to the protein structure used as reference. in situations we observed a mean success ratio % and % in cases among the possible ones. s. aida-hyugaji , h. nakagawa , j. nomura , m. sakurai , d. tokushima , t. takada , u. nagashima , t. ishikawa tokai university, japan, tokyo institute of technology, japan, nec corporation, japan, national institute of advanced industrial science and technology, japan irinotecan is a widely-used antitumor drug that inhibits mammalian dna topoisomerase i. however, overexpression of abcg can confer cancer cells resistance to sn- , that is, the active form of cpt- . in the present study to develop a platform for the molecular modeling to circumvent drug resistance associated with abcg , we have characterized a total of fourteen new sn- analogues by some typical properties, which were evaluated by molecular orbital (mo) calculations and neural network (nn) analysis. the nn was rst applied to estimate hydrophobic properties (logp) of the analogues. thereafter, the electrostatic potential (esp) and the solvation free energy ( g) were evaluated by mo calculation. these indexes were found to be well correlated with the drug resistance ratio experimentally observed in abcg -overexpressing cells. it is suggested that hydrophilic analogues carrying oh-or nh -groups are good substrates for abcg and therefore exported from cancer cells. in contrast, sn- analogues with cl or br atom at those positions have similar logp values and high af nities toward the putative active site of abcg , however they were not substrates of abcg . from these results, it is strongly suggested that hydrogen bond formation with oh-or nh -groups are critically involved in the transport mechanism of abcg . a. agopian , j. depollier , e. gros , g. aldrian-herrada , p. clayette , n. bosquet , g. divita crbm-cnrs, route de mende, montpellier, france., spi-bio-cea fontenais aux roses, france reverse transcriptase (rt) plays an essential role in the replication of hiv and constitutes the main target for the development of aids therapies. the biologically active form of hiv rt is a heterodimer of two subunits, p and p , each consisting of distinct subdomains: the ngers, the palm, the thumb, the connection and the rnase h subdomain, the latter only present in p . we have demonstrated that formation of fully active rt is a two-step process involving rapid association of the two subunits (dimerization) followed by a conformational change (maturation). thanks to the crystal structure of rt we have identi ed a new class of inhibitors based on short peptide motifs derived from the dimer interface. we rst identi ed a short mer peptide (pep- ) derived from the tryptophanrich motif of the connection subdomain that blocks dimerization of rt and ef ciently abolishes hiv- replication. pep- interacts preferentially in a pocket involving residues trp and phe on p . we then designed mer peptides derived from the thumb domain which inhibit rt maturation as well as viral replication when delivered into cells. taking into account these results we propose that dimerization of rt constitutes a potential target for the design of more speci c new antiviral drugs. . the success of gene therapy largely relies on the availability of vectors that would deliver the genetic material ef ciently to the target cells with a minimal toxicity. in this context, our purpose was to evaluate as possible vectors a series of newly synthesized low molecular weight ( kda) chitosan derivatives grafted with dodecenoyl (ddc) groups at different percentages ( , , and %). in the absence of dna, the critical micellar concentration (cmc) of these derivatives in mm mes buffer ph . was found to be strongly dependent on the percentage of ddc but not on ph or salt concentrations. this indicates that the ddc groups confer to the chitosan derivatives the potency to self-assemble probably in micellar structures: a property that may dictate the formation and the structure of their complexes with dna. next, we investigated by quasielastic light scattering the size and the surface charge of complexes of plasmid dna with these derivatives at different ph, salt concentrations and n/p ratios (expressed in charged units of chitosan amines to dna phosphates). we found the smallest and more positively charged complexes were obtained at ph . and n/p= in the absence of salt: a condition where the chitosan derivatives were fully protonated and in excess over the dna phosphate groups. biophysical and biological examination of dna/lipids complexes particles of virus-like structure designed for in vivo gene transfer d. durand , m. schmutz , b. lebleu , a. r. thierry lure, centre universitaire paris sud, orsay, france, institut henri sadron, strasbourg, france, laboratoire des défenses antivirales et antitumorale, umr , montpellier, france the structure of complexes made from dna and suitable lipids (lipoplexes lx) was examined by cryo electron microscopy. we observed a distinct concentric ring-like pattern with striated shells when using plasmid dna. these spherical multilamellar particles have a mean diameter of nm with repetitive spacing of . nm with striation of . nm width. small angle x-ray scattering (saxs) con rmed cryoem data and revealed repetitive ordering of . nm, suggesting a lamellar structure containing at least a dozen layers. this concentric and lamellar structure with different packing regimes was also observed by cryoem with linear dsdna, ssdna and oligodeoxynucleotides. for the rst time, dna chains could be visualized in dna/lipid complexes. such speci c supramolecular organization is the result of thermodynamic forces, which cause compaction to occur through concentric winding of dna in a liquid crystalline phase. cryoem of t phage dna packed either in t capsides or in lipidic particles showed similar patterns. saxs suggested an hexagonal phase in lx-t dna. thus, both lamellar and hexagonal phases may coexist in the same lx preparation or particle and transition between both phases may depend upon equilibrium in uenced by type and length of the dna used. organization of such nucleotidic supramolecular assemblies is relevant for prebiotic chemistry. engineering self-assembly peptides for targeted delivery of therapeutics and imaging agents s. s. dhadwar, m. sung, k. kawamura, j. gariépy department of medical biophysics, university of toronto, canada peptide-mediated delivery systems have recently emerged as a means to substitute or augment conventional drug and gene delivery technologies. these approaches are versatile and easily designed to incorporate a number of speci c attributes required for ef cient delivery of therapeutic and imaging agents. in particular, self-associating peptide domains can be utilized to construct stable and structurally well-de ned protein-like assemblies displaying a series of cell-routing functions. more speci cally, a peptide-based self-assembling intercellular delivery vehicle was designed by incorporating the -residue long tetramerization domain of the human tumor suppressor protein p (hp tet). the resulting peptide tetramer displays termini within its structure that allows for the simultaneous presentation of distinct cell targeting signal or functional domains. the fusion of polycationic sequences to the hp tet domain promotes the cellular import of the resulting constructs into eukaryotic cells. this internalization event was dramatically enhanced for such multivalent peptides in relation to their monomeric counterparts. peptides containing a nuclear localization sequence along with a polycationic sequence were found to shuttle reporter plasmids ef ciently to the nucleus of cells. these results have important implications in the design and construction of novel targeted delivery vehicles. mechanisms of non-covalent peptide mediated cellular delivery of therapeutics: a biophysical study s. deshayes , m. c. morris , a. heitz , p. charnet , g. divita , f. heitz crbm -cnrs fre montpellier france, cbs -cnrs umr -inserm u montpellier france two different cell-penetrating peptides mpg and pep- were shown to promote non-endosomal intracellular delivery of non-covalent bound cargos, namely nucleic acids and proteins; respectively. in order to identify the peptide mediated internalization pathway, we undertook conformational investigations of both peptides with and without associated cargos and checked the conformational consequences of the presence of phospholipids. from the conformational point of view, pep- behaves differently from mpg. cd analysis revealed a transition from a non-structured to a helical conformation upon increase of the concentration while mpg remained nonstructured. determination of the structure by nmr showed that in water, it's a-helical domain extends from residue to . cd and ftir indicated that pep- adopts a helical conformation in the presence of phospholipids while mpg is in a -sheet form. adsorption measurements performed at the air-water interface were consistent with the helical form. pep- did not undergo conformational changes upon formation of a particle with a cargo peptide. in contrast, we observed a partial conformational transition when the complex encountered phospholipids. for mpg, interactions with nucleic acids generated a partial folding into -sheet which was more pronounced in the presence of lipids. electrophysiological measurements showed that both peptides, whether associated or not with their cargo, can induce transmembrane pore-like structures. self-assembly of hydrolysed alpha-lactalbumin into nanotubes j. f. graveland-bikker , k. g. de kruif nizo food reseach, ede, netherlands, van´t hoff laboratory, netherlands nanotubes are formed by self-assembly of partially hydrolysedlactalbumin, a kda milk protein. there are several promising applications of these -lactalbumin tubes, in food, pharmacy and nanotechnology. we studied the mechanism of self-assembly, the structure and the properties of the nanotubes. limited proteolysis of the -lactalbumin (by a serine protease) makes the molecule prone to self-assembly. in the presence of ca tubular structures are formed. other divalent ions like mn and zn can also induce tubular self-assembly, while mg leads to random aggregation. light scattering showed that the self-assembly is reversible, which is of relevance for controlled release applications. on the other hand, we could also make stable tubes by cross linking, which would be a requisite for several other applications. from afm and saxs measurements, we obtained values for the outer diameter: nm; and the inner diameter: nm. afm and cryo-em revealed the helical structure of the tube wall; it is a right-handed helix. by performing nano indentations with afm we determined mechanical properties of the tubes. the tubes were shown to be relatively resilient upon small deformations; the elastic modulus is of the order of . gpa. targeted delivery of photosensitizers into the cancer cell nuclei enhances their cytotoxic ef cacy the search for new pharmaceuticals has raised interest in locallyacting drugs which act over short distances within the cell, and for which different cell compartments have different sensitivities, e.g. photosensitizers used in anticancer therapy should be transported to the most sensitive subcellular compartments where their action is most pronounced. earlier we have produced a number of modular recombinant transporters for locally-acting drugs comprising several functional modules for cell-speci c targeting, internalization, escape from intracellular acidic vesicle, and targeting to the nuclei of melanoma cells overexpressing melanocortin receptors. here we describe new transporters on the basis of epidermal growth factor which are speci c for a wide variety of cancers. these transporters possess all necessary functional activities and deliver photosensitizers into the nuclei of human carcinoma cells to result in photocytotoxic effects almost orders of magnitude greater than those of nonmodi ed photosensitizers. characterization of mixtures of dna and nonionic polymeric agents for gene delivery in muscle j. m. gau , j. lal , l. auvray laboratoire mpi -lrp, umr cnrs , université d´Évry val d´essonne, Évry cedex, france, argonne national laboratory, ipns, south cass avenue, illinois , usa a strategy to cure muscle disease is to introduce genes (dna) into the muscle cell to correct or to add genes. nonionic polymeric agents have emerged as an ef cient vector to deliver dna in the muscle. these polymers protect dna from extracellular nuclease degradation by allowing the dna diffusion throughout the muscle tissue. there is at present no understanding about how nonionic polymers enhance transfection in the muscle. the kind of interactions between these nonionic agents and dna, dna-nonionic polymeric agent mixtures and cell membrane are currently unknown. also the structure of dna-nonionic polymeric agent mixtures is not yet well de ned. more information is needed to improve this delivery system. neutron scattering (contrast variation) and light scattering were used to investigate the interaction between: dna and nonionic polymers (pvp, di-and triblock copolymers). furthermore, electrical measurements with the same polymer complexes and black lipid membrane were also performed. depending on the polymer type there is either direct interaction with dna or in other cases polymers exhibit strong interaction with the lipid membrane. an explanation for transfection ef ciency of these nonionic agents in gene delivery to muscle will be given. high throughput in-silico screening against exible protein receptors b virtual screening of chemical databases to targets of known threedimensional structure is developing into an increasingly reliable method for nding new lead candidates in drug development. based on the stochastic tunneling method (stun) we have developed flexscreen, a novel strategy for high-throughput in-silico screening of large ligand databases. each ligand of the database is docked against the receptor using an all-atom representation of both ligand and receptor. in the docking process both ligand and receptor can change their conformation. the ligands with the best evaluated af nity are selected as lead candidates for drug development. using the thymidine kinase inhibitors as a prototypical example we documented the shortcomings of rigid receptor screens in a realistic system. we demonstrate a gain in both overall binding energy and overall rank of the known substrates when two screens with a rigid and exible (up to sidechain dihedral angles) receptor are compared. we note that the stun suffers only a comparatively small loss of ef ciency when an increasing number of receptor degrees of freedom is considered. flexscreen thus offers a viable compromise between docking exibility and computational ef ciency to perform fully automated database screens on hundreds of thousands of ligands. maturation and inhibitor design of sars-cov cl protease based on a product-bound crystal structure severe acute respiratory syndrome (sars) is an emerging infectious disease caused by a novel human coronavirus. here we report that the cl pro containing n-and/or c-terminal additional in-frame sequences underwent autoactivation to cleave the tags and yielded the mature protease in vitro. the -d structure of the c a mutant protease shows that the active site of one protomer of the dimeric protease is bound with the c-terminal six amino acids of the protomer in another asymmetric unit, suggesting a possible mechanism for maturation. the crystal structure of this product-bound form shows that the active site has a p pocket that binds the gln side chain speci cally. in addition, the p and p sites are clustered together to accommodate large hydrophobic side chains. the tagged c a mutant protein served as a substrate for the wildtype protease and the n-terminus was rst digested ( -fold faster) followed by the c-terminal cleavage as shown by the sds-page analysis. the analysis of t analytical ultracentrifuge experiments reveals the remarkably tighter dimer formation for the mature enzyme (k d = . nm) than for the mutant (c a) containing the n-terminal (k d = . nm) or the c-terminal extra amino acids (k d = . nm). taken together, the study here provides insights to the design of our new structure-based inhibitors. nevertheless, a signi cant proportion of patients do not respond to this therapy, and adverse effects are common. here we report the delivery and expression of recombinant mycobacterial dna vaccines in vivo and demonstrate the ability of multicomponent dna vaccines to enhance th -polarized immune responses. splenocytes from immunized groups of mice were re-stimulated in vitro and examined for cytotoxicity against bladder tumour cells. we used four combined recombinant bcg dna vaccines (multi-rbcg) for electroporative immunotherapy in vivo, and found that tumour growth was signi cantly inhibited and mouse survival was prolonged. increased immune cell in ltration and induction of apoptosis were noted after treatment with multi-rbcg alone, with the interleukin- (il- ) vaccine alone, and-most signi cantly-with their combinations. thus, electroporation immunogene therapy using multi-rbcg plus il- may be an attractive regimen for the treatment of bladder cancer. this approach presents new possibilities for the treatment of bladder cancer using recombinant bcg dna vaccines and il- dna vaccine. the cell-penetrating peptide (cpp) pep- is capable of introducing large proteins into different cell lines, maintaining their biological activity. two mechanisms have been proposed to explain the entrance of other cpps in cells, endosomal-dependent and independent. we evaluated the molecular mechanisms of pep- mediated cellular uptake of -galactosidase ( -gal) from e. coli, in large unilamellar vesicles (luv) and hela cells. fluorescence spectroscopy and immuno uorescence microscopy were used to study the translocation. internalization of -gal into luv and protein functionality in hela cells were detected by enzymatic activity. -gal translocated into luv in a transmembrane potentialdependent manner. likewise, -gal incorporation was extensively decreased in depolarized cells. furthermore, -gal uptake efciency and kinetics were temperature-independent and -gal did not co-localize with endosomes, lysosomes or caveosomes. therefore, -gal translocation was not associated with the endosomal pathway moreover transmembrane pores were not detected. these results indicated that the protein uptake in vitro and in vivo was mainly, if not solely, dependent on a physical mechanism governed by electrostatic interactions between pep- (positively-charged) and membranes (negatively-charged). peptide the dramatic acceleration in the identi cation of new nucleic acidbased therapeutic molecules has provided new perspectives in pharmaceutical research. however, the development of nucleic acidand peptide-based therapeutics is limited by their poor cellular uptake and traf cking. with the aim of addressing these issues, we have designed a family of short amphipatic peptides for delivery of nucleic acids (mpg) and of peptide/pna (pep). these carriers consist of a hydrophobic moiety and a nls-derived hydrophilic domain. they form stable non-covalent complexes with peptides, proteins, sirna or pna without any requirement for prior covalent cross-linking. both mpg and pep carriers enter cells rapidly, in a process involving membrane disorganization, independently of the endosomal pathway. mpg ef ciently delivers short odns and sirna into a wide variety of mammalian cell lines, without interfering with their biological function. pep signi cantly improves delivery of pna and peptides. both carriers were used for the delivery of sirna or antisense pna targeting the cell cycle regulatory protein cyclin b in an animal model and were found to block tumor growth upon intravenous injection. we believe that mpg and pepbased technologies will contribute signi cantly to the development of basic and therapeutic applications. probing the bound conformation of acetylcholinesterase (ache) inhibitor at the binding site c. g. kim, x. zhao, s. goodall, a. watts department of biochemistry, university of oxford, south parks road, oxford, ox qu, uk acetylcholinesterases are the enzymes which preferentially hydrolyze acetyl esters (such as ach or acetyl-â-methylcholine), containing amino acid residues in the eeache form and arranged as a -stranded â-sheet surrounded by á-helices. the protein is ellipsoidal in shape, with approximate dimensions of Å by Å by Å. inhibitors of acetylcholinesterase are of commercial and medical interest as pesticides and as therapeutics in the treatment of alzheimer's disease. an understanding of the conformation of inhibitors in the binding site enables the rational design of novel inhibitors with increased potency and speci city. interaction between the ligand, amino- -methyl- -( tri uoroacetylbenzyl-oxymethyl)quinoline (r ), and ache inhibitor has been studied by advanced solid-state nmr through double-quantum chemical shift and distance measurements. combining solid-state nmr data and docking simulations, conformation of the ache inhibitor at the active site has been predicted. in vivo, heat shock proteins (hsps) being stress-inducible chaperones can attenuate detrimental consequences of ischemic insults, inammation, neurodegenerative diseases, etc. also, intracellular accumulation and chaperone activities of some hsps may contribute to improved cell survival following uv or ionizing radiation. in models of pathological states and their treatment, we used special virusbased vectors for overexpression of hsp or hsp in cell cultures to confer cytoprotection under simulated ischemia/reperfusion. in parallel, similar cytoprotection was achieved after pretreatments of the cells with a pharmacological hsp inducer, geranylgeranylacetone. the cytoprotective effects were manifested in the lesser extent of oxidative modi cation and aggregation of cellular proteins, better preservation of the cytoskeleton, faster restoration of energy metabolism and the improved post-stress cell survival. in the other model, we treated normal and tumor cells with an inhibitor of the chaperone activity of hsp , geldanamycin. only the drug-treated tumor cells became more sensitive to gamma-irradiation; such results characterize this drug as a potentially selective radiosensitizer of tumors. taken together our data demonstrate promising approaches to clinically bene cial manipulating the levels of expression and/or chaperone activity of hsp(s) by means of gene therapy or pharmacotherapy. characterizaton of proteins from human pleural uid r. jain, s. kumar, n. singh, s. sharma, t. p. singh all india institute of medical sciences, new delhi ,india the samples of human pleural uid were obtained from both healthy subjects and patients infected by tuberculosis. after the preliminary processing these samples were run in independent lanes of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page). the two lanes indicated variations in the intensities of a few bands and some new bands were also observed in the infected samples. these were characterized by determining their nterminal sequences. the new bands which had low density were carefully identi ed and cloned. some of the common bands that showed intensity variations were characterized. these were matrix metalloproteins, secretory phospholipasea , transferrin and ceruloplasmin. they were also studied with maldi-tof and their molecular weights have been determined. some of these proteins have been crystallized and their detailed crystal structure determinations are in progress. biophysical study of non-lethal stress response of cultured dc f cells stress factors may induce two kinds of responses in living cells: either cell death or adapting mechanisms. our aim was to search for non-lethal effects of various stress conditions on cultured hamster lung broblasts (dc f cells) as well as to assess the recovery time after stress removal. dc f cells were cultured in standard conditions and were submitted to stress, either by incubation with chemical substances (sodium arsenate, sodium nitroprusiate) and drugs (bleomicine and statins) either by irradiation (uv, he-ne laser). the doses and exposure times were chosen as to avoid cell death. after stress removal, cells were allowed to recover and the recovery time period was measured. structural and functional parameters were evaluated before and after stress, as well as during recovery. by now, experimental models for the in vitro study of non-lethal stress inducing factors have been set up. severe acute respiratory syndrome (sars) is an emerging infectious disease caused by a novel human coronavirus. the viral maturation requires a main protease ( cl pro ) to cleave the virus-encoded polyproteins. we report here that the cl pro containing n-and/or c-terminal additional in-frame sequences underwent autoactivation to cleave the tags and yielded the mature protease in vitro. the -d structure of the c a mutant protease shows that the active site of one protomer of the dimeric protease is bound with the cterminal six amino acids of the protomer in another asymmetric unit, suggesting a possible mechanism for maturation. the tagged c a mutant protein served as a substrate for the wild-type protease and the n-terminus was rst digested ( -fold faster) followed by the c-terminal cleavage as shown by the sds-page analysis. the analysis of the quaternary structures for the tagged and mature proteases by analytical ultracentrifuge experiments reveals the remarkably tighter dimer formation for the mature enzyme than for the mutant (c a) containing the n-terminal or the c-terminal extra amino acids. taken together, the study here provides insights to the design of our new structure-based inhibitors. characterisation of macromolecular transport in physiologically relevant mixed ecm based gels s. lelu, a. pluen school of pharmacy and pharmaceutical sciences, university of manchester (uk) the extracellular matrix (ecm) a complex gel made of hyaluronic acid, collagen and proteoglycans (pg) impedes the penetration of macromolecules especially in tumours, and may compromise the success of novel therapies. though recent in vivo investigations pointed out that, not only ha but brillar collagen its content and organisation and its interactions with pg were involved in macromolecular transport hindrance, transport mechanisms relating the macromolecular drug and these ecm components are unknown. in this study we seek to evaluate the determinants of passive transport mechanisms of biomacromolecules in complex gels made of ha, collagen using uorescence techniques (frap and confocal re ection microscopy (crm)) and rheology. focus was on conditions relevant to tumours and, initially, on low collagen and relatively high ha content. rheology experiments showed that mixed systems containing less than mg/ml of ha present higher elastic modulus ge than pure ha network or pure collagen gels. interestingly crm and frap studies revealed similarities for collagen and mixed gels: the organisation and spacing of the collagen bres did not change and the ratio of the diffusivities (d/d ) of dextran m and igg were not different but higher than those in ha networks. systems with higher collagen content are under investigation to complete the characterisation of transport. encapsulation of clone vector dna by cationic diblock copolymer vesicles for gene delivery a. v. korobko , j. r. van der maarel leiden university, the netherlands, national university of singapore, singapore we will discuss the design, control, and structural characterization of cationic copolymer vesicles loaded with dna. these vesicles serve as a model system for diverse applications such as gene delivery, micro-arraying techniques and packaging of dna in congested states. encapsulation of dna was achieved with a single emulsion technique. for this purpose, an aqueous puc or pegfp-n plasmid solution is emulsi ed in an organic solvent and stabilized by an amphiphilic diblock copolymer. the neutral block of the copolymer forms an interfacial brush, whereas the cationic block complexes with dna. a subsequent change of the quality of the organic solvent results in a collapse of the brush and the formation of a capsule. the capsules are subsequently dispersed in aqueous medium to form vesicles and stabilized with an osmotic agent in the external phase. inside the vesicles, the dna is compacted in a liquid-crystalline fashion as shown by the appearance of birefringent textures under crossed polarisers and the increase in uorescence of labeled dna. the compaction ef ciency and the size distribution of the vesicles were determined by light and electron microscopy, respectively, and the integrity of the dna after encapsulation and subsequent release was con rmed by gel electrophoresis. we demonstrate the gene transfer ability of this new carrier system by the transfection of encapsulated pegfp plasmid into hela cancer cells. cellular transduction of nucleotide kinases to improve the activation of nucleoside analog prodrugs m. konrad , c. monnerjahn , s. ort , a. lavie max-planck-institute for biophysical chemistry, goettingen, germany, university of illinois at chicago, chicago, usa the objective of our study is to improve therapeutic enzymeprodrug systems by generating catalytically superior nucleoside and nucleotide kinases that are essential for activation of nucleoside analogs. compounds, such as azt for the treatment of hiv infections, acv and gcv used against herpes virus, or the anticancer compounds arac and gemcitabine, can enter cells only in the unphosphorylated state (prodrug) and need to be transformed by different kinases to their pharmacologically active triphosphate state that interferes with dna replication. we have rst designed mutants of the human tmp kinase (htmpk) that phosphorylate aztmp up to -fold faster than wildtype. expression of this enzyme in human cells leads to -fold higher intracellular concentrations of azttp and to enhanced hiv inhibition. second, the prodrugs acv and gcv are not phosphorylated by human kinases, but are converted to their monophosphate forms by hsv -tk which is used in enzyme/prodrug-dependent cancer suicide gene therapy. we generated enzyme variants which show selective and ef cient phosphorylation of gcv. third, an engineered human dck variant catalyzes more ef ciently the activation of the prodrugs arac and gemcitabine. thus, the concept of a gene (or enzyme) therapeutic treatment involving expression (or direct intracellular transduction) of a catalytically improved human enzyme may pave the way to the development of novel strategies in nucleoside prodrug-dependent cancer chemotherapy. docking-molecular dynamics studies on the peroxidase site of prostaglandin endoperoxide h synthase prostaglandin endoperoxide h synthases- and (pghs- and ) catalyze the rst step in the biosynthesis of prostaglandins, prostacyclins and thromboxanes. arachidonic acid is transformed into prostaglandin g (pgg ) at the cyclooxygenase site of the enzyme and the -hydroperoxide oxygen-oxygen bond of pgg is subsequently cleaved by reaction with haem at the distinct peroxidase site (pox) to produce prostaglandin h (pgh ). herein we present a plausible productive conformation obtained by docking calculations for the binding of pgg to the pox site of pghs- . the enzyme-substrate complex stability was veri ed by a -ps molecular dynamics simulation. structural analysis unveils the requirements for enzyme-substrate recognition and binding: the pgg -hydroperoxide group is in the proximity of the haem iron and participates in a hydrogen bond network with the invariant his and gln and a water molecule, whereas the carboxylate group establishes salt bridges with the remote lysines and . the interaction of the peptide lah with anionic lipids during dna/rna delivery to eukaryotic cells a. j. mason , a. martinez , c. leborgne , a. kichler , b. bechinger faculté de chimie, université louis pasteur, strasbourg, france, généthon, evry, france the histidine rich amphipathic peptide lah has antibiotic and dna delivery capabilities. the peptide has a strong af nity for anionic lipids found in the outer membrane of bacterial membranes and has shown evidence of higher transfection activity against transformed over healthy tissue in culture. it has been proposed that anionic lipids can ip-op to reach the cytoplasmic monolayer. here they neutralise the cationic transfection complexes thereby causing release of oligonucleotides into the cytoplasm. we were, therefore, particularly interested to test for the role of the acidic lipid phosphatidylserine (ps) in mediating lah -mediated delivery of dna ef ciency. to understand the potential peptide-lipid interactions in more detail, solid-state nmr experiments on model membranes have been performed. p mas nmr on mixed phosphatidylcholine (pc)/ps and pc/phosphatidylglycerol (pg) membranes has been used to investigate speci c lah interactions with anionic lipids. by using deuterated lipids and wide-line h nmr when probing lipid chain order, it is demonstrated that lah preferentially interacts with ps over pc. lah thereby effectively disorders the anionic ps lipid fatty acyl chains. the lipid chain destabilising effect of lah and also lah analogues can then be compared with their transfection ef ciency for dna or sirna in cell culture to aid in rational peptide vector design. virtual screening is now widely accepted as a basis for drug discovery thanks to signi cant improvement and good hit rates [ ]. however, it is still highly cpu-consuming. at the same time, the number of protein-ligand complexes described at the atomic level is rising and the sequence similarity is used for structure and function predictions. new approaches are being developed to take advantage of the available structural data and the huge number of protein sequences in order to allow better tuned virtual screening. new web servers are being built to ease and to speed up the whole process (http://abcis.cbs.cnrs.fr/kindock/). integrating these servers into a pipeline dedicated to molecular modelling (http://abcis.cbs.cnrs.fr/atome/) shall allow both the re ned validation of modelled active sites as well as the oriented screening for the primary caracterization of potential ligands. an ideal drug delivery system should own the following characteristics, the rst is the targeting of therapeutic agent to the speci c site of their action. the second is the controlled delivery of a therapeutic molecule or protein in a pulsatile or staggered fashion. the third is the achieving sustained zero-order release of a therapeutic agent over a prolonged period of time. in this study, a new drug delivery system combined these characteristics was provided, which contains azobenzene derivatives (ab lipid) as an on-off switch incorporated into liposomes. the drastic release of calcein was observed on the rst uv irradiation of ab lipid to the cis isomer, while a suppressed release was observed when irradiated with the rst visible light. after that, the slope of release pro le became coincident. furthermore, calcein release was greatly increased after uv irradiation of ab lipid to the cis isomer and the drug release was greatly suppressed after vis irradiation of ab lipid to the trans isomer. we can control the release rate of calcein from ab lipid/egg pc mixed liposomes by uv or vis light irradiation. tryptophanase (trpase), a bacterial enzyme with no counterpart in eukaryotic cells, produces l-trp pyruvate ammonia and indole. it was suggested that indole is essential for bacteria multiplication and bio lm formation. bio lms destroy equipment and food and cause many illnesses. most synthesized quasi-substrates inhibit trpase at mm range. an optimal and speci c inhibitor of trpase may eliminate indole production and prevent bio lm formation. x-ray crystallography of the holo-wt e. coli trpase soaked with l-trp and the known mechanism of trpase activity should provide the information for the design and synthesis of active-sitespeci c quasi-analogs. utilizing the chromogenic substrate s-(onitrophenyl)-l-cysteine, the following michaelis-menten kinetics analyses determined the mode of trpase inhibition by trp and quinone based quasi-analogues and the corresponding ki values (in µm): dl- -alanyl- , anthraquinone, noncompetitively, ; trypthophan ethylester, competitively, ; acetyltryptophan, uncompetitively, . ; s-phenylbenzoquinone-l-tryptophan, uncompetitively, . plp-l-trp, inhibited irreversibly only the apo form of trpase and may serve for structure-determination purposes. further attempts are being made to synthesize improved trpase inhibitors, i.e., in the nm range. polyelectrolyte multilayer lms (pem) adsorbed on biomaterial surfaces are a new way to create a controlled release system. using biodegradable polymers, the lms can be degraded in vivo and release active molecules. in this work, we demonstrate the possibility of tuning the degradability of polysaccharide pem in vitro and in vivo. chitosan and hyaluronan pem (chi/ha) were either native or cross-linked (cl) using a water soluble carbodiimide (edc) at various concentrations in combination with nhydroxysulfosuccinimide. the in vitro degradation of the lms in contact with enzymes was followed by quartz crystal microbalance measurements and confocal laser scanning microscopy after lm labeling with chi fitc . whereas the native lms were subjected to degradation, the cl lms were more resistant to enzymatic degradation. films made of chitosan of medium molecular weight were indeed more resistant than lms made of chitosan-oligosaccharides. in addition, macrophages could degrade all types of lms and internalize the chitosan in vitro. the native lms implanted in vivo in mouse peritoneal cavity for a week showed an almost complete degradation whereas the cl lms were only partially degraded. these results suggests that the polysaccharides pem are of potential interest for in vivo applications as biodegradable coatings and that degradation can be tuned by controlling lm cross-linking. membrane electroporation -tool for therapeutic electrotransfer of drugs and gene dna e. neumann, s. kakorin physical and biophysical chemistry, faculty of chemistry, university of bielefeld, germany membrane electroporation (mep) is a new electrical high voltage scalpel, transiently opening the cell membranes of tissue for the penetration of foreign substances. due to the enormous complexity of cellular membranes, many fundamental problems of mep have to be studied at rst on model systems, such as the curved bilayer membranes of unilamellar lipid vesicles. electrooptical and conductometrical data of unilamellar liposomes indicate that electric eld pulses cause not only the formation of membrane electropores but also shape deformation of the liposomes, both processes mutually affecting each other. the primary eld effects of mep and cell deformation can trigger a cascade of numerous secondary phenomena, such as pore percolation and transport of small and large molecules across the electroporated membrane. the chemical mep theory represents a molecular physico-chemical approach to electrochemomechanical pore formation, yielding transport parameters, such as permeation coef cients, pore fractions and pore sizes. the pore concept is successfully applied to rationalize optimization strategies for biotechnological and medical applications of mep. in silico elucidation of xenobiotic processing loops k. nakata , y. tanaka , t. nakano , t. ishikawa , h. tanaka , t. kaminuma national institute of health sciences, tokyo, japan, tokyo medical and dental university, tokyo, japan, tokyo institute of technology, yokohama, japan, hiroshima university, hiroshima, japan one of the important challenges for drug designers is to predict and analyze how drugs are absorbed, distributed, metabolized and excreted (adme) in the body. these processes highly correlated with toxicity of drugs and are actively studied in pharmacology. two classes of proteins, the drug metabolizing enzymes such as cytochrome p s (cyps) and transporters, are the target of such adme/tox research. it was relatively recent that these two classes of proteins are synthesized by the genes that are the target genes of the nuclear receptors. nuclear receptors are ligand-activated transcription factors that form a superfamily. in case of humans there are nuclear receptors almost half of whose ligands are identied, leaving some as true orphans. thus it was now recognized that these nuclear receptors play the role of sensors of drugs and other xenobiotic substances including environmental chemical pollutants and nutritional ingredients, while the drug metabolizing enzymes and the transporters are the processors which carry the actual cleaning jobs. we have started to elucidate the feedback loops that are formed by the xenobiotic ligands, nuclear receptors, their target genes, their product proteins, and their feedback actions on the ligands. the work is being carried out on our background database on the ligands and their receptors called kibank, and search programs for target genes of nuclear receptors algorithmically. the most recent results will be presented at the presentation. the major route for drug entry into cells is permeation across lipid bilayers. due to methodological limitations there are only few studies on permeation of drug-like molecules across lipid bilayers. an assay developed in our lab allows the direct measurement of lipid bilayer permeation of aromatic carboxylic acids (acas). tb , which forms a uorescent complex with acas, is entrapped in liposomes and aca entry is determined from luminescence increase. lipid bilayer permeation was ph-dependent, following a henderson-hasselbalch function with a plateau for the neutral and the anionic species, respectively. in contrast to the expectations of the ph-partition hypothesis, permeation of the anionic species was only to magnitudes lower than that of the neutral species, leading to anion-controlled permeation at ph . , independently of bilayer state and lipid composition. permeation across bilayers with a biologically relevant lipid composition was signi cantly slower than across egg-phosphatidylcholine membranes. the in uence of single lipids, such as cholesterol, was dependent on the structure and ionization state of the permeant. permeation coef cients of the neutral species correlated better with the polar surface area (psa) than logp oct , therefore psa is a better predictor for bilayer permeation of the neutral species of small acas than logp oct . interplay between polymerized liposomes physicochemical properties and composition and citotoxicity this study was aimed at investigating whether there is an interplay between diacetylenic polymerized liposomes physicochemical properties and lipid composition affecting citotoxicity in vitro. unsaturated , -bis( , -tricosadiynoyl)-sn-glycero- -phosphocholine with saturated , -dimiristoyl-sn-glycero- phosphocholine in molar ratio : , were combined to give a chemically modi ed membrane by uv-polymerization. biophysical characterization was carried out determining the hydrophobic factor and hydrodynamic radius. citotoxicity was evaluated through haemolytic capacity on bovine red blood cells and indirectly by capacity of induction of lipid peroxidation on microsomes or mitochondrial membranes. the haemolysis percentage in presence of dc , pc/dmpc is less than that induce by polymers used in dentistry. the data obtain suggests that the polymerized lipids can not induce lipid peroxidation on natural membranes. the polymerized diacetylenic liposomes showed less interaction with serum proteins than non polymerized and lower citotoxicity as compared with natural lipids. also cell viability was determined in cell line nih t after exposure to lipids systems under study. the hydrophobic factor showed further augmentation for polymerized liposomes and is discussed in relation to in vitro stability. the above results suggest that polymerized and non-polymerized liposomes would serve as an effective delivery vehicle. s. sonar, s. d´souza, k. p. mishra radiation biology and health sciences division,bhabha atomic research centre,mumbai- , india liposomes offer new approaches for drug delivery through their encapsulation to alter pharmacodynamic properties of loaded drug leading to reduction in toxicities and/ or improved ef cacy. for prolonged systemic circulation, the liposomes size has been shown to be limited to nm or less. the ethanol injection method is an excellent technique for the formation of liposomes of < nm without the need of sonication or extrusion. the present study was aimed to produce liposomes encapsulating doxorubicin in minimum procedural steps. liposomes were prepared using distearoyl phosphatidylcholine and cholesterol, distearoyl phosphatidylcholine, cholesterol and oleic acid. the effects of different operational conditions for vesicle production and drug encapsulation were evaluated, with a view to achieve process cost to a minimum, suitable size and high encapsulation ef ciency. although high ef ciency of doxorubicin encapsulation was obtained by 'active' or 'remote' loading process in dspc/chol system, it was poor in one-step injection method. oleic acid was included to cut down the active loading by ph-gradient. dspc/chol/oa systems spontaneously loaded doxorubicin with encapsulation ef ciency of % and nal drug to lipid molar ratio upto . . the mean diameter of the vesicles was + nm. the method offers liposomes of small size with high loading. design of peptides with consecutive dehydro phenylalanine residues r in order to develop general rules for the design of peptide conformations with consecutive alpha, beta-dehydro phenylalanine-residues, peptides were synthesized, crystallized and crystal structures and molecular conformations were determined. following conclusions were drawn based on the structural data: -peptide unit sequences with two consecutive dehydro-phe residues at (i+ ) and (i+ ) positions adopt an unfolded s -shaped structure with dihedral angles phi-psi centred at , . -the peptides containing two consecutive dehydro-phe residues at (i+ ) and (i+ ) positions • form two overlapping type iii beta -turns (incipient -helix). • with branched beta -carbon residue only at (i+ ) position adopt a conformation with two overlapping types ii and iii beta -turns. • with branched beta-carbon residues such as val and ile at both (i+ ) and (i+ ) positions form two overlapping types ii and i beta -turns. the consistency in the formations of these conformations makes the design of peptides with alpha,beta ?-dehydro -residues a useful and highly predictable method for developing speci c ligands for various biological applications including drug design. binding of cationic porphyrin to isolated double-stranded dna and nucleoprotein complex k. zupán , l. herényi , k. tóth , z. majer , g. csík institute of biophysics and radiation biology, semmelweis univ., budapest, hungary" biophysics of macromolecules, german cancer research center, heidelberg, germany, department of organic chemistry, eötvös loránd univ., budapest hungary the complexation of tetrakis( -n-methylpyridyl)porphyrin (tmpyp) with free and encapsidated dna of t bacteriophage was investigated. to identify binding modes and relative concentrations of bound tmpyp forms, the porphyrin absorption spectra at various base pair/porphyrin ratios were analyzed. spectral decomposition, uorescent lifetime, and circular dichroism measurements proved the presence of two main binding types of tmpyp, e. g., external binding and intercalation both in free and in encapsidated dna. tmpyp binding does not in uence the protein structure and/or the protein -dna interaction. concentrations of tmpyp species were determined by comprehensive spectroscopic methods. our results facilitate a qualitative analysis of tmpyp binding process at various experimental conditions. we analyzed the effect of base pair composition of dna, the presence of protein capsid and the composition of buffer solution on the binding process. protein crystallization and structural study of upa protease domain with active site serine mutation the urokinase (upa) system is composed of upa, its receptor (upar), and inhibitor (pai). it plays important role in various physiologic processes, including brinolysis, cell adhesion, and signal transduction, and has been recognized as a target for intervention in tumor growth and tumor metastasis. we constructed an active site mutant of upa protease domain ( - ) with three mutations (c a, n q, and s a) and expressed it as secreted protein in pichia pastoria with ppicza vector. the secreted mutant was captured from culture medium by a cation exchange column and then further puri ed on a gel ltration column. the puri ed mutant was then crystallized by sitting drop vapor diffusion method with several precipitant conditions: ( ) . - . m ammonium sulphate, - % peg , mm sodium citrate ph . or mm sodium phosphate ph . , . % sodium azide. ( ) . m ammonium sulphate, . m lithium sulphate, mm sodium acetate at ph . , ( ) . - . m sodium formate, mm sodium acetate at ph . . ( ) . - . m sodium chloride, mm sodium acetate at ph . . the crystals were of varying quality but generally diffracted from . Å- . Å with inhouse x-ray source. the structure of this upa mutant and its complex with various inhibitors will provide a platform for rational upa inhibitor design. abstract in this work, the linear interaction energy (lie) method was used to calculate the binding free energies of hiv- integrase (in) and a series of dicaffeoyl -or digalloyl pyrroliding and furan derivatives inhibitors. the model of binding free energy prediction for homogeneous inhibitors of hiv- in has been obtained with a root-mean-square deviation (rmsd) of . kj/mol and estimated to be a precise model with good prediction capability. in addition, the probable binding mode of this series of inhibitors with hiv- in was proposed by using molecular docking and molecular dynamics (md) simulation methods. our results indicate that caffeoyl -or galloyl group of inhibitors have close interaction with a hiv- in conservative dde motif. a speci c non-competitive inhibitor of a small g protein/gef complex on the protective role of selenium and catechin in cadmium toxicity s. Özdemir , s. dursun , s. toplan , n. dariyerli , m. c. akyolcu istanbul university, cerrahpasa medical faculty, department of biophysics, turkey, istanbul university cerrahpasa medical faculty, department of physiology, turkey cadmium as heavy metal is toxic and carcinogenic for organisms. cadmium perform their effects on living organisms by accumulation in blood and various tissues. due to their accumulation in various tissues and in blood, tissue antioxidant enzyme systems are affected. the present study was planned to determine the possible protective roles of selenium and catechin against the toxic effects of considered heavy metals. the study has been performed in wistar albino type rats which divided into four groups as control and cadmium, cadmium+selenium, cadmium+catechin received groups. besides cadmium as heavy metal, selenium concentration determinations were performed in blood, liver and kidney tissues of each group of rats. in the same tissue samples besides lipid peroxidation measurements, glutathione, glutathione peroxidase and superokside dismutase enzyme activity determinations were also performed. the accumulation of heavy metals was determined in blood, liver and kidneys after cadmium administration during experimental period. in the tissue of experimental group animals there was an increased lipid peroxidation but decreased antioxidant enzyme activities were observed. while effects of selenium in decreased toxicity of cadmium have been detected, there was no statistically signicant effect of catechin observed. proposed that motors could dynamically cluster at the tip of tubes when they are individually attached to the membrane. we demonstrate, in a recently designed experimental system, the existence of an accumulation of motors allowing tube extraction. we determine the motor density along a tube by using uorescence intensity measurements. we also perform a theoretical analysis describing the dynamics of motors and tube growth. the only adjustable parameter is the motor binding rate onto microtubules, which we measure to be . +/- . s . in addition, we quantitatively determine, for a given membrane tension, the existence of a threshold in motor density on the vesicle above which nanotubes can be formed. we nd that the number of motors pulling a tube can range from four at threshold to a few tens away from it. the threshold in motor density (or in membrane tension at constant motor density) could be important for the understanding of membrane traf c regulation in cells. kinesin and dynein move a peroxisome in vivo: a tug-ofwar or coordinated movement? c. kural, p. r. selvin, k. hwajin, g. goshima, v. i. gelfand university of illinois at urbana-champaign, usa we have used fluorescence imaging with one nanometer accuracy (fiona) for analysis of organelle movement by conventional kinesin and cytoplasmic dynein in a cell. we can locate a green uorescence protein (gfp)-tagged peroxisome in cultured drosophila s cells to within . nanometer in . milliseconds, a -fold improvement in temporal resolution, suf cient to determine the average step size to be nanometers for both dynein and kinesin. furthermore, we nd that dynein and kinesin do not work against each other in vivo during peroxisome transport. rather, we nd that multiple kinesins or multiple dyneins work together, producing up to times the in vitro speed. engineering a bio-molecular walker h. jankevics, j. e. molloy division of physical biochemistry, mrc national institute of medical research, the ridgeway, mill hill nw aa, london, uk in this work we describe the design and development of a biomolecular walker based on the motile system found in certain ciliated protists. the motile system is driven by the binding of ca ions and in contrast to other commonly studied motor proteins is independent of atpase (amos et al., ) . the motor protein is a kda ca-binding protein called spasmin (maciejewski et al., ; itabashi et al., ) which belongs to the ef-hand family of calcium binding proteins called calmodulins. upon calcium binding, spasmin is thought to undergo a large conformational change as it binds its own target peptide and we wish to exploit this in order to create our own novel molecular walker.we have created a recombinant spasmin with sequence tags to enable speci c immobilization and various conjugate chemistries. cystein mutants have been introduced at speci c points in the protein to enable attachment of other small molecules, for example uorophores. we are now optimising protein expression and puri cation to maximise the yield of active protein on which we can perform the conjugate chemistries. we will characterize the structural changes using biophysical methods such as circular dichroism, analytical ultracentrifugation, electron microscopy, afm and total internal re ection uorescence spectroscopy on single molecules. the force generation in muscle arises from direct interaction of the two main protein components of the muscle, myosin and actin. the process is driven by the energy liberated from the hydrolysis of atp by myosin. the interaction is performed by cyclic interaction of myosin with atp and actin, and at least six intermediates are proposed for actomyosin atpase in solution. the powerful dsc technique allows the derivation of heat capacity of proteins as a function of temperature. from the deconvolution of the thermal unfolding patterns it is possible to characterize the structural domains of the motor protein. in this work we tried to approach the temperature-induced unfolding processes in different intermediate state of atp hydrolysis in striated muscle bres. we have extended the experiments to study the ber system prepared from psoas muscle of rabbit in rigor, strongly binding and weakly binding states of myosin to actin where the inorganic phosphate (p i ) was substituted by the phosphate analogue orthovanadate. the dsc transitions were analyzed in different buffer solutions (tris and mops) to get information about the temperature dependence of ph on the conformational changes. single kinesin motor proteins walking through the searchlight s. verbrugge, l. c. kapitein, e. j. peterman vrije universiteit, de boelelaan , hv, amsterdam, the netherlands the dimeric motor protein kinesin steps by a hand-over-hand mechanism. this means that the centre of mass moves with nm steps, while the two motor domains, the one after the other, move nm to the next binding site on the microtubule. the molecular details of what happens during a step are not fully understood, partly because of lack of time resolution in wide-eld, single-molecule uorescence experiments. we set out to develop an approach to study the motility of kinesin with a time resolution below a millisecond (a single step takes on the order of milliseconds). this approach allows us to look into the mechanochemistry and coupling of the two kinesin motor domains while they are stepping. our method is based on confocal microscopy and we study the uorescent properties of single labeled motors while they walk through the confocal laser spot. we present the experimental details of our approach and show our results on human kinesin constructs that are speci cally labeled in the tail. we show that our approach enables us to study the mechanism of kinesin with a much higher time resolution than what was achieved before with single-molecule uorescence experiments. movement of coupled single-headed kinesins analysed by a brownian-ratchet model . the analysis of this system is expected to provide insights into the mechanism underlying the motility of conventional double-headed kinesin, espetially the roles played by individual heads. we would like to clarify whether the experimentally observed behaviors that are supposed to be caused by a pair of single-headed kinesins can be explained by a simple brownianratchet model, which is successful in describing the motion of an unconventional single-headed kinesin kif a. our model consists of two brownian motors (ratchets) separated by a xed distance r. the velocity and other quantities of the coupled motors are calculated by solving the fokker-planck equation with various choices of r and other parameters. then, assuming a certain probability distribution of r associated with random attachment of kinesin heads on a bead in the experiment, the statistical properties of the motion of the coupled brownian motors are analysed. the force-velocity relation observed experimentally is found to be consistent with the present model with appropriate choices of the model parameters. the adequacy of the parameter choice needs to be con rmed by other experiments. optical trap with fast programmable feedback loop to study rotary molecular motors t. pilizota, f. bai, r. m. berry clarendon laboratory, univeristy of oxford an optical trap with back-focal plane detection and fast programmable feedback has been developed for the study of rotary molecular motors. a helium-neon laser ( nm) is used for position detection and a solid state bre laser ( nm, w cw) forms the trap. acousto-optic de ectors (aods) controlled by a digital signalling processing board are used to achive programmable feedback loops with exible control options and speeds up to khz. several modes of feedback are demonstrated, controlling both bead position (x,y) and angle (r, ). polystyrene beads or bead pairs can be held at set (x,y) or , and the set-point can be changed while the program is running. for example, feedback can be used to move a bead or a bead pair in a circle. results of using the system to study the bacterial agellar motor are presented. a dimeric -d lattice gas as model for molecular motors collective dynamics p. pierobon , t. franosch , e. frey ludwing-maximilian universitaet, münchen, germany, hahn meitner institut, berlin, germany, arnold sommerfeld center for theoretical physics, münchen, germany the transport of molecular motors along microtubules closely resemble the dynamics of a driven lattice gas of dimers without conservation of particles. the unidirectionality, asymmetry and stochasticity of the motion are encoded in the well studied totally asymmetric simple exclusion process (tasep). we extend the model to a more realistic one, including attachment and detachment kinetics and extended (dimeric) particles. we study the stationary phase diagram by means of monte carlo simulations combined with a continuum description (based on an extended mean eld theory). we also evaluate the domain wall theory nding out the effective potential con ning the phase interface into the bulk. a. e. wallin , j. lisal , r. tuma department of physical sciences, pobox , fin- , university of helsinki, finland, institute of biotechnology, university of helsinki, finland molecular motors often consist of two or more subunits that cooperate to convert chemical energy into mechanical motion. hexameric helicases and viral packaging atpases constitute a special class of molecular motors that translocate along nucleic acids. recent structural and spectroscopic characterization of these motors revealed that their enzymatic cooperativity does not result from cooperative binding [ - ]. in order to understand this new type of cooperativity we simulated the kinetics of a single hexameric motor by multiple coupled stochastic reactions using the gillespie algorithm [ ] . in contrast to analytical methods, our direct simulation allowed us to investigate the kinetics with an arbitrary model for cooperativity between the subunits. simulations on the kinetics of the hexameric rna packaging motor p from dsrna bacteriophage [ ] with different cooperativity mechanisms provided insight into the rnamediated cooperativity and yielded a sound theoretical basis for the interpretation of experimental results [ ]. the viral infectivity factor (vif) encoded by hiv- is a small basic protein that strongly modulates the viral replication and is required for pathogenicity. vif is packaged into hiv- particles through a strong interaction with genomic rna and is associated with viral nucleoprotein complexes. moreover vif acts during the early stages of the viral infection (capsid disassembly, reverse transcription) as well as during the late stages of virus replication (virus assembly and maturation of the virion). however the effect on early stages is probably a consequence of a defective assembly and / or virion maturation. understanding the rna-binding properties of vif would contribute to elucidate the role played by vif in the regulation of the genomic rna traf cking in the cytoplasm to unable ef cient packaging, and the prevention of cellular inhibitors from altering hiv- rna. in this context, we have characterised the interactions of recombinant vif with hiv- genomic rna by uorescence spectroscopy, and determined the af nity of the protein for synthetic rnas corresponding to various regions of hiv- genome. taken together our results demonstrate cooperative and speci c binding. in particular, we showed that vif has a high af nity for the 'untranslated region of hiv- genomic rna. s. bernacchi , e. ennifar , k. toth , p. walter , j. langowski , p. dumas cnrs upr -strasbourg (france), dkfz -heidelberg (germany) we have used the dimerization initiation site (dis) of hiv- genomic rna as a model to investigate hairpin-duplex interconversion by using a combination of uorescence, uv-melting, gel electrophoresis and x-ray crystallographic techniques. fluorescence studies with molecular beacons and crystallization experiments with -nucleotide dis fragments showed that the ratio of hairpin to duplex formed after annealing in water essentially depends on rna concentration, and not on cooling kinetics. with natural sequences able to form a loop-loop complex (or 'kissing complex'), concentrations as low as µm in strands are necessary to obtain a majority of the hairpin form. on the contrary, when kissing-complex formation was made impossible by mutation in the loop, a majority of hairpins was obtained even at µm in strands. this mutated sequence also showed that kissing-complex formation is not a prerequisite for an ef cient conversion to duplex in presence of salts. we proved that this happens through hairpins engaged in a cruciform intermediate, but not from free strands after hairpin melting. supporting this view, the very rst step of formation of such a cruciform intermediate could be trapped in a crystal structure. such a mechanism might be biologically signi cant beyond the strict eld of hiv- rna dynamics. generation of rna dimeric form of the human immunode ciency virus type (hiv- ) genome is important for the viral replication. the dimerization initiation site (dis) has been identi ed as a short sequence that can form a stem-loop structure with a selfcomplementary sequence in the loop and a bulge in the stem. a mer dis rna fragment, dis , spontaneously formed "loosedimer" and was converted into "tight-dimer" by supplement of nucleocapsid protein ncp . nmr chemical shift analysis for dis in the kissing-loop and extended-duplex dimers revealed that three dimensional structures of the stem-bulge-stem region were similar between the two types of dimers. therefore, we determined the solution structures of two shorter rna molecules corresponding to the loop-stem region and the stem-bulge-stem region of dis , and the solution structures of dis in the kissing-loop and extendedduplex dimers were determined by combining the parts of structures. the mechanism of conformational conversion will be discussed based on the solution structures and the molecular dynamics analysis. nmr and molecular modelling studies of an rna hairpin containing a g-rich hexaloop the mrna of the pgy /mdr gene encoding the transmembrane p-glycoprotein (p-gp) contains a hairpin that is the target of antisense oligonucleotides, suppressing the p-gp function of multidrug resistance. the solution conformation of this hairpin constituted by the '(gggaug) ' loop closed by a g-u mismatch containing stem is studied by nmr and molecular dynamics in explicit solvent. special attention is given on the sugar and the backbone conformations and the hexaloop intrinsic properties of these two components are carefully investigated. the stem structures obtained by molecular dynamics with and without nmr constraints converge to the same a-type double helix. the wobble g-u mismatch moderately perturbs the overall conformation, despite of c '-endo sugars and unusual backbone conformations located between the mismatch and the loop. in the hexaloop part, the sugar puckers are in majority in c '-endo conformations, probably to extend the strand with the help of unusual backbone angles conformations. the loop appears stabilized by one hydrogen bond and stacking interactions. thus, from the ' to the '-ends, the four purine bases ggga are stacked together, then a u-turn like is observed, and nally, u stacks on the last g that remains rather far from the stem. nmr and molecular modelling studies of an rna hairpin containing a g-rich hexaloop the mrna of the pgy /mdr gene encoding the transmembrane p-glycoprotein (p-gp) contains a hairpin that is the target of antisense oligonucleotides, suppressing the p-gp function of multidrug resistance. the solution conformation of this hairpin constituted by the '(gggaug) ' loop closed by a g-u mismatch containing stem is studied by nmr and molecular dynamics in explicit solvent. special attention is given on the sugar and the backbone conformations and the hexaloop intrinsic properties of these two components are carefully investigated. the stem structures obtained by molecular dynamics with and without nmr constraints converge to the same a-type double helix. the wobble g-u mismatch moderately perturbs the overall conformation, despite of c '-endo sugars and unusual backbone conformations located between the mismatch and the loop. in the hexaloop part, the sugar puckers are in majority in c '-endo conformations, probably to extend the strand with the help of unusual backbone angles conformations. the loop appears stabilized by one hydrogen bond and stacking interactions. thus, from the ' to the '-ends, the four purine bases ggga are stacked together, then a u-turn like is observed, and nally, u stacks on the last g that remains rather far from the stem. aminoglycoside binding to hiv- dis kissing-loop complex: from crystals to cells e. ennifar , j.-c. paillart , a. bodlenner , p. pale , r. marquet , p. dumas cnrs upr , strasbourg -france, cnrs/université louis pasteur strasbourg umr , strasbourg -france all retroviral genomes consist in two homologous single stranded rnas. dimerization is an essential step for viral replication. hiv- dimerization initiation site (dis) is a strongly conserved stem-loop in the ' leader region of the genomic rna. it was shown in vivo that alteration of the dis dramatically reduces viral infectivity. we have previously solved crystal structures of the dis kissing-loop complex. analysis of these structures revealed an unexpected resemblance between the dis kissing-loop and the s ribosomal aminoacyl-trna site (a-site), which is the target of aminoglycoside antibiotics. we have shown that some aminoglycosides specifically bind to the dis kissing-loop complex with an af nity and geometry similar to that observed in the a-site. in agreement with these previous results, we have now solved highresolution crystal structures of the dis kissing-loop complex bound to four aminoglycosides. these structures show that, as expected, two aminoglycosides are bound per kissing-loop complex. importantly, the binding is observed not only in vitro on large hiv- genomic rna fragments, but also on infected cells. moreover, we showed that some of these aminoglycosides stabilize the kissingloop rna dimer, which is consistent with the observation in crystal structures of numerous direct and water-mediated drug-rna contacts. these structures are currently used as starting points for designing potential new drugs targeted against the viral rna. modeling the long range entropy of rna: w. k. dawson , k. fujiwara , k. yamamoto , g. kawai chiba institute of technology, - - tsudanuma, narashino, chiba, japan, international medical center of japan, - - toyama, shinjuku-ku, tokyo, japan non-coding rna appears to make up a large part of the human genome. a reliable rna structure prediction program is needed to understand the structure of this non-coding rna. we recently developed a new way to model the long range entropy in rna and applied it to rna secondary structure prediction. in some of instances, the new approach is able to achieve far better predictions than the state of the art secondary structure programs even given exactly the same parameters. predictions using this method tend to show distributions that are funnel shaped. a new and important parameter in these calculations is the persistence length (a measure of the correlation and exibility of the rna). (url: http://www.rna.it-chiba.ac.jp/ vsfold/vsfold /) this new approach has now been extended to prediction of pseudoknots. the method is a heuristic wherein the hierarchical folding hypothesis is used to nd the pseudoknots as the rna secondary structure is folding, and corrections to that secondary structure are made to accommodate the pseudoknot. it is able to do these searches in roughly n^ time. the model is consistent with the hierarchical hypothesis and it is possible to estimate rna folding times that are of the correct order of magnitude using this model. with further adaptations to account for the size, shape and variablity of amino acid residues (hydrophobicity etc.), the model also appears to be transferable to protein folding problems. a. v. melkikh ural state technical university, ekaterinburg, russia a model of the genome as a gene network capable of receiving information about the environment and performing some operations on genes has been considered. the evolution rate of replicators for the mechanism of random mutations has been estimated. it was shown that the evolution rate under random actions is negligibly small for real dimensions of genomes of replicators [ ]. it was inferred that only a deterministic mechanism of the evolution can explain the known evolution rate of replicators. a deterministic model of the evolution has been proposed. the basic principles of this model include: ) information about the replicators evolution is encoded in the conformational states of proteins; ) the conformational language of proteins is translated into the language of nucleotide sequences during the evolution; ) the structure of genes is controlled such that the transition to a nearest free ecological niche takes a minimum time (at a preset restriction on the control). transfer rnas are synthesized as part of longer primary transcripts that require processing of both their ' and ' extremities in every living organism known. the ' side is matured by the quasiuniversally conserved endonucleolytic ribozyme, rnase p, while removal of the ' tails can be either exonucleolytic or endonucleolytic. the endonucleolytic pathway is catalysed by an enzyme known as rnase z. rnase z cleaves precursor trnas immediately after the discriminator base in most cases, yielding a trna primed for addition of the cca motif by nucleotidyl transferase. rnase z is found in the vast majority eukaryotes and archaea and in about half of the sequenced bacteria. it is often essential for growth and mutations in one of the two genes encoding rnase z (elac ) have been linked with prostate cancer in man. in this poster we present the crystal structure of bacillus subtilis rnase z at . Å resolution ( ) resolved by mad method and propose a model for trna recognition and cleavage. the structure explains the allosteric properties of the enzyme and also sheds light on the mechanisms of inhibition.it also highlights the extraordinary adaptability of the metallohydrolase domain of the b-lactamase family for the hydrolysis of covalent bonds. ( )i. most rnas undergo several steps of post-transcriptional modi cation before carrying out their assigned functions. one of the major modi cations is the splicing process, by which non-coding introns are removed from the coding exons. splicing can be performed by autocatalytic, self-splicing introns (e.g. group ii introns), i.e. catalytic rna or ribozymes. group ii introns, which occur in bacterial genomes and in organellar genes of plants, funghi and lower eukaryotes, consist of a conserved set of six domains. domain recognizes the '-exon through a - base pairing interaction formed by two regions within the intron (exon binding sites, ebs and ebs ) and the last - nucleotides of the '-exon (intron binding sites, ibs and ibs ). as the correct recognition of ibs by ebs is crucial for a successful splicing event we are investigating the structural and metal ion requirements of this part by various spectroscopic techniques, e.g. nmr. our data shows that the hairpin including ebs consists of a helical region followed by an unstructured single stranded part, which is ready for splice site recognition. the results of the structure analysis will be presented. financial support by boehringer ingelheim fonds (fellowship to d. k.) and the swiss national science foundation (snf-förderungsprofessur to r. k. o. s.) is gratefully acknowledged. structural basis for the antigene and antisense properties of modi ed dna:dna and rna:dna duplexes e. c. m. juan , t. kurihara , j. kondo , t. ito , y. ueno , a. matsuda , a. takenaka graduate school of bioscience and biotechnology, tokyo institute of technology, graduate school of pharmaceutical sciences, hokkaido university, faculty of engineering, gifu university oligonucleotides containing polyamines are currently being evaluated as potential antigene and antisense compounds. those with -(n-aminohexyl)carbamoyl- '-deoxyuridine ( n u) and its '-omethyl derivative ( n u m ) exhibit improved nuclease resistance and form stable duplexes with their dna and rna targets. x-ray structures of these duplexes have shown good correlation between the conformational changes and the observed chemotherapeutic properties. the amide groups of the modi ed uracil bases form six-membered rings through the intra-molecular nh-o hydrogen bonds, so that the aminohexyl chains protrude into the major grooves. some of the terminal ammonium groups are involved in intra-duplex interactions with phosphate oxygen anions, whereas the others interact with those of the adjacent duplex. such interactions contribute to the stability of duplex formation. the '-o-methyl modi cation in n u m shifts the ribose ring toward the c '-endo conformation and in uences duplex stability. observed changes in the dimensions of the minor grooves and in the hydration structures are also well correlated to nuclease resistance and duplex stability. group ii intron ribozymes catalyze selfsplicing in bacterial genomes as well as in organellar genes of lower eucaryotes. for correct structure and function these ribozymes need speci c concentrations of monovalent and divalent cations such as k and mg . most of these ions are used for charge screening, but some are also bound to distinct sites ful lling various speci c tasks. the conserved secondary structure of group ii intron ribozymes consists of six domains grouped around a central wheel. here the focus is set on domain (d ) of the yeast mitochondrial intron ai , a hairpin of nucleotides, which is crucial for catalytic activity. the -nucleotide bulge in d is known to be exible and acts as a metal ion binding platform. we have investigated the binding of different metal ions (mg , ca , mn , cd ) to this platform by uorescence spectroscopy. for this the bulge site adenosine in d was replaced by the uorescent nucleotide base analogue aminopurine ( ap). the binding data ts to an equation describing a binding to a single class of sites. the titration experiments not only reveal different dissociation constants for the tested metal ions but also indicate different effects on the bulge structure. financial support by the swiss national science foundation (snf-förderungsprofessur to r.k.o.s) is gratefully acknowledged. modi ed nucleosides and across the anticodon loop interactions in trna u. b. sonavane, k. d. sonawane, r. p. tiwari national chemical laboratory, pune , india in several interesting trna molecules, the ( th ) as well as the ( th ) nucleoside are hyper modi ed. as an example, unique hypermodi ed nucleosides mcm s u and ms t a are crucial in human trna lys , which acts as a primer in hiv replication. modi ed nucleosides may facilitate or hinder across the loop interactions. large substituents in th and th modi ed nucleosides if oriented suitably may also interact with each other. across the loop interactions may lead to unconventional anticodon loop structures also affecting exibility of the anticodon loop. this may restrict or enlarge synonymous codon choice and decoding during protein biosynthesis. except for trna asn (with interacting q and t a ), our studies show conventional 'open' loop structure -free of across the loop interactions, for a number of interesting trna anticodon loops with diverse hyper modi ed nucleosides at both of these locations. molecular dynamics simulations of hydrated anticodon arm of trna asn show persisting interaction involving the diol group of q and carbonyl group of ureido linkage in t a . additionally, the hoogsteen edge of th adenine base participates in hydrogen bonding with watson -crick edge of rd base and thus contributes to unique loop structure of trna asn . resulting suboptimal q:c base pairing leads to unbiased reading of u or c as the third codon letter. absence of queuosine modi cation, q happens to be also associated with uncontrolled rapid proliferation of cells and malignant growth. structural properties of ctg/cag repeats, and preliminary x-ray analysis of cug repeats y. sato, k. kimura, a. takénaka graduate school of bioscience and biotechnology, tokyo institute of technology, yokohama, japan. the human genome contains so many different types of repetitive sequences. some of them are tandem repeats of trinucleotides. their unusual expansions cause genetic diseases such as type myotonic dystrophy (dm ) and huntington's disease (hd), the unit sequences being ctg and cag, respectively. the numbers of repeats of the two complementary sequences change independently during dna replication or repair. the direct origin of dm is, however, the transcribed rna fragments with cug repeats, which forms a speci c structure and inhibits other protein syntheses. in the present study, structural versatilities of such dna and rna fragments has been examined. native pages of (cug) n show that the hairpin structure with even number is more stable than that with odd number. this difference might be ascribed to the structural difference at the hairpin head. the pages also show that duplex formation is dependent on coexisting cationic species and their concentration. crystal data of (cug) (a=b= . , c= . Å, and the space group r ) suggest that the asymmetric unit contains the rna fragment. an approximate crystal structure solved by molecular replacement techniques at . Å resolution shows that the rna fragments form a duplex similar to an a-form rna. context-dependent selection of promoter in a natural selection-type evolution reactor h. nagayasu, y. ageno, x. t. ma, y. husimi saitama university, saitama, japan using an isothermal ampli cation of hairpin dna/rna (developed by g.joyce), we drove a natural selection-type evolution reactor taking the speci c growth rate as the tness. we used hiv- rt and thermot rnap at c. starting from the random promoter pool, we selected the strongest promoter at c, which was separated by hamming distance from the strongest promoter at c. the latter was found to be identical to the natural t promoter. when we used a simple random pool, the selection process showed one-step convergence. when we used a random pool with a speci c short sequence at the upstream anking region of the random region, we observed an evolution process as convergencedivergence-convergence of the promoter sequence, driven by deletion of the speci c short sequence. this context-dependent selection was found to come through a neutral path, judged from the tness measurement. intronic sirna and mirna, and dna methylation gif sur yvette, france. abnormal activation of small g proteins is involved in several human diseases. small g proteins are activated by gdp/gtp exchange, which is stimulated by their guanine nucleotide exchange factors (gefs). thus, small g protein/gef complexes appear as emerging targets for interrupting signalling networks regulated by small g proteins in pathological contexts. the activation of small g proteins of the arf family is initiated by exchange factors which carry a sec catalytic domain stimulating the dissociation of the bound gdp nucleotide. the structure of the arf -gdp-arno reaction intermediate, trapped by a mutation of the catalytic glutamate, was recently solved by x-ray crystallography (pdb code: r s) acknowledgements: the work was supported by contract k- j. q. yin, f. chen, y. tan, t. gu institute of biophysics, chinese academic sciences, beijing, china sirna/mirna can ef ciently induce mrna cleavage or translational repression at the posttranscriptional level in a sequencespeci c manner. recently, it has been shown that these small rnas guide genome modi cation in mammalian cells. however, their ability to direct cognate dna methylation has been con rmed so far only in plants, and their biogenesis, functions, and modes of silencing genes are yet elusive. here, we report that small rnas derived from intron regions of some genes can target homologous dna sequence in promoter, 'utr or 'utr regions of genes in different human tumor cells. surprisingly, we also discovered that endogenous sirnas from introns of genes possessed a large number of target mrnas by using bioinformatics, and con rmed their existence in human cells with northern blot analysis. intronic small rnas generated by sliceosomes can form mature mirnas or sirnas through the processing of drosha and /or dicer. rt-pcr analysis indicated that vector-based small rna repressed expression of homologous genes at the transcriptional and/or translational levels. western blotting demonstrated that the expression of some proteins was greatly reduced or completely inhibited owing to promoter methylation. these ndings reveal that the expression of some genes can incredibly control cell activities at both protein and rna levels. our results also suggest that these small rnas may regulate gene expression in different modes of action. role of stacking in speci c recognition of capped rna by the cbc protein the stacking interaction involving -methylguanine moiety of mrna ' terminal cap (m g) and aromatic amino acid side chains is a common feature of all known cap-binding proteins. the crystal structures of the human cap binding complex (cbc) showed its induced folding upon m gpppg cap analogue binding. stabilization of the cbc-m gpppg complex by sandwich stacking of m g in between y and y is additionally enhanced by stacking of the second base of capped mrna with y . gibbs free energy of the association of various cbc mutants with the synthetic cap analogues, m gpppg, m gpppu, and m gtp, has been determined by uorimetric titration. preference of the wild type (wt) cbc for the dinucleotide analogues is also observed for the y a mutant, with the energy loss of . - . kcal/mol. however, all proteins with the mutated second stacking partner y prefer to bind m gtp compared with m gpppg. the binding of m gtp to the y a mutant is only . kcal/mol less favourable compared with the wt cbc. these divergences may be ascribed to smaller entropic costs of conformational rearrangement of cbc in the case of a smaller ligand, which can nd more favourable contacts when its second part is not ef ciently 'constrained' by stacking with y . supported by kbn p a annealing of the tar dna hairpin to a complementary tar rna hairpin, resulting in the formation of an extended duplex, is an essential step in the minus-strand transfer process of hiv- reverse transcription. in this work, we use gel-mobility-shift analysis to follow the kinetics of this reaction in the absence or presence of hiv- nc prepared by solid-phase peptide synthesis. to elucidate the reaction pathway, we use either the complete -nt tar hairpins or truncated -to -nt minihelices (mini-tar) derived from the top part (i.e. hairpin loop) of tar. assays were also carried out with mutant tar constructs. the annealing kinetics were studied systematically as a function of dna concentration and temperature. we show that the annealing initiates through a weak loop-loop kissing interaction, followed by a much slower conversion step, which results in formation of the extended duplex. nc facilitates both reaction steps, resulting in the overall -fold and -fold rate enhancement for mini-tar and tar annealing, respectively. we show that the kissing step is facilitated by the nc-induced nucleic acid aggregation, which is more pronounced for the longer tar hairpins. at the same time, the conversion steps in tar and mini-tar appear to be very similar and are similarly facilitated by nc - -fold. the later effect relays on the ability of nc to destabilize nucleic acid duplexes, and is equivalent to destabilization of a few base pairs required for the conversion initiation. linking of the n-terminus of a peptide to its encoding mrna s. ueno, h. arai, y. husimi department of functional materials science, saitama university, saitama, japanin evolutionary protein engineering, in vitro selection using a cellfree translation system has advantages of large library size and also of applicability to cytotoxic protein. although many in vitro protein selection techniques such as in vitro virus, ribosome display are developed, most of these are the techniques that link the genotype molecule to the peptide at its cterminus. we developed a method to link the genotype molecules to the nterminus of its encoding peptide. the mrna has dna-linker hybridize region, translation enhancer, initiation codon, single codon, amber stop codon, n-terminus sequence in gfp gene, his-tag and ochre stop codon. the mrna is also linked at '-terminus to sup trna via spacer and the speci c amino acid. the mrna is translated in the cell-free translation system. thus, c-terminus of the protein becomes free. moreover, in this system, the free protein of the full length is never generated. for this reason, the problem in the conventional technique, that is, the competition between the protein displayed on the genotype molecule and the free protein is eliminated. we succeeded to turn one round of the " life cycle " of the in vitro virus, judged by his-tag selection. key: cord- - y kcwu authors: lan, tammy c. t.; allan, matthew f.; malsick, lauren e.; khandwala, stuti; nyeo, sherry s. y.; bathe, mark; griffiths, anthony; rouskin, silvi title: structure of the full sars-cov- rna genome in infected cells date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: y kcwu sars-cov- is a betacoronavirus with a single-stranded, positive-sense, -kilobase rna genome responsible for the ongoing covid- pandemic. currently, there are no antiviral drugs or vaccines with proven efficacy, and development of these treatments are hampered by our limited understanding of the molecular and structural biology of the virus. like many other rna viruses, rna structures in coronaviruses regulate gene expression and are crucial for viral replication. although genome and transcriptome data were recently reported, there is to date little experimental data on predicted rna structures in sars-cov- and most putative regulatory sequences are uncharacterized. here we report the secondary structure of the entire sars-cov- genome in infected cells at single nucleotide resolution using dimethyl sulfate mutational profiling with sequencing (dms-mapseq). our results reveal previously undescribed structures within critical regulatory elements such as the genomic transcription-regulating sequences (trss). contrary to previous studies, our in-cell data show that the structure of the frameshift element, which is a major drug target, is drastically different from prevailing in vitro models. the genomic structure detailed here lays the groundwork for coronavirus rna biology and will guide the design of sars-cov- rna-based therapeutics. severe acute respiratory syndrome coronavirus (sars-cov- ) is the causative agent of the current coronavirus disease , recently declared a global pandemic by the world health organization (who). sars-cov- is an enveloped virus belonging to the genus betacoronavirus, which also includes sars-cov, the virus responsible for the sars outbreak, and middle east respiratory syndrome coronavirus (mers-cov), the virus responsible for the mers outbreak. despite the devastating effects these viruses have had on public health and the economy, currently no effective antivirals treatment or vaccines exist. there is therefore an urgent need to understand their unique rna biology and develop new therapeutics against this class of viruses. coronaviruses (covs) have single-stranded and positive-sense genomes that are the largest of all known rna viruses ( - kb) (masters, ) . previous studies on coronavirus structures have focused on several conserved regions that are important for viral replication. for several of these regions, such as the ' utr, the ' utr, and the frameshift element (fse), structures have been predicted computationally with supportive experimental data from rnase probing and nuclear magnetic resonance (nmr) spectroscopy (plant et al., ; yang and leibowitz, ) . functional studies have revealed the importance of their secondary structures for viral transcription and replication (brierley, digard and inglis, ; liu et al., ; li et al., ; yang and leibowitz, ) . the fse is located near the boundary of orf a and orf b and causes the ribosome to "slip" and shift register by - nt in order to bypass a canonical stop codon at the end of orf a and translate the viral rna-dependent rna polymerase (rdrp) and other proteins in orf b (brierley et al., ; plant et al., ) . studies on multiple viruses have shown that an optimal frameshifting rate is critical, and small differences in percentage of frameshifting lead to dramatic differences in genomic rna production and infection dose (plant et al., ) . therefore, the fse has emerged as a major drug target for binding of small molecules that can influence the rate of ribosome slippage and be used as a treatment against sars-cov- . the structure of the fse of sars-cov (whose sequence differs from the sars-cov- fse by just one nucleotide), was solved by nmr to be a -stem pseudoknot (plant et al., ; rangan, zheludev and das, ) . the prevailing mechanism is that the -stem pseudoknot causes the ribosome to pause at the slippery sequence and backtrack by nt to release mechanical tension (plant and dinman, ) . however, the frameshifting rate of sars-cov- during infection is unknown, and none of the rna structure models for the fse has been validated in cells. over the past ten years, the drawbacks of in silico models have been largely overcome by the development of genome-wide strategies to chemically probe rna structure in cells. the most popular chemical probes are dimethyl sulfate (dms) (rouskin et al., ) , shape (siegfried et al., ) , and icshape (spitale et al., ) , all of which provide critical in vivo measurements that are used by computational algorithms to generate accurate models of rna structures in vivo. in this study, we perform dms mutational profiling with sequencing (dms-mapseq) (zubradt et al., ) on infected vero cells to generate the first experimentally determined genome-wide secondary structure of sars-cov- . our results reveal major differences with in silico predictions and highlight the physiological structures of known functional elements. our work provides experimental data on the structural biology of rna viruses and will inform efforts in the development of rna-based diagnostics and therapeutics for sars-cov- . to determine the intracellular genome-wide structure of sars-cov- , we added dimethyl sulfate (dms) to infected vero cells and performed mutational profiling with sequencing (dms-mapseq) (zubradt et al., ) (figure a) . dms rapidly and specifically modifies unpaired adenines and cytosines in vivo at their watson-crick faces. our results were highly reproducible between independent biological replicates ( r = . ; figure b ). combined, a total of ~ . million paired reads mapped to the coronavirus genome ( figure c ), representing ~ % of total cellular rna (post ribosomal rna depletion). this large fraction of coronavirus reads from total intracellular rna is consistent with previous literature using sars-cov- infected vero cells (kim et al., ) . dms treated samples had high signal to noise ratio, with adenines and cytosines having a mutation rate ~ -fold higher than the background (guanines and uracils). in contrast, in untreated samples the mutation rate on all four bases ( . %) was slightly lower than previously reported average sequencing error rates of . % (pfeiffer et al., ) ( figure d ). we used the dms-mapseq data as constraints in rnastructure (mathews, ) to fold the entire sars-cov- genomic rna (supplementary figure ) . we first examined the results at the nt ' utr ( figure e ), one of the best studied regions in the coronavirus genome (yang and leibowitz, ; madhugiri et al., ) . the ' utrs of multiple coronaviruses have been characterized extensively in terms of their structures and roles in viral replication. in agreement with previously published structures of this highly conserved region (yang and leibowitz, ; madhugiri et al., ) , we found five stem loops (sl - ) within the sars-cov- ' utr and three stem loops (sl - ) near the beginning of orf a. stem loop (sl ) is vital for viral replication. a previous study on murine hepatitis virus (mhv) found that mutations that destabilize the upper portion (near the loop) of sl or stabilize the lower portion (near the base) prevent viral replication (li et al., ) . in addition, viable viruses with a single-base deletion in the lower portion also had mutations in the ' utr. together, these observations suggested that the lower portion of sl must be able to unfold and likely interacts with the ' utr to assist in sgrna transcription (li et al., ) . in our structure, the lower portion of sl has low dms reactivity, consistent with a pairing between these bases either within the stem or with the ' utr. further work is needed to test a potential alternative structure for sl that would involve long range interactions between the ' and the ' utrs of sars-cov- . the most evolutionarily conserved secondary structure in the ' utr is stem loop (sl ) and the structure of the mhv sl has been solved by nmr (liu et al., ) . the sequences of sars-cov- and mhv sl are identical, and our in-cell model of sl is consistent with the nmr structure, with every base in the stem showing low dms reactivity. although c within the loop of sl has low reactivity, this is explained by the nmr structure, in which c and g (as numbered here) are paired. interestingly, the secondary structure but not the sequence of the stem was shown to be essential for replication: in a previous study, disruption of sl by three mutations on either side of the stem prevented mhv-a replication, but compensatory mutations restored nearly wildtype growth (liu et al., ) . mutants of mhv-a with non-functional sl were able to transcribe minus-strand genomic rna in cells but not minus-strand subgenomic rnas, suggesting that sl plays a role in discontinuous transcription. disruption of sl also reduced the rate of in vitro translation of a downstream luciferase reporter (liu et al., ) . to the best of our knowledge, the precise mechanism through which sl is required for viral replication remains unknown. stem loop (sl ) contains the leader trs (trs-l) involved in discontinuous transcription (yang and leibowitz, ) , the structure and function of which will be discussed in figure . stem loop (sl ) is a bulged stem loop downstream of trs-l. a previous study in mhv found that sl was required for sgrna synthesis and that deleting the entire sl was lethal (yang et al., ) . however, mutation or deletion of either the lower (sl a) or upper (sl b) part of sl merely impairs replication, with disruption of sl a causing greater impairment (yang et al., ) . in addition, replacing the entire sl with a shorter, unrelated sequence that was predicted to fold into a stable stem loop impaired mhv growth but was not lethal (yang et al., ) . based on these results, yang et al. proposed that discontinuous transcription requires proper spacing between the stem loops upstream and downstream of the trs, and sl maintains this space (yang et al., ) . sl also contains the start codon of a short open reading frame (nine codons in sars-cov- ) known as the "upstream" orf (uorf) (madhugiri et al., ) . in a study on mhv, disruption of the uorf start codon increased translation of orf a and modestly decreased viral replication (madhugiri et al., ) . stem loop (sl ) contains four branches (sl , sl a, sl b, sl c), and its structure in mhv-a was predicted using chemical probing with shape (yang and leibowitz, ) . disruption of sl c has been shown to prevent replication of viral defective interfering particles (yang and leibowitz, ) but not of whole mhv . sl contains the start codon of orf a, although in different coronaviruses the start codon can be located in sl a, sl b, or the stem ' of sl c (yang and leibowitz, ) . our data support a three-branched sl for sars-cov- , similar to previous models of sars-cov (yang and leibowitz, ) and sars-cov- (rangan, zheludev and das, ) in which the start codon is located in the main sl stem, just downstream of sl c. stem loops , , and (sl - ) lie downstream of the ' utr, within the coding sequence of nsp . the structures of these stem loops vary among betacoronaviruses, with sars-cov predicted to have all three stem loops, mhv only sl and sl , and bovine coronavirus (bcov) a structure in which sl and sl are branches of another stem . as predicted for sars-cov, our model also has all three stem loops, though this contrasts with an in silico model of sars-cov- that features three short stem loops in place of sl (rangan, zheludev and das, ) . mutations of sl that disrupt its secondary structure but preserve the amino acid sequence of nsp were not lethal in mhv, suggesting that sl is not essential to broadly locate regions of potential interest in the sars-cov- genome, we identified all structured and accessible regions, which we define as stretches of at least consecutive paired bases or consecutive unpaired bases, respectively ( figure a ). structured regions could be functional elements of the genome that are potential targets of therapeutics. we identify structured regions covering a total of , bases ( . % of the genome). these regions include structures with known functions, such as sl in the ' utr and a bulged stem loop in the ' utr (yang and leibowitz, ) , as well as many structures whose functions are unknown. the longest stretch of consecutive paired bases is nt (positions , - , ) and falls within a previously uncharacterized set of two bulged stem loops in orf a. these results provide a starting point for characterizing additional functional structures in the viral life cycle. accessible regions in viral genomes are potential targets of antisense oligonucleotide therapeutics (eckardt, romby and sczakiel, ; ding, ; rangan, zheludev and das, ) through the mechanism of rna interference (wilson and doudna, ) . we identify accessible regions covering , bases ( % of the entire genome). as there are such regions within orf-n, which is present in every sgrna, every sgrna may be targetable with antisense oligos. additionally, every open reading frame except orf-e contains at least one of these predicted accessible regions, raising the possibility of targeting individual sgrnas. at a more stringent threshold of consecutive nucleotides predicted to be unpaired, we find accessible regions, one of which is located within orf-n ( nt, positions , - , ) and hence in every genomic and subgenomic rna. the two longest stretches of consecutive predicted unpaired bases are both nt long and occupy positions , - , (within orf a) and , - , (within orf-s). these regions may offer multiple binding sites for antisense oligonucleotides. as the transcription-regulating sequences (trss) are necessary for the synthesis of sgrnas, we analyzed our structural models of the leader trs (trs-l) and the nine body trss (trs-b). the leader trs (trs-l) is the central component of the ' utr involved in discontinuous transcription (sola et al., ) . in silico models for several alpha and betacoronaviruses variously place trs-l in stem loop (sl ) or in an unpaired stretch of nucleotides (liu et al., ; yang and leibowitz, ) . the trs-l of sars-cov and of sars-cov- was predicted to lie in the ' side of the stem of sl , which is consistent with our in-cell model (liu et al., ; yang and leibowitz, ; rangan, zheludev and das, ) . in our data, the stem of sl contains two bases with medium reactivity ( figure b ), which suggests that sl transitions between folded and unfolded states, as is hypothesized for the alphacoronavirus transmissible gastroenteritis virus (tgev) (madhugiri et al., ) . of the nine body trss, we find that seven (all but the trss of orf a and orf b) lie within a stem loop. of these, all but one trs (n) place the core sequence on the ' side of the stem. four body trss (m, orf , orf , and n) are predicted to lie in stem loops with two or three bulges, with the core sequence spanning one of the internal bulges. the other three structured body trss (s, orf a, and e) lie in stem loops without bulges, with the final paired base in the ' side of the stem contained in the core sequence. strikingly, the entire core sequence is paired in two body trss (s and m), and partially exposed in a loop or bulge in the other five ( figure b ). we evaluated the robustness of our in-cell data derived genome-wide model by varying two critical rna folding parameters used by rnastructure: ) the maximum allowed distance for base pairing and ) the threshold for dms signal normalization. a previous in silico approach for folding rna found that limiting base pairs to be to nt apart was optimal to avoid overpredicting structured regions (lange et al., ) . however, some rna viruses contain known essential structures wherein bases over nt apart are paired (e.g. the rev response element in hiv- spans approximately nt (watts et al., ) ). we therefore varied the maximum distance (md) allowed for base pairing from nt to nt. we computed the agreement between the resulting structures using a modified version of the fowlkes-mallows index (fowlkes and mallows, ) that compares base pairing partners as well as unpaired bases (methods). overall, there was high agreement while varying the md from nt to nt, suggesting that long-distance (i.e. > nt) interactions across the sars-cov- rna have a small effect on the identity of local structures. the genome structure folded with an md of nt was . % identical to the structure with an md of nt, and in the latter structure only . % of base pairs spanned > nt. next, we proceeded with the md limit of nt and tested two different dms signal thresholds that normalize reactivity to either the median of the top % or top % of the most reactive bases. we found that the structure models produced with the two normalization approaches were highly similar, with . % identity ( figure a ). thus, within the ranges that we tested, our genome-wide data-derived model was robust to variation in the parameters of rnastructure (mathews, ) . we proceeded with the whole genome structure modelled with a md of nt and a dms signal normalization of % for further analysis (supplementary figure) . previous studies that computationally predicted genome-wide sars-cov- rna structures used ) rnaz, a thermodynamic-based model that additionally takes sequence alignment and considers base pairing conservation (gruber et al., ; rangan, zheludev and das, ) , and ) contrafold, which predicts rna secondary structures without physics-based models and instead uses learned parameters based on known structures (do, woods and batzoglou, ) . these recent studies predicted structures with rnaz with lengths ranging from to nt, and structures with contrafold with lengths ranging from to nt (rangan, zheludev and das, ) . for each of these structures, we computed the agreement between the different models ( figure b ). we report the agreement using the mfmi while either excluding external bases pairs or including these pairs (methods). as expected, agreement with the structures from purely computational prediction is higher when excluding external base pairs (average . % for rnaz, . % for contrafold) than when including them (average . % for rnaz, . % for contrafold). since our goal is to compare the overall similarity of two structures, we chose the inclusion of external base pairs as the more accurate metric for comparing the structures. our predictions overall agreed more with those from rnaz (mean . %, median . %) than contrafold (mean . %, median . %). we report the agreement between our structure and the rnaz structures across the entire genome ( figure c ). most structures are to % identical, with several short regions that disagree substantially. consistency of our in-cell structure models. agreement is given between our structure models predicted using a maximum distance limit of nt and nt between paired bases at % signal normalization and between our predictions using % and % dms normalization at nt maximum allowed base pair distance. (b) agreement of our structure model with all predicted structures from rnaz and contrafold. agreement is given for both excluding and including external base pairs. (c) agreement of our structure with a previous model from rnaz across the genome. at positions for which multiple rnaz model exists, the average agreement with all models is given. (d) agreement of our model with rnaz predicted structures with the three highest p-values in regions with previously unannotated structures. (e) agreement of our model with contrafold predicted structures with the five highest maximum expected accuracies in evolutionarily conserved regions. (f) agreement of our trs structure models to rnaz predicted structures. for trss for which multiple rnaz models exist, agreement with each prediction is shown. in addition, we computed the similarity of our model compared to the structures with the three highest p-values predicted with rnaz that do not overlap known structures in the rfam database (kalvari et al., ; rangan, zheludev and das, ) (figure d ). we noted that in all three cases, the structure at the center of the window was nearly identical to ours, and most of the disagreements arose at the edges, presumably due to the effects of the windows from rnaz being limited to nt. of the five structures predicted with contrafold that had the largest maximum expected accuracies, our agreement ranged from . % to . %, well above the genome-wide mean ( . %), suggesting that these structures are indeed more accurate than the average contrafold structure ( figure e ). finally, we compared the structures at the trs elements to those predicted by rnaz (rangan, zheludev and das, ) (figure f ). to remove the effects of external base pairs, we focused only on the complete structural element (e.g. a stem loop) in which the trs was located. rnaz predicted structure for four trss. our model for trs-l was identical to the first predicted window from rnaz but differed significantly ( . % agreement) from the next prediction of the same trs-l element within a different folding window, indicating that the choice of folding window can have a large effect on the rnaz structure model. for the other three trs elements for which rnaz predicted at least one structure for, our agreement ranges from . % to . %, above the genome-wide average of . %, lending support to both models. the frameshift element (fse) causes the ribosome to slip and shift register by - nt in order to bypass a canonical stop codon and translate the viral rna-dependent rna polymerase (rdrp) (plant and dinman, ) . previous studies on coronaviruses and other viruses have shown that an optimal frameshifting rate is critical and small differences in percentage of frameshifting lead to dramatic differences in genomic rna production and infection dose (plant et al., ) . therefore, the fse has emerged as a major drug target for small molecule binding that could influence the rate of frameshifting and be used as a treatment against sars-cov- . to date, there is little experimental data on the structure of sars-cov- fse and the prevailing model is a -stem pseudoknot forming downstream of the slippery site, which is thought to pause the ribosome and allow frameshifting to occur (plant and dinman, ) . to closely examine the fse structure in cells, we used dms-mapseq target specific protocol (zubradt et al., ) . we designed primers targeting nt surrounding the fse and amplified this region from cells infected with sars-cov- that were treated with dms. our analysis revealed a strikingly different structure than the prevailing model (plant et al., ; rangan, zheludev and das, ) (figure a ). our in-cell model does not include the expected pseudoknot formation downstream of the slippery sequence. instead, half of the canonical stem (figure a , purple) finds an alternative pairing partner (pink) driven by complementary bases upstream of the slippery site ( figure a , pink). we call this pairing alternative stem (as ). the prevailing model of the sars-cov- fse is based on previous studies of the sars-cov fse, as they only differ in sequence by a single nucleotide located in a putative loop (rangan, zheludev and das, ) . nuclease mapping and nuclear magnetic resonance (nmr) analysis of the sars-cov fse solved the structure of an in vitro refolded, truncated nt region starting at the slippery site (plant et al., ) . this structure did not include the sequence upstream of the slippery site and formed a -stem pseudoknot. interestingly, in silico predictions of the rna structure of the sars-cov- genome using rnaz (rangan, zheludev and das, ) and scanfold (andrews et al., ) do not find the -stem pseudoknot but instead support our in-cell model of alternative stem . in sars-cov- , scanfold not only predicted the as but also found that it was more stable relative to random sequences than any other structure in the entire frameshift element (andrews et al., ) . indeed, three conceptually varied methods (dms-mapseq, rnaz, and scanfold) aimed at identifying functional structures, run independently by different research groups all converge on the alternative stem as a central structure at the fse. in order to directly compare our in-cell findings with the reports of the -stem pseudoknot, we in vitro-transcribed, refolded, and dms-probed the same nt sequence as analyzed by nmr (plant et al., ) . our in vitro-data driven model agrees well with the nmr model ( . % identical) and finds all three canonical stems, including the pseudoknot. the major differences we observed in the structure of the fse in cells vs. in vitro could either be due to ) length of the in vitro refolded viral rna or ) factors in the cellular environment that are absent in vitro. to distinguish between these two possibilities, we re-folded the fse in the context of longer native sequences. we found that as we increased the length of the in vitro re-folded construct by including more of its native sequence, from nt to nt to kb, the dms reactivity patterns became progressively more similar to the pattern we observed in cells ( figure b ). indeed, in the context of the full ~ kb genomic rna, the structure of the fse is nearly identical to the structure in physiological conditions during sars-cov- infection in cells (r = . ). these results indicate that the length of the entire rna molecule is important for correctly folding the fse. strikingly, at a length of nt and above, the main structure forming is alternative stem rather than the -stem pseudoknot. our data indicate that given the full range of pairing possibilities in the genome, as is more favorable and the predominant structure in cells. to determine if other coronaviruses may have a similar alternative structure of the frameshift element, we searched for the sequence that pairs with canonical stem in a set of curated coronaviruses (ceraolo and giorgi, ) . this set contains isolates of sars-cov- , other sarbecoviruses (including the sars-cov reference genome), and merbecoviruses. the nt complement (ccgcgaaccc) to a sequence overlapping canonical stem of the fse (ggguuugcgg) was perfectly conserved in all of the sarbecoviruses, six of which were isolated from bats ( figure c ). however, the nt complement was not present in either merbecovirus. aligning the sequences of all betacoronaviruses with complete genomes in refseq revealed that the nt complement was conserved in all of and only the three sarbecoviruses in refseq: sars-cov, sars-cov- , and btcov bm - (data not shown). these results suggest that as is unique to the sarbecoviruses. in the past, rna structures based on experimental data have been limited to a population average structure which assumes that every individual molecule are in folded in the same conformation. however, previous studies have identified the existence of biologically relevant alternative structures in viral genomic rna that play vital roles in the viruses' life cycles. an example of this is the hiv- rev responsive element (rre) and ′ untranslated region (utr) which regulate viral rna export and packaging. as aforementioned, the rate of frameshifting in coronaviruses is critical for the virus and set at a specific percentage. while the mechanism of how this frameshifting rate is maintained remains unidentified, studies have proposed that alternative rna structure may play a role in promoting frameshifting. to determine whether the sars-cov- fse forms alternative structures, we applied the "detection of rna folding ensembles using expectation-maximization" (dreem) algorithm on our in-cell dms-mapseq data (tomezsko et al., ) . in brief, the dreem algorithm groups dms reactivities into distinct clusters by considering the likelihood of the co-occurrence of reactive bases. (i.e. if two bases are highly mutated in the population average but never mutated together on a single read, we can assume that at least two conformations are present). dreem directly clusters experimental data without relying on generating a thermodynamic based model, thus enabling the discovery of new rna structures based purely on chemical probing data from cells. we analyzed modified intracellular rna of a nt region surrounding the fse specifically amplified from two biological replicates. we found two distinct patterns of dms reactivities ( figure a ), suggesting that the rna folds into at least two distinct conformations in this region. in both biological replicates, clusters and (corresponding to structure and ) separate at a reproducible ratio (~ % vs. %) where structure is significantly different from structure (r = . ) but highly similar to the corresponding cluster in biological replicates (r = . ) ( figure b ). both structures have the alternative stem pairing spanning the slippery sequence. however, structure forms a large nt stem immediately downstream of alternative stem whereas structure does not ( figure c ). further studies measuring frameshifting efficiency are needed to determine whether this nt stem specifically promotes frameshifting by stalling the ribosome right at the slippery sequence. when genomic rna folds into alternative structure (top), the slippery site resides within a loop in the middle of a long stem-loop. as the ribosome starts to unwind the rna, it may pause at the base of the stem, but this pause is far from the slippery site. by the time the ribosome reaches the slippery site, the structure in front of it has been unwound. as the ribosome continues it will reach the upstream stop codon and terminate translation. in contrast, alternative structure (bottom) forms a nt stem loop right in front of the slippery site. this stem loop can cause the ribosome to pause, frameshift - nt and bypass the upstream stop codon to continue translation. here we present, to our knowledge, the first secondary structure of the entire rna genome of sars-cov- in infected cells, based on chemical probing with dms-mapseq. importantly, we find that many genomic regions fold differently than in silico-based predictions. we attribute these differences in large parts to the sequence context used for folding (how much native sequence is taken upstream and downstream for a given region of interest). our data underscore the contribution of the full-length rna molecule towards structure formation at local regions. of particular note, our in-cell data reveal an alternative conformation for the frameshift element (fse) in which the ' side of stem of the canonical pseudoknot is paired to a complementary nt sequence that lies shortly upstream. we find that this alternative stem (as ) predominates to the extent that we are not able to detect the canonical pseudoknot. in previous work (tomezsko et al., ) , we estimated our limit of detection for minor conformations at %, suggesting two potential models for frameshifting in sars-cov- . in the first model, the pseudoknot structure causes frameshifting and forms at a level below our limit of detection. as provides a means to downregulate the rate of frameshifting by sequestering the ' side of the canonical stem . in the second model, frameshifting is stimulated by a large nt stem loop immediately downstream of as that blocks the ribosome and causes it to slip. an alternative structure without the nt stem loop does not cause ribosome pausing at the slippery site. these alternative genomic conformations enable sars-cov- to regulate translation of orf ab ( figure ). if the second model is true, then in sarbecoviruses the ability to form a pseudoknot may be vestigial, as the fse sequence is highly conserved among coronaviruses (plant et al., ) . alternative structure (cluster) ribosome does not frameshift previous functional studies on sars-cov report that the pseudoknot increases frameshifting but do not contradict our model of alternative stem (baranov et al., ; plant et al., ; su et al., ) . functional assays use short reporter constructs that are likely to fold into a structure with a pseudoknot, similar to what we observed for the nt construct refolded in vitro. indeed, a large body of literature shows that different types of pseudoknots or stable stems placed shortly after the slippery site increase frameshifting rates in reporter assays (brierley, digard and inglis, ; baranov et al., ) . however, much work is needed to understand which structures play a major role during active infection. to our knowledge, no studies have examined the effects of mutations and measured the translation efficiency of orf ab in cells infected with a sarbecovirus. our data reveal additional structures in the genome that may be involved in regulating gene expression. for example, previous work in mhv and sars-cov suggests that the n protein binds to and unwinds trs structures to regulate sub genomic gene expression (grossoehme et al., ) . importantly, there is evidence that the stability of the trs structure can affect its affinity for the n protein. all together, these results indicate that small molecules or antisense oligoes that alter the stability of the trs structures will disrupt the expression of subgenomic rnas and can serve as therapeutic strategies. so far, we have not identified any structures of minus-strand rnas. of our . million paired reads mapping to sars-cov- , only , ( . %) came from minus-strand rna, insufficient coverage to obtain reliable dms signals. however, as minus-strand rnas must be transcribed to positive-strand genomic and subgenomic rnas, they may contain structures that are important for this process and potentially druggable. higher-throughput genome-wide sequencing or pcr amplification of a targeted region of the minus strand would enable us to discover structures in the minus strands. in this study we obtained structures of rna in infected vero cells. we were limited to studying population average rna structures, with no temporal resolution or measure of heterogeneity. our models of stable structures across the genome represent prominent conformers forming in cells. however, it is possible that some of the open regions we identify, which have evenly distributed dms signal, are in fact composites of alternative structures (tomezsko et al., ) . deeper sequencing data is needed to determine the degree of structure heterogeneity access the coronavirus genome. further studies will determine how rna structures change from the time a virion enters a cell to the time when new virions are released. we show that in vitro rna-refolding of the full-length kb genome can recapitulate the structures formed at the fse in cells. these results provide evidence that some of the structures we find in cells are largely driven by intrinsic rna thermodynamics. we expect such structures will remain the same between different cell types, although more work is needed to establish the extent of structure changes as a function of the abundance of various cellular and viral protein factors. our in-cell data-derived model of sars-cov- presents major rna structures across the entire genome and provides the foundation for further studies. in particular, we propose a new model for the fse in which the predominant structure forms an alternative stem . future work will involve determining by what mechanism and to what extent the alternative structures of the sars-cov- fse regulate translation of orf ab, as well as whether the fse can fold into a pseudoknot in cells. better understanding of the structures and mechanisms of elements of the sars-cov- genome will enable the design of targeted therapeutics. sample buffer (thermofisher scientific) and run on a % tbe-urea gel (thermofisher scientific) at v for h min for size selection of rna that is ~ nt. to dephosphorylate and repair the ends of randomly fragmented rna, μl x cutsmart buffer (new england biolabs), μl shrimp alkaline phosphatase (new england biolabs), μl rnaseout (thermofisher scientific) and water were added to a final volume of μl and °c for h. next, μl % peg- (new england biolabs), μl × t rna ligase buffer (new england biolabs), μl t rna ligase, truncated kq (england biolabs) and μl linker were added to the reaction and incubated for h at °c. the rna was purified with rna clean and concentrator - , following the manufacturer's instructions for recovery of all fragments and eluted in μl water. excess linker was degraded by adding μl × recj buffer (lucigen), μl recj exonuclease (lucigen), μl ′ deadenylase (new england biolabs) and μl rnaseout, then incubating for h at °c. the rna was purified with rna clean and concentrator - , following the manufacturer's instructions and eluted in μl water. for reverse transcription, . μg of rrna subtracted total rna or μg of in vitrotranscribed rna was added to μl × first strand buffer (thermofisher scientific), μl μm reverse primer, μl dntp, μl . m dtt, μl rnaseout and μl tgirt-iii (ingex). the reverse-transcription reaction was incubated at °c for . h. μl m naoh was then added and incubated at °c for min to degrade the rna. the reversetranscription product was mixed with an equal volume × novex tbe-urea sample buffer (thermofisher scientific) and run on a % tbe-urea gel (thermofisher scientific) at v for h min for size selection of cdna that is ~ nt. the size-selected and purified cdna was circularized using circligase ssdna ligase kit (lucigen) following manufacture's protocol. μl of the circularized product was then used for pcr amplification using phusion high-fidelity dna polymerase (neb) for a maximum of cycles. the pcr product was run on an % tbe gel at v for h and size-selected for products ~ nt. the product was then sequenced with iseq (illumina) to produce either × -nt paired-end reads. fastq files were trimmed using trimgalore (github.com/felixkrueger/trimgalore) to remove illumina adapters. trimmed paired reads were mapped to the genome of sars-cov- isolate sars-cov- /human/usa/usa-wa / (genbank: mn . ) (harcourt et al., ) using bowtie (langmead and salzberg, ) with the following parameters: --local --no-unal --no-discordant --no-mixed -l -x . reads aligning equally well to more than one location were discarded. sam files from bowtie were converted into bam files using picard tools samformatconverter (broadinstitute.github.io/picard). for each pair of aligned reads, a bit vector the length of the reference sequence was generated using dreem (tomezsko et al., ) . bit vectors contained a at every position in the reference sequence where the reference sequence matched the read, a at every base at which there was a mismatch or deletion in the read, and no information for every base that was either not in the read or had a phred score < . we refer to positions in a bit vector with a or as "informative bits" and all other positions as "uninformative bits." for each position in the reference sequence, the number of bit vectors covering the position and the number of reads with mismatches and deletions at the position were counted using dreem. the ratio of mismatches plus deletions to total coverage at each position was calculated to obtain the population average mutation rate for each position. in cases indicated below, bit vectors were discarded if they had two mutations closer than bases apart, had a mutation next to an uninformative bit, or had more than an allowed total number of mutations (greater than % of the length of the bit vector and greater than three standard deviations above the median number of mutations among all bit vectors). the average mutation rate for each position was computed from the filtered bit vectors in the same way as described above. the mutation rates for all of the bases in the rna molecule were sorted in numerical order. the greatest % or % of mutation rates (specified where relevant in the main text) were chosen for normalization. the median among these signals was calculated. all mutation rates were divided by this median to compute the normalized mutation rates. normalized rates greater than . were winsorized by setting them to . (dixon, ) . genome-wide coverage ( figure c ) was computed by counting the number of unfiltered bit vectors from the in-cell library that contained an informative bit ( or ) at each position. signal and noise plots ( figure d ) were generated from the unfiltered population average mutation rate. a total of ( . %) positions across the genome were discarded for having a noise mutation rate greater than % in the untreated sample (likely due to endogenous modifications or "hotspot" reverse transcription errors). the signal and noise were computed every nt, starting at nucleotide . for each of these nucleotides, the average mutation rate was computed over the nt window starting bases upstream and ending bases downstream. the "signal" was defined as the average mutation rate of a and c, while the "noise" was defined as the average mutation rate of g and u. the correlation of mutation rates between biological replicates genome-wide ( figure b) was computed using the unfiltered bit vectors. the correlation of mutation rates between different conditions of the fse ( figure b ) was computed using the filtered bit vectors. the correlation of mutation rates between clusters and biological replicates for the fse ( figure b ) was computed using the filtered bit vectors after clustering into two clusters. for all correlation plots, the pearson correlation coefficient is given. a total of ( . %) outliers with > % mutation rate were removed to prevent inflating the pearson correlation coefficients. the unfiltered population average mutation rate was obtained from the in-cell library reads. the , nt genome of sars-cov- was divided into ten segments, each roughly kb the boundaries of which are predicted to be open and accessible by rnaz (rangan, zheludev and das, ) . for each segment, the population average mutation rate was normalized. the segment was then folded using the fold algorithm from rnastructure (mathews, ) with parameters -m to generate the top three structures, -md to specify a maximum base pair distance, and -dms to use the normalized mutation rates as constraints in folding. all mutation rates on g and u bases were set to - (unavailable constraints). connectivity table files output from fold were converted to dot bracket format using ct dot from rnastructure (mathews, ) . the ten dot bracket structures were concatenated into a single genome-wide structure. given two rna structures of the same length (l) in dot-bracket notation, all base pairs in each structure were identified. each base pair was represented as a tuple of (position of ' base, position of ' base). the number of base pairs common to both structures (p ) as well as the number of base pairs unique to the first structure (p ) and to the second structure (p ) were computed. given these quantities, the fowlkes-mallows index (a measure of similarity between two binary classifiers) is defined as fmi = !" ⁄"( !" + ! )( !" + " ) (fowlkes and mallows, ). in the case that ( !" + ! )( !" + " ) = , we let fmi = . as the fowlkes-mallows index does not consider positions at which the structures agree on bases that are unpaired, the index needed to be modified; otherwise regions with few base pairs would tend to score too low. thus, the number of positions at which both sequences contained an unpaired base (u) was computed. two variations of the modified fowlkes-mallows index (mfmi) were tested that differed in their treatment of externally paired bases, defined as bases paired to another base outside of the region of the structure being compared. the version of mfmi excluding external base pairs counted all externally paired bases as unpaired when computing u. the number of positions containing a paired base (p) was computed as p = l -u. in this case, mfmi was defined as mfmi = ⁄ + ⁄ × fmi, which weights the fowlkes-mallows index by the fraction of paired bases and adds the fraction of unpaired bases (u/l), as the structures agree at all unpaired positions. to include external base pairs, any position containing an externally paired base was not counted in u. the number of positions at which both structures contained an externally paired base with the same orientation (i.e. both facing in the ' or ' direction) was computed as the number e. the number of positions at which at least one structure contained a base that was paired, but not externally, was computed as p. then, the mfmi was defined as mfmi = ⁄ + ⁄ + ⁄ × fmi, which weights the fowlkes-mallows index by the fraction of positions containing a paired base and considers positions in which both bases are unpaired as in agreement, but only counts externally paired bases as agreeing if both structures contain an externally paired base at the same position and the base pairs have the same orientation. excel files from the supplemental material of (rangan, zheludev and das, ) were parsed to obtain the coordinates and predicted structures. for each predicted structure, agreement with the region of our structure with the same coordinates was computed using the mfmi, either including or excluding external base pairs (as specified in the text). box plots of the agreement for each window ( figure b ) show the minimum, first quartile, median, third quartile, and maximum; data lying more than . times the interquartile range from the nearest quartile are considered outliers and are plotted as individual points. the numbers of points in each box plot are given in the results section for figure b . reads from rt-pcr of a nt segment of in-cell rna spanning the fse (nucleotides , - , ) were used to generate bit vectors. the bit vectors were filtered as described above, and the filtered average mutation rates were normalized. the rna was folded using the shapeknots algorithm from rnastructure (hajdin et al., ) with parameters -m to generate three structures and -dms to use the normalized mutation rates as constraints in folding. all signals on g and u bases were set to - (unavailable constraints). connectivity table files output from shapeknots were converted to dot bracket format using ct dot from rnastructure (mathews, ) . accession numbers of curated sarbecovirus and merbecovrus genomes were obtained from (ceraolo and giorgi, ) and downloaded from ncbi. the sequences were aligned using the muscle (edgar, ) web service with default parameters. the region of the multiple sequence alignment spanning the two sides of alternative stem was located and the sequence conservation computed using custom python scripts. for the alignment of all betacoronaviruses with genomes in ncbi refseq (o'leary et al., ) , all reference genomes of betacoronaviruses were downloaded from refseq using the query "betacoronavirus[organism] and complete genome" with the refseq source database as a filter. the sequences were aligned using the muscle (edgar, ) web service with default parameters. the subgenus of betacoronavirus to which each virus belonged was obtained from the ncbi taxonomy database (sayers et al., ) . the filtered bit vectors (the same used to fold the frameshift element) were clustered using the expectation maximization algorithm of dreem to allow detection of a maximum of two alternative structures (tomezsko et al., ) . mapped reads from the in-cell library were classified as minus-strand using a custom python script if they had the following sam flags (li et al., ): paired and proper_pair and ({read and mreverse and not reverse} or {read and reverse and not mreverse}) and not (unmap or munmap or secondary or qcfail or dup or supplementary) . rna structures were drawn using varna (darty, denise and ponty, ). the bases were colored using the normalized dms signals. the sars-cov- starting material was provided by the world reference center for emerging viruses and arboviruses (wrceva), with natalie thornburg (nax @cdc.gov) as the cdc principal investigator." we thank t.b. faust for manuscript input and e. smith for illustrator images. this work was supported by the office of naval research award # n - - - and the burroughs wellcome fund. the authors declare no competing interests ) an in silico map of the sars-cov- rna structurome., biorxiv : the preprint server for biology programmed ribosomal frameshifting in decoding the sars-cov genome', virology an efficient ribosomal frame-shifting signal in the polymeraseencoding region of the coronavirus ibv characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot genomic variance of the -ncov coronavirus varna: interactive drawing and editing of the rna secondary structure statistical prediction of single-stranded regions in rna secondary structure and application to predicting effective antisense target sites and beyond simplified estimation from censored normal samples contrafold: rna secondary structure prediction without physics-based models implications of rna structure on the annealing of a potent antisense rna directed against the human immunodeficiency virus type muscle: multiple sequence alignment with high accuracy and high throughput coronavirus n protein n-terminal domain (ntd) specifically binds the transcriptional regulatory sequence (trs) and melts trs-ctrs rna duplexes rnaz . : improved noncoding rna detection accurate shape-directed rna secondary structure modeling severe acute respiratory syndrome coronavirus from patient with coronavirus disease, united states', emerging infectious diseases rfam . : shifting to a genome-centric resource for non-coding rna families the architecture of sars-cov- transcriptome global or local? predicting secondary structure and accessibility in mrnas fast gapped-read alignment with bowtie ' the sequence alignment/map format and samtools structural lability in stem-loop drives a ′ utr- ′ utr interaction in coronavirus replication a u-turn motif-containing stem-loop in the coronavirus ′ untranslated region plays a functional role in replication mouse hepatitis virus stem-loop adopts a uynmg(u)a-like tetraloop structure that is highly functionally tolerant of base substitutions rna structure analysis of alphacoronavirus terminal genome regions structural and functional conservation of cis-acting rna elements in coronavirus '-terminal genome regions', virology the molecular biology of coronaviruses using an rna secondary structure partition function to determine confidence in base pairs predicted by free energy minimization reference sequence (refseq) database at ncbi: current status, taxonomic expansion, and functional annotation systematic evaluation of error rates and causes in short samples in next-generation sequencing a three-stemmed mrna pseudoknot in the sars coronavirus frameshift signal achieving a golden mean: mechanisms by which coronaviruses ensure synthesis of the correct stoichiometric ratios of viral proteins the role of programmed- ribosomal frameshifting in coronavirus propagation rna genome conservation and secondary structure in sars-cov- and sars-related viruses: a first look genome-wide probing of rna structure reveals active unfolding of mrna structures in vivo database resources of the national center for biotechnology information rna motif discovery by shape and mutational profiling (shape-map)', nature methods continuous and discontinuous rna synthesis in coronaviruses structural imprints in vivo decode rna regulatory mechanisms' an atypical rna pseudoknot stimulator and an upstream attenuation signal for - ribosomal frameshifting of sars coronavirus determination of rna structural diversity and its role in hiv- rna splicing', nature architecture and secondary structure of an entire hiv- rna genome' molecular mechanisms of rna interference mouse hepatitis virus stem-loop functions as a spacer element required to drive subgenomic rna synthesis shape analysis of the rna secondary structure of the mouse hepatitis virus ' untranslated region and n-terminal nsp coding sequences', virology the structure and functions of coronavirus genomic ' and ' ends', virus research reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus dms-mapseq for genome-wide or targeted rna structure probing in vivo materials & methods vero cells infection and dms modification sars-cov- total viral rna was extracted from vero cells (atcc ccl- ) cultured in dmem (gibco) supplemented with % fbs (gibco) plated into mm dishes and infected at a moi of . with -ncov/usa-wa / (passage ) or % v/v) was added dropwise to the plated vero cells h post sars-cov- infection and incubated for min at °c. dms was neutralized by adding ml pbs (thermofisher scientific) with % β-mercaptoethanol. the cells were centrifuged at , g for min at °c. the cells were washed twice by resuspending the pellet with ml pbs with % β-mercaptoethanol and centrifugation to pellet then just once with ml pbs. after washes, the pellet was resuspended in ml trizol (thermofisher scientific) and rna was extracted following the manufacturer's specifications dms modification of in vitro-transcribed rna gblocks were obtained from idt for the sars-cov- nt and nt fse which corresponds to nucleotides - and nucleotides on the basis of the dms concentration used in the next step, mm sodium cacodylate buffer (electron microscopy sciences) with mm mgcl + (refolding buffer) was added so that the final volume was μl. (e.g. for . % final dms concentration: add . μl refolding buffer and . μl dms) then, . μl was added and incubated at °c for min while shaking at r.p.m. on a thermomixer. the dms was neutralized by adding μl β-mercaptoethanol (millipore-sigma). the rna was purified using rna thermofisher scientific) and rna was extracted following the manufacturer's specifications. the rna was purified using rna clean and concentrator - kit (zymo) and dms modified as described above. of total rna per reaction was used as the input for rrna subtraction. first, μl rrna subtraction mix ( μg/μl) and μl × hybridization buffer (end concentration: mm nacl, mm tris-hcl, ph . ) were added to each reaction, and final volume was then adjusted with water to μl. the samples were denatured at °c for min and then temperature was reduced by . °c/s until the reaction was at °c. next, μl rnase h buffer and μl hybridase thermostable rnase h (lucigen) preheated to ° were added. the samples were incubated at °c for min. the rna was cleaned with rna clean and concentrator - , following the manufacturer's instructions and eluted in μl water. then, μl turbo dnase buffer and μl turbo dnase (thermofisher scientific) were added to each reaction and incubated for the reverse-transcription reaction was incubated at °c for . h. μl m naoh was then added and incubated at °c for min to degrade the rna. the cdna was purified with oligo clean and concentrator - (zymo) following instructions. pcr amplification was done using advantage hf dna polymerase (takara) for cycles according to the manufacturer's specifications. the pcr product was purified by dna clean and concentrator - (zymo) following manufacturer's instructions. rna-seq library for bp insert size was constructed following the manufacturer's instruction (nebnext ultra™ ii dna library prep kit) library generation with dms-modified sars-cov- rna after rrna subtraction (described above), extracted dms-modified rna from sars-cov- infected vero cells was fragmented using the rna fragmentation kit (thermofisher scientific). . μg of rrna subtracted total rna was fragmented at °c for . min. the fragmented rna was mixed with an equal key: cord- -o kiadfm authors: durojaye, olanrewaju ayodeji; mushiana, talifhani; uzoeto, henrietta onyinye; cosmas, samuel; udowo, victor malachy; osotuyi, abayomi gaius; ibiang, glory omini; gonlepa, miapeh kous title: potential therapeutic target identification in the novel coronavirus: insight from homology modeling and blind docking study date: - - journal: egypt j med hum genet doi: . /s - - - sha: doc_id: cord_uid: o kiadfm background: the -ncov which is regarded as a novel coronavirus is a positive-sense single-stranded rna virus. it is infectious to humans and is the cause of the ongoing coronavirus outbreak which has elicited an emergency in public health and a call for immediate international concern has been linked to it. the coronavirus main proteinase which is also known as the c-like protease ( clpro) is a very important protein in all coronaviruses for the role it plays in the replication of the virus and the proteolytic processing of the viral polyproteins. the resultant cytotoxic effect which is a product of consistent viral replication and proteolytic processing of polyproteins can be greatly reduced through the inhibition of the viral main proteinase activities. this makes the c-like protease of the coronavirus a potential and promising target for therapeutic agents against the viral infection. results: this study describes the detailed computational process by which the -ncov main proteinase coding sequence was mapped out from the viral full genome, translated and the resultant amino acid sequence used in modeling the protein d structure. comparative physiochemical studies were carried out on the resultant target protein and its template while selected hiv protease inhibitors were docked against the protein binding sites which contained no co-crystallized ligand. conclusion: in line with results from this study which has shown great consistency with other scientific findings on coronaviruses, we recommend the administration of the selected hiv protease inhibitors as first-line therapeutic agents for the treatment of the current coronavirus epidemic. the first outburst of pneumonia cases with unknown origin was identified in the early days of december , in the city of wuhan, hubei province, china [ ] . revelation about a novel beta coronavirus currently regarded as the novel coronavirus [ ] came up after a high-throughput sequencing of the viral genome which exhibits a close resemblance with the severe acute respiratory syndrome (sars-cov) [ ] . the -ncov is the seventh member of enveloped rna coronavirus family (subgenus sarbecovirus, orthocoronavirinae) [ ] , and there are accumulating facts from family settings and hospitals confirming that the virus is most likely transmitted from person-to-person [ ] . the -ncov has also recently been declared by the world health organization as a public health emergency of international concern [ ] and as of the th of february , over , cases has been confirmed and documented from laboratories around the world [ ] while more than , of such cases were documented in china through laboratory confirmation as of the th of february [ ] . despite the fast rate of global spread of the virus, the characteristics clinically peculiar to the -ncov acute respiratory disease (ard) remain unclear to a very large extent [ ] . over infections and deaths were recorded worldwide in the summer of before a successful containment of the severe acute respiratory syndrome wave was achieved as the disease itself was also a major public health concern worldwide [ , ] . the infection that led to a huge number of death cases was linked to a new coronavirus also known as the sars coronavirus (sars-cov). coronaviruses are positive-stranded rna viruses and they possess the largest known viral rna genomes. the first major step in containing the sars-cov-lined infection was to successfully sequence the viral genome, the organization of which was found to exhibit similarity with the genome of other coronaviruses [ ] . the main proteinase crystal structure from both the transmissible gastroenteritis virus and the human coronavirus (hcov e) has been determined with the discovery that the enzyme crystal structure exists as a dimer and the orientation of individual protomers making up the dimer has been observed to be perpendicular to each other. each of the protomers is made up of three catalytic domains [ ] . the first and second domains of the protomers have a two-βbarrel fold that can be likened to one of the folds in the chymotrypsin-like serine proteinases. domain iii have five α-helices which are linked to the second domain by a long loop. individual protomers have their own specific region for the binding of substrates, and this region is positioned in the left cleft between the first and second domain. dimerization of the protein is thought to be a function of the third domain [ ] . the main proteinase of the sars cov is known to be a cysteine proteinase which has in its active site, a cysteine-histidine catalytic dyad. conservation of the sars cov main proteinase across the genome sequence of all sars coronaviruses is very high, likewise the homology of the protein to the main proteinase of other coronaviruses. on the basis that high similarity exists between the different coronavirus main proteinase crystal structures and the conservation of almost all the amino acid residue side chains involved in the dimeric state formation, it was proposed that the only biologically functional form coronavirus main proteinase might be is its existence as a dimer [ ] . more recently, chen et al. in his study which involved the application of molecular dynamic simulations and enzyme activity measurements from a hybrid enzyme showed that the only active form of the proteinase is in its dimeric state [ ] . recent studies based on the sequence homology of the coronavirus main proteinase structural model with tgev as well as the solved crystal structure has involved the docking of substrate analogs for the virtual screening of natural products and a collection of synthetic compounds, alongside approved antiviral therapeutic agents in the evaluation of the coronavirus main proteinase inhibition [ ] . some compounds from this study were identified for the inhibitory role played against the viral proteinase. these compounds include the l- , , which is an hiv- protease inhibitor, calanolide a, and nevirapine, both of which are reverse transcriptase inhibitors, an inhibitor of the α-glucosidase named glycovir, sabadinine, which is a natural product and ribavirin, a general antiviral agent [ ] . ribavirin was shown to exhibit an antiviral activity in vitro, at cytotoxic concentrations against the sars coronavirus. at the start of the first outbreak of the sars epidemic, ribavirin was administered as a first-line of defense. the administration was as a monotherapy and in combination with corticosteroids or the hiv protease inhibitor, kaletra [ ] . according to reports from a very recent research conducted by cao et al., where a total of a laboratoryconfirmed sars-cov-infected patients were made to undergo a controlled, randomized, open-labeled trial in which patients were assigned to the standard care group and patients assigned to the lopinavirritonavir group. . % of the patients in the lopinavir-ritonavir group ( patients) and . % of the patients in the standard care group ( patients) exhibited serious adverse events between randomization and the th day. the exhibited adverse events include acute respiratory distress syndrome (ards), acute kidney injury, severe anemia, acute gastritis, hemorrhage of lower digestive tract, pneumothorax, unconsciousness, sepsis, acute heart failure etc. patients in the lopinavir-ritonavir group in addition, specifically exhibited gastrointestinal adverse events which include diarrhea, vomiting, and nausea [ ] . our current study took advantage of the availability of the sars cov main proteinase amino acid sequence to map out the nucleotide coding region for the same protein in the -ncov. two selected hiv protease inhibitors (lopinavir and ritonavir) were then targeted at the catalytic site of the protein d structure which was modeled using already available templates. the predicted activity of the drug candidates was validated by targeting them against a recently crystalized d structure of the enzyme, which has been made available for download in the protein data bank. lopinavir is an antiretroviral protease inhibitor used in combination with ritonavir in the therapy and prevention of human immunodeficiency virus (hiv) infection and the acquired immunodeficiency syndrome (aids). it plays a role as an antiviral drug and a hiv protease inhibitor. it is a member of amphetamines and a dicarboxylic acid diamide (fig. ). the complete genome of the isolated wuhan seafood market pneumonia virus ( -ncov) was downloaded from the genbank database with an assigned accession number of mn . . the nucleotide sequence of the full genome was copied out in fasta format. the gen-bank sequence database is an annotated collection of all nucleotide sequences which are publicly available with their translated protein segments and also open access. this database is designed and managed by the national center for biotechnology information (ncbi) in accordance with the international nucleotide sequence database collaboration (insdc) [ ] . nucleotides between the and sequence of the -ncov genome was selected as the sequence of interest. translation of the nucleotide sequence of interest in the -ncov and the back-translation of the sars cov main proteinase amino acid sequence was achieved with the use of emboss transeq and backtranseq tools, respectively [ ] . transeq reads one or more nucleotide sequences and writes the resulting translated sequence of protein to file while backtranseq makes use of a codon usage table which gives the usage frequency of individual codon for every amino acid [ ] . for every amino acid sequence input, the corresponding most frequently occurring codon is used in the nucleotide sequence that forms the output. the corresponding amino acid sequence generated as a product of the transeq translation of the nucleotide sequence of interest had no stop codons and as such was used directly for protein homology modeling without the need for any deletion. two sets of sequence alignments were carried out in this study. the first was the alignment between the translated nucleotide sequence copy of the -ncov genome which was used for the reference protein homology modeling and the amino acid sequence of the sars cov main proteinase while the second alignment was between the back-translated sars cov main proteinase nucleotide sequence and the -ncov full genome. the latter was used in mapping out the protein coding sequence in the -ncov full genome. these alignments were carried out using the clustal omega software package. clustal omega can read inputs of nucleotide and amino acid sequences in formats such as a m/fasta, clustal, msf, phylip, selex, stockholm, and vienna [ ] . template search with blast and hhblits was performed against the swiss-model template library. the target sequence was searched with blast against the primary amino acid sequence contained in the smtl. a total of templates were found. an initial hhblits profile was built using the procedure outlined in remmert et al. [ ] followed by iteration of hhblits against nr . the obtained profile was then searched against all profiles of the smtl. a total of templates were found. models were built based on the targettemplate alignment using promod . coordinates which are conserved between the target and the template are copied from the template to the model. insertions and deletions are remodeled using a fragment library. side chains were then rebuilt and finally, the geometry of the resulting model, regularized by using a force field [ ] . for the estimation of the protein structure model quality, we used the qmean (qualitative model energy analysis), a composite scoring function that describes the main aspects of protein structural geometrics, which can also derive on the basis of a single model, both global (i.e., for the entire structure) and local (i.e., per residue) absolute quality estimates [ ] . an appreciable number of alternative models have been produced which formed the basis on which scores produced by the final model was selected. the qmean score was thus used in the selection of the most reliable model against which the consensus structural scores were calculated. molprobity (version . ) was used as the structurevalidation tool that produced the broad-spectrum evaluation of the quality of the target protein at both the global and local levels. it greatly relies on the provided sensitivity and power by optimizing the placement of hydrogen and all-atom contact analysis with complementary versions of updated covalent-geometry and torsion-angle criteria [ ] . the torsion angles between individual residues of the target protein were calculated using the ramachandran plot. this is a plot of the torsional angles [phi (φ) and psi (ψ)] of the amino acid residues making up a peptide. in the order of sequence, the torsion angle of n(i- ), c(i), ca(i), n(i) is φ while the torsion angle of c(i), ca(i), n(i), c(i+ ) is ψ. the values of φ were plotted on the x-axis while the values of ψ were plotted on the y-axis [ ] . plotting the torsional angles in this way graphically shows the possible combination of angles that are allowed. the quaternary structure annotation of the template is employed to model the target sequence in its oligomeric state. the methodology as proposed by bertoni et al. [ ] was supported on a supervised machine learning algorithm rule, support vector machines (svm), which mixes conservation of interface, clustering of structures with other features of the template to produce a quaternary structure quality estimate (qsqe). the qsqe score is a number that ranges between and , and it is a reflection of the accuracy expected of the inter-chain contacts for a model engineered based on a given template and its alignment. the higher score is an indication of a more reliable result. this enhances the gmqe score that calculates the accuracy of the d structure of the resulting model. the d structural homology modeling of the -ncov genome translated segment was followed by a structural comparison with the sars cov main proteinase d structure (pdb: uj ). this was achieved using the ucsf chimera which is a highly extensible tool for interactive analysis and visualization of molecular structures and other like data, including docking results, supramolecular assemblies, density maps, sequence alignments, trajectories, and conformational ensembles [ ] . high-quality animation videos were also generated. the amino acid constituents of the target protein secondary structures were colored and visualized in d using the pymol molecular visualzer which uses opengl extension wrangler library (glew) and freeglut. the py aspect of the pymol is a reference to the programming language that backs up the software algorithm which was written in python [ ] . the percentage composition of each component making up the secondary structure was calculated using the chou and fasman secondary structure prediction (cfssp) server. this is a secondary structure predictor that predicts regions of secondary structure from an amino acid input sequence such as the regions making up the alpha helix, beta sheet, and turns. the secondary structure prediction output is displayed in a linear sequential graphical view according to the occurrence probability of the secondary structure component. the cfssp implemented methodology is the chou-fasman algorithm, which is based on the relative frequency analyses of alpha helices, beta sheets, and loops of each amino acid residue on the basis of known structures of proteins solved with x-ray crystallography [ ]. the expasy server calculates protein physiochemical parameters as a part of its sub-function, basically for the identification of proteins [ ] . we engaged the function of the protparam tool in calculating various physiochemical parameters in the model and template protein for comparison purposes. the calculated parameters include the molecular weight, theoretical isoelectric point, amino acid composition, extinction coefficient, instability index, etc. the inference on evolutionary relationship was made utilizing the maximum likelihood methodology which is the basis of the jtt matrix-based model [ ] . the corresponding consensus tree on bootstrap was inferred from a thousand replicates, and this was used to represent the historical evolution of the analyzed taxa. the tree branches forming partitions that were reproduced in bootstrap replicates of less than % were automatically collapsed. next to every branch in the tree is the displayed percentage of tree replicates of clustered associated taxa in the bootstrap test of a thousand replicates. initial trees were derived automatically for the search through the application of the neighbor-join and bionj algorithms to a matrix of pairwise distances calculated using a jtt model and followed by the selection of the most superior log likelihood value topology. the phylogenetic analysis was carried out on amino acid sequences with close identity. the complete dataset contained a total of positions. the whole analysis was conducted using the molecular evolutionary and genetics analysis (mega) software (version ) [ ] . ligand preparation and molecular docking protocol d structures of the experimental ligands were viewed from the pubchem repository and sketched using the chemaxon software [ ] . the sketched structures were downloaded and saved as mrv files which were converted into smiles strings with the openbabel. the compounds prepared as ligands were docked against each of the prepared protein receptors using autodock vina [ ] . blind docking analysis was performed at extra precision mode with minimized ligand structures. after a successful docking, a file consisting of all the poses generated by the autodock vina along with their binding affinities and rmsd scores was generated. in the vina output log file, the first pose was considered as the best because it has stronger binding affinity than the other poses and without any rmsd value. the polar interactions and binding orientation at the active site of the proteins were viewed on pymol and the docking scores for each ligand screened against each receptor protein were recorded. the same docking protocol was performed against the sars-cov main proteinase d structure that was downloaded from the protein data bank with a pdb identity of m n. obtained outputs were visualized, compared, and documented for validation purpose. the full genome of the -ncov (https://www.ncbi. nlm.nih.gov/nuccore/mn . ?report=fasta) consists of nucleotides, but for the purpose of this study, nucleotides between and were considered to locate the protein of interest. the direct translation of this segment of nucleotides produced a sequence of amino acids (fig. ). this amino acid count was reached after the direct translation of the nucleotide sequence of interest as there were no single existing stop codons hence, deletion of any form was needless. as depicted in fig. , few structural differences were noticed. the amino acid sequences making up these nonconserved regions were clearly revealed in fig. . notwithstanding, a % identity was observed between both sequences showing the conserved domains were predominant. figure represents the percentage amino acid sequence identity between the target and the template protein, where the positions with a single asterisk (*) depicts regions of full residue conservation while the segments with the colon (:) indicates regions of conservation between amino acid residues with similar properties. positions with the period (.) show regions of conservation between amino acids with less similar properties. the amino acid sequence of the sars coronavirus main proteinase was back-translated to generate the corresponding nucleotide sequence which was then aligned with the -ncov full genome. this was carried out for the purpose of mapping out the region of the -ncov full genome where the proteinase coding sequence is located. as depicted in fig. , the target protein coding sequence is located between and nucleotides of the viral genome the outcome of a qmean analysis is anchored on the composite scoring function which calculates several features regarding the structure of the target protein. the expression of the estimated absolute quality of the model is in terms of how well the score of the model is in agreement with the values expected of a set of resultant structures from high-resolution experiments. the global score values can either be from qmean or qmean . qmean is a combination of four statistical potential terms represented in a linear form while qmean in addition to the functionality of qmean uses two agreement terms in the consistency evaluation of structural features with sequence-based predictions. both qmean and qmean originally are in the range of to with being the good score and are by default transformed into z-scores (table ) for them to be related to what would have been expected from x-ray structures of high resolution. the local scores are also a combination of four linear potential statistical terms with the agreement terms of evaluation being on a per residue basis. the local scores are also estimated in the range of to , where one is the good score (fig. ) . when compared to the set of non-redundant protein structures, the qmean z-score of the target protein as shown in fig. was . the models located in the dark zone are shown in the graph to have scores less than while the scores of other regions outside the dark zone can either be < the z-score < or z-score > . good models are often located in the dark zone. whenever such values are found, they result in some strains in the polypeptide chain and in cases of such, the stability of the structure will depend greatly on additional interactions but this conformation may be conserved in a protein family for its structural significance. another αand β-regions clustering principle exemption can be viewed on the right side plot of fig. where the distribution of torsion angles for glycine are the only displayed angles on the ramachandran plot. glycine has no side chain, and this gives room for flexibility in the polypeptide chain hence making accessible the forbidden rotation angles. glycine for this reason is more concentrated in the regions making up the loop where sharp bends can occur in the polypeptide. for this reason, glycine is highly conserved in protein families as the presence of turns at specific positions is a characteristic feature of particular structural folds. the comparative physiochemical parameter computation of the template and target proteins by protparam were deduced from the amino acid sequences of the individual proteins. no additional information was required about the proteins under consideration and each of the complete sequences was analyzed. the amino acid sequence of the target protein has not been deposited in the swiss-prot database. for this reason, inputting the standard single-letter amino acid sequence for both proteins into the text field was employed in computing the physiochemical properties as shown in tables , the two hiv protease inhibitors (lopinavir and ritonavir) when targeted at the modeled -ncov catalytic site gave significant inhibition attributes; hence, the in silico study was planned through molecular docking analysis with autodock vina. the binding orientation of the drugs to the protein active site as viewed in the pymol molecular visualizer (fig. ) showed an induced fit model binding conformation. the same compounds were targeted against the active site of the downloaded pdb d structure of the sars-cov main proteinase (pdb m n) for comparison purposes fig. . the active site residues as visualized in pymol are shown in fig. . the binding of lopinavir to the target protein which produced the best binding score was used as the predictive model. residues at the distance of < angstroms to the bound ligand were assumed to form fig. the combined view of the d structural comparison between the modeled target protein and the downloaded pdb structure of the viral protein (left column) and their primary sequence alignment (right column). the target protein is colored in grey while its protein data bank equivalence is colored in red. the high structural similarity between the two proteins was validated through their sequence alignment which produced . % sequence identity score. homology modeling which is a computational method for modeling the d structure of proteins and also fig. depicted here are two ramachandran plots. the plot on the left hand side shows the general torsion angles for all the residues in the target protein while the plot on the right hand side is specific for the glycine residues of the protein fig. the target protein secondary structures with bound lopinavir. at the top is the secondary structure visualization on pymol with regions making up the alpha helix, beta sheets, and loops shown in light blue, purple, and brown, respectively. below is the prediction by cfssp where the red, green, yellow, and blue lines depict regions of the helices, sheets, turns, and coils (loops), respectively. the predicted secondary structure composition shows a high degree of alpha helix and beta sheets, respectively, occupying and % of the total residues with the percentage loop occupancy at % regarded as comparative modeling, constructs atomic models based on known structures or structures that have been determined experimentally and likewise share more than % sequence homology. the backing principle for this is that there is likely to be an existing three-dimensional structure similarity between two proteins with high similarity in their amino acid sequence. with one of the proteins having an already determined d structure, the structure of the unknown one can be copied with a high degree of confidence. there is a higher degree of accuracy for alpha carbon positions in homology modeling than the side chains and regions containing the loop which is mostly inaccurate. as regards the template selection, homologous proteins with determined structures are searched through the protein data bank (pdb) and templates must have alongside a minimum of % identity with the target sequence, the highest possible resolution with appropriate cofactors for selection consideration [ ] . in this study, the target protein was modeled using the sars coronavirus main proteinase as template. this selection was based on the high resolution and its identity with the target protein which is as high as %. qualitative model energy analysis (qmean) is a composite scoring function that describes protein structures on the basis of major geometrical aspects. the scoring function of the qmean calculates the global quality of models on six structural descriptors linear combination basis, where four of the six descriptors are statistical potentials of mean force. the analysis of local geometry is carried out by potential of torsion angle over three consecutive amino acids. in predicting the structure of a protein structure, final models are often selected after the production of a considerable number of alternative models; hence, the prediction of the protein structure is anchored on a scoring function which identifies within a collection of alternative models the best structural model. two distance-dependent interaction potentials are used to assess long-range interactions based on c_β atoms and all atoms, respectively. the burial status of amino acid residues describes the solvation potential of the model while the two terms that reflect the correlation between solvent accessibility, calculated and predicted secondary structure are not excluded [ ] . the resultant target protein can be considered a good model as the z-scores of interaction energy of c_β, pairwise energy of all atoms, solvation energy, and torsion angle energy are − . , − . , − . , and . , respectively, as shown in table . the quality of a good model protein can be compared to high-resolution reference structures that are products of x-ray crystallography analysis through z-score where is the average z-score value for a good model [ ] . according to benkert et al., qmean z-score provides an estimate value of the degree of nativeness of the structural features that can be observed in a model, and this is an indication that the model is of a good quality in comparison to other experimental structures [ ] . our study shows the z-score of the target is " " as indicated in fig. and such a score is an indication of a relatively good model as it possesses the average z-score for a perfect model. properties of the model that is predicted determines the molprobity scores. work initially done on all-atom contact analysis has shown that proteins possess exquisitely well-packed structures with favorable van der waals interactions which overlap between atoms that do not form hydrogen bonds [ ] . unfavorable steric clashes are correlated strongly with the quality of data that are often poor where a near zero occurrence of such steric clashes occurs in the ordered regions of crystal structures with high resolution. therefore, low values of clash scores are indications of a very good model which likewise has been proven by the clash score value exhibited by the target protein that was modeled for the purpose of this study (table ). in addition to the clash score, the protein conformation details are remarkably relaxed, such as staggered χ angles and even staggered methyl groups [ ] . applied forces to a given local motif in environments predominantly made up of folded protein interior can produce a locally strained conformation but significant strain are kept near functionally needed minimum by evolution and this is on the presumption that the stability of proteins is too marginal for high tolerance. in traditional validation measures updates, there has been a compilation of rigorously quality-filtered crystal structures through homology, resolution, and the overall score validation at file level, by b-factor and sometimes at residue level, by all-atom steric clashes. the resulting multi-dimensional distributions generated after an adequate smoothing are used in scoring the "protein-like" nature of each local conformation in relation to known structures either for backbone ramachandran values or the side chain rotamers [ ] . rotamer outliers are equivalent to < % at high resolution while general-case ramachandran outliers to a high-resolution equivalence of < . %, and ramachandran favored to %. in this regard, the definition of the molprobity score (mpscore) was given as mpscore = . *ln( +clashscore) + . *ln( +max( , rota_out|- )) + . *ln( + max( , rama_iffy|- )) + . where the number of unfavorable all-atom steric overlaps ≥ . Å per atoms defines the clashscore [ ] . the rota_out is the side chain conformation percentage termed as the rotamer outliers, from side chains that can undergo possible evaluation while rama_iffy is the backbone ramachandran percentage conformations that allows beyond the favored region, from residues that can undergo possible evaluation. the derivatives of the coefficients are from a log-linear fit to crystallographic resolution on a set of pdb structures that has undergone filtration, so that the mpscore of a model is the resolution at which each of its scores will be the values expected thus, the lower mpscores are the indications of a better model. with a clash score of . and a . % value for the ramachandran favored region as compared to the ramachandran outliers and rotamer outliers with individual values of . % and . % respectively, we arrived at a molprobity score of . which is low enough to indicate the quality of a good model in our experimental protein. the characteristic repetitive conformation attribute of amino acid residues is the basis for the repetitive nature of the secondary structures hence the repetitive scores of φ and ψ. the range of φ and ψ scores can be used in distinguishing the different secondary structural elements as the different φ and ψ values of each secondary structure elements map their respective regions on the ramachandran plot. peptides of the ramachandran plot have the average values of their α-helices clustered about the range of φ = − °and ψ = − °while the average values of °and ψ = + °describes the ramachandran plot clustering for twisted beta sheets [ ] . the core region (green in fig. ) on the plot has the most favorable combinations for the φ and ψ values and has the highest number of dots. the figure also shows in the upper right quadrant, a small third core region. this is known as the allowed region and can be found in the areas of the core regions or might not be associated with the core region. it has lesser data points compared to the core regions. the other areas on the plot are regarded as disallowed. since glycine has only one hydrogen atom as side chain, steric hindrance is not as likely to occur as φ and ψ are rotated through a series of values. the glycine residues having φ and ψ values of + °and − °, respectively [ ] do not exhibit steric hindrance and for that reason positioned in the disallowed region of the ramachandran plot as shown in the right hand side plot in fig. . the extinction coefficient is an indication of the intensity of absorbed light by a protein at specific wavelength. the importance of estimating this coefficient is to monitor a protein undergoing purification in a spectrophotometer. woody in his experiment [ ] has shown the possibility of estimating a protein's molar extension coefficient from knowledge of its amino acid composition which has been presented in table . the extinction coefficient of the proteins (both the template and the target proteins) was calculated using the equation: e(prot) = numb(tyr) × ext(tyr) + numb(trp) × ext(trp) + numb(cystine) × ext(cystine)the absorbance (optical density) was calculated using the following formula: for this equation to be valid, the following conditions must be met: ph . , . m guanidium hydrochloride, . m phosphate buffer. the n-terminal residue identity of a protein is an important factor in the determination of its stability in vivo and also plays a major role in the proteolytic degradation process mediated by ubiquitin [ ] . βgalactosidase proteins with different n-terminal amino acids were designed through site-directed mutagenesis, and the designed β-galactosidase proteins have different half-lives in vivo which is striking, ranging from over a hundred hours to less than min, but this is dependent on the nature of the amino terminus residue on the experimental model (yeast in vivo; mammalian reticulocytes in vitro, e. coli in vivo). the order of individual amino acid residues is thus in respect to the conferred half-lives when located at the protein's amino terminus [ ] . this is referred to as the "n-end rule" which was what the estimated half-life of both the template and target proteins were based on. the instability index provides an estimate of the protein's stability in a test tube. statistical analysis of stable and unstable proteins has shown [ ] that there are specific dipeptides with significantly varied occurrence in the unstable proteins as compared with those in the stable proteins. the authors of this method have assigned a weight value of instability to each of the different dipeptides (diwv). the computation of a protein's instability index is thus possible using these weight values, which is defined as: table amino acid composition table for both template and target protein amino acid residues in one letter codes template target durojaye et al. where l is the sequence length and diwv(x[i]x[i + ]) is the instability weight value for the dipeptide starting from position i. a protein that exhibits an instability index value less than can be predicted as a stable protein while an instability index value that exceeds the threshold is an indication that the protein may be unstable. the comparative instability index values for the template and target proteins were . and . (table ) , respectively, showing both are stable proteins. the relative volume occupied by aliphatic side chains (valine, alanine, leucine, and isoleucine) of a protein is known as its aliphatic index. it may be an indicator of a positive factor for an increment in globular proteins thermostability. the aliphatic index of the experimental proteins was calculated according to the following formula [ ] : where x(ala), x(val), x(ile), and x(leu) are the mole percent ( × mole fraction) of alanine, valine, isoleucine, and leucine. the coefficients "a" and "b" are the relative volume of the valine side chain (a = . ) and of leu/ile side chains (b = . ) to the alanine side chain. the calculated aliphatic index for the experimental protein shows that the thermostability of the target protein is slightly higher than the template. the most common secondary structures are the alpha helices and beta sheets although the beta turns and omega loops also occur. elements of the secondary structures spontaneously form as an intermediate before their folding into the corresponding three-dimensional tertiary structures [ ] . the stability and how robust the α-helices are to mutations in natural proteins have been shown in previous studies. they have also been shown to be more designable than the beta sheets; thus, designing a functional all-α helix protein is likely to be easier than designing proteins with both α helix and strands, and this has recently been confirmed experimentally [ ] . the template and target proteins both have a total of amino acid residues (table ) with the composition of individual residues shown in table . as shown in fig. , the target protein which shares a structural homology with the template (fig. and the animation video) is predominantly occupied by residues forming alpha helix and beta sheets, with very low percentage of the residues forming loops. the stability of these two proteins is revealed in their physiochemical characteristics which can therefore be linked to the high percentage of residues forming alpha helix. the ultimate goal of genome analysis is to understand the biology of organisms in both evolutionary and functional terms, and this involves the combination of different data from various sources [ ] . for the purpose of this study, we compared our protein of interest to similar proteins in the ncbi database to predict the evolutionary relationships between homologous proteins represented in the genomes of each divergent species. this makes the amino acid sequence alignment the most suitable form of alignment for the phylogenetic tree construction. organisms with common ancestors were positioned in the same monophyletic group in the tree, and the same node where the protein of interest (the -ncov main proteinase) is positioned also houses the non-structural polyprotein of the ab bat sars-like coronavirus. this shows that the two viral proteins share a common source with shorter divergence period. bootstrapping allows evolutionary predictions on the level of confidence. one hundred is a very high level of confidence in the positioning of the node in the topology. the lower scores are more likely to happen by chance than it is in the real tree topology [ ] . the bootstrap value of the above-mentioned viral proteins which is exactly is a very high level of statistical support for their positioning at the nodes in the branched part of the tree. the length of the branches is a representation of genetic distance. it is also the measure of the time since the viral proteins diverged, which means, the greater the branch length, the likelihood that it took a longer period of time since divergence from the most closely related protein [ ] . the tw and tjf strains of the sars coronavirus orf a polyprotein and replicase, respectively, are the most distantly related, based on their branch length and as such can be regarded as the out-group in the tree. structure-based drug discovery is the easiest molecular docking methodology as it screens variety of listed ligands (compounds) in chemical library by "docking" them against proteins of known structures which in this study is the modeled d structure of the -ncov main proteinase and showing the binding affinity details alongside the binding conformation of ligands in the enzyme active site [ ] . ligand docking can be specific, that is, focusing only on the predicted binding sites of the protein of interest or can be blind docking where the entire area of the protein is covered. most docking tool applications focus on the predicted primary binding site of ligands; however, in some cases, the information about the target protein binding site is missing. blind docking is known to be an unbiased molecular docking approach as it scans the protein structure in order to locate the ideal binding site of ligands [ ] . the autodock-based blind docking approach was introduced in this study to search the entire surface of the target and template protein for binding sites while optimizing the conformation of peptides simultaneously. for this reason, it was necessary to set up our docking parameters to search the entire surface of the modeled main proteinase of the -ncov. this was achieved using the autogrid to create a very large grid map (center Å × − Å × Å and size Å × Å × Å) with the maximum number of points in each dimension in order to cover the whole protein. we observed a partial overlap in the docking pose of lopinavir to the active site of both template and target protein as compared to the conspicuous difference observed in the binding orientation of ritonavir to the protein active sites. these differential poses can be viewed distinctively in the attached animation video. a keen view of the binding orientation of the two drug candidates to the -ncov virus main proteinase active site (fig. ) is also consistent with the proposed induced fit binding model. in a comparative docking study, the same drug candidates (lopinavir and ritonavir) were docked against the active site of the pdb downloaded version of the viral main proteinase. the docking grid for this purpose was set with precision as the solved pdb structure of the virus included a cocrystalized ligand at the enzyme active site (center - Å × − Å × Å and size Å × Å × Å) and experimental ligands bind to this site with precision and variation in poses (fig. ) . the binding energy results table here, the docking results of lopinavir and ritonavir against the template and target protein are shown. the binding of ritonavir to the template protein produced the highest number of inter model hydrogen bonds while the binding of lopinavir to the target protein formed polar interaction with three residues at the active site as compared to the two formed by the other interactions table the amino acid residues involved in polar interaction, the number of inter-model hydrogen bonds and the docking score of lopinavir and ritonavir upon binding to the d pdb download of the sars-cov main proteinase (pdb m n) showed a difference of − . kcal/mol upon the binding of lopinavir to the template and the pdb d structure of the enzyme (pdb m n), and a difference of − . kcal/ mol between the pdb d structure of the enzyme and the target protein (table and ). the same comparative study was repeated for the binding of ritonavir and a difference of − . and − . kcal/mol was observed upon the binding of drug to the template and target proteins, respectively, in comparison with the binding to the downloaded d structure of the enzyme from the pdb. the observed consistency in the binding energy of the drug candidates can also serve as a reference to the validity and quality of the modeled protein, which has exhibited a high sequence and structural similarity with the downloaded d structure from the protein data bank (fig. ). in an effort to make available potent therapeutic agents against the fast rising novel coronavirus epidemic, we identified from the viral genome the coding region and modeled the main proteinase of the virus coupled with the evaluation of the efficacy of existing hiv protease inhibitors by targeting the protein active site using a blind docking approach. our study has shown that lopinavir displays a broader spectrum inhibition against both the sars coronavirus and -ncov main proteinase as compared to the inhibition profile of ritonavir. the modeled d structure of the enzyme has also provided interesting insights regarding the binding orientation of the experimental drugs and possible interactions at the protein active site. the conclusion from the study of cao et al. as previously discussed however has shown that the administration of the lopinavir-ritonavir therapy might elicit additional health concerns as a result of the extreme adverse events exhibited by the experimental subjects for the purpose of their study. it was also observed that the drugs showed no increased benefit when compared with the standard supportive care. in view of this findings, we therefore suggest a drug modification approach aimed at avoiding the health concerns posed by the lopinavir-ritonavir combined therapy while retaining their proteinase inhibitory activity. supplementary information accompanies this paper at https://doi.org/ . /s - - - . additional file . supplementary information to this article can be found online at https://www.rcsb.org/structure/ m n clinical features of patients with novel coronavirus in wuhan genomic characterization and epidemiology of novel coronavirus: implications of virus origins and receptor binding a novel coronavirus from patients with pneumonia in china a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster importation and human-tohuman transmission of a novel coronavirus in vietnam national health commission of the people's republic of china transmission of -ncov infection 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flat file data libraries issues in bioinformatics benchmarking: the case study of multiple sequence alignment hhblits: lightning-fast iterative protein sequence searching by hmm-hmm alignment the swiss-prot protein knowledgebase and its supplement trembl in toward the estimation of the absolute quality of individual protein structure models molprobity: more and better reference data for improved all-atom structure validation chapter : protein composition and structure modeling protein quaternary structure of homo-and hetero-oligomers beyond binary interactions by homology ucsf chimera-a visualization system for exploratory research and analysis fasman gd ( ) prediction of protein conformation protein identification and analysis tools on the expasy server the rapid generation of mutation data matrices from protein sequences mega : molecular evolutionary genetics analysis version . for bigger datasets chemoinformatics: theory, practice, & products critical assessment of the automated autodock as a new docking tool for virtual screening critical assessment of methods of protein structure prediction (casp) round visualizing and quantifying molecular goodnessof-fit: small-probe contact dots with explicit hydrogen atoms a test of enhancing model accuracy in high-throughput crystallography the penultimate rotamer library protein geometry database: a flexible engine to explore backbone conformations and their relationships to covalent geometry circular dichroism spectrum of peptides in the poly(pro)ii conformation calculation of protein extinction coefficients from amino acid sequence data universality and structure of the n-end rule the n-end rule in bacteria correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence thermostability and aliphatic index of globular proteins alpha helices are more robust to mutations than beta strands global analysis of protein folding using massively parallel design, synthesis, and testing time of the deepest root for polymorphism in human mitochondrial dna intraspecific nucleotide sequence differences in the major noncoding region of human mitochondrial dna limitation of the evolutionary parsimony method of phylogenetic analysis efficient docking of peptides to proteins without prior knowledge of the binding site molecular recognition and docking algorithms we appreciate the leadership of the laboratory of cellular dynamics (lcd), university of science and technology of china, for the all-around support and academic advisory role. we also acknowledge the strong support from the ustc office of international cooperation all through the challenging period of the coronavirus epidemic. the authors received no funding for this project from any organization. ethics approval and consent to participate not applicable the authors declare that they have no competing interests. key: cord- -d jsek authors: eguchi, raphael r.; anand, namrata; choe, christian a.; huang, po-ssu title: ig-vae: generative modeling of immunoglobulin proteins by direct d coordinate generation date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: d jsek while deep learning models have seen increasing applications in protein science, few have been implemented for protein backbone generation—an important task in structure-based problems such as active site and interface design. we present a new approach to building class-specific backbones, using a variational auto-encoder to directly generate the d coordinates of immunoglobulins. our model is torsion- and distance-aware, learns a high-resolution embedding of the dataset, and generates novel, high-quality structures compatible with existing design tools. we show that the ig-vae can be used to create a computational model of a sars-cov -rbd binder via latent space sampling. we further demonstrate that the model’s generative prior is a powerful tool for guiding computational protein design, motivating a new paradigm under which backbone design is solved as constrained optimization problem in the latent space of a generative model. over the past two decades, the field of protein design has made steady progress, with new computational methods providing novel solutions to challenging problems such as enzyme catalysis, viral inhibition, de novo structure generation and more [ , , , , , , , , ] . building on the improvements in force field accuracy and conceptual advancements in understanding structural engineering principles, computational protein design is seemingly ready to take on any engineering challenge. however, designs created computationally rarely match the performance of variants derived from directed evolution experiments; there is still much room for improvement. while design force fields are known to be imperfect, a major limitation of computational design also stems from the difficulty in modeling backbone flexibility, specifically, movements in loops and the relative geometries of the structural elements in the protein. when comparing structural changes between pre-and post-evolution structures, we often find that the polypeptide chain responds to sequence changes in ways not predictable by the design algorithm. this is because current methods, such as rosetta [ ] , tend to restrict backbone conformations to local energy minima. while this issue is usually addressed by sampling backbone torsional angles from known fragments, such a stochastic optimization process is discrete, sparse, time-consuming, and rarely uncovers the true ground state. to address this fundamental challenge in modeling protein structure, we explore the use of deep neural networks to infer a continuous structural space in which we can smoothly interpolate between different backbone conformations. in doing so we seek to capture elements of backbone motility that are otherwise difficult to reflect in rigid body design. we focus on the task of antibody design, as it harbors many challenges common across different protein design problems and is of significant practical importance: applications of antibodies have been found in every sector of biotechnology, from diagnostic procedures to immunotherapy. with recent advances in deep learning technology, machine learning tools have seen increasing applications in protein science, with deep neural networks being applied to tasks such as sequence design [ ] , fold recognition [ ] , binding site prediction [ ] , and structure prediction [ , ] . generative models, which approximate the distributions of the data they are trained on, have garnered interest as a data-driven way to create novel proteins. unfortunately, the majority of protein-generators create d amino acid sequences [ , ] making them unsuitable for problems that require structure-based solutions such as designing protein-protein interfaces. very few machine learning models have been implemented for protein backbone generation [ , ] , and none of the reported methods produce structures comparable in quality to those of experimentally validated tools such as rosettaremodel [ ] . previously, our group was the first to report a generative adversarial network (gan) that generated -residue backbones that could serve as templates for de novo design [ ] . this prior work focused on unconditioned structure generation via a distance matrix representation, and d coordinates were recovered using a convex optimization algorithm [ ] or a learned coordinate recovery module [ ] . despite its novelty, the gan method is accompanied by certain difficulties. the generated distance constraints are not guaranteed to be euclidean-valid, and thus it is not possible to recover d coordinates that perfectly satisfy the generated constraints. we found that the quality of the recovered backbone torsions could often be degraded due to inconsistencies or errors in the generated distance constraints, leading to loss of important biochemical features, such as hydrogen-bonding. moreover, since the gan is trained on peptide fragments, we are not guaranteed to sample constraints for globular structures that can eventually fold. in this study, we use a variational autoencoder (vae) [ ] to perform direct d coordinate generation of full-atom protein backbones, circumventing the need to recover coordinates from pairwise distance constraints, and also avoiding the problem of distance matrix validity [ ] . using a coordinate representation has allowed us to make our model torsion-aware, significantly improving the quality of the generated backbones. importantly, our model motivates a conceptually new way of solving protein design problems. because the ig-vae generates coordinates directly, all of its outputs are fully differentiable through the coordinate representation. this allows us to use the generative prior to constrain structure generation with any differentiable coordinate-based heuristic, such as rosetta energy, backbone shape constraints, packing metrics, and more. by optimizing a structure in the vae latent space, designers are able to specify a set of desired structural features while the model creates the rest of the molecule. as an example of this approach, we use the ig-vae to perform constrained loop generation, towards epitope-specific antibody design. our technology ultimately paves the way for a novel approach to protein design in which backbone construction is solved via constrained optimization in the latent space of a generative model. in contrast to conventional methods [ , ] , we term this approach "generative design." figure : vae training scheme. the flow of data is shown with black arrows, and losses are shown in blue. first, ramachandran angles and distance matrices are computed from the full-atom backbone coordinates of a training example. the distance matrix is passed to the encoder network (e), which generates a latent embedding that is passed to the decoder network (d). the decoder directly generates coordinates in d space, from which the reconstructed ramachandran angles and distance matrix are computed. errors from both the angles and distance matrix are back-propagated through the d coordinate representation to the encoder and decoder. note that both the torsion and distance matrix losses are rotationally and translationally invariant, and that the coordinates of the training example are never seen by the model. the shown data are real inputs and outputs of the vae for the immunoglobulin chain in pdb: yxh(l). all training data were collected from the antibody structure database abdb [ ] . domains that were missing residues were excluded, and sequence-redundant structures were included to allow the network to learn small backbone fluctuations. the final training set is comprised of immunoglobulins spanning non-sequence-redundant structures, including single-domain antibodies. the training set covers close to % of the abdb database. structures in the dataset vary in length from to residues, with most falling between and . since the input of our model was fixed at residues ( atoms), structures larger than were center-cropped. structures smaller than were "structurally padded" by using rosettaremodel to append dummy residues to the n and c termini. the reconstruction loss of the padded regions was down-weighted over the course of training (see supplemental methods). we found that the structural padding step led to slight improvements in local bond geometries at the terminal regions. all structures were idealized and relaxed under the rosetta energy function with constraints to starting coordinates [ ] . this relaxation step was done to remove any potential confounding factors resulting from various crystal structure optimization procedures. a schematic of the model training scheme is shown in figure . like classical vaes [ ] our model minimizes a reconstruction loss and a kl-divergence loss that constrains the latent embeddings to be isotropic gaussian. importantly, the reconstruction loss is comprised of a distance matrix reconstruction loss and a torsion loss. the torsion loss is formulated as a supervised-learning objective, where the network infers the correct torsion distribution from the distance matrix. we found that early in training, the torsion loss must be heavily up-weighted relative to the distance loss in order to achieve correct stereochemistry, as molecular handedness cannot be uniquely determined from pairwise distances alone. decreasing the torsion weight later in training led to improvements in local structure quality. a detailed description of the loss weighting schedule is included in the supplemental methods. we note that both the distance and torsion losses are rotationally and translationally invariant, so the absolute position of the output coordinates is determined by the model itself. coordinates of the training examples are never seen by the model directly. to assess the utility our model, we studied the ig-vae's performance on several tasks. the first of these is data reconstruction, which reflects the ability of the model to compress structural features into a low-dimensional latent space (section . ). this functionality is an underlying assumption of generative sampling, which requires that the latent space capture the scope of structure variation with sufficient resolution. next, we assessed the quality and novelty of the generated structures, characterizing the chemical validity of the samples (section . ), while also evaluating the quality of interpolations between embedded structures (section . ). we visualize the distribution of embeddings within the latent space to better understand its structure, and to determine if the sampling distribution is well-supported (section . ). we ultimately challenge the ig-vae with a real design task; specifically, generation of a novel backbone with high shape-complementarity to the ace epitope of the sars-cov -rbd [ ] (section . . ). to evaluate the general utility of our approach, we investigated whether we could leverage the model's generative prior to perform backbone design subject to a set of local, human-specified constraints (section . . ). a core feature of an effective vae is the model's ability to embed and reconstruct data. high quality reconstructions indicate that a model is able to capture and compress structural features into a low-dimensional representation, which is a prerequisite for generation by latent space sampling. to evaluate this functionality, we reconstructed randomly selected structures and compared the real and reconstructed distributions of backbone torsion angles, pairwise distances, bond lengths, and bond angles ( figure ). the structurally padded "dummy" regions were excluded from this analysis. the distance and torsion distributions are shown in figure a , where we observe that the real and reconstructed data agree well. on average, pairwise distances smaller than Å tended to be reconstructed slightly smaller than the actual distances, while larger distances tended to be reconstructed slightly larger ( figure b , reconstructed). φ and ψ torsions tended within ∼ • of the real angles, while ω angles tended to fall within ∼ • . examples of reconstructed backbones are shown in the top row of figure c . these data demonstrate that the ig-vae accurately performs full-atom reconstructions over a range of loop conformations. in order to use generated backbones in conjunction with existing protein design tools, it is crucial that our model produce structures with near-chemically-valid bond lengths and bond angles. otherwise, large movements in the backbone can occur as a result of energy-based corrections during the design process, leading to the loss of model-generated features. the bond length and bond angle distributions are depicted in figure d . the majority of bond length reconstructions were within ∼ . Å of ideal lengths, while bond angles tended to be within ∼ • of their ideal angles. we found that a constrained optimization step using the rosetta centroid-energy function (see supplemental methods) could be used to effectively refine the outputs. this refinement process kept structures close to their output conformations ( figure c , bottom) while correcting for non-idealities in the bond lengths and bond angles ( figure d , green). refinement did not improve backbone reconstruction accuracy ( figure b , refined), but did improve chemical validity, implying that our model outputs could be refined with rosetta without washing-out generated structural features. our analysis of the reconstructions reveal that the ig-vae can be used to obtain high-resolution structure embeddings that are likely useful in various learning tasks on d protein data. these results support our later conclusion that the ig-vae embedding space can be leveraged to generate structures with high atomic precision, while also showing that the kl-regularization imposed on the latent embeddings does not overpower the autoencoding functionality of the vae. the left panel shows a plot of the post-refinement per-residue centroid energy against normalized nearest neighbor distance for the generated structures. the nearest neighbor distance is computed as the minimum frobenius distance between the generated distance matrix and all distance matrices in the training set. each point is colored based on whether the nearest neighbor is a heavy or light chain ig. the center panel shows an overlay of the generated structures (pink) and their nearest neighbors (blue) in the training set. these six structures were selected using a combination of centroid energy, nearest neighbor distance, heavy/light classification, and manual inspection. the right panel shows sequence design results for structures iii and vi. the energies in the left panel are centroid energies, while the energies in the right panel are full-atom rosetta energies using the ref score function. to determine whether the ig-vae could generate novel, realistic ig backbones, we sampled structures from the latent space of the model and compared their feature distributions to non-redundant structures from the dataset. each generated structure was cropped based on its nearest neighbor in the training set. in figure a we show overlays of the distance and torsion distributions for the real and generated structures. the generated torsions were more variable than the real torsions, with more residues falling outside the range of the training data. the real and generated distance distributions agreed well. visual assessment of the backbone ensembles ( figure b ) revealed that the two were similarly variable, suggesting that our model captures much of the structural variation found in the training set. the generated structures exhibited good chemical-bond geometries ( figure c , red) that were slightly noisier but comparable to those of the reconstructed backbones ( figure d ). once again, we found that constrained refinement using rosetta could improve chemical bond geometries ( figure c , green), with minimal changes to the generated structures. to assess both the novelty and viability of the generated examples, we evaluated structures based on two criteria: ( ) post-refinement energy and ( ) nearest-neighbor distance. energies were normalized by residue-count to account for variable structure sizes. nearest-neighbor distance was computed as a length-normalized frobenius distance between c α -distance matrices. for notational convenience, nearest-neighbor distances were normalized between and . we avoid the use of the classical c α -rmsd, because it is neither an alignment-free nor a length-invariant metric, and because it lacks sufficient precision to make meaningful comparisons between ig structures. we found that there was a positive correlation between energy and nearest-neighbor distance ( figure d ), implying that while our model is able to generate structures that differ from any known examples, there is a concurrent degradation in quality when structures drift too far from the training data. despite this, we found that a significant number of generated structures had novel loop shapes, achieved favorable energies, and retained ig-specific structural features. six of these examples are shown as raw model outputs, overlaid with their nearest neighbors in the center panel of figure d . both heavy and light-chain-like structures exhibited dynamic loop structures, and the model appears to performs well in generating both long and short loops. to assess whether the generated loop conformations could be sustained by an amino acid sequence, we used rosetta fastdesign [ , ] to create sequences for the selected backbones. the outputs of two representative design trajectories are shown in the right panel of figure d . the design process yielded energetically favorable sequences with loops supported by features such as hydrophobic packing, π-π stacking and hydrogen bonding. overall these results suggest that the ig-vae is capable of generating novel, high-quality backbones that are chemically accurate, and that can be used in conjunction with existing design protocols to obtain biochemically realistic sequences. while the results of the preceding section suggest that the ig-vae is able to produce novel structures, an important feature of any generative model is the ability to interpolate smoothly between examples in the latent space. in design applications this functionality allows for dense structural sampling, and modeling of transitions between distinct structural features. a linear interpolation between two randomly selected embeddings is shown in figure a . the majority of interpolated structures adopt realistic conformations, retaining characteristic backbone hydrogen bonds while transitioning smoothly between different loop conformations ( figure c ). structures along the interpolation trajectory were able to achieve negative post-refinement energies ( figure b) , with the highest energy structure corresponding to the most unrealistic portion of the trajectory ( figure a , b, index ). to better understand the structure of the embedding space, we visualized the training data embeddings ( figure d ) using two dimensionality-reduction methods: t-distributed stochastic neighbor embedding (tsne) [ ] and principal components analysis (pca) [ ] . the top panel depicts a tsne decomposition of the embedding means (without variance) for the non-redundant structures in the dataset. k-means clustering (k= ) revealed distinct clusters that roughly correlated with loop structure, suggesting a correspondence between latent space position and semantically meaningful features ( figure d, insert) . in the bottom panel, we visualized sampled embeddings for the same set of structures, sampling embeddings per example. pca revealed a spherical, densely populated embedding space, suggesting that the isotropic gaussian sampling distribution is well supported. the pca results also suggest that the kl-loss was sufficiently weighted during training. overall these results support the conclusions of the previous section, demonstrating that the ig-vae exhibits the features expected of a properly-functioning generative model, and that sampling from a gaussian prior is well motivated. the smooth interpolations agree with the observation that our model is capable of generating novel structures, which are expected to arise by sampling from interpolated regions between the various embeddings. while antibodies are usually comprised of two ig domains, there also exist a large number of single-domain antibodies in the form of camelids [ ] and bence jones proteins [ ] . to test the utility of our model in a real-world design problem, we challenged the ig-vae to generate a single-domain-binder to the ace epitope of the sars-cov receptor binding domain (rbd), an epitope which is of significant interest in efforts towards resolving the / coronavirus pandemic [ ] . to do this, we sampled structures from the latent space of the ig-vae. to find candidates with high shape complementarity to the ace epitope, we used patchdock [ ] to dock each generated structure against the rbd. to make the search sequence agnostic, both proteins were simulated as poly-valines during this step. we then selected ig's that bound the ace epitope specifically, and used fastdesign to optimize the sequences of the binding interfaces. two ig's that exhibited good shape complementarity to the ace epitope and adopted unique loop conformations are shown in figure a . after sequence design these candidates achieved favorable energies and complex ddg's of - . and - . rosetta energy units ( figure a, designed) . using rosettadock [ , ] , we were able to accurately recover the designed interfaces as the energy minimum of a blind global docking trajectory, suggesting that the binders are specific to their cognate epitopes ( figure a, recovered, docking) . these results demonstrate that the latent space of our generative model can be leveraged to create novel binding proteins that are otherwise unobtainable by discrete sampling of real structures. while we believe that designed proteins must be experimentally validated, our data suggest that generative models can provide compelling design candidates by computational design standards, making the method worthy of larger-scale and broader experimental testing. our data also demonstrate the compatibility of the ig-vae with established design suites like rosetta, which have conventionally relied on real proteins as templates. the functional elements of a protein are often localized to specific regions. antibodies are one example of this, where binding is attributable to a set of surface-localized loops, as well as enzymes, which depend on the positioning of catalytic residues to form an active site. despite this apparent simplicity, natural proteins carry a rich set of evolutionoptimized structural features that are required to host the functional elements. the protein design process often seeks to mimic this organization, requiring the manual engineering of supporting elements centered around a desired structural feature. while logical, designing supportive features is almost always a difficult task, requiring large amounts of experience and manual tuning. motivated by these difficulties, we sought to investigate whether the generative prior of our model could be leveraged to create structures that conform to a human-specified feature without specification of other supporting features. to test this, we specified a -residue antibody loop shape as pairwise ca distances. we then sampled random latent initializations and applied the constraints to the generated structures. next, we optimized the structures via gradient descent, backpropagating constraint errors to the latent vectors through the decoder network. from initializations, we were able to recover the target loop shape in trajectories, with the vast majority of structures retaining high quality, realistic features. we visualize one trajectory in the center panel of figure b . while the middle ig-loop ( figure b , blue) is being constrained, the other loops move to adopt sterically compatible conformations and the angles of the β-strands change to support the new loop shapes. we note that the latent-vector optimization problem is non-convex, which is why we require multiple random initializations [ , ] . importantly, the recovered backbones in the generated ensemble differ from the originating structure ( figure b , orange). these data suggest that our model can be used to create backbones that satisfy specific design constraints, while also providing a distribution of compatible supporting elements. we emphasize that this procedure is not limited to distance constraints, and can be done using any differentiable coordinate-based heuristic such as shape complementarity [ ] , volume constraints, rosetta energy [ ] , and more. with a well-formulated loss function, which warrants a study in itself, it is possible to "mold" the loops of an antibody to a target epitope, or even the backbone of an enzyme around a substrate of choice. our example demonstrates a novel formulation of protein design as a constrained optimization problem in the latent space of a generative model. in contrast to methods that require manual curation of each part of a protein, we term our approach "generative design," where the requirement of human-specified heuristics constitutes the "design" element, and using a generative model to fill in the details of the structure constitutes the "generative" element. perhaps the greatest challenge in deep structure generation is that generated backbones must be both globally realistic and chemically valid to be of any practical use. in the case that a generated example is poor in quality, energybased corrections to chemical bond angles, lengths, and torsions will often lead to unintended backbone movements, resulting in loss of model-derived features. this issue is closely tied to that of data representation; while proteins are often represented by d coordinates, these are not invariant under rotations and translations. several groups have experimented with distance matrix representations which provide such invariances [ , , ] ; however, generation of euclidean-valid distance matrices remains non-trivial [ ] and recovery of d coordinates from invalid matrices leads to feature degradation [ , ] . the training scheme that we present has sought to address these challenges by auto-encoding distance matrices while factoring through a d coordinate representation. our approach circumvents the need for coordinate recovery via secondary methods, and avoids the problem of euclidean validity altogether. importantly, the ig-vae loss function does not specify the absolute positions of the atomic coordinates, which are instead learned. factoring through a coordinate representation has also allowed us to back-propagate torsion error to the generative model, significantly improving local structure quality. in the structure prediction field, fragment-sampling has been used for many years as a powerful tool to efficiently search the vast backbone conformational space [ ] . recently, however, several groups discovered that structural priors obtained from coevolution data [ ] can be used to sufficiently restrict the conformational search space to allow for continuous optimization by gradient descent [ , ] . this insight gave alphafold [ ] a significant advantage in casp [ ] , as it enabled sampling of conformations otherwise not accessible by a discrete fragment set. unlike structure prediction, protein design does not often start with a sequence from which a structural prior can be obtained, and remains heavily dependent on stochastic fragment sampling. despite past success, fragment-based design methods can be problematic for two reasons. first, they are unable to model backbone flexibility, limiting the range of accessible conformations, sequences, and thus functions [ ] . second, their stochastic nature means that they use little prior information about global organization (e.g. topology, secondary structure contacts, etc.). while it is possible to filter for global patterns [ ] , such information is difficult to integrate into the sampling process itself, leading to the overwhelming majority of trajectories being dsiscarded. in this study we have sought to address these long-standing difficulties by class-specific generative modeling. because our model provides realistic interpolations within the embedding space, generated backbones capture a range of dynamic motions that are difficult to sample via conventional simulations or fragment-sampling; the model infers a continuous distribution of ig-loop conformations from a set of discrete ones. while our model is classspecific, its generative prior allows us to restrict backbone conformations to the regime of a specific class, allowing for dense sampling while also supplying information about high-level features not provided by quantitative heuristics. additionally, because our approach is not specific to ig's, it can be applied to any fold-class well represented in structural databases. although there is much room to improve our model, in its current form the ig-vae remains a powerful tool for creating single-domain antibodies, and allows for high-throughput construction of epitope-tailored, structure-guided libraries. with such a tool, it may be possible to circumvent screening of fully randomized libraries, a large proportion of which are usually insoluble or fundamentally incompatible with the target of interest [ , , , ] . overall, our work is of significant interest to both protein engineers and machine learning scientists as the first successful example of d deep generative modeling applied to protein design. with the novel paradigm that it offers, we speculate that our scheme will motivate further study of class-specific generative models, as well as development of differentiable loss functions that can be used to create novel, functional proteins. code for the working model will be released on github at a later date. supplemental methods, rosetta commands, and an interpolation movie are available for download at: https://tinyurl.com/y cao h . de novo design of a fluorescence-activating β-barrel de novo design of a four-fold symmetric tim-barrel protein with atomic-level accuracy de novo design of potent and selective mimics of il- and il- a potent and broad neutralizing antibody recognizes and penetrates the hiv glycan shield kemp elimination catalysts by computational enzyme design de novo design of bioactive protein switches computational design of closely related proteins that adopt two well-defined but structurally divergent folds accurate computational design of multipass transmembrane proteins the coming of age of de novo protein design rosetta : an object-oriented software suite for the simulation and design of macromolecules protein sequence design with a learned potential multi-scale structural analysis of proteins by deep semantic segmentation deciphering interaction fingerprints from protein molecular surfaces using geometric deep learning koray kavukcuoglu, and demis hassabis. improved protein structure prediction using potentials from deep learning improved protein structure prediction using predicted interresidue orientations unified rational protein engineering with sequence-based deep representation learning progen: language modeling for protein generation. preprint, synthetic biology generating tertiary protein structures via an interpretative variational autoencoder generative modeling for protein structures rosettaremodel: a generalized framework for flexible backbone protein design fully differentiable full-atom protein backbone generation auto-encoding variational bayes generating valid euclidean distance matrices principles for designing ideal protein structures abdb: antibody structure database-a database of pdb-derived antibody structures potential role of ace in coronavirus disease (covid- ) prevention and management accurate de novo design of hyperstable constrained peptides rosettascripts: a scripting language interface to the rosetta macromolecular modeling suite visualizing data using t-sne on lines and planes of closest fit to systems of points in space. the london, edinburgh, and dublin philosophical magazine camelid single-domain antibodies: historical perspective and future outlook bence jones proteins patchdock and symmdock: servers for rigid and symmetric docking benchmarking and analysis of protein docking performance in rosetta v protein-protein docking with simultaneous optimization of rigid-body displacement and side-chain conformations precise recovery of latent vectors from generative adversarial networks generalized latent variable recovery for generative adversarial networks shape complementarity at protein/protein interfaces the rosetta all-atom energy function for macromolecular modeling and design are there pathways for protein folding large-scale determination of previously unsolved protein structures using evolutionary information. elife critical assessment of methods of protein structure prediction (casp)-round xiii rosettabackrub-a web server for flexible backbone protein structure modeling and design evaluation of protein engineering and process optimization approaches to enhance antibody drug manufacturability predictive tools for stabilization of therapeutic proteins post-translational modifications of recombinant proteins: significance for biopharmaceuticals general strategy for the generation of human antibody variable domains with increased aggregation resistance we thank sergey ovchinnikov for helpful discourse during early phases of this project, and for contributing initial code that became part of the torsion-reconstruction loss function. this project was supported by startup funds from the stanford schools of engineering and medicine, the stanford chem-h chemistry/biology interface predoctoral training program and the national institute of general medical sciences of the national institutes of health under award number t gm . additionally, this project was supported by the u.s. department of energy, office of science, office of advanced scientific computing research, scientific discovery through advanced computing (scidac) program. this project is also based upon work supported by google cloud. key: cord- -d rz dc authors: webb, b.; eswar, n.; fan, h.; khuri, n.; pieper, u.; dong, g.q.; sali, a. title: comparative modeling of drug target proteins date: - - journal: reference module in chemistry, molecular sciences and chemical engineering doi: . /b - - - - . - sha: doc_id: cord_uid: d rz dc in this perspective, we begin by describing the comparative protein structure modeling technique and the accuracy of the corresponding models. we then discuss the significant role that comparative prediction plays in drug discovery. we focus on virtual ligand screening against comparative models and illustrate the state-of-the-art by a number of specific examples. structure-based or rational drug discovery has already resulted in a number of drugs on the market and many more in the development pipeline. [ ] [ ] [ ] [ ] structure-based methods are now routinely used in almost all stages of drug development, from target identification to lead optimization. [ ] [ ] [ ] [ ] central to all structure-based discovery approaches is the knowledge of the threedimensional ( d) structure of the target protein or complex because the structure and dynamics of the target determine which ligands it binds. the d structures of the target proteins are best determined by experimental methods that yield solutions at atomic resolution, such as x-ray crystallography and nuclear magnetic resonance (nmr) spectroscopy. while developments in the techniques of experimental structure determination have enhanced the applicability, accuracy, and speed of these structural studies, , structural characterization of sequences remains an expensive and time-consuming task. the publicly available protein data bank (pdb) currently contains $ structures and grows at a rate of approximately % every years. on the other hand, the various genome-sequencing projects have resulted in over million sequences, including the complete genetic blueprints of humans and hundreds of other organisms. , this achievement has resulted in a vast collection of sequence information about possible target proteins with little or no structural information. current statistics show that the structures available in the pdb account for less than % of the sequences in the uniprot database. moreover, the rate of growth of the sequence information is more than twice that of the structures, and is expected to accelerate even more with the advent of readily available next-generation sequencing technologies. due to this wide sequence-structure gap, reliance on experimentally determined structures limits the number of proteins that can be targeted by structure-based drug discovery. fortunately, domains in protein sequences are gradually evolving entities that can be clustered into a relatively small number of families with similar sequences and structures. , for instance, - % of the sequences in the uniprot database have been grouped into fewer than domain families. , similarly, all the structures in the pdb have been classified into about distinct folds. , computational protein structure prediction methods, such as threading and comparative protein structure modeling, , strive to bridge the sequence-structure gap by utilizing these evolutionary relationships. the speed, low cost, and relative accuracy of these computational methods have led to the use of predicted d structures in the drug discovery process. , the other class of prediction methods, de novo or ab initio methods, attempts to predict the structure from sequence alone, without reliance on evolutionary relationships. however, despite progress in these methods, [ ] [ ] [ ] especially for small proteins with fewer than amino acid residues, comparative modeling remains the most reliable method of predicting the d structure of a protein, with an accuracy that can be comparable to a low-resolution, experimentally determined structure. the basis of comparative modeling the primary requirement for reliable comparative modeling is a detectable similarity between the sequence of interest (target sequence) and a known structure (template). as early as , chothia and lesk showed that there is a strong correlation between sequence and structural similarities. this correlation provides the basis of comparative modeling, allows a coarse assessment of model errors, and also highlights one of its major challenges: modeling the structural differences between the template and target structures (figure ). comparative modeling stands to benefit greatly from the structural genomics initiative. structural genomics aims to achieve significant structural coverage of the sequence space with an efficient combination of experimental and prediction methods. this goal is pursued by careful selection of target proteins for structure determination by x-ray crystallography and nmr spectroscopy, such that most other sequences are within 'modeling distance' (e.g., > % sequence identity) of a known structure. , , , the expectation is that the determination of these structures combined with comparative modeling will yield useful structural information for the largest possible fraction of sequences in the shortest possible timeframe. the impact of structural genomics is illustrated by comparative modeling based on the structures determined by the new york structural genomics research consortium. for each new structure without a close homolog in the pdb, on average, protein sequences without any prior structural characterization could be modeled at least at the level of the fold. thus, the structures of most proteins will eventually be predicted by computation, not determined by experiment. in this review, we begin by describing the various steps involved in comparative modeling. next, we emphasize two aspects of model refinement, loop modeling and side-chain modeling, due to their relevance in ligand docking and rational drug discovery. we then discuss the errors in comparative models. finally, we describe the role of comparative modeling in drug discovery, focusing on ligand docking against comparative models. we compare successes of docking against models and x-ray structures, and illustrate the computational docking against models with a number of examples. we conclude with a summary of topics that will impact on the future utility of comparative modeling in drug discovery, including an automation and integration of resources required for comparative modeling and ligand docking. comparative modeling consists of four main steps (figure (a)): ( ) fold assignment that identifies similarity between the target sequence of interest and at least one known protein structure (the template); ( ) alignment of the target sequence and the template(s); ( ) building a model based on the alignment with the chosen template(s); and ( ) predicting model errors. although fold assignment and sequence-structure alignment are logically two distinct steps in the process of comparative modeling, in practice almost all fold assignment methods also provide sequence-structure alignments. in the past, fold assignment methods were optimized for better sensitivity in detecting remotely related homologs, often at the cost of alignment accuracy. however, recent methods simultaneously optimize both the sensitivity and alignment accuracy. therefore, in the following discussion, we will treat fold assignment and sequence-structure alignment as a single protocol, explaining the differences as needed. as mentioned earlier, the primary requirement for comparative modeling is the identification of one or more known template structures with detectable similarity to the target sequence. the identification of suitable templates is achieved by scanning structure databases, such as pdb, scop, dali, and cath, with the target sequence as the query. the detected similarity is usually quantified in terms of sequence identity or statistical measures, such as e-value or z-score, depending on the method used. sequence-structure relationships are coarsely classified into three different regimes in the sequence similarity spectrum: ( ) the easily detected relationships characterized by > % sequence identity; ( ) the 'twilight zone,' corresponding to relationships figure average model accuracy as a function of sequence identity. as the sequence identity between the target sequence and the template structure decreases, the average structural similarity between the template and the target also decreases (dashed line, triangles). structural overlap is defined as the fraction of equivalent c a atoms. for the comparison of the model with the actual structure (filled circles), two c a atoms were considered equivalent if they belonged to the same residue and were within . Å of each other after least-squares superposition. for comparisons between the template structure and the actual target structure (triangles), two c a atoms were considered equivalent if they were within . Å of each other after alignment and rigid-body superposition. the difference between the model and the actual target structure is a combination of the target-template differences (green area) and the alignment errors (red area). the figure was constructed by calculating comparative models based on a single template of varying similarity to the targets. all targets had known (experimentally determined) structures. with statistically significant sequence similarity in the - % range; and ( ) the 'midnight zone,' corresponding to statistically insignificant sequence similarity. for closely related protein sequences with identities higher than - %, the alignments produced by all methods are almost always largely correct. the quickest way to search for suitable templates in this regime is to use simple pairwise sequence alignment methods such as ssearch, blast, and fasta. brenner et al. showed that these methods detect only $ % of the homologous pairs at less than % sequence identity, while they identify more than % of the relationships when sequence identity is between % and %. another benchmark, based on reference structural alignments with - % sequence identity, indicated that blast is able to correctly align only % of the residue positions. the sensitivity of the search and accuracy of the alignment become progressively difficult as the relationships move into the twilight zone. , a significant improvement in this area was the introduction of profile methods by gribskov and co-workers. the profile of a sequence is derived from a multiple sequence alignment and specifies residue-type occurrences for each alignment position. the information in a multiple sequence alignment is most often encoded as either a position-specific scoring matrix (pssm) , , or as a hidden markov model (hmm). , to identify suitable templates for comparative modeling, the profile of the target sequence is used to search against a database of template sequences. the profile-sequence methods are more sensitive in detecting related structures in the twilight zone than the pairwise sequence-based methods; they detect approximately twice the number of homologs under % sequence identity. , , the resulting profile-sequence alignments correctly align approximately - % of residues in the - % sequence identity range; , this number is almost twice as large as that of the pairwise sequence methods. frequently used programs for profile-sequence alignment are psi-blast, sam, hmmer, hhsearch, hhblits, and build_profile. as a natural extension, the profile-sequence alignment methods have led to profile-profile alignment methods that search for suitable template structures by scanning the profile of the target sequence against a database of template profiles, as opposed to a database of template sequences. these methods have proven to include the most sensitive and accurate fold assignment and figure comparative protein structure modeling. (a) a flowchart illustrating the steps in the construction of a comparative model. (b) description of comparative modeling by extraction of spatial restraints as implemented in modeller. by default, spatial restraints in modeller include: ( ) homology-derived restraints from the aligned template structures; ( ) statistical restraints derived from all known protein structures; and ( ) stereochemical restraints from the charmm- molecular mechanics force field. these restraints are combined into an objective function that is then optimized to calculate the final d model of the target sequence. alignment protocols to date. , [ ] [ ] [ ] profile-profile methods detect $ % more relationships at the superfamily level and improve the alignment accuracy by - % compared to profile-sequence methods. , there are a number of variants of profile-profile alignment methods that differ in the scoring functions they use. , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] however, several analyses have shown that the overall performances of these methods are comparable. , [ ] [ ] [ ] some of the programs that can be used to detect suitable templates are ffas, sp , salign, hhblits, hhsearch, and ppscan. sequence-structure threading methods as the sequence identity drops below the threshold of the twilight zone, there is usually insufficient signal in the sequences or their profiles for the sequence-based methods discussed above to detect true relationships. sequence-structure threading methods are most useful in this regime as they can sometimes recognize common folds, even in the absence of any statistically significant sequence similarity. these methods achieve higher sensitivity by using structural information derived from the templates. the accuracy of a sequence-structure match is assessed by the score of a corresponding coarse model and not by sequence similarity, as in sequence comparison methods. the scoring scheme used to evaluate the accuracy is either based on residue substitution tables dependent on structural features such as solvent exposure, secondary structure type, and hydrogen bonding properties, , - or on statistical potentials for residue interactions implied by the alignment. [ ] [ ] [ ] [ ] [ ] the use of structural data does not have to be restricted to the structure side of the aligned sequence-structure pair. for example, sam-t makes use of the predicted local structure for the target sequence to enhance homolog detection and alignment accuracy. commonly used threading programs are genthreader, , d-pssm, fugue, sp , sam-t multitrack hmm, , , and muster. iterative sequence-structure alignment yet another strategy is to optimize the alignment by iterating over the process of calculating alignments, building models, and evaluating models. such a protocol can sample alignments that are not statistically significant and identify the alignment that yields the best model. although this procedure can be time-consuming, it can significantly improve the accuracy of the resulting comparative models in difficult cases. regardless of the method used, searching in the twilight and midnight zones of the sequence-structure relationship often results in false negatives, false positives, or alignments that contain an increasingly large number of gaps and alignment errors. improving the performance and accuracy of methods in this regime remains one of the main tasks of comparative modeling today. it is imperative to calculate an accurate alignment between the target-template pair. although some progress has been made recently, comparative modeling can rarely recover from an alignment error. after a list of all related protein structures and their alignments with the target sequence have been obtained, template structures are prioritized depending on the purpose of the comparative model. template structures may be chosen purely based on the targettemplate sequence identity or a combination of several other criteria, such as experimental accuracy of the structures (resolution of x-ray structures, number of restraints per residue for nmr structures), conservation of active-site residues, holo-structures that have bound ligands of interest, and prior biological information that pertains to the solvent, ph, and quaternary contacts. it is not necessary to select only one template. in fact, the use of several templates approximately equidistant from the target sequence generally increases the model accuracy. , model building once an initial target-template alignment is built, a variety of methods can be used to construct a d model for the target protein. , , [ ] [ ] [ ] [ ] the original and still widely used method is modeling by rigid-body assembly. , , this method constructs the model from a few core regions, and from loops and side chains that are obtained by dissecting related structures. commonly used programs that implement this method are composer, - d-jigsaw, rosettacm, and swiss-model. another family of methods, modeling by segment matching, relies on the approximate positions of conserved atoms from the templates to calculate the coordinates of other atoms. [ ] [ ] [ ] [ ] [ ] an instance of this approach is implemented in segmod. the third group of methods, modeling by satisfaction of spatial restraints, uses either distance geometry or optimization techniques to satisfy spatial restraints obtained from the alignment of the target sequences with the template structures. , [ ] [ ] [ ] [ ] specifically, modeller, , , our own program for comparative modeling, belongs to this group of methods. modeller implements comparative protein structure modeling by the satisfaction of spatial restraints that include: ( ) homologyderived restraints on the distances and dihedral angles in the target sequence, extracted from its alignment with the template structures; ( ) stereochemical restraints such as bond length and bond angle preferences, obtained from the charmm- molecular mechanics force field; ( ) statistical preferences for dihedral angles and nonbonded interatomic distances, obtained from a representative set of known protein structures; and ( ) optional manually curated restraints, such as those from nmr spectroscopy, rules of secondary structure packing, cross-linking experiments, fluorescence spectroscopy, image reconstruction from electron microscopy, site-directed mutagenesis, and intuition ( figure (b) ). the spatial restraints, expressed as probability density functions, are combined into an objective function that is optimized by a combination of conjugate gradients and molecular dynamics with simulated annealing. this model-building procedure is similar to structure determination by nmr spectroscopy. accuracies of the various model-building methods are relatively similar when used optimally. , other factors, such as template selection and alignment accuracy, usually have a larger impact on the model accuracy, especially for models based on less than % sequence identity to the templates. however, it is important that a modeling method allows a degree of flexibility and automation to obtain better models more easily and rapidly. for example, a method should allow for an easy recalculation of a model when a change is made in the alignment; it should be straightforward to calculate models based on several templates; and the method should provide tools for incorporation of prior knowledge about the target (e.g., cross-linking restraints and predicted secondary structure). protein sequences evolve through a series of amino acid residue substitutions, insertions, and deletions. while substitutions can occur throughout the length of the sequence, insertions and deletions mostly occur on the surface of proteins in segments that connect regular secondary structure segments (i.e., loops). while the template structures are helpful in the modeling of the aligned target backbone segments, they are generally less valuable for the modeling of side chains and irrelevant for the modeling of insertions such as loops. the loops and side chains of comparative models are especially important for ligand docking; thus, we discuss them in the following two sections. loop modeling is an especially important aspect of comparative modeling in the range from % to % sequence identity. in this range of overall similarity, loops among the homologs vary while the core regions are still relatively conserved and aligned accurately. loops often play an important role in defining the functional specificity of a given protein, forming the active and binding sites. loop modeling can be seen as a mini protein folding problem because the correct conformation of a given segment of a polypeptide chain has to be calculated mainly from the sequence of the segment itself. however, loops are generally too short to provide sufficient information about their local fold. even identical decapeptides in different proteins do not always have the same conformation. , some additional restraints are provided by the core anchor regions that span the loop and by the structure of the rest of the protein that cradles the loop. although many loop-modeling methods have been described, it is still challenging to correctly and confidently model loops longer than approximately - residues. , , two classes of methods there are two main classes of loop-modeling methods: ( ) database search approaches that scan a database of all known protein structures to find segments fitting the anchor core regions , ; and ( ) conformational search approaches that rely on optimizing a scoring function. [ ] [ ] [ ] there are also methods that combine these two approaches. , database-based loop modeling the database search approach to loop modeling is accurate and efficient when a database of specific loops is created to address the modeling of the same class of loops, such as b-hairpins, or loops on a specific fold, such as the hypervariable regions in the immunoglobulin fold. , there are attempts to classify loop conformations into more general categories, thus extending the applicability of the database search approach. [ ] [ ] [ ] however, the database methods are limited because the number of possible conformations increases exponentially with the length of a loop, and until the late s only loops up to residues long could be modeled using the database of known protein structures. , however, the growth of the pdb in recent years has largely eliminated this problem. optimization-based methods there are many optimization-based methods, exploiting different protein representations, objective functions, and optimization or enumeration algorithms. the search algorithms include the minimum perturbation method, dihedral angle search through a rotamer library, , molecular dynamics simulations, , genetic algorithms, monte carlo and simulated annealing, [ ] [ ] [ ] multiple-copy simultaneous search, self-consistent field optimization, robotics-inspired kinematic closure and enumeration based on graph theory. the accuracy of loop predictions can be further improved by clustering the sampled loop conformations and partially accounting for the entropic contribution to the free energy. another way of improving the accuracy of loop predictions is to consider the solvent effects. improvements in implicit solvation models, such as the generalized born solvation model, motivated their use in loop modeling. the solvent contribution to the free energy can be added to the scoring function for optimization, or it can be used to rank the sampled loop conformations after they are generated with a scoring function that does not include the solvent terms. , [ ] [ ] [ ] side-chain modeling two simplifications are frequently applied in the modeling of side-chain conformations. first, amino acid residue replacements often leave the backbone structure almost unchanged, allowing us to fix the backbone during the search for the best side-chain conformations. second, most side chains in high-resolution crystallographic structures can be represented by a limited number of conformers that comply with stereochemical and energetic constraints. this observation motivated ponder and richards to develop the first library of side-chain rotamers for the types of residues with dihedral angle degrees of freedom in their side chains, based on high-resolution protein structures determined by x-ray crystallography. subsequently, a number of additional libraries have been derived. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] rotamers rotamers on a fixed backbone are often used when all the side chains need to be modeled on a given backbone. this approach reduces the combinatorial explosion associated with a full conformational search of all the side chains, and is applied by some comparative modeling and protein design approaches. however, $ % of the side chains cannot be represented well by these libraries. in addition, it has been shown that the accuracy of side-chain modeling on a fixed backbone decreases rapidly when the backbone errors are larger than . Å . earlier methods for side-chain modeling often put less emphasis on the energy or scoring function. the function was usually greatly simplified, and consisted of the empirical rotamer preferences and simple repulsion terms for nonbonded contacts. nevertheless, these approaches have been justified by their performance. for example, a method based on a rotamer library compared favorably with that based on a molecular mechanics force field, and new methods continue to be based on the rotamer library approach. [ ] [ ] [ ] the various optimization approaches include a monte carlo simulation, simulated annealing, a combination of monte carlo and simulated annealing, the dead-end elimination theorem, , genetic algorithms, neural network with simulated annealing, mean field optimization, and combinatorial searches. , , several studies focused on the testing of more sophisticated potential functions for conformational search , and development of new scoring functions for side-chain modeling, reporting higher accuracy than earlier studies. the major sources of error in comparative modeling are discussed in the relevant sections above. the following is a summary of these errors, dividing them into five categories (figure ) . this error is a potential problem when distantly related proteins are used as templates (i.e., less than % sequence identity). distinguishing between a model based on an incorrect template and a model based on an incorrect alignment with a correct template is difficult. in both cases, the evaluation methods (below) will predict an unreliable model. the conservation of the key functional or structural residues in the target sequence increases the confidence in a given fold assignment. the single source of errors with the largest impact on comparative modeling is misalignments, especially when the target-template sequence identity decreases below %. alignment errors can be minimized in two ways. using the profile-based methods discussed above usually results in more accurate alignments than those from pairwise sequence alignment methods. another way of improving the alignment is to modify those regions in the alignment that correspond to predicted errors in the model. segments of the target sequence that have no equivalent region in the template structure (i.e., insertions or loops) are one of the most difficult regions to model. again, when the target and template are distantly related, errors in the alignment can lead to incorrect positions of the insertions. using alignment methods that incorporate structural information can often correct such errors. once a reliable alignment is obtained, various modeling protocols can predict the loop conformation, for insertions of fewer than - residues. , , , distortions and shifts in correctly aligned regions as a consequence of sequence divergence, the main-chain conformation changes, even if the overall fold remains the same. therefore, it is possible that in some correctly aligned segments of a model, the template is locally different (< Å ) from the target, resulting in errors in that region. the structural differences are sometimes not due to differences in sequence, but are a consequence of artifacts in structure determination or structure determination in different environments (e.g., packing of subunits in a crystal). the simultaneous use of several templates can minimize this kind of error. , figure typical errors in comparative modeling. shown are the typical sources of errors encountered in comparative models. two of the major sources of errors in comparative modeling are due to incorrect templates or incorrect alignments with the correct templates. the modeling procedure can rarely recover from such errors. the next significant source of errors arises from regions in the target with no corresponding region in the template, i.e., insertions or loops. other sources of errors, which occur even with an accurate alignment, are due to rigid-body shifts, distortions in the backbone, and errors in the packing of side chains. as the sequences diverge, the packing of the atoms in the protein core changes. sometimes even the conformation of identical side chains is not conserved -a pitfall for many comparative modeling methods. side-chain errors are critical if they occur in regions that are involved in protein function, such as active sites and ligand-binding sites. the accuracy of the predicted model determines the information that can be extracted from it. thus, estimating the accuracy of a model in the absence of the known structure is essential for interpreting it. as discussed earlier, a model calculated using a template structure that shares more than % sequence identity is indicative of an overall accurate structure. however, when the sequence identity is lower, the first aspect of model evaluation is to confirm whether or not a correct template was used for modeling. it is often the case, when operating in this regime, that the fold assignment step produces only false positives. a further complication is that at such low similarities the alignment generally contains many errors, making it difficult to distinguish between an incorrect template on one hand and an incorrect alignment with a correct template on the other hand. there are several methods that use d profiles and statistical potentials, , , which assess the compatibility between the sequence and modeled structure by evaluating the environment of each residue in a model with respect to the expected environment, as found in native high-resolution experimental structures. these methods can be used to assess whether or not the correct template was used for the modeling. they include verify d, and tsvmod. even when the model is based on alignments that have > % sequence identity, other factors, including the environment, can strongly influence the accuracy of a model. for instance, some calcium-binding proteins undergo large conformational changes when bound to calcium. if a calcium-free template is used to model the calcium-bound state of the target, it is likely that the model will be incorrect, irrespective of the target-template similarity or accuracy of the template structure. the model should also be subjected to evaluations of self-consistency to ensure that it satisfies the restraints used to calculate it. additionally, the stereochemistry of the model (e.g., bond lengths, bond angles, backbone torsion angles, and nonbonded contacts) may be evaluated using programs such as procheck and whatcheck. although errors in stereochemistry are rare and less informative than errors detected by statistical potentials, a cluster of stereochemical errors may indicate that there are larger errors (e.g., alignment errors) in that region. when multiple models are calculated for the target based on a single template or when multiple loops are built for a single or multiple models, it is practical to select a subset of models or loops that are judged to be most suitable for subsequent docking calculations. if some known ligands or other information for the desired model is available, model selection should be guided by this known information. if this extra information is not available, model selection should aim to select the most accurate model. while models or loops can be selected by the energy function used for guiding the building of comparative models or the sampling of loop configurations, using a separate statistical potential for selecting the most accurate models or loops is often more successful. , , , it is crucial for method developers and users alike to assess the accuracy of their methods. an attempt to address this problem has been made by the critical assessment of techniques for proteins structure prediction (casp) and in the past by the critical assessment of fully automated structure prediction (cafasp) experiments, which is now integrated into casp. however, casp assesses methods only over a limited number of target protein sequences, and is conducted only every years. , to overcome this limitation, the new cameo web server continuously evaluates the accuracy and reliability of a number of comparative protein structure prediction servers, in a fully automated manner. every week, cameo provides each tested server with the prerelease sequences of structures that are to be shortly released by the pdb. each server then has days to build and return a d model of these sequences. when pdb releases the structures, cameo compares the models against the experimentally determined structures, and presents the results on its web site. this enables developers, non-expert users, and reviewers to determine the performance of the tested prediction servers. cameo is similar in concept to two prior such continuous testing servers, livebench and eva. , there is a wide range of applications of protein structure models ( figure ) . , [ ] [ ] [ ] [ ] [ ] [ ] [ ] for example, high-and medium-accuracy comparative models are frequently helpful in refining functional predictions that have been based on a sequence match alone because ligand binding is more directly determined by the structure of the binding site than by its sequence. it is often possible to predict correctly features of the target protein that do not occur in the template structure. , for example, the size of a ligand may be predicted from the volume of the binding site cleft and the location of a binding site for a charged ligand can be predicted from a cluster of charged residues on the protein. fortunately, errors in the functionally important regions in comparative models are many times relatively low because the functional regions, such as active sites, tend to be more conserved in evolution than the rest of the fold. even low-accuracy comparative models may be useful, for example, for assigning the fold of a protein. fold figure accuracy and applications of protein structure models. shown are the different ranges of applicability of comparative protein structure modeling, threading, and de novo structure prediction, their corresponding accuracies, and their sample applications. assignment can be very helpful in drug discovery, because it can shortcut the search for leads by pointing to compounds that have been previously developed for other members of the same family. , the remainder of this review focuses on the use of comparative models for ligand docking. [ ] [ ] [ ] comparative protein structure modeling extends the applicability of virtual screening beyond the atomic structures determined by x-ray crystallography or nmr spectroscopy. in fact, comparative models have been used in virtual screening to detect novel ligands for many protein targets, including the g-protein coupled receptors (gpcr), , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] protein kinases, [ ] [ ] [ ] [ ] nuclear hormone receptors, and many different enzymes. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] nevertheless, the relative utility of comparative models versus experimentally determined structures has only been relatively sparsely assessed. , , , [ ] [ ] [ ] the utility of comparative models for molecular docking screens in ligand discovery has been documented with the aid of protein targets selected from the 'directory of useful decoys' (dud). for each target sequence, templates for comparative modeling were obtained from the pdb, including at least one holo (ligand bound) and one apo (ligand free) template structure for each of the eight % sequence identity ranges from % to %. in total, models were generated based on templates for the test proteins using modeller v . dud ligands and decoys ( molecules) were screened against the holo x-ray structure, the apo x-ray structure, and the comparative models of each target using dock . . . the accuracy of virtual screening was evaluated by the overall ligand enrichment that was calculated by integrating the area under the enrichment plot (logauc). a key result was that, for % and % of the targets, at least one comparative model yielded ligand enrichment better or comparable to that of the corresponding holo and apo x-ray structure. this result indicates that comparative models can be useful docking targets when multiple templates are available. however, it was not possible to predict which model, out of all those used, led to the highest enrichment. therefore, a 'consensus' enrichment score was computed by ranking each library compound by its best docking score against all comparative models and/or templates. for % and % of the targets, the consensus enrichment for multiple models was better or comparable to that of the holo and apo x-ray structures, respectively, suggesting that multiple comparative models can be useful for virtual screening. despite problems with comparative modeling and ligand docking, comparative models have been successfully used in practice in conjunction with virtual screening to identify novel inhibitors. we briefly review a few of these success stories to highlight the potential of the combined comparative modeling and ligand-docking approach to drug discovery. comparative models have been employed to aid rational drug design against parasites for more than years. , , , as early as , ring et al. used comparative models for computational docking studies that identified low micromolar nonpeptidic inhibitors of proteases in malarial and schistosome parasite lifecycles. li et al. subsequently used similar methods to develop nanomolar inhibitors of falcipain that are active against chloroquine-resistant strains of malaria. in a study by selzer et al. comparative models were used to predict new nonpeptide inhibitors of cathepsin l-like cysteine proteases in leishmania major. sixty-nine compounds were selected by dock . as strong binders to a comparative model of protein cpb, and of these, had experimental ic values below mmol l À . finally, in a study by que et al., comparative models were used to rationalize ligand-binding affinities of cysteine proteases in entamoeba histolytica. specifically, this work provided an explanation for why proteases acp and acp had substrate specificity similar to that of cathepsin b, although their overall structure is more similar to that of cathepsin d. enyedy et al. discovered new inhibitors of matriptase by docking against its comparative model. the comparative model employed thrombin as the template, sharing only % sequence identity with the target sequence. moreover, some residues in the binding site are significantly different; a trio of charged asp residues in matriptase correspond to tyr and trp residues in thrombin. thrombin was chosen as the template, in part because it prefers substrates with positively charged residues at the p position, as does matriptase. the national cancer institute database was used for virtual screening that targeted the s site with the dock program. the best-scoring compounds were manually inspected to identify positively charged ligands (the s site is negatively charged), and compounds were experimentally screened for inhibition, identifying the inhibitors. one of them, hexamidine, was used as a lead to identify additional compounds selective for matriptase relative to thrombin. the wang group has also used similar methods to discover seven new, low-micromolar inhibitors of bcl- , using a comparative model based on the nmr solution structure of bcl-x l . schapira et al. discovered a novel inhibitor of a retinoic acid receptor by virtual screening using a comparative model. in this case, the target (rar-a) and template (rar-g) are very closely related; only three residues in the binding site are not conserved. the icm program was used for virtual screening of ligands from the available chemicals directory (acd). the high-scoring compounds identified in the first round were subsequently docked into a full atom representation of the receptor with flexible side chains to obtain a final set of good-scoring hits. these compounds were then manually inspected to choose the final for testing. two novel agonists were identified, with -nanomolar activity. zuccotto et al. identified novel inhibitors of dihydrofolate reductase (dhfr) in trypanosoma cruzi (the parasite that causes chagas disease) by docking into a comparative model based on $ % sequence identity to dhfr in l. major, a related parasite. the virtual screening procedure used dock for rigid docking of over selected compounds from the cambridge structural database (csd). visual inspection of the top hits was used to select compounds for experimental testing. this work identified several novel scaffolds with micromolar ic values. the authors report attempting to use virtual screening results to identify compounds with greater affinity for t. cruzi dhfr than human dhfr, but it is not clear how successful they were. following the outbreak of the severe acute respiratory syndrome (sars) in , anand et al. used the experimentally determined structures of the main protease from human coronavirus (m pro ) and an inhibitor complex of porcine coronavirus (transmissible gastroenteritis virus, tgev) m pro to calculate a comparative model of the sars coronavirus m pro . this model then provided a basis for the design of anti-sars drugs. in particular, a comparison of the active site residues in these and other related structures suggested that the ag inhibitor of the human rhinovirus type c protease is a good starting point for design of anticoronaviral drugs. comparative models of protein kinases combined with virtual screening have also been intensely used for drug discovery. , , [ ] [ ] [ ] the > kinases in the human genome, the relatively small number of experimental structures available, and the high level of conservation around the important adenosine triphosphate-binding site make comparative modeling an attractive approach toward structure-based drug discovery. g protein-coupled receptors are another interesting class of proteins that in principle allow drug discovery through comparative modeling. , [ ] [ ] [ ] [ ] approximately % of current drug targets belong to this class of proteins. however, these proteins have been extremely difficult to crystallize and most comparative modeling has been based on the atomic resolution structure of the bovine rhodopsin. despite this limitation, a rather extensive test of docking methods with rhodopsin-based comparative models shows encouraging results. the applicability of structure-based modeling and virtual screening has recently been expanded to membrane proteins that transport solutes, such as ions, metabolites, peptides, and drugs. in humans, these transporters contribute to the absorption, distribution, metabolism, and excretion of drugs, and often, mediate drug-drug interactions. additionally, several transporters can be targeted directly by small molecules. for instance, methylphenidate (ritalin) inhibiting the norepinephrine transporter (net) and, consequently, inhibiting the reuptake of norepinephrine, is used in the treatment of attention-deficit hyperactivity disorder (adhd). schlessinger et al. predicted putative ligands of human net by docking drugs from the kyoto encyclopedia of genes (kegg drug) into a comparative model based on $ % sequence identity to leucine transporter (leut) from aquifex aeolicus. of these predicted ligands, ten were validated by cis-inhibition experiments; five of them were chemically novel. close examination of the predicted primary binding site helped rationalize interactions of net with its primary substrate, norepineprhine, as well as positive and negative pharmacological effects of other net ligands. subsequently, schlessinger et al. modeled two different conformations of the human gaba transporter (gat- ), using the leut structures in occluded-outward-facing and outward-facing conformations. enrichment calculations were used to assess the quality of the models in molecular dynamics simulations and side-chain refinements. the key residue, glu , interacting with the substrate was identified during the refinement of the models and validated by site-directed mutagenesis. docking against two conformations of the transporter enriches for different physicochemical properties of ligands. for example, top-scoring ligands found by docking against the outward-facing model were bulkier and more hydrophobic than those predicted using the occluded-outward-facing model. among twelve ligands validated in cis-inhibition assays, six were chemically novel (e.g., homotaurine). based on the validation experiments, gat- is likely to be a high-selectivity/low-affinity transporter. following these two studies, a combination of comparative modeling, ligand docking, and experimental validation was used to rationalize toxicity of an anti-cancer agent, acivicin. the toxic sideeffects are thought to be facilitated by the active transport of acivicin through the blood-brain-barrier (bbb) via the large-neutral amino acid transporter (lat- ). in addition, four small-molecule ligands of lat- were identified by docking against a comparative model based on two templates, the structures of the outward-occluded arginine-bound arginine/agmatine transporter adic from e. coli and the inward-apo conformation of the amino acid, polyamine, and organo-cation transporter apct from methanococcus jannaschii. two of the four hits, acivicin and fenclonine, were confirmed as substrates by a trans-stimulation assay. these studies clearly illustrate the applicability of combined comparative modeling and virtual screening to ligand discovery for transporters. although reports of successful virtual screening against comparative models are encouraging, such efforts are not yet a routine part of rational drug design. even the successful efforts appear to rely strongly on visual inspection of the docking results. much work remains to be done to improve the accuracy, efficiency, and robustness of docking against comparative models. despite assessments of relative successes of docking against comparative models and native x-ray structures, , relatively little has been done to compare the accuracy achievable by different approaches to comparative modeling and to identify the specific structural reasons why comparative models generally produce less accurate virtual screening results than the holo structures. among the many issues that deserve consideration are the following: • the inclusion of cofactors and bound water molecules in protein receptors is often critical for success of virtual screening; however, cofactors are not routinely included in comparative models • most docking programs currently retain the protein receptor in a rigid conformation. while this approach is appropriate for 'lock-and-key' binding modes, it does not work when the ligand induces conformational changes in the receptor upon binding. a flexible receptor approach is necessary to address such induced-fit cases , • the accuracy of comparative models is frequently judged by the c a root mean square error or other similar measures of backbone accuracy. for virtual screening, however, the precise positioning of side chains in the binding site is likely to be critical; measures of accuracy for binding sites are needed to help evaluate the suitability of comparative modeling algorithms for constructing models for docking • knowledge of known inhibitors, either for the target protein or the template, should help to evaluate and improve virtual screening against comparative models. for example, comparative models constructed from holo template structures implicitly preserve some information about the ligand-bound receptor conformation • improvement in the accuracy of models produced by comparative modeling will require methods that finely sample protein conformational space using a free energy or scoring function that has sufficient accuracy to distinguish the native structure from the nonnative conformations. despite many years of development of molecular simulation methods, attempts to refine models that are already relatively close to the native structure have met with relatively little success. this failure is likely to be due in part to inaccuracies in the scoring functions used in the simulations, particularly in the treatment of electrostatics and solvation effects. a combination of physics-based energy function with the statistical information extracted from known protein structures may provide a route to the development of improved scoring functions • improvements in sampling strategies are also likely to be necessary, for both comparative modeling and flexible docking given the increasing number of target sequences for which no experimentally determined structures are available, drug discovery stands to gain immensely from comparative modeling and other in silico methods. despite unsolved problems in virtually every step of comparative modeling and ligand docking, it is highly desirable to automate the whole process, starting with the target sequence and ending with a ranked list of its putative ligands. automation encourages development of better methods, improves their testing, allows application on a large scale, and makes the technology more accessible to both experts and non-specialists alike. through large-scale application, new questions, such as those about ligand-binding specificity, can in principle be addressed. enabling a wider community to use the methods provides useful feedback and resources toward the development of the next generation of methods. there are a number of servers for automated comparative modeling (table ) . however, in spite of automation, the process of calculating a model for a given sequence, refining its structure, as well as visualizing and analyzing its family members in the sequence and structure spaces can involve the use of scripts, local programs, and servers scattered across the internet and not necessarily interconnected. in addition, manual intervention is generally still needed to maximize the accuracy of the models in the difficult cases. the main repository for precomputed comparative models, the protein model portal, , , begins to address these deficiencies by serving models from several modeling groups, including the swiss-model and modbase databases. it provides access to web-based comparative modeling tools, cross-links to other sequence and structure databases, and annotations of sequences and their models. a number of databases containing comparative models and web servers for computing comparative models are publicly available. the protein model portal (pmp) , , centralizes access to these models created by different methodologies. the pmp is being developed as a module of the protein structure initiative knowledgebase (psi kb) and functions as a meta server for comparative models from external databases, including swiss-model and modbase, additionally to being a repository for comparative models that are derived from structures determined by the psi centers. it provides quality estimations of the deposited models, access to web-based comparative modeling tools, cross-links to other sequence and structure databases, annotations of sequences and their models, and detailed tutorials on comparative modeling and the use of their tools. the pmp currently contains . million comparative models for . million uniprot sequences (august ). a schematic of our own attempt at integrating several useful tools for comparative modeling is shown in figure . , modbase is a database that currently contains $ million predicted models for domains in approximately . million unique sequences from uniprot, ensembl, genbank, and private sequence datasets. the models were calculated using modpipe , and modeller. the web interface to the database allows flexible querying for fold assignments, sequence-structure alignments, models, and model assessments. an integrated sequence-structure viewer, chimera, allows inspection and analysis of the query results. models can also be calculated using modweb, , a web interface to modpipe, and stored in modbase, which also makes them accessible through the pmp. other resources associated with modbase include a comprehensive database of multiple protein structure alignments (dbali), structurally defined ligand-binding sites, structurally defined binary domain interfaces (pibase), , predictions of ligand-binding sites, interactions between yeast proteins, and functional consequences of human nssnps (ls-snp). , , a number of associated web services handle modeling of loops in protein structures (modloop), , evaluation of models (modeval), fitting of models against small angle x-ray scattering (saxs) profiles (foxs), - modeling of ligand-induced protein dynamics such as allostery (allosmod), , prediction of the ensemble of conformations that best fit a given saxs profile (allosmod-foxs), prediction of cryptic binding sites, scoring protein-ligand complexes based on a , compared to protein structure prediction, the attempts at automation and integration of resources in the field of docking for virtual screening are still in their nascent stages. one of the successful efforts in this direction is zinc, , a publicly available database of commercially available drug-like compounds, developed in the laboratory of brian shoichet. zinc contains more than million 'ready-to-dock' compounds organized in several subsets and allows the user to query the compounds by molecular properties and constitution. the shoichet group also provides a dockblaster service that enables end-users to dock the zinc compounds against their target structures using dock. , in the future, we will no doubt see efforts to improve the accuracy of comparative modeling and ligand docking. but perhaps as importantly, the two techniques will be integrated into a single protocol for more accurate and automated docking of ligands against sequences without known structures. as a result, the number and variety of applications of both comparative modeling and ligand docking will continue to increase. cameo http://cameo d.org/ casp http://predictioncenter.llnl.gov figure an integrated set of resources for comparative modeling. various databases and programs required for comparative modeling and docking are usually scattered over the internet, and require manual intervention or a good deal of expertise to be useful. automation and integration of these resources are efficient ways to put these resources in the hands of experts and non-specialists alike. we have outlined a comprehensive interconnected set of resources for comparative modeling and hope to integrate it with a similar effort in the area of ligand docking made by the shoichet group. , drug disc comparative protein structure modeling modeller comparative protein structure modeling and its applications to drug discovery comparative protein structure modeling this article is partially based on papers by jacobson and sali, fiser and sali, and madhusudhan et al. we also acknowledge the funds from sandler family supporting foundation, nih r gm , p gm , p a , and u gm , as well as sun, ibm, and intel for hardware gifts. key: cord- -q qw authors: leão, rozires p.; cruz, josiane v.; da costa, glauber v.; cruz, jorddy n.; ferreira, elenilze f. b.; silva, raí c.; de lima, lúcio r.; borges, rosivaldo s.; dos santos, gabriela b.; santos, cleydson b. r. title: identification of new rofecoxib-based cyclooxygenase- inhibitors: a bioinformatics approach date: - - journal: pharmaceuticals (basel) doi: . /ph sha: doc_id: cord_uid: q qw the cyclooxygenase- receptor is a therapeutic target for planning potential drugs with anti-inflammatory activity. the selective cyclooxygenase- (cox- ) inhibitor rofecoxib was selected as a pivot molecule to perform virtual ligand-based screening from six commercial databases. we performed the search for similarly shaped rapid overlay of chemical structures (rocs) and electrostatic (eon) compounds. after, we used pharmacokinetic and toxicological parameters to determine the best potential compounds, obtained through the softwares qikprop and derek, respectively. then, the compounds proceeded to the molecular anchorage study, which showed promising results of binding affinity with the hcox- receptor: lmqc (∆g = − . kcal/mol), lmqc (∆g = − . kcal/mol), and lmqc (∆g = − . kcal/mol). lmqc and lmqc showed higher binding affinity compared to rofecoxib (∆g = − . kcal/mol). finally, molecular dynamics (md) simulations were used to evaluate the interaction of the compounds with the target hcox- during ns. in all md simulation trajectories, the ligands remained interacting with the protein until the end of the simulation. the compounds were also complexing with hcox- favorably. the compounds obtained the following affinity energy values: rofecoxib: Δgbind = − . kcal/mol; lmqc : Δgbind = − . kcal/mol; lmqc : Δgbind = − . kcal/mol; and lmqc : Δgbind = − . kcal/mol. the selected lmqc , lmqc , and lmqc structures showed satisfactory pharmacokinetic results related to absorption and distribution. the toxicological predictions of these compounds did not display alerts for possible toxic groups and lower risk of cardiotoxicity compared to rofecoxib. therefore, future in vitro and in vivo studies are needed to confirm the anti-inflammatory potential of the compounds selected here with bioinformatics approaches based on rofecoxib ligand. cyclooxygenases are an important and thoroughly studied group of enzymes present in two isoforms in mammals: constitutive form cyclooxygenase- (cox- ) and an inducible form cyclooxygenase- (cox- ) [ ] . the cox- enzyme is expressed in most tissues and is responsible for maintaining homeostasis and production of prostaglandins (pgs) [ ] . cox- is found predominantly in the brain, renal, and endothelial cells and is significantly increased through various acute and chronic inflammatory infections [ , ] . the inhibition of the cox- enzyme through selective anti-inflammatory drugs has been an important strategy to contain the inflammation process. many selective cox- inhibitors achieve the market as anti-inflammatory drugs, such as celecoxib (celebrex ® ), valdecoxib (bextra ® ), and rofecoxib (vioxx ® ) [ ] , once it was thought that more selective drugs leads to less gastric side-affects-see figure . nevertheless, some of these selective inhibitors of cox- also depress prostacyclin (pgi ), an atheroprotective agent, which might predispose patients to heart attack and stroke [ ] . pharmaceuticals , , x for peer review of cyclooxygenases are an important and thoroughly studied group of enzymes present in two isoforms in mammals: constitutive form cyclooxygenase- (cox- ) and an inducible form cyclooxygenase- (cox- ) [ ] . the cox- enzyme is expressed in most tissues and is responsible for maintaining homeostasis and production of prostaglandins (pgs) [ ] . cox- is found predominantly in the brain, renal, and endothelial cells and is significantly increased through various acute and chronic inflammatory infections [ , ] . the inhibition of the cox- enzyme through selective anti-inflammatory drugs has been an important strategy to contain the inflammation process. many selective cox- inhibitors achieve the market as anti-inflammatory drugs, such as celecoxib (celebrex ® ), valdecoxib (bextra ® ), and rofecoxib (vioxx ® ) [ ] , once it was thought that more selective drugs leads to less gastric side-affects-see figure . nevertheless, some of these selective inhibitors of cox- also depress prostacyclin (pgi ), an atheroprotective agent, which might predispose patients to heart attack and stroke [ ] . thus, the side effects promoted by these therapeutic agents directed the search for new compounds, whose anti-inflammatory potential is accompanied by greater selectivity and specificity, minimal side effects, and lower cost [ ] . rofecoxib (vioxx ® ) was approved by the food and drug administration (fda) for human use in may , and withdrawn from the market on september, [ ] . this drug, from the coxibs family, presents risks of cardiovascular events; however, it presents anti-inflammatory effects and properties similar to traditional non-steroidal anti-inflammatory drugs (nsaids) with reduced gastrointestinal toxicity, which would have the potential [ ] [ ] [ ] [ ] [ ] without side-effects, such as ulcers and gastrointestinal problems [ ] . since then, rofecoxib became an important prototype for the design of new promising nsaids for the cox- target and with possible minor side effects in humans [ ] . in this work, we used a virtual screening ligand-based methodology to identify new potential cox- inhibitors based on the rofecoxib structure [ ] [ ] [ ] . the virtual screening strategy was chosen once it has been widely applied in the early phase of drug discovery, being able to accelerate hit discovery and reducing drug development costs. thus, the similarity and electrostatic potential of the selected structures were performed using computer programs and commercial databases of compounds [ ] and then we performed the filtering of the results considering the pharmacokinetic and toxicological properties [ ] [ ] [ ] [ ] [ ] . furthermore, the docking simulation evaluated the binding affinity of compounds to cox- in comparison with rofecoxib [ , ] . biological target prediction was used as a screening step through the web server swiss [ ] and the bioactivity was determined on the molinspiration web server [ ] . finally, we used molecular dynamics to investigate interaction over time in cox- of the promising compounds. general scheme of the methodological steps in this article is presented in figure (see more details in the materials and methods section). thus, the side effects promoted by these therapeutic agents directed the search for new compounds, whose anti-inflammatory potential is accompanied by greater selectivity and specificity, minimal side effects, and lower cost [ ] . rofecoxib (vioxx ® ) was approved by the food and drug administration (fda) for human use in may , and withdrawn from the market on september, [ ] . this drug, from the coxibs family, presents risks of cardiovascular events; however, it presents anti-inflammatory effects and properties similar to traditional non-steroidal anti-inflammatory drugs (nsaids) with reduced gastrointestinal toxicity, which would have the potential [ ] [ ] [ ] [ ] [ ] without side-effects, such as ulcers and gastrointestinal problems [ ] . since then, rofecoxib became an important prototype for the design of new promising nsaids for the cox- target and with possible minor side effects in humans [ ] . in this work, we used a virtual screening ligand-based methodology to identify new potential cox- inhibitors based on the rofecoxib structure [ ] [ ] [ ] . the virtual screening strategy was chosen once it has been widely applied in the early phase of drug discovery, being able to accelerate hit discovery and reducing drug development costs. thus, the similarity and electrostatic potential of the selected structures were performed using computer programs and commercial databases of compounds [ ] and then we performed the filtering of the results considering the pharmacokinetic and toxicological properties [ ] [ ] [ ] [ ] [ ] . furthermore, the docking simulation evaluated the binding affinity of compounds to cox- in comparison with rofecoxib [ , ] . biological target prediction was used as a screening step through the web server swiss [ ] and the bioactivity was determined on the molinspiration web server [ ] . finally, we used molecular dynamics to investigate interaction over time in cox- of the promising compounds. general scheme of the methodological steps in this article is presented in figure (see more details in the materials and methods section). in this initial stage, the pivot molecule rofecoxib was used as a research model for the virtual screening in six commercial molecule databases: chembridge diversetexp, diverset core library (https://www.chembridge.com) [ ] , maybridge collections (www.maybridge.com) [ , ] , zinc drug database, zinc natural stock (http://zinc.docking.org) [ ] , and drug fda bindingdb (http://www.bindingdb.org) [ ] using the programs rapid overlay of chemical structures (rocs) and electrostatic similarity (eon). in the rocs software [ ] [ ] [ ] , we used a virtual screening tool for searching three-dimensional ( d) structures with chemical similarity and shape with the pivot molecule rofecoxib [ , ] . the rofecoxib molecule was used as a comparison model with each of the molecules in the databases looking for chemical similarity [ , ] , according to the structural characteristics and molecular volume fractions of the pivot molecule, observing the maximum overlap in relation to the shape (chemical structure), using as a parameter the gaussian functions [ ] implemented in the rocs software. the compounds were selected and classified by means of an algorithm that generated relative scores for the overlapping of forms in the databases according to the pharmacophoric characteristics of rofecoxib [ , ] . this stage of virtual screening identified the most similar two thousand ( ) molecules in each database (top_ ), resulting in twelve thousand ( , ) tracked structures, which exhibited highest scores of chemical similarities. in the sequence, the selected compounds were submitted to electrostatic correlations of aligned molecules based on the tanimoto electrostatic score in eon software [ , ] . this electrostatic potential is calculated using openeye's poisson-boltzmann (pb) electrostatic calculation [ , ] . the top molecules by database (top_ ), led to six hundred structures ( ) hits with best alignment based on the electrostatic potential [ ] . the remaining six hundred structures ( ) were then evaluated for their pharmacokinetic properties (the absorption, distribution, metabolism) using the qikprop software [ ] [ ] [ ] . structures submitted for pharmacokinetic study, resulted in two hundred and thirty-three ( ) hits that presented satisfactory pharmacokinetic properties, especially electronic affinity, lipinski's rule, and the central nervous system (cns) parameter, when compared with the properties of rofecoxib. in this initial stage, the pivot molecule rofecoxib was used as a research model for the virtual screening in six commercial molecule databases: chembridge diversetexp, diverset core library (https://www.chembridge.com) [ ] , maybridge collections (www.maybridge.com) [ , ] , zinc drug database, zinc natural stock (http://zinc.docking.org) [ ] , and drug fda bindingdb (http://www. bindingdb.org) [ ] using the programs rapid overlay of chemical structures (rocs) and electrostatic similarity (eon). in the rocs software [ ] [ ] [ ] , we used a virtual screening tool for searching three-dimensional ( d) structures with chemical similarity and shape with the pivot molecule rofecoxib [ , ] . the rofecoxib molecule was used as a comparison model with each of the molecules in the databases looking for chemical similarity [ , ] , according to the structural characteristics and molecular volume fractions of the pivot molecule, observing the maximum overlap in relation to the shape (chemical structure), using as a parameter the gaussian functions [ ] implemented in the rocs software. the compounds were selected and classified by means of an algorithm that generated relative scores for the overlapping of forms in the databases according to the pharmacophoric characteristics of rofecoxib [ , ] . this stage of virtual screening identified the most similar two thousand ( ) molecules in each database (top_ ), resulting in twelve thousand ( , ) tracked structures, which exhibited highest scores of chemical similarities. in the sequence, the selected compounds were submitted to electrostatic correlations of aligned molecules based on the tanimoto electrostatic score in eon software [ , ] . this electrostatic potential is calculated using openeye's poisson-boltzmann (pb) electrostatic calculation [ , ] . the top molecules by database (top_ ), led to six hundred structures ( ) hits with best alignment based on the electrostatic potential [ ] . the remaining six hundred structures ( ) were then evaluated for their pharmacokinetic properties (the absorption, distribution, metabolism) using the qikprop software [ ] [ ] [ ] . structures submitted for pharmacokinetic study, resulted in two hundred and thirty-three ( ) hits that presented satisfactory pharmacokinetic properties, especially electronic affinity, lipinski's rule, and the central nervous system (cns) parameter, when compared with the properties of rofecoxib. the "surviving structures" were submitted to derek software [ ] to evaluate toxicological properties, having as reference the properties of the commercial drug rofecoxib. thus, only seventy-nine structures were selected because they did not present toxicity alerts and toxicophoric groups [ ] . subsequently, these structures were subjected to a molecular study to assess binding mode and affinity with hcox- receptor. at the end of this process, only three structures (lmqc , lmqc , and lmqc ) were selected, for having binding affinity with the cox- molecular target and good pharmacokinetic and toxicological profile. therefore, this study discusses the main selected structures (lmqc , lmqc , and lmqc ) that offer promising results with the therapeutic ligand of interest. in silico prediction of absorption, distribution, metabolism, excretion, and toxicity (admet) properties are fundamental for the selection of the most promising molecules for further development. the selected structures were subjected to predictions of pharmacokinetic properties absorption, distribution, metabolism, and elimination using the qikprop software. to evaluate these properties, nine parameters (see table ) were used, related to the inflammatory process, and based on the compound rofecoxib. the #star parameter compares results obtained with properties of drugs present in database of the qikprop software [ ] . an alert is given when a result is outside the % range of values similar to commercially available drugs. this parameter takes into account a set of properties and descriptors such as: molecular weight (mw), dipole moment, electron affinity (ea), total solvent accessible surface area (sasa), hydrophobic component of the sasa (fosa), hydrophilic component of the sasa (fisa), π (carbon and attached hydrogen) component of the sasa (pisa), weakly polar component of the sasa (halogens, p, and s) (wpsa), polar surface area (psa), molecular volume, number of rotatable bonds (#rotor), number of hydrogen bond donor groups (donorhb), number of hydrogen bond acceptor groups (accpthb), predicted polarizability in cubic angstroms (qppolrz), predicted hexadecane/gas partition coefficient (qplogpc ), predicted octanol/gas partition coefficient (qplogpoct), predicted water/gas partition coefficient (qplogpw), predicted octanol/water partition coefficient (qplogpo/w), predicted aqueous solubility (logs), prediction of binding to human serum albumin (qplogkhsa), predicted brain/blood partition coefficient (qplogbb), number of likely metabolic reactions (#metabol) [ ] . these results for the three selected compounds are shown in table . the pharmacokinetic predictions for lmqc , lmqc , and lmqc show no violations in the descriptors and properties analyzed, which indicates that its properties are similar to commercial drugs (#star = ). however, rofecoxib has an alert (#star = ) in the molecular descriptor electronic affinity (ea), which is out of range (− . to . ), with a value of . ev. ea is an essential characteristic for intermolecular interactions and charge transfer complex [ ] [ ] [ ] [ ] . lipinski's (ro ) investigation are based on molecular weight (mw), lipophilicity (represented by the partition coefficient, logp) and hydrophilicity (represented by the number of hydrogen bond donors and acceptors groups) descriptors. ro represents a well-established form of limits for the absorption and permeability of a drug [ ] . in this study, lmqc , lmqc , and lmqc showed no violations to ro , indicating that these compounds would make it a likely orally active drug in humans. rofecoxib is an orally administered drug and in consonance, its properties did not violate the rule of lipinski (ro ). thus, this result predicts similarity to biological activity designed for oral administration [ , ] . the percentage of human oral absorption (%hoa) was evaluated through a set of properties based on number of metabolites (#metab), number of rotating bonds (#rotor), solubility and cell permeability in comparison within the standards [ ] . the prediction %hoa of the selected compounds showed excellent results, once lmqc , lmqc , and lmqc exhibited values of % hoa. moreover, rofecoxib showed a value of . % hoa, which indicates a better oral absorption of the novel compounds. ; e apparent permeability of compound between octanol/water (qplogpo/w); f permeability of the differentiated cells of intestinal epithelium caco- (qppcaco); g madin-darby canine kidney (qppmdck); h activity in the central nervous system; i apparent permeability of compound in the blood-brain barrier [ ] . pharmaceuticals , , of the apparent permeability between octanol/water (qplogpo/w) is a parameter used in drug design processes to estimate solubility, membrane permeability, and bioavailability [ ] [ ] [ ] [ ] . the calculated values regarding qlogpo/w for lmqc , lmqc , and lmqc are higher than the value found for rofecoxib (qplogpo/w = . ). lmqc , lmqc , and lmqc values ranged from . ≥ qlogpo/w ≥ . , considered more lipophilic compounds (logpo/w ≥ ). thus, this means that the novel compounds are mainly absorbed by passive transcellular processes in the intestine. lmqc , lmqc , and lmqc are within the limits indicated in ranges to , favoring better absorption, that is, easily overcome the lipid bilayer of biological membranes [ ] . models predictive of intestinal drug absorption are important in drug development to identify compounds with promising biopharmaceutical properties [ ] . in this study, the intestinal absorption was estimated by caco- and madin-darby canine kidney (mdck) cell values [ ] . predictions values of these cells make it possible to evaluate the cell permeability of potential drug candidates and routes of drug transport (e.g., passive versus carrier mediated) [ , ] . descriptors used for the prediction of passive transport should have values above nm/s to be considered good, whereas values less than nm/s are considered poor. lmqc , lmqc , and lmqc showed values between . and . nm/s for caco- cells and between . and . nm/s for mdck cells. thus, the compounds showed good results, indicating a promising intestinal absorption and even better in comparison with rofecoxib. the blood-brain barrier (bbb) is a critical factor in drug design. high penetration is needed for cns-active drugs, while negligible penetration may be desirable in order to minimize cns-related side-effects of drugs with a peripheral site of action [ , ] it is a selective barrier formed by narrow junctions between endothelial cells, to limit the penetration of different blood substances in the brain [ , ] . in our study, compounds lmqc , lmqc , and lmqc were evaluated by the brain-blood partition coefficient (qplogbb). the parameter established to indicate inactivity for penetration into the blood-brain barrier and consequent cns activity includes values below (c brain /c blood < ) and, for values greater than , it suggests activity in the central nervous system [ , ] . evaluation of the penetration capacity (qplogbb) of the lmqc , lmqc , and lmqc exhibited negative values (< ) which reveals low penetrability to cns [ ] . then, the prediction of the central nervous system activity of the selected compounds was performed. the established cns activity parameter ranges from − (inactive) to + (active). in our study, lmqc , lmqc , and lmqc exhibited values equal to zero ( ), which indicates that they are inactive and do not produce cns side effects in humans [ ] . therefore, these results are similar to the pivot compound rofecoxib, which has values of below (inactive) for the parameters: qplogbb and cns. in terms of pharmacokinetic properties, one may evaluate that the new hcox- inhibitors show better pharmacokinetic performance without violations in their descriptors and molecular properties when compared to rofecoxib. the seventy-nine compounds selected here by the toxicological studies followed the study of molecular docking to assess the binding mode and affinity with the hcox- receptor. to validate the molecular docking protocol, the crystallographic ligand was re-docked in the hcox- with the protein data bank (pdb) id kir structure with resolution . Å [ ] . the root mean square deviation (rmsd) obtained by re-docking, and the bonding pose found in the complex was . Å [ ] . the comparison between the crystallographic ligand and the pose predicted by docking overlap of the ligand can be visualized in figure . according to literature, the binding mode prediction using docking should present rmsd value < . Å when superimposed to the crystallographic pose of the ligand [ , , ] . we also evaluated the interaction affinity of rofecoxib to hcox- . the binding affinity value obtained in re-docking was ∆g = − . kcal/mol. it was considered close to the experimental value (∆g = − . kcal/mol). thus, our protocol showed satisfactory performance in predicting the interaction conformation once the interaction affinity value was close to the observed experimentally, see table . the seventy-nine compounds selected here by the toxicological studies followed the study of molecular docking to assess the binding mode and affinity with the hcox- receptor. to validate the molecular docking protocol, the crystallographic ligand was re-docked in the hcox- with the protein data bank (pdb) id kir structure with resolution . Å [ ] . the root mean square deviation (rmsd) obtained by re-docking, and the bonding pose found in the complex was . Å [ ] . the comparison between the crystallographic ligand and the pose predicted by docking overlap of the ligand can be visualized in figure . according to literature, the binding mode prediction using docking should present rmsd value < . Å when superimposed to the crystallographic pose of the ligand [ , , ] . we also evaluated the interaction affinity of rofecoxib to hcox- . the binding affinity value obtained in re-docking was ∆g = − . kcal/mol. it was considered close to the experimental value (∆g = − . kcal/mol). thus, our protocol showed satisfactory performance in predicting the interaction conformation once the interaction affinity value was close to the observed experimentally, see table . protein-ligand binding affinity is essential for biological processes, as these physical and chemical interactions determine biological recognition at the molecular level. in this way, it is possible to look for a ligand capable of inhibiting or activating a specific target protein through its interaction. in such a way, it is important to find a ligand that binds to a target protein with high affinity [ ] . all compounds that showed good pharmacokinetic and toxicological profiles were subjected to the molecular docking simulations in order to verify the binding affinity at the target receptor binding site (hcox- , pdb kir). binding affinity values of the compounds with higher affinity to hcox- compounds are shown in figure . protein-ligand binding affinity is essential for biological processes, as these physical and chemical interactions determine biological recognition at the molecular level. in this way, it is possible to look for a ligand capable of inhibiting or activating a specific target protein through its interaction. in such a way, it is important to find a ligand that binds to a target protein with high affinity [ ] . all compounds that showed good pharmacokinetic and toxicological profiles were subjected to the molecular docking simulations in order to verify the binding affinity at the target receptor binding site (hcox- , pdb kir). binding affinity values of the compounds with higher affinity to hcox- compounds are shown in figure . tables and . molecular docking studies (autodock/vina) [ ] also allowed us to determine the types of interactions between the target receptor's binding site with the promising compounds lmqc , lmqc and lmqc . table shows the interactions between the hcox- inhibitor rofecoxib (pdb id kir). in comparison with the pivot, table shows the types of interactions and amino acid residues between hcox- and lmqc , lmqc , and lmqc . crystallographic complex of rofecoxib with hcox- deposited in the pdb under the code kir exhibits the main interactions in the region of monomer b of the protein. rofecoxib methyl sulfone group binds to the active site of the enzyme, specifically with residues: his and arg in the α helix of the hydrophilic part of hcox- (chain b) [ , ] . figure a shows the amino acid residues phe , leu , ala , ser , val , and val of hcox- interacting with rofecoxib [ ] . experimental data shows that the selected compounds share the following interactions with hcox- : lmqc makes a hydrophobic and pi-alkyl type interaction with residues val and ala , respectively in the α-helix and β-leaf regions of the protein ( figure b ). lmqc makes a hydrophobic and pi-alkyl interactions with ala residue located in the α-helix region of the protein ( figure c ). lmqc interacts with three amino acid residues that are present in the hcox- interaction with rofecoxib, which are val (pi-alkyl), phe (pi-pi stacked) and arg (hydrogen bond), in the α-helix region of the protein ( figure d ). therefore, the evaluation carried out through the autodock vina program enables us to affirm that the selected compounds are close to the interactions made with the rofecoxib (rcx) ligand ( kir) at the active site of hcox- , as the molecules share the main interactions in the hydrophobic part with the and amino acid residues val , val , ala , phe , and arg linked by b chain. table summarizes the chemical information from the selected structures resulting from the molecular docking study. the three remaining compounds were subjected to the bioactivity prediction, through the molinspiration server (https://www.molinspiration.com/). in this prediction, table summarizes the chemical information from the selected structures resulting from the molecular docking study. the three remaining compounds were subjected to the bioactivity prediction, through the molinspiration server (https://www.molinspiration.com/). in this prediction, biological activity measured by the bioactivity score for enzyme inhibitor was evaluated enzyme (see table ), which are classified into three different ranges: molecule having bioactivity score more than . is most likely to possess considerable biological activities, while values − . to . are expected to be moderately active, and if score is less than − . , it is presumed to be inactive [ ] . the bioactivity scores of the lmqc , lmqc , and lmqc structures were calculated for different parameters, as receptor binding of the ligand to the g protein coupled (gpcr) and nuclear receptor ligand, modulating ion channel, kinase inhibition, protease inhibition, and inhibition of enzyme activity. then, compared with the bioactivity score of the pivot molecule rofecoxib [ ] . a web swiss target prediction: probability (%) for the query molecule-assumed as bioactive-to have this enzyme as target [ ] . the bioactivity scores of the lmqc , lmqc , and lmqc structures were calculated for different parameters, as receptor binding of the ligand to the g protein coupled (gpcr) and nuclear receptor ligand, modulating ion channel, kinase inhibition, protease inhibition, and inhibition of enzyme activity. then, compared with the bioactivity score of the pivot molecule rofecoxib [ ] . the bioactivity scores for the g protein-coupled receptor ligand (gpcr) are most active for the lmqc , lmqc and rofecoxib structures with values greater than . . meanwhile, the lmqc has a moderately active score between − . to . . the score values of the lmqc , lmqc , and lmqc structures are considered good because they are close to the pivot compound with probable biological activity (see table ). this estimated property, the binding to the ligand by the gpcr receptors, act as the main responsible for the mediation of inflammatory (and anti-inflammatory) responses and can contribute to the regulation of the vascular permeability process [ ] . the results of the ion channel modulators' scores for the lmqc , lmqc , and rofecoxib structures are estimated score values between − . to . considered moderately active and the lmqc structure with a score value above . considered biologically active. these ionic pharmaceuticals , , x for peer review of table . selected structures resulting from the molecular docking. a web swiss target prediction: probability (%) for the query molecule-assumed as bioactive-to have this enzyme as target [ ] . the bioactivity scores of the lmqc , lmqc , and lmqc structures were calculated for different parameters, as receptor binding of the ligand to the g protein coupled (gpcr) and nuclear receptor ligand, modulating ion channel, kinase inhibition, protease inhibition, and inhibition of enzyme activity. then, compared with the bioactivity score of the pivot molecule rofecoxib [ ] . the bioactivity scores for the g protein-coupled receptor ligand (gpcr) are most active for the lmqc , lmqc and rofecoxib structures with values greater than . . meanwhile, the lmqc has a moderately active score between − . to . . the score values of the lmqc , lmqc , and lmqc structures are considered good because they are close to the pivot compound with probable biological activity (see table ). this estimated property, the binding to the ligand by the gpcr receptors, act as the main responsible for the mediation of inflammatory (and anti-inflammatory) responses and can contribute to the regulation of the vascular permeability process [ ] . the results of the ion channel modulators' scores for the lmqc , lmqc , and rofecoxib structures are estimated score values between − . to . considered moderately active and the lmqc structure with a score value above . considered biologically active. these ionic a web swiss target prediction: probability (%) for the query molecule-assumed as bioactive-to have this enzyme as target [ ] . the bioactivity scores of the lmqc , lmqc , and lmqc structures were calculated for different parameters, as receptor binding of the ligand to the g protein coupled (gpcr) and nuclear receptor ligand, modulating ion channel, kinase inhibition, protease inhibition, and inhibition of enzyme activity. then, compared with the bioactivity score of the pivot molecule rofecoxib [ ] . the bioactivity scores for the g protein-coupled receptor ligand (gpcr) are most active for the lmqc , lmqc and rofecoxib structures with values greater than . . meanwhile, the lmqc has a moderately active score between − . to . . the score values of the lmqc , lmqc , and lmqc structures are considered good because they are close to the pivot compound with probable biological activity (see table ). this estimated property, the binding to the ligand by the gpcr receptors, act as the main responsible for the mediation of inflammatory (and anti-inflammatory) responses and can contribute to the regulation of the vascular permeability process [ ] . the results of the ion channel modulators' scores for the lmqc , lmqc , and rofecoxib structures are estimated score values between − . to . considered moderately active and the lmqc structure with a score value above . considered biologically active. these ionic the bioactivity scores for the g protein-coupled receptor ligand (gpcr) are most active for the lmqc , lmqc and rofecoxib structures with values greater than . . meanwhile, the lmqc has a moderately active score between − . to . . the score values of the lmqc , lmqc , and lmqc structures are considered good because they are close to the pivot compound with probable biological activity (see table ). this estimated property, the binding to the ligand by the gpcr receptors, act as the main responsible for the mediation of inflammatory (and anti-inflammatory) responses and can contribute to the regulation of the vascular permeability process [ ] . the results of the ion channel modulators' scores for the lmqc , lmqc , and rofecoxib structures are estimated score values between − . to . considered moderately active and the lmqc structure with a score value above . considered biologically active. these ionic modulators are important for planning potential anti-inflammatory drugs because they participate in the protection of tissues against lesions induced by the inflammatory process, they carry charged particles across cell membranes and their activity can be directed towards the discovery of new potential drugs for the regulation of the depolarization of ionic charges [ , ] . the lmqc and lmqc structures have score values for kinase inhibitors greater than . considered biologically active. meanwhile, the compound rofecoxib and lmqc have moderately active score values (see table ) for protein kinase inhibitors, as cyclooxygenase- is induced by various extracellular signals including pro-inflammatory stimuli and growth promoters. a cyclooxygenase- is induced by several extracellular signals, including pro-inflammatory and stimulating growth promoters. thus, all of the signals converge for the activation of mitogen-activated protein kinases (mapk) that regulate cyclooxygenase- mrna and contribute to the infection treatment process [ ] . moreover, the nuclear receptor score values (nrs), in the lmqc and lmqc structures, are considered moderately active, as they have score values between − . to . . lmqc and rofecoxib are considered biologically active, with a score value above . , according to the classification ranges of smant and chowdhary. the bioactivity of nuclear receptors (nrs) is important because they are involved in several physiological processes, including homeostasis, an important process that regulates inflammation [ ] . the lmqc , lmqc , lmqc structures have moderately active score values between − . and . for protease inhibitors. already, the dynamic compound of rofecoxib has an estimated value greater than . considered active. therefore, the results of the lmqc and lmqc structures are considered to have biological activity (active), enzyme inhibitor, since they had score values greater than zero, such as the compound rofecoxib. while lmqc is expected to be moderately active with a score between − . to . . the activity score profile of the selected structures demonstrates the probability that they are biologically active and that they have the necessary properties to act with potential enzyme inhibitors of cyclooxygenase- (cox- ) [ ] . compounds lmqc , lmqc , and lmqc were also submitted to web server swiss target prediction (http://www.swisstargetprediction.ch) [ ] . to identify the likelihood of bioactivity through similarity based on chemical structure and molecular form (electroshape) [ ] . the server uses a database of molecules: chembl [ , ] , drugbank [ ] , pubchem [ ] , and zinc [ ] to track sets of molecules and identify proteins with ligands similar to bioactive molecules and also uses species selection for virtual screening (top_ homo sapiens). the results of the virtual bioactivity screening for the enzymatic target performed by the swiss targetprediction [ ] server issued a summary displayed in percentages with the probability of being the enzymatic target [ ] . the table shows the percentage probability values for enzyme inhibition. prediction analysis of enzymatic inhibition for rofecoxib was %; while the selected compounds exhibited the following probability of reaching the enzyme: lmqc %, lmqc %, and lmqc %. thus, it is observed that rofecoxib did not reach % of the enzyme and the selected compounds had lower enzyme values. however, the results were assessed as likely for possible bioactivity and the structures proceeded with analysis taking into account the pharmacokinetic and toxicological profiles in which they presented favorable results. to evaluate the conformational changes in the receptor-ligand complexes along the time, the md simulations were applied in ns simulation nodes, for each complex hcox and ligand: rofecoxib, lmqc , lmqc , and lmqc . the simulations also allowed the evaluation of the conformational changes in the structure of the ligand and the protein backbone. these conformational changes in the backbone and ligand were evaluated from the root mean square deviation plot (rmsd). to plot the rmsd of the backbone, cα atoms were used, while to plot the rmsd of the ligand, all heavy atoms were used. in addition, the fluctuation of the residues from the protein backbone was evaluated, for this, the cα atoms were also used. this analysis was performed to evaluate the difference in the structural fluctuation of the protein during the interaction with the different ligands, throughout the ns md simulation (see figure ) [ , [ ] [ ] [ ] [ ] [ ] . the rmsd plot reveals that the ligands showed small conformational variations when interacting with the protein along the time. their rmsd graphs show slight variations, which suggests that the ligands remained interacting with the active site of the protein undergoing minor conformational changes. this conformational stability over ns of md simulations demonstrates a good interaction of the ligands with the molecular target, thus, remained in a favorable conformation to inhibit the biological receptor. conformational changes. this conformational stability over ns of md simulations demonstrates a good interaction of the ligands with the molecular target, thus, remained in a favorable conformation to inhibit the biological receptor. the low rmsd fluctuation of the ligands is also related to the interactions established in the binding pocket. all ligands showed interactions with residues observed in the results of molecular docking, which are summarized in tables and . these interactions were able to keep the ligands interacting with the active site throughout the entire trajectory, allowing the maintenance of the receptor-ligand. the different ligands were able to impact the flotation of the atoms of the hcox- backbone in different ways, as can be seen from the differences of the root-mean-square fluctuation (rmsf) plot. the greatest differences in fluctuations in protein residues are observed at residues - . this region of the protein corresponds to the n-terminal portion; in addition, it is initially composed of a small alpha-helice, followed by two beta-leaves that will connect to another alpha-helice through a region's relatively large loop. finally, the residue gap is formed by three more alpha helices that are connected by loop regions (see figure ). tables and . these interactions were able to keep the ligands interacting with the active site throughout the entire trajectory, allowing the maintenance of the receptor-ligand. the different ligands were able to impact the flotation of the atoms of the hcox- backbone in different ways, as can be seen from the differences of the root-mean-square fluctuation (rmsf) plot. the greatest differences in fluctuations in protein residues are observed at residues - . this region of the protein corresponds to the n-terminal portion; in addition, it is initially composed of a small alpha-helice, followed by two beta-leaves that will connect to another alpha-helice through a region's relatively large loop. finally, the residue gap is formed by three more alpha helices that are connected by loop regions (see figure ) . colors. (a) general view of the protein structure with emphasis on the binding pocket occupied by the ligands. the protein was represented in cyan color and ligand protein was represented in spheres (red color). (b) rmsds of hcox- -rofecoxib-system, (c) rmsds of hcox- -lmqc system, (d) rmsds of hcox- -lmqc system, (e) rmsds of hcox- -lmqc system. ligands and residues were represented in sticks. the greatest fluctuation of residues - was observed in the complex established with the lmqc ligand. apparently, this greater fluctuation should impair the stability of the ligand at the active site, since residues - correspond to a region of the protein that is close to the active site. however, this behavior was not observed, since the rmsd plot of the ligand shows that the maintenance of the molecule in the binding pocket with conformational stability along the ns trajectory. additionally, the affinity energy value (Δgbind = − . kcal/mol) demonstrates that the ligand was able to interact favorably with the protein. this result demonstrates that this region of the protein, despite showing high fluctuation, was not able to impair the interaction of the ligand with the active site. this suggests that the residues around the active site are sufficient to keep the ligands complexed to the protein, despite fluctuations conformations observed in the region of the protein formed by the residues - . to evaluate the interaction energy of the selected compounds with hcox- , the molecular mechanics/generalized born surface area (mm/gbsa) method was applied and the obtained results are summarized in table . the greatest fluctuation of residues - was observed in the complex established with the lmqc ligand. apparently, this greater fluctuation should impair the stability of the ligand at the active site, since residues - correspond to a region of the protein that is close to the active site. however, this behavior was not observed, since the rmsd plot of the ligand shows that the maintenance of the molecule in the binding pocket with conformational stability along the ns trajectory. additionally, the affinity energy value (∆gbind = − . kcal/mol) demonstrates that the ligand was able to interact favorably with the protein. this result demonstrates that this region of the protein, despite showing high fluctuation, was not able to impair the interaction of the ligand with the active site. this suggests that the residues around the active site are sufficient to keep the ligands complexed to the protein, despite fluctuations conformations observed in the region of the protein formed by the residues - . to evaluate the interaction energy of the selected compounds with hcox- , the molecular mechanics/generalized born surface area (mm/gbsa) method was applied and the obtained results are summarized in table . according to the values of affinity energy (∆gbind), all ligands selected by molecular docking are able to establish stable complexes with hcox- . rofecoxib achieved the free energy value of ∆gbind= − . kcal/mol. the other compounds reached the following affinity energy values: lmqc : ∆gbind = − . kcal/mol; lmqc : ∆gbind = − . kcal/mol; and lmqc : ∆gbind = − . kcal/mol. the compounds lmqc , lmqc , and lmqc showed favorable values of affinity energy for formation of the complexes. van der waals (∆evdw) interactions showed the greatest contributions to the formation of the different systems of this study. in addition, electrostatic (∆eele) and non-polar (∆gnp) interactions also contributed to complexes being formed spontaneously. the values of affinity energy for the three selected compounds were promising, as the values were relatively close to the obtained for rofecoxib. this demonstrates that the selected substances can be considered as putative hcox- inhibitors, being promising leads for new anti-inflammatory drugs project. the chemical structures of cox- inhibitors are heterogenic and can be classified into tricyclics and non-tricyclics compounds. contrary to the classic nsaids, this new class of enzyme inhibitors is lacking a carboxylic group, thus effecting cox- affinity by a different orientation within the enzyme without formation of a salt bridge in the hydrophobic channel of the enzyme [ ] . celecoxib, rofecoxib, valdecoxib share in common the same structural features of the selected compounds lmqc , lmqc , and lmqc , which exhibit a tricyclic scaffold, and a , -diarylsubstitution on a central hetero ring system. in addition, these compounds show characteristic groups on one of the aryl rings that plays a crucial role on cox- selectivity. all selected compounds present five membered core heterocycles, even though all different from rofecoxib, which shows a furanone ring (see figure ) . compound lmqc present a pharmacophore-based , , triazole group, which increases a certain degree of conformational rigidity to compound, which can be seen in the binding free energy essay. lmqc shows a pyrazole moiety, the same core as celecoxib (figures and ) , which favors a hydrogen bond interaction with hcox- ( figure b ). moreover, lmqc presents a -sulfonylmethylphenyl substitution at position on the pyrazole ring which increases the inhibitory effects against cox- enzyme [ ] . according to the values of affinity energy (Δgbind), all ligands selected by molecular docking are able to establish stable complexes with hcox- . rofecoxib achieved the free energy value of Δgbind= − . kcal/mol. the other compounds reached the following affinity energy values: lmqc : Δgbind = − . kcal/mol; lmqc : Δgbind = − . kcal/mol; and lmqc : Δgbind = − . kcal/mol. the compounds lmqc , lmqc , and lmqc showed favorable values of affinity energy for formation of the complexes. van der waals (Δevdw) interactions showed the greatest contributions to the formation of the different systems of this study. in addition, electrostatic (Δeele) and non-polar (Δgnp) interactions also contributed to complexes being formed spontaneously. the values of affinity energy for the three selected compounds were promising, as the values were relatively close to the obtained for rofecoxib. this demonstrates that the selected substances can be considered as putative hcox- inhibitors, being promising leads for new anti-inflammatory drugs project. the chemical structures of cox- inhibitors are heterogenic and can be classified into tricyclics and non-tricyclics compounds. contrary to the classic nsaids, this new class of enzyme inhibitors is lacking a carboxylic group, thus effecting cox- affinity by a different orientation within the enzyme without formation of a salt bridge in the hydrophobic channel of the enzyme [ ] . celecoxib, rofecoxib, valdecoxib share in common the same structural features of the selected compounds lmqc , lmqc , and lmqc , which exhibit a tricyclic scaffold, and a , -diarylsubstitution on a central hetero ring system. in addition, these compounds show characteristic groups on one of the aryl rings that plays a crucial role on cox- selectivity. all selected compounds present five membered core heterocycles, even though all different from rofecoxib, which shows a furanone ring (see figure ) . compound lmqc present a pharmacophore-based , , triazole group, which increases a certain degree of conformational rigidity to compound, which can be seen in the binding free energy essay. lmqc shows a pyrazole moiety, the same core as celecoxib (figure and figure ), which favors a hydrogen bond interaction with hcox- ( figure b ). moreover, lmqc presents a -sulfonylmethylphenyl substitution at position on the pyrazole ring which increases the inhibitory effects against cox- enzyme [ ] . lmqc shows an isoxazole ring such as valdecoxib ( figure and figure ), linked to the aryl ring by an amide group, which also confers rigidity to the structure and favors an additional pi-sigma interaction with hcox ( figure c ). compound lmqc present an imidazole ring, favoring a pi-pi stacking interaction with hcox- ( figure d ) [ ] . according to previous studies, imidazole, triazole, ozaxol, benzene sulfonamide, and pyrazole favors the formation of hydrogen bonds capable of introducing a certain degree of conformational rigidity, indicating a wide range of pharmacological activity as a desired, anti-inflammatory activity ( figure ) [ , ] . lmqc shows an isoxazole ring such as valdecoxib (figures and ) , linked to the aryl ring by an amide group, which also confers rigidity to the structure and favors an additional pi-sigma interaction with hcox ( figure c ). compound lmqc present an imidazole ring, favoring a pi-pi stacking interaction with hcox- ( figure d ) [ ] . according to previous studies, imidazole, triazole, ozaxol, benzene sulfonamide, and pyrazole favors the formation of hydrogen bonds capable of introducing a certain degree of conformational rigidity, indicating a wide range of pharmacological activity as a desired, anti-inflammatory activity ( figure ) [ , ] . due to the aforementioned facts, the compounds with the most promising results (figure ) , were submitted to an investigation in scifinder ® , available on the internet, and linked to the chemical abstract service (cas) (https://scifinder.cas.org/), in order to verify additional information about structures and/or experiments with biological activities (patents). no additional information on the promising structures was found in the search. this demonstrates that the molecules mentioned above, with great potential for inhibition in cox- , still do not have in vitro or in vivo studies that evaluate this activity. therefore, these are important findings for future research and development studies of cox- selective anti-inflammatory drugs. lmqc , lmqc , and lmqc were also submitted for evaluation of their toxicological properties using derek software. this assessment was carried out to investigate whether these compounds had a profile of adverse toxicological effects on humans, mice, and rats. according to the results (table ) , the selected compounds did not present any toxicity alert. results of the pivot compound rofecoxib, on the other hand, were flagged as "plausible", since it presented a warning of hepatotoxicity (humans, mice and rats) for derivatives of the furanone group [ ] . information on the promising structures was found in the search. this demonstrates that the molecules mentioned above, with great potential for inhibition in cox- , still do not have in vitro or in vivo studies that evaluate this activity. therefore, these are important findings for future research and development studies of cox- selective anti-inflammatory drugs. lmqc , lmqc , and lmqc were also submitted for evaluation of their toxicological properties using derek software. this assessment was carried out to investigate whether these compounds had a profile of adverse toxicological effects on humans, mice, and rats. according to the results (table ) , the selected compounds did not present any toxicity alert. results of the pivot compound rofecoxib, on the other hand, were flagged as "plausible", since it presented a warning of hepatotoxicity (humans, mice and rats) for derivatives of the furanone group [ ] . [ ] . table shows also the oral lethal dose prediction (ld ) based on mg/kg body weight and toxicity class ranging from i to vi, performed on the protox-ii web server (http://tox.charite.de/protox_ii). ld of the lmqc structure was mg/kg and of the lmqc mg/kg both with iv classification was considered harmful if ingested ( < ld ≤ ), however, they showed higher lethal dose when compared to rofecoxib. lmqc presented a ld value of mg/kg and classification vi, which is non-toxic if ingested, estimated as the best result of an oral lethal dose. therefore, the results for ld of the investigated compounds are better than the commercial compound and may present greater safety in use [ ] . the compounds were also submitted to the preadmet [ , ] software to assess the cardiotoxicity. drug candidates often cause an unwanted blockage of the potassium ion channel of the human ether-a-go-go-related gene (herg). the blockage leads to long qt syndrome (lqts), which is a severe life-threatening cardiac side effect [ ] . the evaluation of this parameter was by means of herg ( [ ] takes into account the electro-affinity calculation (ea) of the compounds. the results of the evaluation of the cardiotoxicity capacity for lmqc , lmqc , lmqc , and rofecoxib can be seen in table . [ ] . table shows also the oral lethal dose prediction (ld ) based on mg/kg body weight and toxicity class ranging from i to vi, performed on the protox-ii web server (http://tox.charite.de/protox_ii). ld of the lmqc structure was mg/kg and of the lmqc mg/kg both with iv classification was considered harmful if ingested ( < ld ≤ ), however, they showed higher lethal dose when compared to rofecoxib. lmqc presented a ld value of mg/kg and classification vi, which is non-toxic if ingested, estimated as the best result of an oral lethal dose. therefore, the results for ld of the investigated compounds are better than the commercial compound and may present greater safety in use [ ] . the compounds were also submitted to the preadmet [ , ] software to assess the cardiotoxicity. drug candidates often cause an unwanted blockage of the potassium ion channel of the human ether-a-go-go-related gene (herg). the blockage leads to long qt syndrome (lqts), which is a severe life-threatening cardiac side effect [ ] . the evaluation of this parameter was by means of herg ( [ ] takes into account the electro-affinity calculation (ea) of the compounds. the results of the evaluation of the cardiotoxicity capacity for lmqc , lmqc , lmqc , and rofecoxib can be seen in table . lmqc , lmqc , lmqc , and rofecoxib showed a medium risk of cardiotoxicity in the electro-affinity calculation. this pharmacokinetic property that is related to drug-receptor interaction and electron transfers, we consider an aspect of paramount importance for therapeutic activity and in determining toxicity [ ] . the human ether-a-go-go-related gene (herg) is codified for a protein that forms a voltage-dependent potassium ion channel found in heart and nervous system [ ] [ ] [ ] ; a myocardial conduction disorder (electrical conduction) can alter ventricular repolarization and, consequently, increase the vulnerability for the development of a cardiac action [ ] . therefore, lmqc , lmqc , and lmqc have a lower risk of cardiotoxicity when compared to rofecoxib, since they do not present violation in the electro-affinity parameter. crystallographic structure (pdb kir at . Å resolution) of human cyclooxygenase- (hcox- ) was obtained as pdb file from the protein data bank (pdb) (https://www.rcsb.org/pdb) complexed with the pivot rofecoxib [ , ] . in this step, we used six commercial databases for virtual screening based of rofecoxib ligand: chembridge diverset™-express-pick™ collection (diverset™-exp), diverset core library (diverset™-cl), zinc drug database, zinc natural stock e zinc drug@fda bindingdb, and maybridge. for each molecule in the database, we obtained conformers using the mmff molecular force fields were generated [ ] , running on omega v . . . software (open eye scientific software, santa fe, nm, http://www.eyesopen.com) for windows operating system and intel core i machine of . ghz. initially, for each molecule in the database, the fast conformer generation method was used with a maximum energy tolerance of kcal.mol − and mean square deviation (rmsd) of . Å [ , , ] . in this study, rapid overlay of chemical structures (rocs) v . . . (openeye) software was used as a tool for three-dimensional ( d) molecular similarity research. we used six databases to select chemical compounds through the rocs software (https://www.eyesopen.com/rocs) [ ] , with gaussian function algorithm located in atoms that proposes the best overlap between molecules in a characteristic set that can be a steric volume or the molecular interaction, called comboscore. this was done to generate and score three-dimensional ( d) overlays of the database with the pivot compound (rofecoxib) in order to seek better compounds for the cox- receptor, to get the highest rated structures (top_ ) of each base, totaling , compounds [ , , , ] this software generates input files for the eon program. eon v . . . (openeye) software is an electrostatics comparison program (https://www.eyesopen. com/eon) [ ] -it compared the electrostatic potential maps of pre-aligned molecules and determined the tanimoto measures for the comparison of the six databases. moreover, it calculated the new partial load to minimize energy using the mmff force field [ ] . electrostatic classification was based on tanimoto's electrostatic scores; the electrostatic arrangement was obtained from the overlapping of positive and negative charges when completing the variation of an identical to negative values. in this study, a lower energy of rofecoxib conformer was used to perform electrostatic comparisons (more rigid conformation, based on the available crystallographic structure). the output files were grouped according to the scores and the results were classified based on "et combo" analogous to "tanimoto combo". in the end, only the " best compounds/base" were selected, affording molecules [ , , , , , , ] . the assessment of a number of key physicochemical properties, pharmacokinetic parameters, and toxicity endpoints was carried out for the compounds that passed the virtual screening step-the top of each database. pharmacokinetic (#star, "rule of five", human intestinal absorption, qppcaco, qppmdck, qplogpo/w, cns, and qplogbb) properties were predicted using the schrodinger's suite qikprop v. . , and derek nexus software . [ , ] . the toxicity of the compounds with the best pharmacokinetic profiles was assessed using (derek) . . nexus program [ , ] . deductive estimation of risk from existing knowledge (derek) predicts potential toxicity and toxicophoric groups and also includes the following toxicological parameters: carcinogenicity, mutagenicity, genotoxicity, skin sensitization, teratogenicity, irritation, respiratory sensitization, reproductive toxicity [ , ] . this software analyses qualitative predictions and, in this way, generates alerts about the possible toxic action of the chemical compounds analyzed. in this step, the compounds were evaluated in aspects involving the types of toxicity and possible toxicophoric effects [ , ] . we have considered derek toxicity alerts involving the human species and also classified as plausible in mammals, but compounds containing any toxicophoric groups were also discarded [ ] , through visual inspection using the maestro . program. the selected compounds were submitted to the protox web server (http://tox.charite.de/protox_ ii) [ ] , which identifies lethal oral doses (ld ) [ ] . the prediction method is based on the analysis of the two-dimensional ( d) similarity to compounds with known ld values and the identification of fragments over-represented in toxic compounds. the results are generated instantly on the server page, showing the predicted average lethal dose (ld ) in mg/kg of weight and the toxicity classes (i, ii, iii, iv, v and vi) [ , ] . the prediction of cardiotoxicity was determined using the online server preadmet (https: //preadmet.bmdrc.kr/) [ ] and qikprop [ ] software. the prediction method for the risk of cardiac toxicity is based on the inhibition property of the human ether-to-go-go (herg) gene based on the electron affinity of the compounds. preadmet instantly generates alerts on the server page classified as: low risk, medium risk, and high risk for the herg property [ ] . this screening step for the prediction of the biological target was performed via web servers: molinspiration and swisstargetprediction. the bioactivity score of selected compounds was evaluated using the molinspiration server cheminformatics tool (http://www.molinspiration.com) [ ] . the prediction made was based on the enzyme inhibition score, taking into account the pivot molecule. the results are analyzed according to roy; samant; chowdhary [ ] . therefore, it is recommended that if the value is equal to or greater than . , the more active it will be, while if the values are between − . and . , it is moderately active, and, if the score is less than − . , it will be considered inactive [ , ] . then, the query structures were submitted to the swisstargetprediction web server (http://www. swisstargetprediction.ch), to predict small molecule protein targets in homo sapiens (top_ ). targets are classified according to their percentage probability on the assumption that if the molecule is active, it is likely to bind to some protein. the investigation of the bioactivity target was based on the value of the enzymatic target of the pivot molecule rofecoxib with known bioactivity. the results of the server prediction via swisstargetprediction web server are presented as a percentage in a pie chart [ , ] . molecular docking simulations were based on fitting the ligand to the active site of an enzyme. this simulation is called re-docking which aims to recover, from computer simulation, the original position of a ligand present in a crystallographic structure of a protein-ligand complex [ ] . for the determination of this protocol an approach called validation is used, where we used as reference a crystallographic structure already determined [ , ] . for this study, the crystallographic structure of hcox- complexed with rofecoxib ligand deposited in the pdb was used with code kir (homo sapiens) and a resolution of . Å [ , ] . the enzyme structure was prepared by removing water and binders, and adding hydrogen atoms, using discovery studio . software. then, the autodock/vina software was subjected to molecular coupling [ , , ] autodock is a set of tools that allow the interaction between ligand and macromolecule and provides combinations with algorithm options: simulated annealing (sa), genetic algorithm (ga), and lamarckian genetic algorithm (lga). in this work, the search algorithm used was lga (lamarckian genetic algorithm), that presents the best results in the search for the global minimum [ , ] . the interactions between inhibitors and the receptor were visualized using the discovery studio . software with standard parameters. the evaluation of the molecular coupling was determined by means of the ligand obtained experimentally and the theoretical conformation performed with the molecular coupling in the pdb ( kir), and were validated by the rmsd value. table shows the x, y and z coordinates according to the interaction between cox- and the standard ligand. the x, y and z coordinates of the receivers were determined according to the average region of the active site. moreover, ten solutions were calculated for each ligand and minimum conformations of binding energy were analyzed [ , , , , ] . the energy scoring function was used to assess the free binding energy (∆g) of interactions between cox and ligands in pyrx . . . the analysis of the poses (conformation + orientation) of the binders was also taken into account in the selection of the best binding free energy and binding affinity calculations in autodock . /vina . . in order to assess selectivity towards homo sapiens as a function of binding affinity at the cox- receptor. the initial structure for the system was obtained from molecular docking methods, as described in the previous section. the restrained electrostatic potential (resp) protocol with the hf/ - g* basis sets was applied to obtain the charges of the atoms of each ligand [ ] [ ] [ ] atomic charge calculated using gaussian [ , , ] the parameters of the ligand were constructed with the antechamber module, available in the amber package [ ] [ ] [ ] . the protonation state of ionizable residues of protein structure was analyzed using the propka [ ] server in the neutral ph before performing the md simulations. the ligand was treated with the general amber force field (gaff) and protein was treated with the ff sb [ ] . the force field parameters developed by giammona were used for the heme group [ ] . the system was constructed for the simulation using the tutorial for the leap (tleap) of amber package. the system was solvated in an octahedron periodic box containing water molecules in the tip p model [ ] . the partial charges of the systems were neutralized by adding counterions. we used the sander.mpi for the four stages of energy minimization. in each of these stages, it took cycles using the steepest descent method and cycles using the conjugate gradient algorithm. in the first stage the hydrogen atoms of the water molecules were optimized; then, the ions and the water molecules were minimized; in the third stage, the hydrogen atoms of the protein, and in the last step, the solute and the solvent, underwent the process of energy minimization. three heating steps were used for a total time of picoseconds to raise the system temperature to k. first, the solute was restricted with a constant harmonic force of kcal mol − . Å − , so only the solvent and the counter ions moved. in the next step, the constant harmonic force was removed. to equilibrate the systems, we performed ns simulations with no restriction at constant temperature. finally, for each system, we performed ns of molecular dynamics of production. particle mesh ewald method [ ] was used for the calculation of electrostatic interactions and the bonds involving hydrogen atoms were restricted with the shake algorithm [ ] . temperature control was performed with the langevin thermostat [ ] within collision frequency of ps- . to estimate the binding affinity (∆g bind ), we used the molecular mechanics/generalized born surface area (mm-gbsa) method [ , , ] the affinity energy (∆gbind) is the summation of the interaction energy of the gas phase between protein-ligand (∆emm), desolvation free energy (∆gsolv) and system entropy (-t∆s). ∆emm is the result of the sum of internal energy (∆einternal, sum of the energies of connection, angles, and dihedral) electrostatic contributions (∆eele) and the van der waals term (∆evdw). ∆gsolv is the sum of the polar (∆ggb) and non-polar (∆gnp) contributions. ∆gsa was determined from the solvent accessible surface area (sasa) estimated by the linear combination of pairwise overlaps (lcpo) algorithm. the mm/gbsa method was used to determine the energy contribution of each protein residue, thus, rendering it possible to determine which residues are most important for the ligand interaction with the active site. the interaction energy of residues with the inhibitor can be described from four terms: van der waals contribution (∆e vdw ), electrostatic contribution (∆e ele ), polar solvation contribution (∆g pol ), and nonpolar solvation contribution (∆g nonpol ), according to the equation [ , , ] : ∆g ligand-residue = ∆e vdw + ∆e ele + ∆g pol + ∆g nonpol ( ) in this study, a computational strategy was applied to identify new potential selective hcox- inhibitors base conclusions d on the known drug rofecoxib. compounds from six databases were filtered by a ligand-based virtual screening study, followed by pharmacokinetic, toxicological, and molecular dynamic studies. the selected structures lmqc , lmqc , and lmqc have aspects strictly related to physical-chemical properties and biological activity. therefore, such selected structures reproduce values within the limits established in the pharmacokinetic predictions: absorption and distribution in the human body. moreover, in the prediction of toxicity the structures lmqc , lmqc , and lmqc did not present alerts for possible toxic groups. through the study of molecular dynamics, lmqc , lmqc , and lmqc were identified as promising due to values of affinity energy relatively close to those obtained for rofecoxib. along the trajectories of molecular dynamics simulations, the selected compounds showed conformational stability, as well as the pivot compound. lmqc , lmqc , and lmqc showed satisfactory pharmacokinetic results related to absorption and distribution. the toxicological predictions of these compounds did not display alerts for possible toxic groups and lower risk of cardiotoxicity compared to rofecoxib. this demonstrates that lmqc , lmqc , and lmqc can be considered as putative hcox- inhibitors, in addition to serving as the basis for the new anti-inflammatory drug project. cyclooxygenase isozymes: the biology of prostaglandin synthesis and inhibition distinct functions of cox- and cox- molecular inflammatory mediators in peripheral nerve degeneration and regeneration regulation of cyclo-oxygenase- optimization and validation of a docking-scoring protocol; application to virtual screening for cox- inhibitors selective cox- inhibitors: a review of their structure-activity relationships cyclooxygenase- : a therapeutic target rofecoxib (vioxx) voluntarily withdrawn from market selective cox- inhibitors development and testing of a general amber force field the protein data bank protein data bank (pdb): the single global macromolecular structure archive crystal structure of rofecoxib bound to human cyclooxygenase- ligand-and structure-based virtual screening of -((diiso-butylamino)methyl)- α-hydroxyvouacapane- β, β-lactone, a compound with potential anti-prostate cancer activity preadmet | prediction of adme/tox-just another bmdrc sites preadmet version . ; bioinformatics and molecular design research center identification of novel protein kinase receptor type inhibitors using pharmacophore and structure-based virtual screening silico evaluation of ibuprofen and two benzoylpropionic acid derivatives with potential anti-inflammatory activity andrographolide as a potential inhibitor of sars-cov- main protease: an in silico approach determination of molecular property, bioactivity score and binding energy of the phytochemical compounds present in cassia auriculata by molinspiration and dft method identification of novel parasitic cysteine protease inhibitors using virtual screening. . the chembridge database potential inhibitors of the enzyme acetylcholinesterase and juvenile hormone with insecticidal activity: study of the binding mode via docking and molecular dynamics simulations virtual screening, identification and experimental testing of novel inhibitors of pbef /visfatin/nmprtase for glioma therapy a web-accessible database of experimentally determined protein-ligand binding affinities rocs-derived features for virtual screening rocs openeye | rocs software | virtual screening|lead hopping comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis gaussian shape methods integration of ligand-and target-based virtual screening for the discovery of cruzain inhibitors hppd: ligand-and target-based virtual screening on a herbicide target discovery of novel ppar ligands by a virtual screening approach based on pharmacophore modeling, d shape, and electrostatic similarity screening hierarchical virtual screening of potential insectides inhibitors of acetylcholinesterase and juvenile hormone from temephos identification of new inhibitors with potential antitumor activity from polypeptide structures via hierarchical virtual screening qikprop . user manual qikprop user manual web services as applications' integration tool: qikprop case study in silico tools for sharing data and knowledge on toxicity and metabolism: derek for windows, meteor, and vitic free energy calculations to estimate ligand-binding affinities in structure-based drug design the calculation of the electron affinity of atoms and molecules dft study on ground state electronic structures of simple to complex molecular specimens calculation of absolute binding free energies between the herg channel and structurally diverse drugs experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings drug metabolites and their effects on the development of adverse reactions: revisiting lipinski's rule of five effect of atomic charges on octanol-water partition coefficient using alchemical free energy calculation medicinal chemical properties of successful central nervous system drugs the log p parameter as a molecular descriptor in the computer-aided drug design-an overview octanol water partition coefficients of simple organic compounds predicting a drug's membrane permeability: a computational model validated with in vitro permeability assay data predicting apparent passive permeability of caco- and mdck cell-monolayers: a mechanistic model correlation between human ether-a-go-go-related gene channel inhibition and action potential prolongation the human intestinal epithelial cell line caco- ; pharmacological and pharmacokinetic applications variability in caco- and mdck cell-based intestinal permeability assays computational approaches to the prediction of the blood-brain distribution admet in silico modelling: towards prediction paradise? blood-brain barrier: from physiology to disease and back an in silico study of the antioxidant ability for two caffeine analogs using molecular docking and quantum chemical methods molecular modeling approaches of selective adenosine receptor type a agonists as potential anti-inflammatory drugs calculation of protein-ligand binding affinities unit using autodock for ligand-receptor docking toward of safer phenylbutazone derivatives by exploration of toxicity mechanism swisstargetprediction: a web server for target prediction of bioactive small molecules role of g protein-coupled receptors in inflammation ion channels in inflammatory processes: what is known and what is next? mediat. inflamm potassium channel modulators as anti-inflammatory agents kinase inhibitors for the treatment of inflammatory and autoimmune disorders structural overview of the nuclear receptor superfamily: insights into physiology and therapeutics signalling networks regulating cyclooxygenase- improving the accuracy of ultrafast ligand-based screening: incorporating lipophilicity into electroshape as an extra dimension chembl: a large-scale bioactivity database for drug discovery using chembl web services for building applications and data processing workflows relevant to drug discovery drugbank . : a comprehensive resource for 'omics' research on drugs nuclear receptors and the adaptive response of the heart zinc: a free tool to discover chemistry for biology swisstargetprediction: updated data and new features for efficient prediction of protein targets of small molecules systems biology shaping the interaction landscape of bioactive molecules identification of novel chemical entities for adenosine receptor type a using molecular modeling approaches in silico study to identify new antituberculosis molecules from natural sources by hierarchical virtual screening and molecular dynamics simulations molecular dynamics simulation and binding free energy studies of novel leads belonging to the benzofuran class inhibitors of mycobacterium tuberculosis polyketide synthase computational design of new protein kinase inhibitors for the treatment of inflammatory diseases using qsar, pharmacophore-structure-based virtual screening, and molecular dynamics oil from the fruits of pterodon emarginatus vog.: a traditional anti-inflammatory. study combining in vivo and in silico recent advancement in the discovery and development of cox- inhibitors: insight into biological activities and sar studies synthesis of some thiophene, imidazole and pyridine derivatives exhibiting good anti-inflammatory and analgesic activities pharmacological significance of triazole scaffold pharmacological significance of triazole scaffold evolution of nonsteroidal anti-inflammatory cyclooxygenase (cox) inhibition and beyond drugs (nsaids) protox-ii: a webserver for the prediction of toxicity of chemicals computational determination of herg-related cardiotoxicity of drug candidates benzodiazepines: electron affinity, receptors and cell signaling-a multifaceted approach the human ether-a-go-go-related gene (herg) potassium channel represents an unusual target for protease-mediated damage +) channels: structure, function, and clinical significance hidden cardiotoxicity of rofecoxib can be merck molecular force field. ii. mmff van der waals and electrostatic parameters for intermolecular interactions similarity for lead-hopping akritopoulou-zanze, i. the use of three-dimensional shape and electrostatic similarity searching in the identification of a melanin-concentrating hormone receptor antagonist bringing the mmff force field to the rdkit: implementation and validation computer prediction of possible toxic action from chemical structure protox: a web server for the in silico prediction of rodent oral toxicity release - : maestro calculation of molecular properties and bioactivity score research article in silico pharmacokinetics analysis and admet of phytochemicals of datura in silico pharmacokinetic, bioactivity and toxicity evaluation of some selected anti-ulcer agents designing quorum sensing inhibitors of pseudomonas aeruginosa utilizing fabi: an enzymic drug target from fatty acid synthesis pathway application of resp charges to calculate conformational energies, hydrogen bond energies, and free energies of solvation exploring the potentiality of natural products from essential oils as inhibitors of odorant-binding proteins: a structure-and ligand-based virtual screening approach to find novel mosquito repellents spectroscopic methods and in silico analyses using density functional theory to characterize and identify piperine alkaloid crystals isolated from pepper (piper nigrum l.) chemical profile of lippia thymoides, evaluation of the acetylcholinesterase inhibitory activity of its essential oil, and molecular docking and molecular dynamics simulations the amber biomolecular simulation programs naphthoquinones isolated from eleutherine plicata herb: in vitro antimalarial activity and molecular modeling to investigate their binding modes phytochemical profile, antioxidant activity, inhibition of acetylcholinesterase and interaction mechanism of the major components of the piper divaricatum essential oil obtained by supercritical co pdb pqr: an automated pipeline for the setup of poisson-boltzmann electrostatics calculations improving the accuracy of protein side chain and backbone parameters from ff sb comparison of simple potential functions for simulating liquid water particle mesh ewald: an n·log(n) method for ewald sums in large systems numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes langevin stabilization of molecular dynamics calculating structures and free energies of complex molecules: combining molecular mechanics and continuum models insight into the interaction mechanism of nicotine, nnk, and nnn with cytochrome p a based on molecular dynamics simulation insights into protein-protein binding by binding free energy calculation and free energy decomposition for the ras-raf and ras-ralgds complexes measuring the structural impact of mutations on cytochrome p a , the major steroid -hydroxylase related to congenital adrenal hyperplasia the authors declare no conflict of interest. key: cord- -s d di authors: westhof, eric; jaeger, luc title: rna pseudoknots date: - - journal: current opinion in structural biology doi: . / - x( ) -r sha: doc_id: cord_uid: s d di abstract rna pseudoknots result from watson-crick base pairing involving a stretch of bases located between paired strands and a distal single-stranded region. recently, significant advances in our understanding of their structural and functional aspects have been accomplished. at the structural level, modelling and nmr studies have shown that a defined subset of pseudoknots may be considered as tertiary motifs in rna foldings. at the functional level, there is evidence that the realm of functions encompassed by rna pseudoknots extends from the control of translation in prokaryotes, retroviruses and coronaviruses to the control of catalytic activity in ribozymes and the control of replication in some plant viruses. the possibility of pseudoknot formation has been suggested ever since the early thinking on the folding of single-stranded rna molecules [ ]. it was not until the experimental and theoretical work of pleij and his collaborators [ ] , however, that the existence of pseudoknots in rna molecules became recognized. recently, a variety of biological functions have been attributed to pseudoknotting in rna single strands. in the first part of this review, we will examine the problems related to the definition of pseudoknots and will try to convey the message that pseudoknots with at least some approximate co-axial stacking between two adjacent helices are of particular value for understanding and modelling rna three-dimensional foldings. in the second part, we will discuss the various functional aspects of pseudoknotting and the advances made during the past year. finally, we will comment on the difficulties in predicting and recognizing pseudoknots in rna sequences. in the folding of a single-stranded rna molecule, there are only three ways in which two base-paired segments can be related to each other: two consecutive hairpins; two helices separated by an internal bulge; and, pseudoknots [ ]. the first two motifs can be represented as two-dimensional graphs without self-intersections whereas pseudoknots cannot, as they are fundamentally three-dimensional structures in which the four base-paired strands alternate along the sequence of the rna molecule. this general definition of pseudoknotting biology , : - is illustrated in fig. . in other words, pseudoknots can be most generally defined as standard watson-crick base pairing involving a stretch of bases located between paired strands and an outlying parmer (e.g. watson-crick interactions between two hairpin loops or between a loop and a bulge) [ , ", "]. in an important piece of work, pleij and his collaborators [ ] demonstrated that because of the special geometry of rna helices, it is possible for the two helical stems in the pseudoknot to be co-axially stacked. this arrangement constitutes a very important subset of pseudoknots for biological functions. the definition portrayed in fig. is general and does not set any constraint on either the lengths of the connecting segments or the helices. a full turn of rna double helix requires bp. when each of the helices forming a pseudoknot makes a full rum, a topologically knotted structure is obtained. in all proposed pseudoknot structures, however, the helical stems are shorter than a full helical turn. further, the minimum length of the connecting segments bridging the two grooves depends clearly on the length of $ and $ , i.e. on the number of base pairs that each contains. thermodynamic studies have been carried out only on a pseudoknot of type (ii) which contained bp in helix s and bp in helix $ [ o, ]. in the presence of mg +, a stable pseudoknot structure is obtained the three types of 'classic' pseudoknots with co-axial stacking of the two base-paired helices and with single-stranded segments crossing the rna grooves. rna segments forming double-stranded helices are labelled $ and $ ; the connecting rna segments are labeled l , l and l (same colour code as in fig. ). right-handed rna helices (similar to a-form dna helices) are characterized by a deep but narrow groove (equivalent to the major groove of b-dna) and a shallow but wide groove (equivalent to the minor groove of b-dna). in the central drawing of a hypothetical circular rna, the connecting segment (i) crosses the deep groove whereas segment (iii) crosses the shallow groove. segment (ii) bridges the whole length of the co-axial helices. when l is equal or larger than three nucleotides and l is equal or larger than four nucleotides [ ] . with six base pairs in s , however, one could expect that a single nucleotide in l is sutt]cient to obtain a stable pseudoknot structure. when the connecting segments contain a large number of nucleotides, they may be stmctured and partially basepaired. several situations can be envisaged. if l or l is large, a type (ii) pseudoknot becomes equivalent to either a type (i) or type (iii), respectively. types (i) and (iii) could still be described as motifs even if the segment crossing the whole helical stem is long and structured. where there is no short loop over either groove, however, the notion of structural motif is obviously lost. figure illustrates schematically another type of rna tertiary motif, which also is the outcome of the particular geometry of rna helices and which exhibits stacking of two helices. it results from the formation of base triples between a single strand and a double-stranded helix (either in the deep groove or in the shallow groove of the helix) and can lead to a segment of small triple helix. fig. . two-dimensional and three-dimensional diagrams illustrating how a pseudoknot can be related to a triple helix. instead of crossing the deep groove, the single-stranded ' end of helix h (in black) interacts with the side of the bases exposed in the deep groove of helix h (in grey). similarly, instead of crossing the shallow groove, the single-stranded ' end of helix h (in grey) interacts with the shallow groove of helix h (in black). a three-dimensional example of such a double triple-helix motif has been proposed in the core of the catalytic introns of group i [ ]. the formation of classic pseudoknots leads to a compact structure, the stability of which can be affected by the size of the loops and the number of base pairs. thermodynamic data on these aspects are still scarce. recent work has indicated that pseudoknots are only marginally more stable than their constituent hairpins (by less than kcal mol- ) [ ", "" ], if upheld by further evidence, this observation suggests a role for pseudoknots as conformational switches or control elements in several biological functions [ ]. in molecules that lack an overall three-dimensional fold, pseudoknots fold locally and their positions along the sequence reflect their function [ . ]. for example, pseudoknots that are folded at the ' end of mrnas tend to be involved in translational control whereas those at the ' end maintain intact signals for replication. in molecules with catalytic and mechanistic activities, pseudoknots are located at the core of the tertiary fold and involve nucleotides that are far apart in the sequence. pseudoknots appear to adopt two roles in the control of mrna translation: either specific recognition of a pseudoknot by some protein is required for control, as described for the ' end of mrnas in some prokaryotic systems the presence of three pseudoknots in s rrna has been suggested on the basis of comparative sequence analysis (reviewed in [ ]). the first pseudoknot lies between the ' end and the region around nucleotide . the second is located at a phytogeneticalty conserved tertiary interaction between positions and , also in the ' domain [ ] . the third pseudoknot involves the stem-loop structure [ ] , which is known to be important for the binding of trna to the ribosomal a site [ ] , and was recently shown to be essential for ribosomal function [ .o] . interestingly, some mismatches such as a.c or c.a in the third pseudoknot were lethal, whereas the wobble base pairs (g.u, u.g) conferred resistance to streptomycin, an antibiotic that perturbs the control of translational accuracy [ ..] . the same region interacts with protein $ [ ] . mutations in $ lead to streptomycin resistance and give rise to hyperaccurate ribosomes [ .-] . these observations are particularly interesting in view of the suggested conformational switch that involves two pseudoknots, one at the ' end and one in the region around nucleotide [ ] . the equilibrium between the two states would be controlled by $ and $ (a protein which has effects opposite to those of $ with respect to translational accuracy) [ ] . clearly, further experiments are required to prove this model. there has, however, been suggestion of an alternative conformational switch between the pseudoknot at position and a new one that involves the anti-shine-dalgamo sequence [ ] . like the pseudoknot at the ' end of the s rrna, the pseudoknots at the core of the catalytic rnas of group i introns [ ] and ribonucleasep [ , , . ] are highly conserved. a pseudoknot in the satellite rna of barley yellow dwarf virus, which belongs to the 'hammerhead' class, has been characterized [ "']. it has been suggested that pseudoknotting stabilizes the structure required for the self-cleavage of the hepatitis delta vires rna [ ] . secondary structures for genomic and antigenomic sequences that share a common stem-loop axehead motif could be developed without a pseudoknot [ " ], indicating that pseudoknotting might not be needed for the catalytic action. in support of this, a tran~active ribozyme was engineered from the normally c~active molecule [ -., ..]. though not capable of catalytic activity in the absence of protein [ ] , the mitochondrial-rna-processing rnase could, as the rna component of rnase p, possess a cage-shaped structure centered around a pseudoknot [ ] . the existence of two pseudoknots in eubacterial ribonudease p rna is attested by phylogenetic and mutational analyses [ . ]. the second pseudoknot (which base pairs nucleotides - with - , using escherichia coli nomenclature) is absent in bacillus subtilis and it was suggested, on the basis of molecular models, that an additional helix present in bacillus (and absent in e. coli) could hold an equivalent architectural function [ . ]. although these two pseudoknots impose topological constraints on the three-dimensional fold, neither is considered absolutely essential for catalytic activity in vitro [ ] . similarly, in the self-splicing introns of group i, a new pseudoknot, pll, was shown to be not essential for self-splicing activity in vitro, however, mutants with disrupted pll required a higher concentration of mgci for self-splicing to occur [ "]. by contrast, mutants that strengthen base pairing in pll self-splice more efficiently than the wild type at high temperature [ "]. this peripheral pseudoknot, which is present only in the subgroup ia, assists therefore the formation and the maintenance of an active conformation without itself being necessary for catalysis. two similar pseudoknots, involving one terminal loop and a distal section of the molecule, have already been proposed for intron--exon interactions in group ii introns [ , ] . the ribozyme in the satellite rna of barley yellow dwarf virus [ "'] undergoes self-cleavage at a low rate which was attributed to the presence of a pseudoknot. base substitutions that prevented folding of the pseudoknot increased the self-cleavage rate up to fold, whereas compensatory mutations reduced the selfcleavage rate in proportion to the helical stability of the helix responsible for pseudoknot formation [ "']. pseudotmotting underlies the trna-like motifs at the ' end of several groups of plant viral rna genomes (for a review, see [ °]). this structural similarity is paralleled in biological function as the trna-like motifs are recognized by many trna-specilic enzymes such as aminoacyl-trna synthetases, nucleotidyl transferase, or rnasep [ "]. the trna-like structure has been shown to be necessary for the initiation of replication of a positivesense rna vires, brome mosaic vires (bmv) [ .]. a telomeric function of the trna-like structure of bmv was also demonstrated in vivo [ ] , in agreement with the 'genomic tag model' associated with such '-terminal trna-like motifs [ ] . the dependence of replication on aminoacylation appears to be variable. the turnip yellow mosaic virus (tymv) depends on valylation for replication [ ] whereas bmv, which is normally tyrosylated, does not [ "'] . on the basis of this observation, transcripts bearing mutations in the trna-like domain on one of the three genomic rnas of bmv (which lead to decreased tyrosylation function in vitro) are able to interfere in tram with the replication of the other viral rnas when inoculated in barley protoplasts [ "] . a mutational analysis of the pseudoknot in tymv concluded that mutations in the loop bridging the shallow groove has stronger effects on valytation efficiency than those in the segment crossing the deep groove [ "*]. recently, the stretch of three pseudoknots preceding the trna-like structure in tobacco mosaic virus was shown to act as the functional equivalent of a poly(a) tail, stabi-lizing a reporter mrna and increasing gene expression up to -fold [ •'] . the formation of pseudoknots is a three-dimensional process and is central to the rna folding pathway. yet, at present, pseudoknot formation is incompatible with the dynamic programming method used in the present algorithms for predicting rna secondary structures [ , ] . nevertheless, two programs for the prediction of pseudoknot formation have been developed recently [ , ] . both simulate the rna fold by a sequential selection around the most stable stems [ , ] and, further, by sequential addition of folding domains ( - bp) during rna .synthesis [ ] . an implicit assumption in the prediction of secondary structures is that all structural elements co-exist in the final folded form; however, the possibility of alternative or transient pairings during the folding pathway or the function of the rna should be kept in mind. generally, it is difficult to prove the existence of pseudoknots merely on the basis of phylogenetic comparisons of only a small number of sequences. for example, a phylogenetically conserved sequence for pseudoknots does not necessarily mean that it is an important structural signal for the bicoid mrna [ ] , or the rna template in telomerase [ , ] , or the rna in the signal recognition particle [ ] . in these instances, systematically directed mutagenesis (with double mutants) coupled to chemical probing and functional tests would be required in order to reach consistent and biologically relevant structures. in contrast, with a large number of sequences, phylogenetic comparisons can be the most powerful approach for determining both the two-dimensional structure and the possible pseudoknots, which are then considered as those watson--crick pairings that are not contained in the secondary pairings [ , , , ] . achieving these goals requires rigorous investigations on the frequency of compensatory base changes and the frequency of the underlying mutational events in the helical stems. additionally, it is necessary to develop a new method, such as that described in [ ] , with which to distinguish those coordinated changes that result from authentic molecular constraints from those that arise from historical contingencies. in addition to demonstrating the requirements of both a pseudoknot and a heptameric shift site for frameshifting, the authors investigate the influence of the sequence at the shift site and especially of the seventh nucleotide of the generalized x xxx yyz shift sequence (where x and y are often a or u, and yyz is usually aac or uua). thus, the human immunodeficiency virus (h v) shift site has the same frameshifting ef ficiencies as mmtv and ibv shift sites in the mmtv context ( %), whereas it is sixfold more efficient in the mmtv context than in its native hiv context. also, changing the seventh nucleotide of the shift site from a c to u, a, or g provokes a -fold decrease in frameshifting efficiency. this last result points to a possible role of modified bases at position in the antic'don loop of the trna binding to the yyz loop. a compelling demonstration that a pseudoknot located eight nu cleotides ' of the uag stop codon is required ff)r translational read-through in mulv. although the nucleotide sequence in the helical stems of the pseudoknot is inconsequential for read-through, base substitutions in l diminish or stimulate read-through whereas insertion of three nucleotides in l or deletion of three nucleotides in l abolish read through. interestingly, replacement of the mulv pseudo knot by the mmtv pseudoknot eight nucleotides from the uag stop c(xton resulted in detectable ( %) read through. evidence for several higher order structural elements in ribosomal rna transfer rna shields specific nucleotides in s ribosomal rna from attack by chemical probes a functional pseudoknot in s ri-• . bosomal rna interaction of ribosomal proteins $ , $ , sll, s , s , and $ with s rrna gtngras k a conformational switch involving the region of escherichia coli s alternative base pairing between '-and '-terminal sequences of small subunit rna may provide the basis of a conformational switch of the small ribosomal subunit similar cage-shaped structures for the rna component of all ribonuclease p and ribonuclease mrp enzymes structure and evolution of ribonuclease p rna nr: • long-range structure in ribonuclease p rna a first attempt at finding equivalent three-dimensional motifs in rn& alternative tertiary structure atten-• . uates self-cleavage of the ribozyme in the satellite rna of barley yellow dwarf virus a pseudoknot-like structure required for efficient serf-cleavage of hepatitis delta virus rna efficient tran~cleavage and a • * common structural motif for the ribozymes of the human hepatitis delta agent analysis of rna secondary structures, together with in vitro rna transcripts, leads to the production of a trans-active ribozyme which could be used as a therapeutic agent. the results suggest also that some rna structures facilitate the folding process to an active ribozyme without themselves being involved in the catalytic reactions sequence • . and structure of the catalytic rna of hepatitis delta virus genomic rna enzymatic and chemical probing indicate that at least two stem-loop structures are required for catalysis. the structure of the region involved in pseudoknotting by [ ] is uncertain but its tertiary structure affects the efficiency of cleavage secondary structure of the rna component of a nuclear mitochondrial ribonucleoprotein multiple exon-binding sites in class ii self-splicing introns comparative and functional anatomy of group ii catalytic introns--a review telomeric function of the trna-like structure of brome mosaic virus rna trna-like structures tag the ' ends of genomic rna molecules for replication: implications for the origin of protein synthesis turnip yellow mosaic virus rnas with antic.don loop substitutions that result in decreased valylation fail to replicate efficiently halt tc: interference in trans with brome mo each of the three genomic rnas of bmv carries a '-end trna like domain. rna- mutants with a deficiency in tyrosylation activity of the '-end trna-like domain are able to interfere in trans with the synthesis and accumulation of the viral rnas when inoculated in barley protoplasts tobacco mosaic virus trna-like structure in cytoplasmic gene regulation on finding all suboptimal folding of an rna molecule the equilibrium partition function and base pair binding probabilities for rna secondary structure the computer simulation of rna folding involving pseudoknot formation bicoid mrna localization signal: phylogenetic conservation of function and rna secondary structure a conserved secondary structure for telomerase rna a conserved pseudoknot in telomerase rna srp-rna sequence alignment and secondary structure me r. descartes, f- we thank our colleagues for sharing with us reprints and we thank especially f michel and y-m hou for their constructive and critical reading of the manuscript. references and recommended reading a state-of-the-art mutational analysis of a functional pseudoknot. the authors investigate the frameshift signal which is comprised of a heptameric shift site and a downstream rna pseudoknot. they show that base pair formation at the junctions between the two helices forming the pseudoknot is not a pre-requisite for efficient frameshifting. the primary structure is not determinant as long as the overall pseudoknot structure is maintained. although small insertions or deletions in the loops of the pseudoknot have no effect on frameshifting, insertion or deletion of three nucleotides in the six nucleotides separating the end of the shift site from the first helix of the pseudoknot abolishes frameshifting. it is further shown that the pseudoknot cannot be replaced by an equivalent stem-loop structure. key: cord- -msicix authors: nan title: virus structure & assembly date: - - journal: biophys j doi: . /s - ( ) - sha: doc_id: cord_uid: msicix nan one-dimensional ( d) polyaniline/tobacco mosaic virus (tmv) composite nanofibers were synthesized by the self-assembly of rod like tmv particles assisted by the polymerization of aniline on its surface. at near-neutral reaction ph ( . - . ), branched polyaniline formed on the surface of tmv prevented lateral association and therefore long d nanofibers were observed with high aspect ratios and excellent processibility. at a lower ph ( . - . ), transmission electron microscopy (tem) analysis revealed that initially long nanofibers were formed and upon long reaction time resulted in bundled structures. this association of single nanofibers to form bundled structures is presumably mediated by the hydrophobic interaction of polyaniline on the surface of these composite nanofibers. in-situ time-resolved small-angle x-ray scattering study on the formation of polyaniline/tmv composite nanofibers at two different reaction ph conditions ( . and . ) supported our mechanism. the polyaniline layer formed on the external surface of tmv is thicker for reaction at ph . than for reaction at ph . . this novel strategy to assemble tmv into well-organized composites could be utilized in the fabrication of advanced materials for potential applications including electronics, optics, sensing, and biomedical engineering. work benefited from the use of ipns and aps funded by doe, bes under contract de-ac - ch to the uchicago argonne, llc and funding from u.s. aro-muri program, dod-durip and the w. m. keck foundation to qw. wouter h. roos from theoretical considerations on the response of viral capsids to deformation one expects that next to (i) complete elastic deformation and (ii) capsid failure upon deformation, there is a third possibility: (iii) plastic deformation. whereas the first two regimes have already experimentally been observed ( , ) , the plastic deformation regime has not. here we report on experiments in this intermediate response regime. the studies were performed with nanoindentation techniques using an atomic force microscope (afm), an approach which is becoming a standard method to measure the mechanical properties of viral particles ( , ) . capsids of the hepatitis b virus (hbv) were used as a model system. this virus was chosen because its capsid is made up out of many copies of a single protein and it can form in either a t¼ or a t¼ morphology. after a certain indentation both capsid types show permanent plastic deformations in their structures and the mechanics of this deformation can be described by the f€ opplvon k arm an (fvk) number. it is shown that the experimental observations match the expected behaviour as deduced from simulations ( ) . overall the two morphologies possess the same mechanical characteristics, but a slight difference in deformation characteristics is predicted by theoretical considerations on the difference in fvk number. we are able to observe this difference experimentally, which illustrates the resolution of our approach. matthias popp, helmut grubm€ uller mpi for biophysical chemistry, g€ ottingen, germany. phi bacteriophage infects the bacterium b. subtilis. during infection phi packs its newly produced dna into an empty capsid, against a very high pressure of ca. atm. for this purpose, a packaging machine containing five atpase units, six prnas and a head-tail connector translocates the dna into the capsid. the atpase units provide energy for the translocation, the connector has a portal to the capsid and the prnas connect the atps with the connector. for the role of the connector during packaging three mechanisms have been proposed so far. one suggests a nut-bolt like packaging of the dna. in the second one, the connector acts as a torrsion spring driving the dna in a ratchet like fashion. according to the third proposed mechanism, the connector is a valve preventing the packed dna from leaving, without any active parts at all. using molecular dynamics simulations of the connector in complex with dna, and aiming at distinguishing between these three models, we calculated mechanical properties of this system. first we calculated a spring constant and potential for the torsion around the connector axis. comparing the potential energy of the torsion with atp hydrolysis energy help to decide whether the connector is too rigid for the torrsion spring mechanism or not. second we have pulled the dna through the connector's portal to decide whether the connector has an active part in dna translocation. gabriel c. lander , alex evilevitch , meerim jeembaeva , bridget carragher , clint potter , jack johnson the scripps research inst., la jolla, ca, usa lund university, lund, sweden. the bacteriophage lambda is composed of an icosahedral capsid, into which a . kbp double-stranded dna genome is packaged, and a long non-contractile tail consisting of disk-like structures. while models describing the organization of these isolated structures have been proposed, there is a lack of complementary structural evidence in the literature. through cryo-em analysis of this phage, we are gaining a better understanding of its assembly and organization. there is a prominent surface feature present at the quasi -fold axes corresponding to the cementing protein gpd that is positioned directly over the covalent cross-linking portion of the docked hk crystal structure, suggesting an evolutionary replacement of this gene product by a chemical autocatalytic cross-link in hk . asymmetric reconstructions of both the empty and fully packaged lambda phage have provided three-dimensional views of the phage interior in the presence and absence of packaged dna, as well as how the tail assembly is attached to the capsid. a comparison between central slices of the asymmetric wild type and empty particles show clear density depicting the dsdna and portal proteins, along with evidence that upon completion of packaging, a plug-like gene product or conformational change of the portal closes the portal opening. tom chou ucla, los angeles, ca, usa. infection by membrane-enveloped viruses requires the binding of receptors on the target cell membrane to glycoproteins, or ''spikes,'' on the viral membrane. the initial entry is usually classified as fusogenic or endocytotic. however binding of viral spikes to cell surface receptors not only initiates the viral adhesion and the wrapping process necessary for internalization, but can simultaneously initiate direct fusion with the cell membrane. both fusion and internalization have been observed to be viable pathways for many viruses. we develop a stochastic model for viral entry that incorporates a competition between receptor mediated fusion and endocytosis. the relative probabilities of fusion and endocytosis of a virus particle initially nonspecifically adsorbed on the host cell membrane are computed as functions of receptor concentration, binding strength, and number of spikes. we find different parameter regimes where the entry pathway probabilities can be analytically expressed. experimental tests of our mechanistic hypotheses are proposed and discussed. was observed. a high salt buffer rinse reversed the conformational change. these results suggest a mechanism by which gag extension is possible only once bound to the plasma membrane and in the presence of the viral genome. this provides a picture consistent with earlier in vivo and solution studies. a detailed understanding of the viral particle assembly process may elucidate susceptible points providing opportunities to inhibit proper virus formation. nankai university, tianjin, china. the outbreak of severe acute respiratory syndrome (sars), caused by a previously unknown coronavirus called sars-cov, had profound social and economic impacts worldwide. since then, structure-function studies of sars-cov proteins have provided a wealth of information that increases our understanding of the underlying mechanisms of sars. we have adopted a structural proteomics approach targeting the sars-cov non-structural proteins that assemble to form a sophisticated machinery required for viral replication/transcription. we have determined protein and complex structures from sars-covand other coronaviruses to date, including: the first structure of the sars-cov mpro and its complex with an inhibitor; broadspectrum inhibitor design targeting coronavirus mpro; the sars-cov nsp -nsp super-complex; sars-cov nsp , a novel zincfinger protein; and mhv nsp , an endoribonuclease. the structure of another replicase protein was recently determined by our group. furthermore, we have been working towards understanding the molecular interactions between the various non-structural protein components of the replicase machinery. no effective therapy against sars is currently available, and considerable efforts have been made by our group towards preventing sars-cov infection. we have used a structure-guided drug discovery approach to design potent inhibitors of the coronavirus mpro, and have also developed a novel method for structure-based drug design using a library of natural mixtures. anton s. petrov, stephen c. harvey georgia institute of technology, atlanta, ga, usa. packaging of the genetic material inside bacteriophages is aided by atp-driven motors and leads to the arrangement of dna into highly-packed and ordered structures. using a coarse-grained model for dna and capsid we performed simulations of dna packaging inside bacteriophages of different shape and size. we found that the final dna conformations depend on the size and shape of the capsid, as well as the size of the protein portal, if any. in particular, isometric capsids with small or absent portals tend to form concentric spools, whereas the presence of a large portal favors coaxial spooling; slightly and highly elongated capsids result in folded and twisted toroidal conformations, respectively. by introducing several order parameters and analyzing the conformations of linear polymers confined inside irregular closed cavities over a wide range of packing densities we found that the ordering of dna to its fully packed structure occurs through two transitions: the first transition is associated with the formation of the global patterns. the second transition corresponds to the loss of mobility and disappearance of the undulations of dna strands. helical symmetry is an architectural schema that is widely imitated in the world. a helical structural is particularly suitable for electron cryo-microscopy study in that it displays a multitude of copies of its constituent asymmetric unit in diversified views. here we report the structural determination by helical reconstruction of a biological structure that was previously considered not suitable for this approach. vesicular stomatitis virus is a negative strand rna virus that has long served as a model system for influenza viruses. the system has not previously been subjected to helical cryoem reconstruction due to several concerns. first, there is insufficient confidence that this macromolecular assembly has a unique helicity and diameter among individual particles. second, the axial rise per subunit for this helix is particularly small, whilst having a large diameter. moreover, the naturally occurring structure is short in its length, not even twice its diameter. these properties have obstructed previous attempts for helical reconstruction. however, using an integrative approach with techniques from single particle reconstruction and helical reconstruction, we have pursued to outwit these technical barriers. the resulting structure is a helix of an unbelievably exact repeat number, . units per turn. in this structure, the viral rna genome is encapsidated by n nucleoproteins which in turn align in a left handed helical cylinder. this core is further reinforced by m matrix proteins interleaved into a chain-mail mash. on the opposite side, m proteins introduce the nucleocapsid to the envelope. in contrast with the nucleocapsid of other negative-strand rna viruses, only one form of helix was captured with d helical reconstruction among the working population of virions, suggestive of internal measures to control particle modality. from the density map, some interesting features of the viruses have been discussed. as revealed by techniques of structural biology and single-molecule experimentation, the capsids of viruses are some of nature's best examples of highly symmetric multiscale self-assembled structures with impressive mechanical properties of strength and elasticity. we present a novel method for creating three-dimensional finite element meshes of viral capsids from both atomic data from pdb files and electron density data from em files. the meshes capture heterogeneous geometric features and are used in conjunction with threedimensional continuum elasticity to simulate nanoindentation experiments as performed using atomic force microscopy. the method is extremely flexible; able to produce meshes with varying levels of coarseness, in addition to being able to capture varying levels of detail in the three-dimensional structure. meshes and nanoindentation simulations are presented for several viruses: hepatitis b, ccmv, hk , and phi . in addition to purely continuum elastic models, for those viruses with atomic coordinate information available, a multiscale technique is developed that combines the coarseness of the three-dimensional meshes with the atomic level forces calculated from an atomic force-field, using a mathematical mapping scheme. in this way, large-scale deformations such as nanoindentation are simulated without the cumbersome nature of other all-atom simulations, such as molecular dynamics. simulations of these capsid deformation experiments provide a testing ground for the techniques, as well as insight into the strengthdetermining mechanisms of capsid deformation. these methods can be extended as a framework for modeling other proteins and macromolecular structures in cell biology. a major challenge in examining the structural aspects of viral assembly is it has been mostly unexplored within the context of the host. much of what we understand today has been generated from computational models and in vitro crystallographic and electron microscopy analyses. the advent of electron cryo-tomography has now made it possible to obtain full three-dimensional reconstructions of unique, asymmetric objects such as whole cells and viruses (lucic et al., ; jensen & briegel, ) . here, we report on the three-dimensional reconstructions of intact caulobacter phage fcb infecting and assembling inside whole caulobacter crescentus cells. through this analysis we have been able to examine individual phage ultrastrucutre, phage attachment to the host cells, and phage dna packing within the cellular context. figure . a nm slice through a tomogram of a fcb infected caulobacter crescentus cell that was frozen in vitreous ice. scale bar nm. values, while at ph's lower than . the virus particles reversibly aggregate without any noticeable structural change, as analyzed under transmission electron microscopy, static and dynamic light scattering and fluorescence spectroscopy. in this study, we also compute at different ph values trv surface electrostatic potential and we postulate that the virus aggregation is due to electrostatic effects. the influenza virus is an enveloped virus from the orthomyxovirus family. the protein rich lipid membrane of the virus particle needs to persist in the often hostile extra-cellular environment when the virus transmits from host to host organism, but it also needs to permit membrane fusion with the target cell to allow infection. we set out to investigate how this virus negotiates these apparently conflicting demands on its membrane rigidity during its lifecycle. we have developed methods to image this relatively large (diameter~ nm) virus and to measure its mechanical properties using an atomic force microscope (afm). we have measured the stiffness of the viral membrane under conditions mimicking the different stages of the viral life-cycle including different ph levels. we have further compared the complex response of the viral membrane with the behavior of simplified model systems to understand the role of the various part of the viral structure for its mechanical properties. (supported by mrc; i.a.t.s. is supported by the european community through a marie curie fellowship) figure, afm scan of an influenza virus attached to a substrate structural studies by electron tomography: from cells to molecules -pos methods for multiscale stochastic simulation of molecular self-assembly tiequan zhang carnegie mellon university, pittsburgh, pa, usa. macromolecular self-assembly is a crucial component of nearly every major cellular process. simulation methods provide a valuable adjunct to experimental work in understanding self-assembly by allowing us to perform model-assisted interpretation of assembly systems too complex for detailed experimental dissection and to extrapolate results from in vitro experimental systems to the cellular environment. unfortunately, it remains computationally intractable to simulate detailed self-assembly dynamics within several biologically important parameter domains, including long time scales needed to understand some in vitro assembly models and dense crowding conditions relevant to in vivo assembly. we have developed simulation methods based on stochastic models of reaction chemistry aimed at extending feasible parameter domains in these biologically important directions. we have applied several heuristics based on markov model and spectral graph theory to acceler-ate the stochastic simulation algorithm for some hard conditions that arise at extremes of in vitro assembly conditions. we have also developed simulations based on green's function reaction dynamics models of spatially heterogeneous environments to better capture some difficult high-concentration domains. we demonstrate these methods using coarse-grained models of the assembly of icosahedral virus capsids as well as several simpler models of generic assembly chemistry. the genome packing in hundreds of viruses is investigated by analyzing the chemical sequences of the genomes and the corresponding capsid proteins, in combination with experimental facts on the structures of the packaged genomes. based on statistical mechanics arguments and computer simulations, we have derived a universal model, based simply on non-specific electrostatic interactions. our model is able to predict the essential aspects of genome packing in diversely different viruses, such as the genome size and its density distribution. our result is in contrast to the long-held view that specific interactions between the sequenced amino acid residues and the nucleotides of the genome control the genome packing. implications of this finding in the evolution and biotechnology will be discussed. triatoma virus (trv) is a þssrna, non-enveloped virus of the insect virus family dicistroviridae. trv capsid has icosahedral t¼ pseudo t¼ symmetry, and is composed of three proteins named vp , vp and vp , which mw are , and kda respectively. we report that the trv capsid remains assembled at very acidic ph feng li , frederic pincet , eric perez , william eng , thomas j. melia , james e. rothman , david tareste laboratoire de physique statistique, paris, france columbia university, new york, ny, usa. membrane fusion occurs when snarepins fold up between lipid bilayers. how much energy is generated during snarepin folding and how this energy is coupled to the fusion of apposing membranes is still a mystery. we have utilized the surface forces apparatus to determine the energetics and dynamics of snarepins formation and characterize the different intermediate structures sampled by cognate snares in the course of their assembly. the force versus distance profiles of assembling snarepins reveal that snare motifs begin to interact when the membranes are nm apart. even after very close approach of the bilayers ( - nm), the snar-epins remain partly unstructured in their membrane-proximal region. the energy stabilizing a single snarepin in this configuration ( kbt) corresponds closely with the energy needed to fuse outer but not inner leaflets (hemifusion) of pure lipid bilayers ( - kbt). the cooperative effect of a few snarepins at the site of exocytosis would thus be sufficient to overcome the high energetic barriers of membrane fusion. marc baaden ibpc, cnrs upr , paris, france. the snare protein complex is central to membrane fusion, a ubiquitous process in biology. modeling this system in order to better understand its guiding principles is a challenging task. this is mainly due to the complexity of the environment: two adjacent membranes and a central bundle of four helices made up by vesicular and plasma membrane proteins as shown in the figure below.we have modeled this system at several levels of detail, reaching from coarse grained representations of the synaptobrevin transmembrane helix in a single lipid bilayer up to an atomistic model of the full membrane-embedded synaptic fusion complex shown above. molecular dynamics simulations of these models were carried out to characterize the conformational dynamics and key interactions in these systems. no evidence for directionality in the key: cord- -rh g jk authors: wigginton, krista rule; kohn, tamar title: virus disinfection mechanisms: the role of virus composition, structure, and function date: - - journal: curr opin virol doi: . /j.coviro. . . sha: doc_id: cord_uid: rh g jk drinking waters are treated for enteric virus via a number of disinfection techniques including chemical oxidants, irradiation, and heat, however the inactivation mechanisms during disinfection remain elusive. owing to the fact that a number of significant waterborne virus strains are not readily culturable in vitro at this time (e.g. norovirus, hepatitis a), the susceptibility of these viruses to disinfection is largely unknown. an in-depth understanding of the mechanisms involved in virus inactivation would aid in predicting the susceptibility of non-culturable virus strains to disinfection and would foster the development of improved disinfection methods. recent technological advances in virology research have provided a wealth of information on enteric virus compositions, structures, and biological functions. this knowledge will allow for physical/chemical descriptions of virus inactivation and thus further our understanding of virus disinfection to the most basic mechanistic level. virus disinfection mechanisms: the role of virus composition, structure, and function krista rule wigginton drinking waters are treated for enteric virus via a number of disinfection techniques including chemical oxidants, irradiation, and heat, however the inactivation mechanisms during disinfection remain elusive. owing to the fact that a number of significant waterborne virus strains are not readily culturable in vitro at this time (e.g. norovirus, hepatitis a), the susceptibility of these viruses to disinfection is largely unknown. an in-depth understanding of the mechanisms involved in virus inactivation would aid in predicting the susceptibility of non-culturable virus strains to disinfection and would foster the development of improved disinfection methods. recent technological advances in virology research have provided a wealth of information on enteric virus compositions, structures, and biological functions. this knowledge will allow for physical/chemical descriptions of virus inactivation and thus further our understanding of virus disinfection to the most basic mechanistic level. obtaining a mechanistic understanding of virus disinfection is a pressing need in environmental engineering owing to the enduring occurrence of waterborne and food-borne virus outbreaks. many important enteric viruses remain non-culturable to date (e.g. norovirus, hepatitis a); therefore, their susceptibility to disinfection cannot be experimentally tested. non-culturable virus disinfection kinetics must be either determined with human charge studies or predicted using surrogate viruses that can be cultured in vitro but that differ in composition, structure, and function. a framework that enables the accurate prediction of virus inactivation behavior based on a detailed understanding of the processes involved would assist in the development of effective disinfection strategies. scientists have long sought to provide mechanistic descriptions of virus inactivation during drinking water disinfection [ ] . in the - s, researchers employed scintillation spectroscopy and electron microscopy techniques to detect modifications in viral genomes and proteins and typically reported one of two conclusions: ) inactivation is the result of damage to the virus proteins or ) inactivation is the result of damage to the genome [ ] [ ] [ ] [ ] [ ] . although these early studies investigated the molecular mechanisms as much as technologically possible, more recent research has focused less on elucidating mechanisms and more on comparing inactivation kinetics with various virus strains, disinfectants, and water chemistries [ ] [ ] [ ] . this is despite the fact that recent technological advances have provided improved tools for probing molecular mechanisms. collectively, the proposed virus inactivation mechanisms by common water disinfectants vary widely and are often contradictory. for example, the inactivation of poliovirus by chlorine has been attributed to rna degradation [ ] and to capsid protein modifications [ ] . at this time, the fundamental questions of what modifications do or do not cause inactivation remain elusive. herein, we discuss how the combined knowledge of virus composition, structure and biological function will further our understanding of virus disinfection at the most basic mechanistic level. a physical/chemical description of inactivation is more feasible today than it was ten years ago thanks to advances in genome sequencing, protein mass spectrometry, and structural virology techniques. we focus the majority of our discussion on the disinfection of waterborne enteric viruses [ ] , in general, and poliovirus, in particular. enteric viruses are the most relevant to water treatment and poliovirus has been the focus of numerous disinfection studies over the past several decades [ ] [ ] [ ] . it should be noted that this discussion could be extrapolated to other settings where disinfection is used to mitigate virus transmission, such as food safety or medical equipment sterilization. such as chlorine, ozone, or uv irradiation are fairly well characterized (table ) . consequently, established reaction rate constants for amino acid and nucleotide monomers can be summed based on their known abundance within virus particles to provide predictions of the relative reaction rates of genome and protein targets [ ] . when this is done for poliovirus mahoney, chemical disinfectants such as chlorine and ozone are much more reactive with viral protein material than with genomic material (figure ). on the contrary, uvc radiation affects the genomic material more than the protein material. unfortunately, such predictions are inaccurate owing to the influence of protein and genome higherlevel organization on reaction rates [ ] [ ] [ ] . for example, when treated with chlorine dioxide, denatured poliovirus genomes degraded at a different rate than native poliovirus genomes [ ] . reactions that take place in the genome and proteins during disinfection can form byproducts that further react with amino acids and nucleotides [ ] ; this makes reactivity predictions even more complicated. unlike chemical oxidants, uvc radiation will lead to direct photolysis of photolabile virus components regardless of their solvent accessibility. a genome-size based approach to predict the sensitivity of virus strains to uvc has been proposed [ ] , although others have reported that genome-size does not always correlate with virus susceptibility to uv disinfection [ , ] . similar to the genome-size based approach, pyrimidine doublet prevalence in virus genomes was suggested as a framework to predict uvc susceptibility [ ] . indeed, a plot of the number of potential dimerization sequences in a virus genome versus effective uv dose suggested a correlation. the presence of outlier virus strains, however, indicates that alternative pathways play a role in some uv inactivation mechanisms. taken together, these discrepancies demonstrate that virus component information (i.e. genome and protein sequences) alone will not allow for an accurate prediction of susceptibility. a prior knowledge of capsid and genome structure should therefore aid in interpreting and predicting virus particle reactivity and in identifying the particle's most susceptible regions. virus disinfection mechanisms wigginton and kohn table reported second-order rate constants and photochemical constants for the most reactive amino acid and nucleoside monomers with common disinfectants in aqueous solutions at ph $ chlorine predicted relative reaction rates for poliovirus protein and genome components calculated with rate constants and photochemical constants presented in table . coupling structure and composition information aids in our understanding of virus reactivity x-ray crystal structures have been published for numerous enteric viruses [ , , ] and with these reports have come a windfall of valuable information including the location and orientation of capsid protein residues. cryo-electron microscopy (cryoem) has expanded our knowledge of virus structures even further, as it allows virologists to study virus particles that are difficult to crystallize due to either complex capsid shapes, inadequate purification, or intermediate, metastable structures (e.g. virus binding or cell entry processes [ , ] ). in fact, recent advances in cryoem have lead to viral reconstructions at resolutions comparable to those obtained with x-ray crystallography [ , ] . in addition to ordered capsid protein visualization, cryoem studies have demonstrated that some viruses have ordered genomes [ ] and have described specific interactions between capsid proteins and packaged genomes [ ] . it should be noted that resolved near-atomic structures are not yet available for some important enteric viruses such as hepatitis a and human norovirus, although recombinant norovirus-like particles have been reconstructed [ ] . some caution must be taken when using virus structural data to identify the location and solvent accessibility of functional groups. virus capsids can be fluid in nature and thus functional groups that are normally protected from oxidants in the solvent can be periodically exposed to the capsid surface [ ] . in human rhinovirus, for example, certain regions are static and in agreement with the crystal structure, while other regions are more fluid [ ] . together, composition data and structure data will provide an improved framework to describe the reactivity of virus components with disinfectants. indeed, a number of studies on nonviral proteins have used structural data to explain the site-specific reactivity of protein components [ , , , ] . at this time, however, only a few reports on virus disinfection have mentioned resolved virus structures in the interpretation of results [ , , ] . a study on adenovirus inactivation with chlorine did employ adenovirus structural data to suggest that damaged met or his residues near a critical motif may contribute to inactivation [ ] . in another study, protein mass spectrometry was used to identify specific residues in ms 's capsid proteins as the virus was inactivated by uv and singlet oxygen [ ] . residues on the outside surface of the capsid were modified with o treatment while residues on the inside surface of the capsid near the viral genome were modified with uv treatment. more research is clearly needed to elucidate the effect of virus structure on the reactivity of virus components. composition and structure information aids in describing where modifications are most likely to occur in a virus particle during disinfection; however, modifications do not always cause inactivation. for example, although ozonation of poliovirus altered viral proteins vp and vp , inactivation was ultimately attributed to genome damage [ ] . solely identifying susceptible virus regions based on composition and structure will be insufficient to describe and predict the effect of a disinfectant on virus infectivity. one must also consider the effect that particular modifications have on fundamental virus functions (e.g. host-cell binding, genome entry, etc.). in order to do this, the fundamental functions and virus components involved in those functions must be well defined. virus structural and dynamic information has provided an improved understanding of the biological function of viral domains, including virus-host cell interaction [ , ] , virus assembly [ ] , capsid-rna interaction sites [ , ] and rna release ( [ ] and references therein). this new knowledge provides valuable insight into critical structures and biological functions of the virus and thus identifies regions and functions that should be targeted in virus neutralization or inactivation strategies. the promise of a structure/function-based approach for disinfection is underscored by the fact that the medical field has exploited this method to develop vaccines [ ] and antiviral drugs [ ] as well as to better understand the mechanism of previously developed vaccines [ ] . this type of approach has great benefits over the traditional method of screening and isolating fortuitously emerged, non-virulent virus strains or neutralizing antibodies. namely, it enables the rational design of site-specific antivirals and vaccines that target relevant virus structures common to several strains or species. for example, coronavirus strains were long believed to have host receptors that were too diverse and too prone to mutation to be susceptible to a broad-spectrum antiviral. recently, however, yang et al. [ ] used function and structure information of all three genetic coronavirus clusters to determine that the main protease has a highly conserved substrate-recognition pocket. based on this structural information combined with compositional information of conserved amino acids within this region, a protease inhibitor was designed that successfully prevented virus replication of two coronaviruses. ultimately, the authors suggest that the knowledge of the conserved structure and biochemistry of the protease will lead to the development of a single, broad spectrum antiviral that targets all coronaviruses. a similar approach to water disinfection will lead to the rational design of novel disinfectants. an important difference between water disinfection and antiviral drugs, however, is that traditional disinfection does not operate by physically blocking a virus site or particular function via the addition of an external chemical or antibody, but by chemically or structurally altering one or several important sites. we therefore expect that the intrinsic reactivities of the various virus components are of greater importance to disinfection than to drug development. as discussed above, this intrinsic reactivity is dictated by both specific chemical composition and the structure. as such, the structure/function approach used in the medical field would have to be expanded to a composition/structure/function approach in water treatment. as an example of how structural commonalities and compositional differences may be used to predict susceptibilities to disinfectants, we look at poliovirus brunhilde and poliovirus mahoney. the brunhilde strain was reported to be twice as resistant to chlorination as the mahoney strain [ , ] . this discrepancy in inactivation kinetics is intriguing as the two strains have nearly identical capsid protein sequences ($ %) and have similarly sized genomes. inactivation of the mahoney strain by chlorine was attributed to a loss in ability to attach to the host cell [ ] , however this has not been examined for the brunhilde strain. cryoem analysis has provided a three-dimensional structure of the poliovirus particle bound to its host cell receptor, spvr [ ] , and a number of capsid protein residues were implicated in the virus capsid-host cell interaction. interestingly, six of these implicated residues differ in the mahoney and brunhilde strains, including mahoney vp met (thr in brunhilde) and his (tyr in brunhilde) ( figure ). methionine and histidine side chains readily react with chlorine; threonine and tyrosine sidechains, on the contrary, are much less reactive. it is thus possible that the lack of met and his residues at the binding site is responsible for the greater resistance of the brunhilde strain to chlorine disinfection. this is only a hypothetical explanation and will need to be confirmed through experimentation; it does, however, demonstrate the manner in which composition, structure, and function information can assist in deducing virus inactivation mechanisms. it also demonstrates how such knowledge aids in predicting how a new strain will behave based on mutations in the binding site. suppose, for example, a new poliovirus strain emerges with fewer reactive functional groups in its binding site. the new strain would be expected to be more resistant to disinfection than the well-characterized strain. for fast-mutating viruses like human norovirus, this type of predictive tool would be particularly valuable. research is needed to assess exactly how similar virus strains should be in structure, composition, and function for such a predictive tool to work. to reach a point where a holistic understanding of virus inactivation is in hand, a number of questions will first need to be addressed. specific questions include: ) which virus protein residues are involved with fundamental functions and how do these vary amongst different strains and species; ) what specific chemical modifications take place in the genome and capsid during disinfection and what effects do these modifications have on virus structure and function; ) how similar are disinfectant-induced modifications amongst various enteric viruses? answering these questions is a lofty goal given the numerous virus-disinfectant pairs that will need to be examined. in conclusion, the study of virus fate in water treatment is entering an exciting new phase thanks to the advent of tools that provide insight into virus structure and function. as a result, virus inactivation predictive tools and the design of highly efficient disinfectants may be a reality in the not-so-distant future. papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest virus disinfection mechanisms wigginton and kohn current opinion in virology poliovirus mahoney strain capsid. vp met is highlighted in green and vp his is highlighted in red. structural visualization with pymol software (http:/www.pymol.org). molecular mechanisms of viral inactivation by water disinfectants structural and compositional changes associated with chlorine inactivation of polioviruses mechanism of disinfectionincorporation of cl- into f virus mechanism of ozone inactivation of bacteriophage-f mechanism of enteroviral inactivation by ozone mechanisms of inactivation of poliovirus by chlorine dioxide and iodine effects of source water quality on chlorine inactivation of adenovirus, coxsackievirus, echovirus, and murine norovirus inactivation of adenoviruses, enteroviruses, and murine norovirus in water by free chlorine and monochloramine inactivation of enteric adenovirus and feline calicivirus by chlorine dioxide capsid functions of inactivated human picornaviruses and feline calicivirus enterically infecting viruses: pathogenicity, transmission and significance for food and waterborne infection inactivation by bromine of single poliovirus particles in water relative resistance to chlorine of poliovirus and coxsackievirus isolates from environmental sources and drinking water reduction of norwalk virus, poliovirus , and bacteriophage ms by ozone disinfection of water singlet oxygen-mediated damage to proteins and its consequences oxidative modification of cytochrome c by singlet oxygen reactivity of singlet oxygen toward proteins: the effect of structure in basic pancreatic trypsin inhibitor and in ribonuclease a photoinduced dna cleavage via electron transfer: demonstration that guanine residues located ' to guanine are the most electron-donating sites degradation of the poliovirus genome by chlorine dioxide this study compared the susceptibility of extracted viral genomes with viral genomes inside an intact virus when treated with chlorine dioxide. the results offered evidence that the structure of the viral genome plays a role in the genome's susceptibility to oxidant attack hypochlorous acidmediated protein oxidation: how important are chloramine transfer reactions and protein tertiary structure? predicted inactivation of viruses of relevance to biodefense by solar radiation inactivation of bacteriophages in water by means of non-ionizing (uv- . nm) and ionizing (gamma) radiation: a comparative approach inactivation of poliovirus and f-specific rna phages and degradation of their genomes by uv irradiation at nanometers a genomic model for predicting the ultraviolet susceptibility of viruses this study proposed a mathematical model to predict the susceptibility of viruses to inactivation by uv . the model was based on the prevalence of viral genome sequences that readily undergo dimerization with uv radiation the crystal structure of coxsackievirus a : new insights into the uncoating mechanisms of enteroviruses. structure biological and immunological characteristics of hepatitis e virus-like particles based on the crystal structure this research solved the first crystal structure of hepatitis e virus-like particles at . -Å resolution and identified a putative cellular binding region in the capsid via mutational analyses crystal structure of human adenovirus at . Å resolution . Å cryo-em structure of a nonenveloped virus reveals a priming mechanism for cell entry viral life cycles captured in threedimensions with electron microscopy tomography an effective review of recent electron cryotomography studies that have elucidated the complex structure and life cycles of viruses near-atomic resolution using electron cryomicroscopy and single-particle reconstruction near-atomic resolution cryo-em for molecular virology a review highlighting several recent cryo-em virus studies and the novel insights they have provided visualization of ordered genomic rna and localization of transcriptional complexes in rotavirus atomic structure reveals the unique capsid organization of a dsrna virus x-ray crystallographic structure of the norwalk virus capsid viral capsid mobility: a dynamic conduit for inactivation this study provided evidence that virus inactivation by small alkylating agents is due to reactions in the viral genome; it therefore demonstrated that certain chemicals can diffuse through virus protein capsids even when the capsids are void of holes or major crevices capsid structure and dynamics of a human rhinovirus probed by hydrogen exchange mass spectrometry hypochlorous acid oxidizes methionine and tryptophan residues in myoglobin site-specific hypochlorous acid-induced oxidation of recombinant human myoglobin affects specific amino acid residues and the rate of cytochrome b -mediated heme reduction mechanistic aspects of adenovirus serotype inactivation with free chlorine this study examined the mechanisms responsible for adenovirus type inactivation with free chlorine disinfection. inactivation was attributed to damage in virus proteins that control functions post host cell attachment oxidation of virus proteins during uv and singlet oxygen mediated inactivation this study used protein mass spectrometry to detect modifications in virus capsid proteins as virus were inactivated and demonstrated that singlet oxygen and uvc affect different virus components crystal structure of measles virus hemagglutinin provides insight into effective vaccines this study solved the crystal structure of measles virus hemagglutinin-the attachment protein responsible for virus entry into the host cell and a target for neutralizing antibodies. the authors suggested that the solved molecular structure can aid in structure-based measles vaccine development catching a virus in the act of rna release: a novel poliovirus uncoating intermediate characterized by cryo-electron microscopy emerging antiviral targets for influenza a virus design of wide-spectrum inhibitors targeting coronavirus main proteases irreversible inhibitors for coronavirus strains were designed using the structure of a conserved substrate-recognition pocket. the results demonstrate that it is possible to develop a single antiviral agent effective against a number of coronavirus strains inactivation of poliovirus-i (brunhilde) single particles by chlorine in water inactivation by chlorine of single poliovirus particles in water threedimensional structure of poliovirus receptor bound to poliovirus the structure of poliovirus type bound to its cellular receptor, spvr, was determined at -Å resolution with cryo-em. capsid protein residues involved in the poliovirus-spvr interactions were identified and were in general agreement with previous mutagenesis results hypochlorous acid interactions with thiols, nucleotides, dna, and other biological substrates degradation of nucleic-acids with ozone. v. mechanism of action of ozone on deoxyribonucleoside '-monophosphates rate constants of ozone reactions with dna, its constituents and related compounds photochemcadz: a computer-aided design and research tool in photochemistry dna excited-state dynamics: from single bases to the double helix gorner h: photochemistry of dna and related biomoleculesquantum yields and consequences of photoionization interactions of hypochlorous acid with pyrimidine nucleotides, and secondary reactions of chlorinated pyrimidines with gsh, nadh, and other substrates absolute rate constants for the reaction of hypochlorous acid with protein side chains and peptide bonds kinetics of ozonation. . amino-acids and model compounds in water and comparisons to rates in nonpolar-solvents numerical values of absorbances of aromatic amino acids in acid neutral and alkaline solutions uv photolysis of aromatic amino acids and related dipeptides and tripeptides key: cord- -u ycqelc authors: rossmann, michael g.; battisti, anthony j.; plevka, pavel title: future prospects date: - - journal: adv protein chem struct biol doi: . /b - - - - . - sha: doc_id: cord_uid: u ycqelc cryo-electron microscopy (cryo-em) in combination with single-particle analysis has begun to complement crystallography in the study of large macromolecules at near-atomic resolution. furthermore, advances in cryo-electron tomography have made possible the study of macromolecules within their cellular environment. single-particle and tomographic studies will become even more useful when technologies for improving the signal-to-noise ratio such as direct electron detectors and phase plates become widely available. automated image acquisition has significantly reduced the time and effort required to determine the structures of macromolecular assemblies. as a result, the number of structures determined by cryo-em is growing exponentially. however, there is an urgent need for improved criteria for validating both the reconstruction process and the atomic models derived from cryo-em data. another major challenge will be mitigating the effects of anisotropy caused by the missing wedge and the excessively low signal-to-noise ratio for tomographic data. parallels between the development of macromolecular crystallography and cryo-em have been used to tentatively predict the future of cryo-em. increasing importance of electron microscopy the growing numbers of cryo-electron microscopy (cryo-em) and cryoelectron tomography (cryo-et) reconstructions deposited with the protein data bank (pdb) (fig. ) indicate that these methods have become frequently used techniques for determining structures of macromolecular complexes. nevertheless, x-ray crystallographic structure determinations cryo-em nmr x-ray crystallography , , still dominate both electron microscopy (em) and nmr spectroscopy. because x-ray crystallography has had the longest history, the initial part of this chapter will trace the development of macromolecular crystallography and compare it with the more recent development of cryo-em in order to extrapolate and divine the future of this latest tool of structural biology. in in munich, germany, paul ewald, together with two students, w. friedrich and p. knipping, demonstrated that a crystal of cu so could diffract x-rays, consistent with von laue's predictions (ewald, ) . the experiment was performed at night to avoid the wrath of the senior professors who were of the opinion that the experiment was worthless. yet, this small episode changed science and the world. less than years later, the same technology had been used to determine the first atomic structures of two proteins (kendrew et al., ; kendrew et al., ; perutz et al., ; hadfield et al., ) , and by the time another years had gone by the structure of two human viruses had been determined (hogle et al., ; rossmann et al., ) . even more recently, the structure and mechanism of the protein synthesizing ribosomal machine (http://nobelprize.org/nobel_prizes/chemistry/laureates/ /) were established. from its beginning, x-ray crystallography was a method that was capable of resolving individual atoms, but its power to view large, complex biological molecules grew only gradually. by the end of the twentieth century, pharmaceutical companies were eagerly employing crystallographers in the hopes that structural information would lead to cures for numerous human diseases. yet, just like a financial bubble, the good times are now coming to an end, as the proteins whose structures have not yet been determined are ever more difficult to crystallize. the projects have grown more ambitious, aiming at membrane proteins and large protein complexes. fortunately, em has now advanced to a point where it is rapidly replacing crystallography as the major pioneering tool of structural biology (harrison, ) . the experiment showed that x-rays were waves, that the repeating units in crystals were of roughly the same dimensions as the wavelength of x-rays, and confirmed the chemical ideas of the atomicity of matter. this news spread rapidly to england where lawrence bragg interpreted the mathematical expressions of von laue to an easier to comprehend physical concept. he went on to use his physical insight to show that the pattern of intensities caused by the diffraction of x-rays from crystals of nacl and kcl were consistent with spherical metal ions filling the spaces between the larger spherical, cubic close packed, cl À ions (bragg, ) . chemists of the time found this difficult to comprehend as they thought that nacl was a compound consisting of individual nacl molecules. when eventually the heated discussion was settled in favor of the cubic arrangement of ions, this discovery turned out to be the birth of structural chemistry. after the end of the first world war in , there reemerged an interest in structural crystallography, led by sir lawrence bragg. initially, this was directed to the investigation of centric structures, mainly minerals, many of which had interesting symmetries, but gradually crystallographers turned to organic compounds. for instance, kathleen lonsdale (later to be elected the first female fellow of the royal society) was able to confirm that hexamethylbenzene was a flat molecule (lonsdale, ) . notwithstanding the difficulties of the second world war, dorothy crowfoot hodgkin had been able to establish the structure of the asymmetric penicillin molecule, the first antibiotic used with much success during the second world war (crowfoot, ) . by this time, crystallographers had gone beyond the ''trial and error'' approach of guessing at atomic positions and were starting to use a variety of computational tools including the patterson function (patterson, (patterson, , and the heavy atom technique. in addition, crystallographers had discovered the use of fourier syntheses to represent electron density as presented on stacked sheets of glass plates (bragg, ) . these new techniques required lengthy calculations, helped by such tools as beevers lipson ''strips'' (beevers and lipson, ) for the manual summation of a fourier series. fortunately, the first home-built, and then commercial, electronic computers became available in the s. many of the early crystallographic structures had been determined with only one-or twodimensional data, but with the advent of better computational devices there was a desire to extend data collection into three dimensions. this set the stage, as mentioned above, for the structural determinations of biological macromolecules and molecular assemblies in the second half of the twentieth century. the structure determinations of large molecules also required large machines, such as robots, for producing crystals, synchrotrons to produce intense, coherent, almost monochromatic x-rays, and fast computer graphics piggy-backed on the development of gaming machines. the most recent emphasis has been on ever-smaller crystals, as the difficulty of growing crystals has increased in proportion to the size of the object being crystallized. in some experiments, the crystals are only a few unit cells in each direction, while others have been even more audacious by looking at the diffraction pattern of single molecules (bogan et al., ) with the help of extremely intense x-ray lasers (kirian et al., ) . indeed, the increasing demands on instrumentation are pushing x-ray imaging techniques to the limits of what is achievable (rossmann and arnold, ) . there are, however, alternative simpler and less expensive ways for obtaining structural results at the boundaries of structural biology. x-ray detectors measure only intensities, from which the amplitude of the diffracted rays can be evaluated. the relative phase of the diffracted rays is the central ''phase problem'' of any x-ray structure determination. the phases need to be determined indirectly in order to be able to calculate the electron density distribution in the crystal. in contrast, an electron microscope uses the wave properties of the electrons to form an image of the object in the same way as does a light microscope. unlike x-rays, scattered electrons can be focused with magnetic lenses, allowing an image to be formed and thereby solving the phase problem directly. the average wavelength of fast electrons is around . Å , of x-rays roughly Å , compared to - Å for visible light. thus, unlike visible light, electrons and x-rays can be used to resolve individual atoms (covalently bonded atoms are separated by about . Å ). nevertheless, the development of atomic-resolution em has lagged behind the development of x-ray crystallography. the first electron microscope was constructed by ernst ruska in . this was a time of rapid advances in basic physics when the properties of elementary particles were being debated. however, the early microscopes were not much of an advance on light microscopes and had the additional disadvantage of requiring the specimen to be in a vacuum. development accelerated after the end of the second world war with the introduction of negative stains using heavy metal dyes to enhance contrast between water and the almost equally dense biological samples. unfortunately, the staining process also caused distortions and artifacts in the samples. de rosier and klug ( ) suggested combining different projections of isolated molecular particles in order to create a three-dimensional image. this led to the three-dimensional structure determinations of small plant viruses using negatively stained samples (crowther and amos, ). an important breakthrough came in with the use of flash freezing techniques for embedding the specimen in vitreous (amorphous) ice (dubochet et al., ) . this technique preserved the specimen in a near-native state, free of the artifacts of staining. immediately, there was a flurry of new reconstructions, particularly of viruses but also of ribosomes, chaperones, and other molecular assemblies. large complexes that could not be crystallized, such as viruses interacting with cellular receptors or viruses interacting with antibodies (smith et al., ; wikoff et al., ) , could now be studied by combining cryo-em of the complex with crystallography of the individual component molecules (rossmann, ; rossmann et al., ) to generate ''pseudo-atomic'' models. cryo-em was used to obtain the lower resolution ''large picture'' into which the higher resolution crystal structures of the components were fitted (rossmann, ; rossmann et al., ) , enabling analysis of interactions between the component structures. another use of cryo-em structures can be to provide a low-resolution model for molecular replacement to initiate x-ray crystal structure determinations by means of phase extension, as was the case for a ribosomal subunit (ban et al., ) or phix virus particle (dokland et al., ) . in contrast to x-ray crystallography, cryo-em was initially used primarily to study complex homogeneous objects at rather limited resolution. another milestone was passed in with the subnanometer structure of hepatitis b cores determined by two independent groups (böttcher et al., ; conway et al., ) . in the past or years, cryo-em has been able to produce, in favorable cases, three-dimensional images that are comparable to x-ray crystal structures of icosahedral viruses ( jiang et al., ; zhang et al., ) . that has been an important development as it has become apparent that larger viruses with lipid envelopes cannot usually be crystallized or give only very poorly diffracting crystals (harrison et al., ; bamford et al., ; kaufmann et al., ) . but, like crystallography, this form of cryo-em is also reaching its limits because large biological objects tend to be heterogeneous. for this reason, cryo-et is becoming a more frequently used tool lučić et al., ) . in this technique, a three-dimensional image is created by combining many images taken of the same sample oriented by a succession of tilt angles about a common axis (or multiple axes). because the same sample is repeatedly exposed to the electron beam, the electron dose per image has to be kept very low to avoid radiation damage, lowering the feasible resolution. currently, numerous schemes are being developed to mitigate the effects of the low dose, the physical limitations of the tilting equipment, and the increased specimen thickness with increased tilt angle. these techniques are making inroads not only for the study of large heterogeneous molecular assemblies but also for bacteria and eukaryotic cells (lučić et al., ) . de rosier and klug proposed two methods for rendering objects in three dimensions using transmission em (de rosier and klug, ) . one approach involved combining several images of randomly oriented particles and has evolved into what is now known as the ''single-particle method.'' the other proposed technique is much like modern-day electron tomography (et), as it involved obtaining a series of sequentially tilted images. although the single-particle method requires homogeneity of the macromolecules under study, the method using sequential tilts is applicable to unique structures. series of tilted micrographs were used to generate three-dimensional reconstructions of macromolecules beginning in the late s and early s (hart, ; hoppe et al., ) , but acquiring a series of tilted images was an immense technical challenge at the dawn of the digital age, and development of this technique remained slow for decades. progress was further hindered because applying doses greater than e À /Å to the same portion of a specimen (unwin and henderson, ) , as would be required for a tilted series, would destroy high-resolution information. thus, tomography was not viewed as a viable method for obtaining reliable three-dimensional structures of macromolecules, and was therefore restricted to the examination of larger cellular components (frank, ) . unlike the sequential tilt method, single-particle methods enjoyed rapid development after , and have been immensely successful in the study of large symmetric or asymmetric homogeneous macromolecules (crowther et al., ; frank, ) . single-particle studies rely upon averaging between a number of macromolecules which sit in random orientations, eliminating the technical difficulties associated with specimen tilting. it is also possible to limit the electron dose, as it is not necessary to repeatedly expose the same portion of the specimen. however, because this technique relies upon the assumption of particle homogeneity, it is limited in its application. structural biology has largely ignored truly unique or heterogeneous molecules until the rebirth of the tilt series and et following the introduction of automated image acquisition (koster et al., ) , the availability of affordable ccd cameras, and the use of cryomicroscopy (dubochet et al., ) , which limits specimen beam damage and preserves the specimen in a near-native state. there are still several technical limitations that make cryo-et a relatively low-resolution tool when compared to x-ray crystallography or even singleparticle cryo-em. however, cryo-et not only allows the analysis of heterogeneous structures, but it can also be used to study macromolecules within their cellular context. cryo-et has been a useful tool for the study of heterogeneous animal viruses. harris et al. achieved a resolution at which the hemagglutinin (ha) and neuraminidase (na) glycoprotein spikes can be distinguished in tomographic reconstructions of pleomorphic influenza virions (harris et al., ) . by fitting the known ha and na structures into the tomographic densities, three-dimensional models were built showing the distribution of spikes on the influenza virion surface. cryo-et has also enabled structural studies of hiv core assembly as well as provided the first in situ structures of the trimeric spikes of hiv and other retroviruses zanetti et al., ; zhu et al., ) . cryo-et has also provided the first insights into the three-dimensional structures of intact coronaviruses (bárcena et al., ) , bunyaviruses (freiberg et al., ; overby et al., ; huiskonen et al., ) , poxviruses (cyrklaff et al., ) , and paramyxoviruses (loney et al., ), all of which are known to cause serious disease in humans. nevertheless, there are technical issues that need to be overcome before the structures of pleomorphic viruses approach the resolution of icosahedral viruses studied by single-particle or crystallographic methods. the goals of structural biology do not involve only the determination of macromolecular structures to the highest possible resolution, but rather an investigation of these molecules within the context of the cell. to this end, baumeister and coworkers have pioneered methods for the identification and mapping of macromolecules such as proteasomes and ribosomes within tomographically imaged cells medalia et al., ; ortiz et al., ) . as the library of macromolecular structures grows, so will increase the knowledge of the interactions among the components of the cell. this might be achieved by using the library of cellular component structures to interpret tomograms of whole cells in a way analogous to the use of molecular folds in crystallographic molecular replacement. the large size of eukaryotic cells is an obstacle that limits the applicability of cryo-et. however, with improvements in cryo-sectioning techniques (leis et al., ) , larger, more complicated cells can be rendered threedimensionally. another potential use of et is the identification of symmetry elements. for instance, freiberg et al. extracted a number of rift valley fever virions from tomograms, some of which could then be aligned and averaged without any assumption of symmetry (freiberg et al., ) . self-rotation functions (tong and rossmann, ) of the averaged density showed the presence of icosahedral symmetry, for a virus previously thought to be pleomorphic. similar analysis could also be used to study objects that possess some local symmetry. one obstacle in cryo-et is the limited range of the tilted images, which results in a ''missing wedge'' of data. a second obstacle is the electron sensitivity of biological specimens that requires the electron dose to be minimized, resulting in a low signal-to-noise ratio. these problems can be alleviated in some situations by averaging homogeneous structures or by the application of symmetry. for example, huiskonen et al. have used cryo-et to observe that the gn-gc spikes on the surface of tula hantavirus are tetrameric and are arranged into localized arrays huiskonen et al., ) . the spikes were then extracted from the tomogram and iteratively aligned and averaged to generate a structure of the spike to an isotropic resolution of . nm. the averaged spike was reintroduced into the original tomogram to give a global distribution of the spikes within the membrane. one approach to reduce the size of the missing wedge has involved the use of dual-axis tomography (iancu et al., ) . alternatively, embedding a specimen in rod-shaped vitreous ice could be used to generate a reconstruction without a systematic region of missing data and without the specimen thickening issues that occur with slab geometry. rod-like specimens have previously been explored kawase et al., ; hayashida et al., ) , but achieving such a geometry with a frozen-hydrated specimen will likely prove to be more of a challenge. kreysing et al. ( ) have developed an optical trap in which large eukaryotic cells can be rotated about a chosen axis and imaged with a light microscope. if a similar tool could be operated within a transmission electron microscope, objects could be rendered with isotropic resolution at the nanometer scale. an alternative approach to transmission et is ion-abrasion scanning em (heymann et al., , , in which a focused ion beam is used to ablate a fraction of the surface of a specimen that is subsequently imaged with scanning em. repeating this process leads to a stack of images that represent the three-dimensional structure of the object. the resolution limit for this technique is comparable to that of transmission et ($ nm in-plane and $ nm in depth). if methods were developed to ablate the surface of a specimen in more finite increments than approximately nm, such a technique would allow entire cells to be rendered in three dimensions in an isotropic fashion. applying this technique to frozen-hydrated specimens requires further development. cryo-negative staining has been introduced as a means to improve the signal-to-noise ratio of projected images as well as provide further protection against radiation damage above and beyond traditional cryo-em (adrian et al., ; de carlo et al., ) . however, this technique has not yet achieved widespread usage in cryo-et because the penetration of the stain may not be uniform throughout the specimen. furthermore, cryo-negative staining may not preserve fine structural details and has been shown to dissociate the subunits of some specimens (de carlo and harris, ) . a phase plate inserted in the back-focal plane of the objective lens is capable of shifting the phase of the diffracted electron beam by p/ , changing the contrast transfer function from a sine-like to a cosine-like function. this allows the electron microscope to be operated close to focus. under these conditions, the contrast transfer function is maximal at low resolution and the first node of the contrast transfer function occurs at a much higher resolution. initial results have indicated improved signalto-noise ratios for both single-particle and tomographic data . however, contamination of the phase plate can affect the phase shift. this issue will need to be overcome before phase plates are commonplace (danev et al., ). recently, there have been some notable successes in determining structures of icosahedral viruses to a resolution that is high enough to enable model building based solely on the cryo-em map, the amino acid sequence, and geometric constraints (yu et al., ; harrison, ; liu et al., a liu et al., , b zhang et al., ) . obtaining near-atomic-resolution results for less symmetric or asymmetric objects will require a proportionate amount of additional projection images, making experimental data collection daunting. zhang et al. ( ) used approximately  virus particles to determine the structure of aquareovirus to . Å resolution using both icosahedral averaging and -fold averaging among quasiequivalent subunits within the icosahedral asymmetric unit. an additional advantage of built-in icosahedral symmetry is that the relative orientations of symmetrically related orientations are known exactly for each particle, whereas the relative orientations of asymmetric particles are subject to experimental error. thus, obtaining a reconstruction with comparable resolution for an asymmetric object of similar size would require at least .  particle images. another obstacle to high-resolution structure determination is sample heterogeneity due to varying conformations of molecular assemblies. although this can be tackled by the careful selection of images that correspond to closely related structures (scheres et al., ) , the need for additional data increases proportionately. improving the resolution in these difficult cases will require efficient data acquisition techniques. the development of the leginon software for automated data collection marks a significant achievement in this area (suloway et al., ) . the use of charge-coupled device (ccd) detectors instead of films further expedites the data collection process (clare and orlova, ) . currently, in development are ''direct detectors'' that produce images with an improved signal-to-noise ratio by avoiding the conversion of electrons into photons required for ccd detectors (milazzo et al., ) . collection of protein x-ray diffraction patterns used to be a major challenge, sometimes requiring years to obtain a full data set. now, with dedicated synchrotron radiation sources, only a few hours are required to collect complicated multiple wavelength data sets. similarly, it is anticipated that future cryo-em data acquisition will be much faster and less labor intensive. with improved data collection devices and improved computational techniques to classify heterogeneous samples, it may become possible to observe dynamic processes. in the future, it may be possible to place an enzyme-substrate mixture into a microscope and obtain a movie describing the reaction mechanism in atomic detail. one goal of structural biology is the determination of molecular models that can be interpreted with respect to their function. because macromolecular complexes can be difficult to crystallize, it is sometimes easier to determine the structure of the individual components of the complex. however, structures of individual proteins may not be sufficient for functional interpretation of molecular interactions in the absence of knowledge of their spatial relationships. further, the structural details of component proteins within functionally relevant complexes are likely to differ in detail from individually determined structures. cryo-em maps of such complexes, though not yet typically determined to atomic resolution, can be interpreted at near-atomic resolution using ''pseudo-atomic'' models. in current practice, these models are constructed by rigid-body fitting of component structures, previously determined by x-ray crystallography or nmr (cheng et al., ) , into cryo-em maps. the models are either treated as a single rigid body or split into a few domains that are each fitted separately (zhang et al., ; hafenstein et al., ; cherrier et al., ). this approach is based on the observation that conformational changes of proteins are usually limited to the relative positioning of domains whose tertiary structure remains much the same. at low resolution, the number of parameters that can be refined might be merely the orientation and position of a known three-dimensional rigid structure. however, as the resolution of the cryo-em map improves, it may be possible to refine the orientation and position of individual structural elements such as a-helices and b-sheets. finally, as the resolution improves beyond Å , it becomes possible to refine ramachandran and rotary dihedral angles, as is usually the case for a crystallographic refinement. care must be taken that the number of parameters describing the structure is appropriate to the number of experimental measurements. for instance, the molecular structure of a eukaryotic ribosome (taylor et al., ) was determined by combining a -Å cryo-em reconstruction with molecular dynamics. the results are impressive given the complexity of the structure. nevertheless, such a structural determination requires validation, as the number of parameters describing the structure might be larger than the number of independent observations. at this time, there is no set of tools that adequately describes either the quality of a cryo-em reconstruction or the accuracy of the resultant model. the only criterion reported for most cryo-em maps is the estimated resolution based on the fourier shell correlation between two randomly selected half-sets. whereas most papers estimate the resolution by the point at which the correlation falls below . (böttcher et al., ; van heel and schatz, ) , it has been suggested that this coefficient should be . (rosenthal and henderson, ) if the estimate of the resolution is to be compared to that of a crystallographic map. standard methods to evaluate the quality and validity of cryo-em reconstructions need to be developed and accepted. also needed are criteria evaluating the molecular properties of the model as well as measures describing how well the model represents the cryo-em map. to evaluate quality of the cryo-em reconstruction, a series of indicators should be developed documenting the whole pathway from data acquisition to calculation of the final model similar to current practices in crystallography. thus, there should be indicators describing the quality of the collected images (similar to crystallographic r merge ; rossmann et al., ) , the accuracy of the contrast transfer function (ctf) determination for each image, and the accuracy of the particle orientation determination. further statistics should describe the resulting map-what is its resolution (i/s(i) as a function of resolution)? how well does the map correspond to original experimental images (baker et al., ) ? perhaps some of the experimental images could be excluded from the calculation of the reconstruction and used as a test set for an unbiased validation of the final map (r map free ) (brünger, ) . the fitting of atomic structures into cryo-em maps for the purpose of evaluating pseudo-atomic models can be accomplished by the equivalent of a six-dimensional search. whether an unambiguous solution can be found will depend on whether the best fit is significantly better than all other fits. however, if this is not the case, additional chemical and physical criteria can be used to identify the best fit (rossmann et al., ) . once a reasonable fit has been obtained, the number of refinable parameters that describe the atomic model needs to be chosen according to the resolution future prospects of the map. a parameter analogous to the crystallographic r model free would be a suitable measure to validate that the number of parameters does not exceed what is useful (brü nger, ) . once an initial position and orientation of the model has been determined, it will be necessary to determine the accuracy of the parameters that describe the position of the model within the cryo-em map (rossmann et al., ; volkmann and hanein, ; volkmann, ) . the uncertainty in model fitting could be expressed by considering the various possible conformational changes that fit the observed data equally well. such a multitude of conformations might be separately reported or can be represented as a ''temperature'' factor that describes the root mean square displacement of each atom from the mean, analogous to crystallographic practices. the knowledge of the error in the parameters will establish the validity of the subsequent interpretations. when it is deemed possible to refine the individual atomic coordinates, statistics describing how well the refinement procedure preserved the geometric properties of the model should be given, including the ramachandran plot distribution and the deviation of angles and bond lengths from their average values. hopefully, the cryo-em community will not repeat the history of protein crystallography in the s when almost all models were described by their authors as ''better than average'' (kleywegt and jones, ) . to forecast the development of the validation criteria for cryo-em, parallels with x-ray crystallography can be drawn. the crucial x-ray structure validation criteria r free was introduced in (brü nger, ). by that time, the pdb archive contained approximately structures (fig. ) . it took several more years before the r free and other structure validation criteria became generally applied and accepted (kleywegt and jones, ) . the number of deposited cryo-em structures will reach in - years time. thus, it should be expected that universally accepted validation criteria will soon be developed. the future of cryo-em will almost certainly bring an obligatory model and data deposition policy analogous to that now widely practiced for x-ray crystallography. the pdb was established in , years after determination of the first x-ray structure of myoglobin in (kendrew et al., ) . by , there had been deposited structures. since then, the number of entries has grown exponentially (fig. ) . initially, the x-ray structure deposition was not an obligatory prerequisite for publication of the results and only over time did it become a general requirement enforced by journal policies. it was only in that experimental data deposition became an obligatory component of atomic coordinate deposition to the pdb. the first model based on cryo-em reconstruction was deposited with the pdb in . since then, the number of deposited models has grown exponentially and will be expected to be around by the end of (fig. ). it will probably not be long until cryo-em reconstruction deposition is generally mandated for publication. based on the rate of crystallographic structures accumulated by the pdb, the rate of deposition of cryo-em reconstructions is likely to continue growing exponentially for about a decade. it would have been interesting to see michael faraday's reaction had somebody predicted that electromagnetic radiation would be used years hence for instantaneously locating an acquaintance on the opposite side of the earth, and then holding a casual conversation face-to-face as if both individuals were in the same room. surely, faraday would have laughed at what he would have thought to be just a silly joke. similarly, predictions of future technical developments that are more than a lifetime ahead are likely to fall on deaf ears, but that is the task given to the authors of this chapter. the technology of structural biology is changing rapidly. crystallography, cryo-em, and cryo-et are becoming automatic, but maybe someday a single technique will supersede all current tools. perhaps a tunable laser that can examine structure at all levels of detail, covering a resolution from m to . nm will become available. further, much progress will have been made to quickly gather large amounts of data and render structures in three dimensions almost instantaneously. for instance, all structural data on one hepatic cell might be collected within minutes. the automatic interpretation of the cell structure would then yield everything from the overall organization of the nucleus, cytoplasm, plasma membrane, and so forth, down to the positions of all atoms within every molecule in the cell. this could be repeated numerous times in order to determine the dynamic properties of the cell. clearly, such achievements will be an enormous boon for understanding molecular interactions and following the life cycle of individual cells. the effect of medication on the cell will be determined at the blink of an eye, allowing immediate correction of dosage. such structural monitoring will become the passion of every doctor. cryo-negative staining adding the third dimension to virus life cycles: three-dimensional reconstruction of icosahedral viruses from cryo-electron micrographs. microbiol diffraction quality crystals of prd , a -mda dsdna virus with an internal membrane a Å resolution x-ray crystallographic map of the large ribosomal subunit cryo-electron tomography of mouse hepatitis virus: insights into the structure of the coronavirion a numerical method for two-dimensional fourier synthesis single particle x-ray diffractive imaging determination of the fold of the core protein of hepatitis b virus by electron cryomicroscopy the structure of some crystals as indicated by their diffraction of x-rays the determination of parameters in crystal structures by means of fourier series the mechanism of hiv- core assembly: insights from three-dimensional reconstructions of authentic virions free r value: a novel statistical quantity for assessing the accuracy of crystal structures nucleocapsid and glycoprotein organization in an enveloped virus structural basis for the preferential recognition of immature flaviviruses by a fusion-loop antibody . Å cryo-em reconstruction of tobacco mosaic virus from images recorded at kev on a k  k ccd camera visualization of a -helix bundle in the hepatitis b virus capsid by cryo-electron microscopy x-ray crystallographic studies of compounds of biochemical interest three-dimensional image reconstructions of some small spherical viruses threedimensional reconstructions of spherical viruses by fourier synthesis from electron micrographs cryo-electron tomography of vaccinia virus practical factors affecting the performance of a thin-film phase plate for transmission electron microscopy cryo-negative staining reduces electron-beam sensitivity of vitrified biological particles negative staining and cryo-negative staining of macromolecules and viruses for tem reconstruction of three dimensional structures from electron micrographs structure determination of the fx closed procapsid cryo-electron microscopy of vitrified specimens oosthoek's uitgeversmaatschappij for the international union of crystallography retrovirus envelope protein complex structure in situ studied by cryo-electron tomography identification of macromolecular complexes in cryoelectron tomograms of phantom cells image analysis of single macromolecules electron tomography: methods for three-dimensional visualization of structures in the cell three-dimensional organization of rift valley fever virus revealed by cryo-electron tomography laue diffraction studies of human rhinovirus and canine parvovirus the interaction of decay-accelerating factor with coxsackievirus b influenza virus pleiomorphy characterized by cryoelectron tomography virology. looking inside adenovirus crystallization of sindbis virus and its nucleocapsid electron microscopy of unstained biological material: the polytropic montage automatic coarse-alignment for tem tilt series of rod-shaped specimens collected with a full angular range interactions and oligomerization of hantavirus glycoproteins site-specific d imaging of cells and tissues with a dual beam microscope d imaging of mammalian cells with ion-abrasion scanning electron microscopy three-dimensional structure of poliovirus at . Å resolution threedimensional reconstruction of individual negatively stained yeast fatty-acid synthetase molecules from tilt series in the electron microscope electron cryotomography of tula hantavirus suggests a unique assembly paradigm for enveloped viruses a ''flip-flop'' rotation stage for routine dual-axis electron cryotomography backbone structure of the infectious e virus capsid revealed by electron cryomicroscopy crystallization and preliminary x-ray diffraction analysis of west nile virus transmission electron microtomography without the ''missing wedge'' for quantitative structural analysis a three-dimensional model of the myoglobin molecule obtained by x-ray analysis structure of myoglobin. a three-dimensional fourier synthesis at Å . resolution femtosecond protein nanocrystallography-data analysis methods where freedom is given, liberties are taken automated microscopy for electron tomography the optical cell rotator visualizing cells at the nanoscale atomic structure of human adenovirus by cryo-em reveals interactions among protein networks structural changes in a marine podovirus associated with release of its genome into prochlorococcus paramyxovirus ultrastructure and genome packaging: cryo-electron tomography of sendai virus the structure of the benzene ring structural studies by electron tomography: from cells to molecules macromolecular architecture in eukaryotic cells visualized by cryoelectron tomography characterization of a direct detection device imaging camera for transmission electron microscopy zernike phase contrast cryo-electron microscopy and tomography for structure determination at nanometer and subnanometer resolutions structure of a human rhinovirus complexed with its receptor molecule mapping s ribosomes in intact cells by cryoelectron tomography and pattern recognition insights into bunyavirus architecture from electron cryotomography of uukuniemi virus a fourier series method for the determination of the components of interatomic distances in crystals a direct method for the determination of the components of interatomic distances in crystals structure of haemoglobin. a three-dimensional fourier synthesis at . -Å resolution, obtained by x-ray analysis optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy fitting atomic models into electron microscopy maps international tables for crystallography volume f: crystallography of biological macromolecules structure of a human common cold virus and functional relationship to other picornaviruses combining electron microscopic with x-ray crystallographic structures processing and post-refinement of oscillation camera data disentangling conformational states of macromolecules in d-em through likelihood optimization structure of human rhinovirus complexed with fab fragments from a neutralizing antibody automated molecular microscopy: the new leginon system comprehensive molecular structure of the eukaryotic ribosome rotation function calculations with glrf program molecular structure determination by electron microscopy of unstained crystalline specimens fourier shell correlation threshold criteria confidence intervals for fitting of atomic models into lowresolution densities docking of atomic models into reconstructions from electron microscopy the structure of a neutralized virus: canine parvovirus complexed with neutralizing antibody fragment . Å structure of cytoplasmic polyhedrosis virus by cryo-electron microscopy cryoelectron tomographic structure of an immunodeficiency virus envelope complex in situ . Å cryo-em structure of a nonenveloped virus reveals a priming mechanism for cell entry distribution and three-dimensional structure of aids virus envelope spikes we thank sheryl kelly for her help in preparation of this chapter. writing of this chapter was made possible by support from nih (r ai , r ai , and p ai ) and nsf (mcb- ) grants to m. g. r. key: cord- - te nu authors: croll, tristan; diederichs, kay; fischer, florens; fyfe, cameron; gao, yunyun; horrell, sam; joseph, agnel praveen; kandler, luise; kippes, oliver; kirsten, ferdinand; müller, konstantin; nolte, kristoper; payne, alex; reeves, matthew g.; richardson, jane; santoni, gianluca; stäb, sabrina; tronrud, dale; williams, christopher; thorn, andrea title: making the invisible enemy visible date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: te nu during the covid- pandemic, structural biologists have rushed to solve the structures of the proteins encoded by the sars-cov- genome in order to understand the viral life cycle and enable structure-based drug design. in addition to the structures from sars-cov previously solved, structures covering of the viral proteins have been released in the span of only months. these structural models serve as basis for research worldwide to understand how the virus hijacks human cells, for structure-based drug design and to aid in the development of vaccines. however, errors often occur in even the most careful structure determination - and are even more common among these structures, which were solved under immense pressure. from the beginning of the pandemic, the coronavirus structural taskforce has categorized, evaluated and reviewed all of these experimental protein structures in order to help downstream users and original authors. our website also offers improved models for many key structures, which have been used by folding@home, openpandemics, the eu jedi covid- challenge, and others. here, we describe our work for the first time, give an overview of common problems, and describe a few of these structures that have since acquired better versions in the worldwide protein data bank, either from new data or as depositor re-versions using our suggested changes. introduction sars-cov- , the coronavirus responsible for covid- , has a single-stranded rna genome that encodes proteins. these macromolecules fulfil essential roles in the viral life cycle, enabling sars-cov- to infect, replicate, and suppress the immune system of its host. for example, the characteristic spikes that protrude from its envelope and allow it to bind to host cells, are a trimer of the surface glycoprotein ( fig. ) . knowing the atomic structures of these macromolecules is vital for understanding the lifecycle of the virus and helping design specific pharmaceutical compounds that bind and inhibit their functions, with the goal of stopping the cycle of infection. since the covid- pandemic hit in the beginning of this year, the structural biology community has swung into action very efficiently and is now strongly engaged to establish the atomic structures of these macromolecules as fast as possible by use of nuclear magnetic resonance (nmr), cryo-electron microscopy (cryo-em) and crystallographic methods ( ) . all of these methods require an interpretation of the measured and processed data with a structural model and cannot be fully automated. the resulting structures are made freely and publicly available in the world wide protein data bank (wwpdb), structural biology's archive of record ( ) . unfortunately, the fit between model and data is never perfect and errors from measurement, post-processing and modelling are a given. structures solved in a hurry to address a pressing medical and societal need are even more prone to mistakes. however, as these structures are used for biological interpretation, small errors can have severe consequences -in particular, in structure-based drug discovery, structural bioinformatics, and computational chemistry; a current focus of sars-cov- research across the world. as of the writing of this publication, macromolecular structures from sars-cov and sars-cov- have been deposited, covering parts of of the proteins. in this time of crisis, it is therefore vital to ensure that the structural data made available to the wider research community are the best they can be in every regard. pushing the methods to the limit the wwpdb ( ) is an invaluable tool, but once released, a structure in the databank can only be reversioned by the original depositors, who, after any associated papers are published, may have little or no motivation to correct their structures. third parties may only deposit new models based on others' deposited data under new accession ids when accompanied by peer reviewed publications. in such cases, there is no explicit link from the original entry to the new one. importantly, % of structure downloads from the pdb are not by experimentalists, but scientists from other fields ( ) . as a consequence, errors can lead to a large waste of resources and time by those making use of the data obtained from the pdb and may even be misinterpreted as biologically and pharmaceutically relevant information. we, the authors of this manuscript, develop computational methods for the solution of macromolecular structures. by being expert users of our own software tools, we were well placed to help in this unprecedented situation. this is why, since the first signs of a possible pandemic arose at the beginning of this year, we joined forces to assess and, where necessary, improve upon the published macromolecular structures from sars-cov and sars-cov- . in cases where we believe we have significantly improved the macromolecular models, we offered them back to the original authors and the scientific community. raw data are not deposited in the wwpdb and are not mandatory for publication; as a consequence, they are difficult to obtain. their absence in the public record is detrimental for reanalysis and validation, and the development of new methods. in an effort to make the most out of the experimental results, we invited authors to send us their raw experimental data, which are key to validation of the entire structure determination process from start-to-finish. we thus offer to help authors deposit these data in a non-pdb public repository if the authors wished to do so. after we began our validation efforts of macromolecular structures from sars-cov and sars-cov- , we were approached by our colleagues in in-silico drug screening, folding@home ( , ) , openpandemics ( ) , and the eu joint european disruptive initiative (jedi) ( ). these initiatives needed the best structures they could get for studies of the virus and had already lost much computing time and resources to suboptimal structure solutions. all macromolecular structures from sars-cov and sars-cov- in the pdb are downloaded into our repository and assessed automatically in the first hours after release. for crystallographic and cryo-em structures, we check the quality of the deposited merged data, and how well the model fits these data. an automatic evaluation of nmr data is forthcoming. then, all structures are checked according to chemical prior knowledge. evaluation specific to crystallographic data and structure solutions as crystal structures make up . % of our data, these are evaluated most thoroughly. crystal diffraction can, for example, stem from more than one crystal lattice (twinning), be contaminated by ice crystal diffraction (ice rings) or be incomplete due to radiation damage or suboptimal measurement strategy. these issues cannot be resolved after data collection, but treating data accordingly can yield a better structural model. deducing such problems from the deposited structure factors (mandatory in wwpdb) can be difficult; raw data allow a much more complete analysis of the experiment. another source of errors is data processing (integration and scaling), which nowadays is often done automatically. assuming the wrong crystal lattice symmetry or including, for example, diffraction spots obscured by the beam stop, can lead to lower quality or even unsolvable structures. if raw data are available, data can be re-processed and these problems can be resolved manually. to analyse crystallographic data for twinning, completeness and overall diffraction quality, we used phenix.xtriage ( ); furthermore we ran auspex ( ) which automatically identifies ice rings and produces plots from which several other pathologies, like a "bad" beam stop mask, can be recognized quickly. the completeness of most datasets is satisfying, with only out of datasets below %. all the datasets have an acceptable strength with intensity/sigma(intensity) above . ice rings were detected in datasets and problems with the beam stop masking in ; crystal structures were indicated as potentially resulting from twinned crystals. a general indication of how well the atomic model fits the measurement data can be obtained by comparing the deposited r-factors to results from pdb-redo ( ) (including whatcheck ( )) to determine the overall density fit as well as many other diagnostics. while the deposited structures are often improved by pdb-redo, they need to be checked and should not be viewed as "more correct" purely on basis of a lower r value. in addition to this, a high r value does not indicate a single type of error and hence should be used with caution. only two structures in the repository present an alarmingly high rfree value above %, although problems can be found in other structures by looking in more detail. pdb-redo improves the rfree for most of the structures, and the only cases where we found a huge degradation pointed to major issues with the pdb entry; this was especially true for older sars-cov structures. evaluation specific to structures from single-particle cryo-em cryo-em structures make up . % of our data. as with crystallographic structures, raw data are not available from the wwpdb, but the three-dimensional map reconstructed from the microscopic single particle images is deposited, allowing the calculation of the fit between model and map in the form of a fourier shell correlation (fsc). the model-map fsc is plotted as a curve, which estimates agreement between features resolvable at different resolutions. for a well-fitted model, a model-map fsc of . roughly corresponds to the cryo-em map resolution (which is determined as where the fsc between two half-maps drops below . ). to calculate fscs, we use the ccp-em ( ) model validation task which utilizes refmac ( ) and calculates real-space cross-correlation coefficient (ccc), mutual information (mi) and segment manders' overlap coefficient (smoc) ( ) . while mi is a single value score to evaluate how well model and map agree, the smoc score evaluates the fit of each modelled residue individually and can help to find regions where errors occur in the model in relation to the map. z-scores highlight residues with a low score relative to their neighbours and point to potential misfits. out of structures, structures had an average model-fsc below . and seven have a mi score below . , indicating a bad overall agreement between map and model and potential for further improvement. the smoc score indicates for twelve structures that more than % of the residues fit poorly with the map, while the other % of structures had a relatively good density fit. however, most modelling errors could only be corrected manually (see below). in addition to this validation, we run haruspex ( ), a neural network to annotate reconstruction maps to evaluate which secondary structures can be recognized automatically in the map. molecular geometry is constrained by the nature of its chemical bonds and steric hindrance between the atoms. in order to evaluate the model quality with respect to chemical prior knowledge we run molprobity ( , ) , which checks covalent geometry, conformational parameters of protein and rna and steric clashes. however, it is unfortunately possible to use some of these traditional indicators of model quality as additional restraints during refinement, which invalidates them to a certain degree -we therefore also used the molprobity cablam score ( ) , which can pinpoint local errors at - Å resolution even if traditional criteria have been used as restraints. cablam scores higher than % outliers indicated that of the structures have many incorrect backbone conformations. during the crisis the molprobity webservice has been pushed to the limit of its capacity, as many different drug developers have screened the very same coronavirus structures many times. we have developed a bespoke molprobity pipeline to make these results available online and to decrease the workload on the webservice. in addition to this, the sequence of each structure is also aligned and checked against the known genome to highlight misidentified residues. every wednesday, after the new pdb structures have been released, an automatic pipeline runs to organize the new structures according to the genetic information and then to assess the quality of models and the experimental results. these results, along with the original structures, are immediately available from our online repository which is accessible via our website insidecorona.net. to facilitate access and to get an overview of structures, we supply an sql database of key statistics and quality indicators along with the results. as a community, for decades, we have aspired to automate structural biology as much as possible. however, neither structure solution nor validation have been fully automated due to the complexity of interpreting low-quality maps that have poor fit between experimental data and structural models. this task requires detailed knowledge of macromolecular/small molecule structure and chemical interactions. even with state-of-the-art automatic methods at hand, experienced human inspection residue-byresidue remains the best way to judge the quality of a structure, highlighting the continuing need for expert structure solvers. given the flood of new sars-cov- structures, resources have not permitted us to check all structures manually. therefore, we have selected representative structures. certain errors were surprisingly common, such as peptide bond flips, rotamer outliers and mis-identification of small molecules, such as water as magnesium, chloride as zinc, and a multi-zinc site modelled as poly ethylene glycols. zinc plays an important role in many viral infections, and is coordinated by many of the sars-cov- protein structures. we also found a large number of cys-zn sites being mismodelled, with the zinc ion missing or pushed out of density, and/or erroneous disulphide bonds between the coordinating cysteine residues. many coronavirus proteins are linked on certain asparagine residues ("n-linked") to carbohydrate chains called glycans. their exact composition depends on the host cell in question, and their main function is to deter the host immune system. in some structures, where the sample was produced in eukaryotic cells, the "stem" sugars of these n-linked glycans were evident in the map. however, in many cases these sugars were flipped approximately degrees from their correct orientation around the n-glycosidic bond. out of the structures we checked manually, we were able to significantly improve which are available from insidecorona.net. in the following, we will give two examples: once sars-cov- infects a cell, the first protein produced is a long polypeptide chain which is cleaved into functional proteins, the non-structural proteins (nsps) ( ) . these are essential for the production of new viruses in the host cell. nsp is a large protein molecule by any measure, amino acid residues in total. its segments have a variety of functions, among them the papain-like protease domain which cleaves the first five nsps from the polypeptide chain ( ) . without cleavage of the polyprotein, the virus cannot replicate and infection is halted. hence, the papain-like protease domain represents an important potential drug target ( ) . the first sars-cov- structure of this domain was pdb w c (released st april ). it was immediately used as the basis for structure-based drug design around the world. the resolution was . Å and rwork/rfree were . % / . %. however, the overall completeness of the measured data was only . %. why were just over half of all reflections that could have been measured at this resolution recorded? the raw data for this entry are available from proteindiffraction.org ( ) . they revealed that the diffraction data had been measured with a very high x-ray intensity, which led to a swift deterioration in diffracting power due to radiation damage -something which could not have been learned from the data deposited in the pdb, underlining the importance of the availability of raw data. typically, crystallographers aim for a dose of mgy or lower. here, we estimated a diffraction weighted dose of . mgy with raddose- d, with a maximum dose value of mgy, which likely completely destroyed parts of the crystal ( ) . we based this calculation on assumption of a x x μm plates, given that the crystals were described as such. in addition, the measurement covered ° sample rotation followed by an additional ° rotation starting from the same angular position, covering the first ° twice, further increasing the dose while recording little additional information. the angular range per image, . °, was also surprisingly wide for the high-throughput pixel detector used (a dectris pilatus m), and the diffraction was highly anisotropic. we re-processed the images using xds ( ) , omitting the final ° of the first sweep and final ° of the second where the radiation damage affected the data too severely. an elliptical resolution cut-off was applied with staraniso ( ) to account for anisotropy. careful manual intervention could improve the resolution to . Å with better data quality overall. the revised ellipsoidal completeness was . %. the structure has -fold non-crystallographic rotational symmetry; with three monomers coordinating a central zinc ion within the asymmetric unit. the second, functionally important, zinc ion is far removed from the three-fold rotation axis and coordinated by four cysteines. this zinc finger domain is essential for activity, in addition to the papain-like cysteine-histidine-aspartate catalytic triad ( ) , but it is poorly resolved and incompletely and differently modelled in each of the three monomers of this structure. this disorder may be the result of radiation damage. only two of the three zinc sites were modelled, and here, the bond lengths between each of the four sulphur atoms and the zinc varied from . Å to . Å, and the cß-sg-zn angles between ° and °. the third site had no zinc (fig. ) , instead being modelled as a disulphide bond. prior knowledge about coordination chemistry dictates that the bond lengths between cys and zn should all be approximately . Å and the angles about °. adding zincs to all sites and restraining the bond lengths and angles to these expected values, adding non-crystallographic symmetry restraints (requiring the copies to look similar), an overall higher weighting of ideal geometry, and the reassessment of side chains and water molecules improved the electron density maps and lowered the r values to . %/ . % at . Å resolution. this example shows the importance of optimised data collection strategy, data processing and model building, the quality of which is interconnected. in this case, even though the data were radiation damaged, by adjusting the data processing to take this into account, and by modifying the model refinement to include stronger restraints and to take full advantage of the non-crystallographic symmetry, this structure could be drastically improved. a new structure of the c s mutant of the same protein (pdb code wrh) came out a month later, in which the zinc site was clearly resolved. by this time, however, the structure had already been widely used as a target in in-silico drug design: for example, % of participants in the eu jedi covid- challenge have used this structure to design potential drugs. the availability of a better structure a month earlier would not only have increased their chances of success but also saved much computing and man hours in computer aided drug development. when sars-cov- replicates, its single-stranded rna genome needs to be copied. this is achieved by a macromolecular complex of rna-dependent rna polymerase (nsp , rdrp), nsp and nsp ( ) . coronaviruses, including sars-cov- , have some of the largest genomes among rna viruses (approximately kilobases), suggesting their polymerase complexes possess proof-reading functionality. this sets coronaviruses apart from other rna viruses ( ) . the first structure of sars-cov rna polymerase (pdb entry nus) was solved in by cryo-em ( ) , before the pandemic began. in this structure, a loop close to the c-terminus (residues - ) was not resolved in the reconstruction map and hence not modelled. following this loop, the polymerase has an irregular helix followed by a flexible tail. density for this helix was poorly resolved -coupled with its short length and the lack of any information from the preceding and following loops, this led to difficulty in assigning register (the identity of the amino acid at each site). nevertheless, the overall validation statistics (clashscore, ramachandran outliers, sidechain outliers) provided by the wwpdb for this model appeared exceptionally good. we inspected one of the first available structures of the sars-cov- rna polymerase complex ( btf) using isolde ( ) , a program used for interactively visualising and remodelling proteins in their experimental density. the higher resolution at this c-terminal tail of the structure made it clear that the c-terminal helix was severely incorrect, with the assigned sequence being nine residues upstream of the correct residues for this site (see fig. ). this error was present in all the structures of this complex from both sars-cov and sars-cov- , presumably propagated due to each subsequent structure using the previous models as the starting point for their modelling, as is standard practice. for each affected rdrp structure we immediately contacted the original authors. of the sars-cov- rna polymerase complexes in the wwpdb now have the corrected sequence alignment at the cterminus, and those also include many of our other changes described below. these pdb re-versioned corrections allow modelling efforts for drugs against sars-cov- rna polymerase to start from a much better model. notably, the authors of a later cryo-em structure of the rna polymerase/rna complex (pdb entry yyt) used one of our corrected models as the starting point for their new model ( ) . the structure of sars-cov- rna polymerase in pre-translocation state and bound to template-primer rna and remdesivir (pdb entry bv ) ( ) represents a useful basis to investigate the inhibitory effect of remdesivir and the rational design of other nucleoside triphosphate (ntp) analogues ( ) . however, we found that this structure has some issues, which may provide misleading information to people who are conducting such studies. apart from the register shift described above, there are three magnesium ions modelled in the active site, a number which is contradictory to our common knowledge of this class of proteins. magnesium ions play an essential role in catalysis in rna polymerase (binding the incoming ntp, positioning ntp for incorporation and stabilizing the leaving group after catalysis) ( , ) . one of the magnesium ions is shown coordinated by a pyrophosphate, which implies that the pyrophosphate ion release in sars-cov- rdrp is relatively slow and may even couple with the translocation ( ) . however, all three magnesium ions as well as the pyrophosphate are poorly supported by the map reconstructed from the experimental data or by local geometry if these ions were included as fixed components of the binding site, this may have severely impacted in-silico docking and drug design studies. in addition to the above, we corrected the conformations of three rna residues close to the remdesivir site including an adenosine base (t ) modelled "backwards", fixed "backwards" peptides flagged by cablam, added several residues and water molecules with good density and geometry, and corrected two proline residues that had been erroneously flipped from cis to trans. our remodelled structure is offering a valuable structural basis for future studies, such as in-silico docking and drug design targeting at sars-cov- rdrp ( ), as well as for computational modelling or simulations to investigate the molecular mechanism of viral replication ( , , ) . many specialists in structural biology and in silico design are now tackling sars-cov- research, but may not be familiar with the wider body of coronavirus research. in addition to improved models and evaluation results, we also supply context on insidecorona.net. this covers literature reviews centred on the structural aspects of the viral life cycle, host interaction partners, illustrations, and evaluation criteria for selecting the best starting models for in silico projects. furthermore, we added entries about the sars-cov- proteins to proteopedia ( ) and molssi ( , ) , as well as d-bionotes ( ) deep-link into our data base. finally, as sars-cov- has had an unprecedented impact on the world at large, we have also tried to make our, and others', research on the topic accessible to the general public. this has included a number of posts on our homepage aimed at non-scientists and live streaming the reprocessing of data on twitch, as well as the design, production, and public release of an accurate d printed model of sars-cov- based on deposited structures for use as a prop for outreach activities. in the last five months, we have done a weekly automatic post-analysis as well as a manual re-processing and re-modelling of representative structures from each of the structurally known macromolecules of sars-cov or sars-cov- . in this global crisis, where the community aims to get structures out as fast as possible, we aim to ensure that structure interpretations available to downstream users are as solid as possible. we provide these results as a free resource to the community in order to aid the hunt for a vaccine or anti-viral treatment. our results are constantly updated and can be found online at insidecorona.net. new contributors to this effort are very welcome. in the last years, structural biology has become highly automated, and methods have advanced to the point that it is now feasible to solve a new structure from start to finish in a matter of months with little specialist knowledge. the extremely rapid and timely solution of these structures is a remarkable achievement during this crisis and, despite some shortcomings, these structures have enabled downstream work on therapeutics to rapidly progress. the downside is that errors at all points during a structure determination are not only common, but can also remain undetected, and if they are detected, this is usually seen as individual failure. however, no individual researcher is fully conversant in all the details of structure determination, protein and nucleic acid structure, chemical properties of interacting groups, catalytic mechanisms, and viral life cycle. the result is that the first draft of a molecular model often contains errors like the ones pointed out above. while any molecular model could benefit from an examination by multiple experts, during this time it is important to bring such inspection to coronavirusrelated structures as quickly as possible. we believe that, as a community, we need to change how we all see, address and document errors in structures to achieve the best possible structures from our experiments. we are scientists: in the end, truth should always win. visualizing an unseen enemy; mobilizing structural biology to counter covid- rcsb protein data bank: sustaining a living digital data resource that enables breakthroughs in scientific research and biomedical education announcing the worldwide protein data bank xtriage and fest: automatic assessment of x-ray data and substructure structure factor estimation auspex: a graphical tool for x-ray diffraction data analysis the pdb_redo server for macromolecular structure model optimization errors in protein structures recent developments in the ccp-em software suite vagin, refmac for the refinement of macromolecular crystal structures refinement of atomic models in high resolution em reconstructions using flex-em and local assessment haruspex: a neural network for the automatic identification of oligonucleotides and protein secondary structure in cryo-electron microscopy maps molprobity: all-atom structure validation for macromolecular crystallography molprobity: more and better reference data for improved all-atom structure validation new tools in molprobity validation: cablam for cryoem backbone, undowser to rethink "waters," and ngl viewer to recapture online d graphics nsp of coronaviruses: structures and functions of a large multi-domain protein identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity a public database of macromolecular diffraction experiments estimate your dose: raddose- d. protein science staraniso (global phasing ltd the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity implications of altered replication fidelity on the evolution and pathogenesis of coronaviruses. current opinion in virology structure of the sars-cov nsp polymerase bound to nsp and nsp co-factors isolde: a physically realistic environment for model building into low-resolution electron-density maps structure of replicating sars-cov- polymerase structural basis for inhibition of the rnadependent rna polymerase from sars-cov- by remdesivir a mechanism for all polymerases the structural mechanism of translocation and helicase activity in t rna polymerase structural basis of the potential binding mechanism of remdesivir to sars-cov- rna-dependent rna polymerase remdesivir and sars-cov- : structural requirements at both nsp rdrp and nsp exonuclease active-sites perspective: computational chemistry software and its advancement as illustrated through three grand challenge cases for molecular science covid- molecular structure and therapeutics hub key: cord- - fduzqyb authors: strauss, james h.; strauss, ellen g. title: the structure of viruses date: - - journal: viruses and human disease doi: . /b - - - - . - sha: doc_id: cord_uid: fduzqyb nan virus particles, called virions, contain the viral genome encapsidated in a protein coat. the function of the coat is to protect the genome of the virus in the extracellular environment as well as to bind to a new host cell and introduce the genome into it. viral genomes are small and limited in their coding capacity, which requires that three-dimensional virions be formed using a limited number of different proteins. for the smallest viruses, only one protein may be used to construct the virion, whereas the largest viruses may use or more proteins. to form a three-dimensional structure using only a few proteins requires that the structure must be regular, with each protein subunit occupying a position at least approximately equivalent to that occupied by all other proteins of its class in the final structure (the principle of quasi-equivalence), although some viruses are now known to violate the principle of quasi-equivalence. a regular three-dimensional structure can be formed from repeating subunits using either helical symmetry or icosahedral symmetry principles. in the case of the smallest viruses, the final structure is simple and quite regular. larger viruses with more proteins at their disposal can build more elaborate structures. enveloped viruses may be quite regular in construction or may have irregular features, because the use of lipid envelopes allows irregularities in construction. selected families of vertebrate viruses are listed in table . grouped by the morphologies of the virions. also shown for each family is the presence or absence of an envelope in the virion, the triangulation number (defined later) if the virus is icosahedral, the morphology of the nucleocapsid or core, and figure numbers where the structures of members of a family are illustrated. electron micrographs of five dna viruses belonging to different families and of five rna viruses belonging to different families are shown in fig. . . the viruses chosen represent viruses that are among the largest known and the smallest known, and are all shown to the same scale for comparison. for each virus, the top micrograph is of a virus that has been negatively stained, the middle micrograph is of a section of infected cells, and the bottom panel shows a schematic representation of the virus. the structures of these and other viruses are described next. helical viruses appear rod shaped in the electron microscope. the rod can be flexible or stiff. the best studied example of a simple helical virus is tobacco mosaic virus (tmv). the tmv virion is a rigid rod nm in diameter and nm long ( fig. . b ). it contains copies of a single capsid protein of . kda. in the right-hand helix, each protein subunit has six nearest neighbors and each subunit occupies a position equivalent to every other capsid protein subunit in the resulting network ( fig. . a) , except for those subunits at the very ends of the helix. each capsid molecule binds three nucleotides of rna within a groove in the protein. the helix has a pitch of Å and there are / subunits per turn of the helix. the length of the tmv virion ( nm) is determined by the size of the rna ( . kb). many viruses are constructed with helical symmetry and often contain only one protein or a very few proteins. the popularity of the helix may be due in part to the fact that the length of the particle is not fixed and rnas or dnas of different sizes can be readily accommodated. thus the genome size is not fixed, unlike that of icosahedral viruses. virions can be approximately spherical in shape, based on icosahedral symmetry. since the time of euclid, there have been known to exist only five regular solids in which each face of the solid is a regular polygon: the tetrahedron, the cube, the octahedron, the dodecahedron, and the icosahedron. the icosahedron has faces, each of which is a regular triangle, and thus each face has threefold rotational symmetry ( fig. . a ). there are vertices where faces meet, and thus each vertex has fivefold rotational symmetry. there are edges in which faces meet, and each edge possesses twofold rotational symmetry. thus the icosahedron is characterized by twofold, threefold, and fivefold symmetry axes. the dodecahedron, the next simpler regular solid, has the same symmetry axes as the icosahedron and is therefore isomorphous with it in symmetry: the dodecahedron has faces which are regular pentagons, vertices where three faces meet, and edges with twofold symmetry. the three remaining regular solids have different symmetry axes. the vast majority of regular viruses that appear spherical have icosahedral symmetry. in an icosahedron, the smallest number of subunits that can form the three-dimensional structure is ( subunits at each of the vertices, or viewed slightly differently, units on each of the triangular faces). some viruses do in fact use subunits, but most use more subunits in order to provide a larger shell capable of holding more nucleic acid. the number of subunits in an icosahedral structure is t, where the permissible values of t are given by t = h + hk + k , where h and k are integers and t is called the triangulation number. permissible triangulation numbers are , , , , , , , , and so forth. a subunit defined in this way is not necessarily formed by one protein molecule, although in most cases this is how a structural subunit is in fact formed. some viruses that form regular structures that are constructed using icosahedral symmetry principles do not possess true icosahedral symmetry. in such cases they are said to have pseudo-triangulation numbers. examples are described later. a protein subunit, shown as blue trapezoids labeled "a." the twofold, threefold, and fivefold axes of symmetry are shown in yellow. this is the largest assembly in which every subunit is in an identical environment. (b) schematic representation of the subunit building block found in many rna viruses, known as the eightfold β barrel or β sandwich. the β sheets, labeled b through i from the n terminus of the protein, are shown as yellow and red arrows; two possible α helices joining these sheets are shown in green. some proteins have insertions in the c-d, e-f, and g-h loops, but insertions are uncommon at the narrow end of the wedge (at the fivefold axis). from granoff and webster ( ) vol. , color plate . [originally from j. johnson ( ) ]. structural studies of viruses have shown that the capsid proteins that form the virions of many plant and animal icosahedral viruses have a common fold. this fold, an eightstranded antiparallel β sandwich, is illustrated in fig. . b . the presence of a common fold suggests that these capsid proteins have a common origin even if no sequence identity is detectable. the divergence in sequence while maintaining this basic fold is illustrated in fig. . , where capsid proteins of three viruses are shown. sv (family polyomaviridae), poliovirus (family picornaviridae), and bluetongue virus (family reoviridae) are a dna virus, a single-strand rna virus, and a double-strand rna virus, respectively. their capsid proteins have insertions into the basic eight-stranded antiparallel β-sandwich structure that serve important functions in virus assembly. however, they all possess a region exhibiting the common β-sandwich fold and may have originated from a common ancestral protein. thus, once a suitable capsid protein arose that could be used to construct simple icosahedral particles, it may ultimately have been acquired by many viruses. the viruses that possess capsid proteins with this fold may be related by descent from common ancestral viruses, or recombination may have resulted in the incorporation of this successful ancestral capsid protein into many lines of viruses. because the size of the icosahedral shell is fixed by geometric constraints, it is difficult for a change in the size of a viral genome to occur. a change in size will require a change in the triangulation number or changes in the capsid proteins sufficient to produce a larger or smaller internal volume. in either case, the changes in the capsid proteins required are relatively slow to occur on an evolutionary timescale and the size of an icosahedral virus is "frozen" for long periods of evolutionary time. for this reason, as well as for other reasons, most viruses have optimized the information content in their genomes, as will be clear when individual viruses are discussed in the following chapters. cryoelectron microscopy has been used to determine the structure of numerous icosahedral viruses to a resolution of to Å. for this, a virus-containing solution on an electron microscope grid is frozen very rapidly so that the sample is embedded in amorphous frozen water. the sample must be maintained at liquid nitrogen temperatures so that ice crystals do not form and interfere with imaging. unstained, slightly out-of-focus images of the virus are captured on bluetongue virus vp t = figure . structure of three vertebrate virus protein subunits that assemble into icosahedral shells. the n termini and c termini are labeled with the residue number in parenthesis. the β barrels are shown as red arrows, α helices are gray coils, and the subunit regions involved in quasi-symmetric interactions that are critical for assembly are colored green. sv and pv have triangulation numbers of "pseudo-t= " or p = and "pseudo-t= " or p = , respectively. adapted from granoff and webster ( ), vol. , plate . film, or more recently captured electronically, using a low dose of electrons. these images are digitized and the density measured. mathematical algorithms that take advantage of the symmetry of the particle are used to reconstruct the structure of the particle. a gallery of structures of viruses determined by cryoelectron microscopy is shown in fig. . . all of the images are to scale so that the relative sizes of the virions are apparent. the largest particle is the nucleocapsid of herpes simplex virus, which is Å in diameter and has t= symmetry (the virion is enveloped but only the nucleocapsid is regular figure . gallery of three-dimensional reconstructions of icosahedral viruses from cryoelectron micrographs. all virus structures are surface shaded and are viewed along a twofold axis of symmetry except ross river, which is viewed along a three-fold axis. all of the images are of intact virus particles except for the herpes simplex structure, which is of the nucleocapsid of the virus. most of the images are taken from baker et al. ( ) , except the images of ross river virus and of dengue virus, which were kindly provided by drs. r. j. kuhn and t. s. baker. rest are not enveloped). b parvovirus has t= . the general correlation is that larger particles are constructed using higher triangulation numbers, which allows the use of larger numbers of protein subunits. larger particles accommodate larger genomes. because the simplest viruses are regular structures, they will often crystallize, and such crystals may be suitable for x-ray diffraction. many viruses formed using icosahedral symmetry principles have been solved to atomic resolution, and a discussion of representative viruses that illustrate the principles used in construction of various viruses is presented here. among t= viruses, the structures of several plant viruses, including tomato bushy stunt virus (tbsv) (genus tombusvirus, family tombusviridae), turnip crinkle virus (tcv) (genus carmovirus, family tombusviridae), and southern bean mosaic virus (sbmv) (genus sobemovirus, not yet assigned to family), have been solved. all three of these viruses have capsid proteins possessing the eight-stranded antiparallel β sandwich. t= means that identical molecules of capsid protein are utilized to construct the shell. the structures of two insect viruses that are also simple t= structures have also been solved. as an example of these simple structures, the t= capsid of the insect virus, flock house virus (family nodaviridae), is illustrated in fig. . . the subunits in these t= structures interact with one another in one of two different ways, such that the protein shell can be thought of as being composed of an assembly of ab dimers and cc dimers (fig. . a ). the bond angle between the two subunits of the dimer is more acute in the ab dimers than in the cc dimers (figs. . b and c). for the plant viruses, there are n-terminal and c-terminal extensions from the capsid proteins that are involved in interactions between the subunits and with the rna. the n-terminal extensions have a positively charged, disordered domain for interacting with and neutralizing the charge on the rna and a connecting arm that interacts with other subunits. in the case of the cc dimers, the connecting arms interdigitate with two others around the icosahedral threefold axis to form an interconnected internal framework. in the case of the ab conformational dimer, the arms are disordered, allowing sharper curvature. for flock house virus, the rna plays a role in controlling the curvature of the cc dimers, as illustrated in fig. . . b. proteins that make up a triangular face are only quasi-equivalent. the angle between the a and b units (shown with a red oval and in diagram (c) is more acute than that along the c-c edge, shown with a blue oval, and diagram (b). this difference in the angles is due to the presence of an rna molecule located under the c-c edge. from johnson ( ) , with permission. the structures of several picornaviruses and of a plant comovirus (cowpea mosaic virus) have also been solved to atomic resolution. the structures of these viruses are similar to those of the plant t= viruses, but the subunits that form the virion are not all identical. a comparison of the structure of a t= virus with those of poliovirus and of cowpea mosaic virus is shown in fig. . . poliovirus has copies of each of three different proteins, whereas the comovirus has copies of an l protein (each of which fills the niche of two units) and copies of an s protein. all three poliovirus capsid proteins have the eight-stranded antiparallel β-sandwich fold. in the comoviruses, the l protein has two β-sandwich structures fused to form one large protein, and the s protein is formed from one sandwich. the structures of the picornavirus and comovirus virions are called pseudo-t= or p= , since they are not true t= structures. the picornavirus virion is Å in diameter. the molecules of each of the three different proteins have different roles in the final structure, as illustrated in fig. . , in which the structure of a rhinovirus is shown. notice that five copies of vp are found at each fivefold axis (compare fig. . with fig. . ) . vp , vp , and vp are structurally related to one another, as stated, all possessing the common βsandwich fold. there exists a depression around each fivefold axis of rhinoviruses that has been termed a "canyon." this depression is believed to be the site at which the virus interacts with the cellular receptor during entry, as illustrated in fig. . . this interaction is thought to lead to conformational changes that open a channel at the fivefold axis, through which vp is extruded, followed by the viral rna. the structures of both mouse polyomavirus and of sv virus, two members of the family polyomaviridae, have been solved to atomic resolution. both viruses possess pseudo-t= icosahedral symmetry. although t= l vp vp vp vp vp vp vp vp vp vp vp vp vp vp vp vp vp vp vp vp vp vp vp vp vp vp vp vp vp vp vp vp vp vp vp vp figure . arrangement of the coat protein subunits of comoviruses compared with those of simple t= viruses and picornaviruses. in simple viruses, the asymmetric unit contains three copies of a single protein β sandwich, labeled a, b, and c in order to distinguish them. in picornaviruses such as poliovirus the asymmetric unit is made up of three similar but not identical proteins, all of which have the β-sandwich structure. in comoviruses such as cowpea mosaic virus, two of the β-sandwich subunits are fused to give the l protein. adapted from granoff and webster ( ) , p. . symmetry would require subunits, these viruses contain only copies of a major structural protein known as vp . these copies are assembled as pentamers. twelve of the pentamers lie on the fivefold axes and the remaining fill the intervening surface in a closely packed array ( fig. . ). these latter pentamers are thus sixfold coordinated and the proteins in the shell are not all in quasi-equivalent positions, a surprising finding for our understanding of the principles by which viruses can be constructed. the pentamers are stabilized by interactions of the β sheets between adjacent monomers in a pentamer ( fig. . c ). the pentamers are then tied together by c-terminal arms of vp that invade monomers in an adjacent pentamer (figs. . b and c). because each pentamer that is sixfold coordinated has five c-terminal arms to interact with six neighboring pentamers, the interactions between monomers in different pentamers are not all identical ( fig. . b ). flexibility in the c-terminal arm allows it to form contacts in different ways. members of the reovirus family are regular t= icosahedral particles. they are composed of two or three concentric protein shells. cryoelectron microscopy has been used to solve the structure of one or more members of three genera within the reoviridae, namely reovirus, rotavirus, and orbivirus, to about -Å resolution. structures of a reovirus and of a rotavirus are shown in fig. . . the complete structure of virions has not been determined because of their large size, but in a remarkable feat the atomic structure of the core of bluetongue virus (genus orbivirus) has now been solved. this is the largest structure determined to atomic resolution to date. solution of the structure was possible because the virus particle had been solved to Å by cryoelectron microscopy, and the structures of a number of virion proteins had been solved to atomic resolution by x-ray diffraction. fitting the atomic structure of the proteins into the -Å structure gave a preliminary reconstruction at high resolution, which allowed the interpretation of the x-ray data to atomic resolution. core particles are formed following infection, when the outer layer is proteolytically cleaved (described in more detail in chapter ). the structure of the inner surface of the bluetongue virus core is shown in fig. . a and of the outer surface in fig. . b. the outer surface is formed by copies of a single protein, called vp , in a regular t= icosahedral lattice. the inner surface is surprising, however. it is formed by copies of a single protein, called vp . these copies have been described as forming a t= five-fold axis lattice. because t= is not a permitted triangulation number, these copies, strictly speaking, form a t= lattice in which each unit of the lattice is composed of two copies of vp . however, the interactions are not symmetrical, leading to the suggested terminology of t= . it has been suggested that the inner core furnishes a template for the assembly of the t= outer surface. the reasoning is that a t= structure may have difficulty in forming, whereas the t= (or t= ) structure could form readily. in this model, the threefold symmetry axis of the inner surface could serve to nucleate vp trimers and organize the t= structure. cryoelectron microscopy has also been applied to adenoviruses, which have a triangulation number of or pseudo- . various interpretations of the structure of adenoviruses, both schematic and as determined by microscopy or crystallography, are shown in fig. . . three copies of a protein called the hexon protein associate to form a structure called a hexon ( fig. . c ). the hexon is the basic building block of adenoviruses. five hexons, called peripentonal hexons, surround each of the vertices of the icosahedron (which, as has been stated, have fivefold rotational symmetry). between the groups of peripentonal hexons are found groups of hexons, which are sixfold coordinated. each group of hexons forms the surface of one of the triangular faces. thus, there are peripentonal hexons and hexons in groups of nine. the structure of the hexon trimer has been solved to atomic resolution by x-ray crystallography ( fig. . c ). the hexon protein has two eight-strand β sandwiches to give the trimer an approximately sixfold symmetry (and each hexon protein fills the role of two symmetry units). there are long loops that intertwine to form a triangular top. these structures can be fitted uniquely into the envelope of density determined by cryoelectron microscopy, which produces a structure refined to atomic resolution for most of the capsid. the minor proteins can be fitted into this structure. from the vertices of the icosahedron project long fibers that are anchored in the surface by a unit called the penton base. each fiber terminates in a spherical extension that forms an organ of attachment to a host cell (fig. . a ). the length of the fiber differs in the different adenoviruses. in addition to the nonenveloped viruses that possess relatively straightforward icosahedral symmetry or helical symmetry, many viruses possess more complicated symmetries made possible by the utilization of a large number of structural proteins to form the virion. the tailed bacteriophages are prominent examples of this ( fig. . ) . some of the tailed bacteriophages possess a head that is a regular icosahedron (or, in at least one case, an octahedron) connected to a tail that possesses helical symmetry. other appendages, such as baseplates, collars, and tail fibers, may be connected to the tail. other tailed bacteriophages have heads that are assembled using more complicated patterns. for example, the t-even bacteriophages have a large head, which can be thought of as being formed of two hemi-icosahedrons possessing regular icosahedral symmetry, which are elongated in the form of a prolate ellipsoid by subunits arranged in a regular net connecting the two icosahedral ends of the head of the virus. many animal viruses and some plant viruses are enveloped; that is, they have a lipid-containing envelope surrounding a nucleocapsid. the lipids are derived from the host cell. although there is some selectivity and reorganization of lipids during virus formation, the lipid composition in general mirrors the composition of the cellular membrane from which the envelope was derived. however, the proteins in the nucleocapsid, which may possess either helical or icosahedral symmetry, and the proteins in the envelope are encoded in the virus. the protein-protein interactions that are responsible for assembly of the mature enveloped virions differ among the different families and the structures of the resulting virions differ. the virions of alphaviruses, and of flaviviruses, are uniform the essential features of the orbivirus native core particle. the asymmetric unit is indicated by the white lines forming a triangle and the fivefold, threefold, and twofold axes are marked. (a) the inner capsid layer of the bluetongue virus (btv) core is composed of molecules of vp , arranged in what has been called t= symmetry. note the green subunit a and the red subunit b which fill the asymmetric unit. (b) the core surface layer is composed of copies of vp arranged as trimers, with t= symmetry. the asymmetric unit contains copies of vp , arranged as five trimers, labeled p, q, r, s, and t, with each trimer a different color. trimer "t" in blue sits on the icosahedral threefold axis and thus contributes only a monomer to the asymmetric unit. from granoff and webster ( ) , color plate . structures that possess icosahedral symmetry. poxviruses, rhabdoviruses, and retroviruses also appear to have a regular structure, but there is flexibility in the composition of the particle and the mature virions do not possess icosahedral symmetry. the herpesvirus nucleocapsid is a regular icosahedral structure ( fig. . ), but the enveloped herpesvirions are not regular. other enveloped viruses are irregular, often pleiomorphic, and are heterogeneous in composition to a greater or lesser extent. the structures of different enveloped viruses that illustrate these various points are described next. the nucleocapsids of enveloped rna viruses are fairly simple structures that contain only one major structural protein, often referred to as the nucleocapsid protein or core protein. this protein is usually quite basic or has a basic domain. it binds to the viral rna and encapsidates it to form the nucleocapsid. for most rna viruses, nucleocapsids can be recognized as distinct structures within the infected cell and can be isolated from virions by treatment with detergents that dissolve the envelope. the nucleocapsids of alphaviruses, and probably flaviviruses and arteriviruses as well, are regular icosahedral structures, and there are no other proteins within the nucleocapsid other than the nucleocapsid protein. in contrast, the nucleocapsids of all minus-strand viruses are helical and contain, in addition to the major nucleocapsid protein, two or more minor proteins that possess enzymatic activity. as described, the nucleocapsids of minus-strand rna viruses remain intact within the cell during the entire infection cycle and serve as machines that make viral rna. the coronaviruses also have helical nucleocapsids, but being plus-strand rna viruses they do not need to carry enzymes in the virion to initiate infection. the helical nucleocapsids of (−) rna viruses appear disordered within the envelope of all viruses except the rhabdoviruses, in which they are coiled in a regular fashion (see later). the nucleocapsids of retroviruses also appear to be fairly simple structures. they are formed from one major precursor protein, the gag polyprotein, that is cleaved during maturation into four or five components. the precursor nucleocapsid is spherically symmetric but lacks icosahedral symmetry. the mature nucleocapsid produced by cleavage of gag may or may not be spherical symmetric. the nucleocapsid also contains minor proteins, produced by cleavage of gag-pro-pol, as described in chapter . these minor proteins include the protease, rt, rnase h, and integrase that are required to cleave the polyprotein precursors, to make a cdna copy of the viral rna, and to integrate this cdna copy into the host chromosome. the two families of enveloped dna viruses that we consider here, the poxviruses and the herpesviruses, contain large genomes and complicated virus structures. the nucleocapsids of herpesviruses are regular icosahedrons but those of poxviruses are complicated structures containing a core and associated lateral bodies. the external proteins of enveloped virions are virusencoded proteins that are anchored in the lipid bilayer of the virus or whose precursors are anchored in the lipid bilayer. in the vast majority of cases these proteins are glycoproteins, although examples are known that do not contain bound carbohydrate. these proteins are translated from viral mrnas and transported by the usual cellular processes to reach the membrane at which budding will occur. when budding is at the cell plasma membrane, the glycoproteins are transported via the golgi apparatus to the cell surface. some enveloped viruses mature at intracellular membranes, and in these cases the glycoproteins are directed to the appropriate place in the cell. both type i integral membrane proteins, in which the n terminus of the protein is outside the lipid bilayer and the c terminus is inside the bilayer, and type ii integral membrane proteins, which have the inverse orientation with the c terminus outside, are known for different viruses. many viral glycoproteins are produced as precursor molecules that are cleaved by cellular proteases during the maturation process. following synthesis of viral glycoproteins, during which they are transported into the lumen of the endoplasmic reticulum (er) in an unfolded state, they must fold to assume their proper conformation, and assume their proper oxidation state by formation of the correct disulfide bonds. this process often occurs very quickly, but for some viral glycoproteins it can take hours. folding is often assisted by chaperonins present in the endoplasmic reticulum. it is believed that at least one function of the carbohydrate chains attached to the protein is to increase the solubility of the unfolded glycoproteins in the lumen of the er so that they do not aggregate prior to folding. during folding, the solubility of the proteins is increased by hiding hydrophobic domains within the interior of the protein and leaving hydrophilic domains at the surface. the glycoproteins possess a number of important functions in addition to their structural functions. they carry the attachment domains by which the virus binds to a susceptible cell. this activity is thought to be related to the ability of many viruses, nonenveloped as well as enveloped, to bind to and agglutinate red blood cells, a process called hemagglutination. the protein possessing hemagglutinating activity is often called the hemagglutinin or ha. the viral glycoproteins also possess a fusion activity that promotes the fusion of the membrane of the virus with a membrane of the cell. the protein possessing this activity is sometimes called the fusion protein, or f. the glycoproteins, being external on the virus, are also primary targets of the humoral immune system, in which circulating antibodies are directed against viruses; many of these are neutralizing antibodies that inactivate the virus. the glycoproteins of some enveloped viruses also contain enzymatic activities. many orthomyxoviruses and paramyxoviruses possess a neuraminidase that will remove sialic acid from glycoproteins. the primary receptor for these viruses is sialic acid. the neuraminidase may allow the virus to penetrate through mucus to reach a susceptible cell. it also removes sialic acid from the viral glycoproteins so that these glycoproteins or the mature virions do not aggregate, and from the surface of an infected cell, thereby preventing released virions from binding to it. the viral protein possessing neuraminidase activity may be called na, or in the case of a protein that is both a neuraminidase and hemagglutinin, hn. the structure of most enveloped viruses is not as rigorously constrained as that of icosahedral virus particles. the glycoproteins are not required to form an impenetrable shell, which is instead a function of the lipid bilayer. they appear to tolerate mutations more readily than do proteins that must form a tight icosahedral shell and appear to evolve rapidly in response to immune pressure. however, the integrity of the lipid bilayer is essential for virus infectivity, and enveloped viruses are very sensitive to detergents. in some enveloped viruses, there is a structural protein that underlies the lipid envelope but which does not form part of the nucleocapsid. several families of minus-strand rna viruses possess such a protein, called the matrix protein. this protein may serve as an adapter between the nucleocapsid and the envelope. it may also have regulatory functions in viral rna replication. the herpesviruses also have proteins underlying the envelope that form a thick layer called the tegument. the thickness of the tegument is not uniform within a virion, giving rise to some irregularity in its structure. the tegument proteins perform important functions early after infection of a cell by a herpesvirus (see chapter ). the alphaviruses, a genus in the family togaviridae, are exceptional among enveloped rna viruses in the regularity of their virions, which are uniform icosahedral particles. virions of two alphaviruses have been crystallized and the crystals are regular enough to diffract to - -Å resolution. higher resolution has been obtained from cryoelectron microscopy, which has been used to determine the structures of several alphaviruses to - Å (fig. . ) . more detailed reconstructions of sindbis virus and ross river virus (rrv) have been derived from a combination of cryoelectron microscopy of the intact virion and x-ray crystallography of alphavirus structural proteins. a cutaway view of rrv at about -Å resolution is shown in fig. . a . the nucleocapsid, shown in red and yellow, has a diameter of Å, and is a regular icosahedron with t= symmetry. it is formed from copies of a single species of capsid protein of size kda. note the fivefold and sixfold coordinated pedestals in yellow that rise above the red background of rna and unstructured parts of the protein. each of these pedestals is formed by the ordered domains of one capsid protein molecule. the lipid bilayer is shown in green and is positioned between the capsid and the external shell of glycoproteins, shown in blue. the glycoproteins are also icosahedrally arranged with t= symmetry. the complete structure is therefore quite regular and the virion has been described as composed of two interacting protein shells with a lipid bilayer sandwiched between. the structure of the ordered part of the capsid protein of sindbis virus has been solved to atomic resolution by conventional x-ray crystallography and this structure is shown in fig. . b. the first residues are disordered and the structure is formed by residues - . this ordered domain has a structure that is very different from the eightfold β sandwich described earlier (compare fig. . b with figs. . b and . ). instead, its fold resembles that of chymotrypsin, and it has an active site that consists of a catalytic triad whose geometry is identical to that of chymotrypsin. the capsid protein is an active protease that cleaves itself from a polyprotein precursor. after cleavage, the c-terminal tryptophan- remains in the active site and the enzymatic activity of the protein is lost. the interactions between the capsid protein subunits that lead to formation of the t= icosahedral lattice have been deduced by fitting the electron density of the capsid protein at . -Å resolution into the electron density of the nucleocapsid found by cryoelectron microscopy. such a reconstruction, based on a cryoem structure of sindbis virus at a resolution of better than Å, is shown in fig. . c . the fit of the capsid protein is unique and the combined approaches of x-ray crystallography and cryoelectron microscopy thus define the structure of the shell of the nucleocapsid to atomic resolution. the envelopes of alphaviruses contain copies of each of two virus-encoded glycoproteins, called e and e . e is first produced as a precursor called pe . e and pe form a heterodimer shortly after synthesis, and both span the lipid bilayer as type i integral membrane proteins (having a membrane-spanning anchor at or near the c terminus). the c-terminal cytoplasmic extension of pe interacts in a specific fashion with a nucleocapsid protein so that there is a one-to-one correspondance between a capsid protein and a glycoprotein heterodimer. the glycoprotein heterodimers form a t= icosahedral lattice on the surface of the particle by interacting with one another and with the capsid proteins. because of the glycoprotein-capsid protein interactions, the icosahedral lattices of the nucleocapsid and the glycoproteins are coordinated. at some time during transport of the glycoprotein heterodimers to the cell surface, pe is cleaved by a cellular protease called furin to form e . e and e remain associated as a heterodimer. if cleavage is prevented, noninfectious particles are produced that contain pe and e . in the virion, three glycoprotein heterodimers associate to form a trimeric structure called a spike, easily seen in figs. . and . a. it is not known if the spike assembles during virus assembly or if heterodimers trimerize during their transport to the cell surface. a reconstruction of a spike of sindbis virus at a resolution better than Å is shown in fig. . a. in this reconstruction, the electron density of e has been replaced by the e structure of the related semliki forest virus determined to atomic resolution by x-ray crystallography. the three copies of e project upwards at an angle of about ° and are shown in three colors because they have slightly dif- b. dengue virus figure . comparison of (a) the spike structure of mature sindbis virus (an alphavirus) with (b) the spike of immature dengue virus (a flavivirus). the cα backbones of the three e (sindbis) and e (dengue) glycoprotein ectodomains are shown in red, green, and blue, as they were fitted into the cryoelectron density envelope. the e and e densities have been zeroed out, leaving the gray envelope that corresponds to e for sindbis and prm for dengue. the density corresponding to the lipid bilayer is shown in bright green. adapted from figure in y. with permission. ferent environments. the electron density in gray that remains after subtracting the density due to e is thus the electron density of e . e projects further upward than does e and covers the apex of e , which has the fusion peptide. thus, e covers the fusion peptide with a hydrophobic pocket so that it does not interact with the hydrophilic environment. the apex of the e spike contains the domains that attach to receptors on a susceptible cell. both e and e have c-terminal membrane-spanning anchors that traverse the lipid bilayer shown in green. the c-terminal domain of e is not present in the protein whose structure has been determined because hydrophobic domains do not easily crystallize. thus, the electron density shown traversing the lipid bilayer arises from both e and e and shows that the two membrane spanning anchors go through as paired α helical structures (fig. . ) . upon entry of an alphavirus into a cell, the acidic ph of endosomal vesicles causes disassembly of e /e heterodim-ers and trimerization of e to form homotrimers. the fusion peptide is exposed and penetrates the target bilayer of the host endosomal membrane. fusion follows by methods discussed in chapter . flaviviruses also possess a regular icosahedral structure (fig. . ) that has been solved by methods similar to those used to determine the structure of alphaviruses. the structures of alphaviruses and flaviviruses are related and have descended from a common ancestral structure. like alphaviruses, flaviviruses produce two structural glycoproteins, called e and prm (for precursor to m). e is homologous to e of alphaviruses. although no sequence identity is detectable, the structures of the two proteins are virtually identical and are formed with a similar fold (fig. . ). prm and e form a heterodimer and immature particles can be formed if cleavage of prm is prevented. the glycoprotein heterodimers in these immature particles trimerize to form spikes whose structure is very similar to the spikes of alphaviruses (fig. . b ). the arrangement of the glycoproteins on the immature virus particle is illustrated in fig. . a and a cryoem reconstruction that illustrates the surface of the immature dengue virus particle is shown in fig. . d . the major differences between the immature flavivirus particle and the alphavirus particle are that there are copies of the heterodimer in the flavivirus particle arranged in a t= icosahedral structure rather than heterodimers arranged in a t= structure in alphaviruses; that prm is a smaller molecule than pe so that in the flavivirus spike there is but a thin trace of density that projects downward, parallel to e, from the cap that shields the fusion peptide ( fig. . b ) rather than a substantial trace of density in the alphavirus spike ( fig. . a) ; and that the c-terminal regions of prm and e that enter the membrane do so independently and do not emerge from the internal side of the membrane (illustrated in fig. . b ), unlike the alphavirus membrane spanning regions (fig. . ). there is no evidence that the membrane glycoproteins interact with the nucleocapsid in flaviviruses, and the flavivirus nucleocapsid, assuming it is a regular icosahedral structure, is not coordinated with the icosahedral structure formed by the spikes, which is again different from alphaviruses where the c-terminal domain of pe interacts with the nucleocapsid. following cleavage of prm by furin to form m, there is a dramatic rearrangement of the flavivirus glycoproteins such that the final virion structure is very different from the alphavirus structure. the heterodimers dissociate and e-e homodimers are formed that collapse over the lipid bilayer (fig. . ) . the e-e homodimers occur in two totally different environments, either perpendicular to the twofold axis where they interact with homodimers in sideto-side interactions, forming a herringbone pattern, or parallel to a twofold axis where they interact at fivefold and threefold axes (fig. . b) . a comparison of the immature flavivirus particle, t figure . the e and e transmembrane helices of sindbis (an alphavirus) determined from a Å resolution cryoelectron microscopy reconstruction. shown are e residues from to and e residues to fitted into the transmembrane density. this is figure from mukhopadhyay et al. ( ) , reprinted with permission. the mature flavivirus particle, and the alphavirus particle is shown in fig. . . the mature flavivirus is smooth, with no surface projections, and is nm in diameter (fig. . e ). the immature particle is ragged in appearance and is nm in diameter (fig. . d ). the alphavirus virion shows conspicuous spikes and is nm in diameter (fig. . f ). the arrangement of e or e in the three particles is also shown (figs. . a, b, c) , illustrating the differences in their arrangement. entry of flaviviruses follows pathways similar to those used by alphaviruses. the acidic ph of the endosome causes the e-e homodimers to reorganize to form e homotrimers. these trimers must reorient so that the exposed fusion peptide is projected upwards where it penetrates the host membrane. the arteriviruses possess icosahedral nucleocapsids, but the mature virion does not appear to be regular in structure. detailed structures of these particles are not available. the herpesviruses are large dna viruses that have a t= icosahedral nucleocapsid (fig. . ) . a schematic diagram of an intact herpesvirion is shown in fig. . a . underneath the envelope is a protein layer called the tegument. the tegument does not have a uniform thickness, and thus the virion is not uniform. two electron micrographs of herpesvirions are shown in figs. . b and . c that illustrate the irregularity of the particle and the differing thickness of the tegument in different particles. the retroviruses have a nucleocapsid that forms initially using spherical symmetry principles. cleavage of gag during virus maturation results in a nucleocapsid that is not icosahedral and that is often eccentrically located in the virion. fig. . a presents a schematic of a retrovirus particle that illustrates the current model for the location of the various proteins after cleavage of gag and gag-pol. figs. . b, c, and d show electron micrographs of budding virus particles and of mature extracellular virions for three genera of retroviruses. betaretrovirus particles usually mature by the formation of a nucleocapsid within the cytoplasm that then buds through the plasma membrane. this process is shown in fig. . b for mouse mammary tumor virus. in the top micrograph in fig. . b, preassembled capsids are seen in the cytoplasm. in the middle micrograph, budding of the capsid through the plasma membrane is illustrated. in the bottom micrograph, a mature virion with an eccentrically located capsid is shown. in gammaretroviruses, the capsid forms during budding, and the nucleocapsid is round and centrally located in the mature virion. this process is illustrated in fig. . c for murine leukemia virus. the top micrograph shows a budding particle with a partially assembled capsid. the bottom micrograph shows a mature virion. in the lentiviruses, of which hiv is a member, the capsid also forms as a distinct structure only during budding. in the top panel of fig. . d is shown a budding particle of bovine immunodeficiency virus. after cleavage of gag to form the mature virion, the capsid usually appears cone shaped or bar shaped (bottom panel of fig. . d) . mason-pfizer monkey virus is a betaretrovirus whose capsid is cone shaped and centrally located in the mature virion. a single amino acid change in the matrix protein ma determines whether the capsid preassembles and then buds, or whether the capsids assemble during budding. thus, the point at which capsids assemble does not reflect a fundamental difference in retroviruses. preassembly of capsids or assembly during budding appears to depend on the stability of the capsid in the cell. stable capsids can preassemble. unstable capsids require interactions with other viral components to form as a recognizable structure. the coronaviruses and the minus-strand rna viruses have nucleocapsids with helical symmetry. the structures of the mature virions are irregular, with the exception of the rhabdoviruses, and the glycoprotein composition is not invariant. because of the lack of regularity in these viruses, as well as the lack of symmetry, detailed structural studies of virions have not been possible. the lack of regularity arises in part because in these viruses there is no direct interaction between the nucleocapsid and the glycoproteins. the lack of such interactions permits these viruses to form pseudotypes, in which glycoproteins from other viruses substitute for those of the virus in question. pseudotypes are also formed by retroviruses. the structures of paramyxoviruses and orthomyxoviruses are illustrated schematically in fig. . . the helical nucleocapsids contain a major nucleocapsid protein called n or np, and the minor proteins p (ns ) and l (pb , pb , pa), as shown. there is a matrix protein m (m ) lining the inside of the lipid bilayer and also two glycoproteins anchored in the bilayer that form external spikes. the two glycoproteins, called f and hn in paramyxoviruses and ha and na in orthomyxoviruses, do not form heterodimers but rather form homooligomers so that there are two different kinds of spikes on the surface of the virions. ha in the orthomyxoviruses forms homotrimers whereas na forms homotetramers, and the two types of spikes can be distinguished in the electron microscope if the resolution is high enough. as occurs in many enveloped rna viruses, f and ha are produced as precursors that are cleaved by furin during transport of the proteins. cleavage is required to activate the fusion peptide of the virus, which is found at the n terminus of the c-terminal product (see fig. . ). electron micrographs of virions are shown in figs. . c and d. the particles in the preparations shown are round and reasonably uniform, but in other preparations the virions are pleomorphic baglike structures that are not uniform in appearance. in fact, clinical specimens of some orthomyxoviruses and paramyxoviruses are often filamentous rather than round, illustrating the flexible nature of the structure of the virion. the micrograph of the paramyxovirus measles virus shown in fig. . c is a thin section and illustrates the lack of higher order structure in the internal helical nucleocapsid. the micrograph of the orthomyxovirus influenza a virus shown in fig. . d is of a negative-stained preparation and illustrates the spikes that decorate the virus particle. the structures of rhabdoviruses and filoviruses are illustrated in fig. . . the rhabdoviruses assemble into bullet-shaped or bacilliform particles in which the helical nucleocapsid is wound in a regular elongated spiral conformation (figs. . b and c). the virus encodes only five proteins (fig. . a) , all of which occur in the virion (fig. . b ). the nucleocapsid contains the major nucleocapsid protein n and the two minor proteins l and ns. the matrix protein m lines the inner surface of the envelope, and g is an external glycoprotein that is anchored in the lipid bilayer of the envelope. budding is from the plasma membrane ( fig. . d) . the filoviruses are so named because the virion is filamentous. a schematic diagram of a filovirus is shown in fig. the structure of viruses the poxviruses, large dna-containing viruses, also have lipid envelopes. in fact, they may have two lipid-containing envelopes. the structures of poxviruses belonging to two different genera, orthopox and parapox, are illustrated in fig. . . electron micrographs of the orthopox virus vaccinia virus and of a parapox virus are also shown. vaccinia virus has been described as brick shaped. the interior of the virion consists of a nucleoprotein core and two (in vertebrate viruses) proteinaceous lateral bodies. surrounding these is a lipid-containing surface membrane, outside of which are several virus-encoded proteins present in structures referred to as tubules. this particle is called an intracellular infectious virion. as its name implies, it is present inside an infected cell, and if freed from the cell it is infectious. a second form of the virion is found outside the cell and is called an extracellular enveloped virion. this second form has a second, external lipid-containing envelope with which is associated five additional vaccinia proteins. this form of the virion is also infectious. parapox virions are similar to orthopox virions. however, their morphology is detectably different, as illustrated in fig. . . virions self-assemble within the infected cell. in most cases, assembly appears to begin with the interaction of one or more of the structural proteins with an encapsidation signal in the viral genome, which ensures that viral genomes are preferentially packaged. after initiation, encapsidation continues by recruitment of additional structural protein molecules until the complete helical or icosahedral structure has been assembled. thus, packaging of the viral genome is coincident with assembly of the virion, or of the nucleocapsid in the case of enveloped viruses. the requirement for a packaging signal may not be absolute. in many viruses that contain an encapsidation signal, rnas or dnas lacking such a signal may be encapsidated, but with much lower efficiency. for some viruses, there is no evidence for an encapsidation signal. assembly of the tmv rod ( fig. . ) has been well studied. several coat protein molecules, perhaps in the form of a disk, bind to a specific nucleation site within tmv rna to initiate encapsidation. once the nucleation event occurs, additional protein subunits are recruited into the structure and assembly proceeds in both directions until the rna is completely encapsidated. the length of the virion is thus determined by the size of the rna. the assembly of the icosahedral turnip crinkle virion has also been well studied. assembly of this t= structure is initiated by formation of a stable complex that consists of six capsid protein molecules bound to a specific encapsidation signal in the viral rna. additional capsid protein dimers are then recruited into the complex until the structure is complete. it is probable that most other viruses assemble in a manner similar to these two well-studied examples. at least some viruses deviate from this model, however, and assemble an empty particle into which the viral genome is later recruited. it is also known that many viruses will assemble empty particles if the structural proteins are expressed in large amounts in the absence of viral genomes, even if assembly is normally coincident with encapsidation of the viral genome in infected cells. the nucleocapsids of most enveloped viruses form within the cell by pathways assumed to be similar to those described above. they can often be isolated from infected cells, and for many viruses the assembly of nucleocapsids does not require viral budding or even the expression of viral surface glycoproteins. after assembly, the nucleocapsids bud through a cellular membrane, which contains viral glycoproteins, to acquire their envelope. budding retroviruses were illustrated in fig. . and budding rhabdoviruses in fig. . . a gallery of budding viruses belonging to other families is shown in fig. . . the membrane chosen for budding depends on the virus and depends, in part if not entirely, on the membrane to which the viral glycoproteins are directed by signals within those glycoproteins. many viruses bud through the cell plasma membrane (figs. . b-f); in polarized cells, only one side of the cell may be used. other viruses, such as the coronaviruses and the bunyaviruses, use the endoplasmic reticulum or other internal membranes. the herpesviruses replicate in the nucleus and the nucleocapsid assembles in the nucleus; in this case, the first budding event is through the nuclear membrane (fig. . a) . although the nucleocapsid of most enveloped viruses assembles independently within the cell and then buds to acquire an envelope, exceptions are known. the example of retroviruses, some of which assemble a nucleocapsid during virus budding, was discussed earlier. in these viruses, morphogenesis is a coordinated event. the forces that result in virus budding are not well understood for most enveloped viruses. in the case of the alphaviruses, there is evidence for specific interactions between the cytoplasmic domains of the glycoproteins and binding sites on the nucleocapsid proteins. the model for budding of these viruses is that the nucleocapsid first binds to one or a few glycoprotein heterodimers at the plasma membrane. by a process of lateral diffusion, additional glycoprotein heterodimers move in and are bound until a full complement is achieved and the virus is now outside the cell. additional free energy for budding is furnished by lateral interactions between the glycoproteins, which form a contiguous layer on the surface (fig. . c ). this model accounts for the regularity of the virion, the one-to-one ratio of the structural proteins in the virion, and the requirement of the virus for its own glycoproteins in order to bud. in other enveloped viruses, however, there is little evidence for nucleocapsid-glycoprotein interactions. the protein composition of the virion is usually not fixed, but can vary within limits. in fact, glycoproteins from unrelated viruses can often be substituted. in the extreme case of retroviruses, noninfectious virus particles will form that are completely devoid of glycoprotein. the matrix proteins appear to play a key role in the budding process, as do other protein-protein interactions that are yet to be determined. for most animal viruses, there are one or more cleavages in structural protein precursors during assembly of virions that are required to activate the infectivity of the virion. interestingly, these cleavages may either stabilize or destabilize the virion in the extracellular environment, depending on the virus. many of these cleavages are effected by viral proteases, whereas others are performed by cellular proteases present in subcellular organelles. virions are formed by the spontaneous assembly of components in the infected cell, sometimes with the aid of assembly factors ("scaffolds") that do not form components of the mature virion. for most nonenveloped viruses, complete assembly occurs within the cell cytoplasm or nucleoplasm. for enveloped viruses, final assembly of the virus occurs by budding through a cellular membrane. in either case, the virion must subsequently disassemble spontaneously on infection of a new cell. the cleavages that occur during assembly of the virus potentiate penetration of a susceptible cell after binding of the virus to it, and the subsequent disassembly of the virion on entry into the cell. a few examples will be described that illustrate the range of cleavage events that occur in different virus families. in the picornaviruses, a provirion is first formed that is composed of the viral rna complexed with three viral proteins, called vp , vp , and vp . during maturation to form the virion, vp is cleaved to vp and vp . no protease has been found that performs this cleavage, and it has been postulated that the virion rna may catalyze it. cleavage to produce vp , which is found within the interior of the capsid shell, as illustrated schematically in fig. . , is required for the virus to be infectious. as described in chapter , vp appears to be required for entry of the virus into the cell. this maturation cleavage has another important consequence. whereas the provirion is quite unstable, the mature virion is very stable. the poliovirus virion will survive treatment with proteolytic enzymes and detergents, and survives exposure to the acidic ph of less than that is present in the stomach. only on binding to its receptor (figs. . and . ) is poliovirus destabilized such that vp can be released for entry of the viral rna into the host cell. similarly, the insect nodaviruses first assemble as a procapsid containing the rna and copies of a single protein species called α ( kda). over a period of many hours, spontaneous cleavage of α occurs to form β ( kda) and γ ( kda). this cleavage is required for the particle to be infectious. these events in nodaviruses have been well studied because it has been possible to assemble particles in vitro, and the structures of both cleaved and uncleaved particles have been solved to atomic resolution. rotaviruses, which form a genus in the family reoviridae, must be activated by cleavage with trypsin after release from an infected cell in order to be infectious. trypsin is present in the gut, where the viruses replicate, and activation occurs normally during the infection cycle of the virus in animals. when the viruses are grown in cultured cells, however, trypsin must be supplied exogenously. a different type of cleavage event occurs during assembly of retroviruses and adenoviruses, as well as of a number of other viruses. during assembly of retroviruses, the gag and gag-pol precursor polyproteins are incorporated, together with the viral rna, into a precursor nucleocapsid. these polyproteins must be cleaved into several pieces by a protease present in the polyprotein if the virus is to be infectious. these cleavages often visibly alter the structure of the particle as seen in the electron microscope (fig. . ). an analogous situation occurs in adenoviruses, where a viral protease processes a protein precursor in the core of the immature virion. in most enveloped viruses, one of the envelope proteins is produced as a precursor whose cleavage is required to activate the infectivity of the virus. this cleavage may occur prior to budding, catalyzed by a host enzyme called furin, or may occur after release of the virus, catalyzed by other host enzymes. the example of the hemagglutinin of influenza virus was described in chapter . this protein is produced as a precursor called ha , which is cleaved to ha and ha ( fig. . ). cleavage is required to potentiate the fusion activity present at the n terminus of ha . as a second example, alphaviruses produce two envelope glycoproteins that form a heterodimer and one of the glycoproteins is produced as a precursor, as described before. the heterodimer containing the uncleaved precursor is quite stable, so that a particle containing uncleaved heterodimer is not infectious. the cleaved heterodimer, which is required for virus entry, is much less stable and dissociates readily during infection. thus, in contrast to the poliovirus maturation cleavage, the alphavirus cleavage makes the virion less stable rather than more stable. maturation cleavages also occur in the envelope glycoproteins of retroviruses, paramyxoviruses, flaviviruses, and coronaviruses. dna or rna has a high net negative charge, and there is a need for counterions to neutralize this charge in order to form a virion. in many viruses, positively charged polymers are incorporated that neutralize half or so of the nucleic acid charge. the dna in the virions of the polyomaviruses is complexed with cellular histones. the viral genomes in these viruses have been referred to as minichromosomes. in contrast, the adenoviruses encode their own basic proteins that complex with the genome in the core of the virion. another strategy is used by the herpesviruses, which incorporate polyamines into the virion. herpes simplex virus has been estimated to incorporate , molecules of spermidine and , molecules of spermine, which would be sufficient to neutralize about % of the dna charge. among rna viruses, the nucleocapsid proteins are often quite basic and neutralize part of the charge on the rna. as one example, the n-terminal amino acids of the capsid protein of sindbis virus have a net positive charge of . the positive charges within this domain of the capsid proteins in a nucleocapsid would be sufficient to neutralize about % of the charge on the rna genome. this charged domain is thought to penetrate into the interior of the nucleocapsid and complex with the viral rna. virions differ greatly in stability, and these differences are often correlated with the means by which viruses infect new hosts. viruses that must persist in the extracellular environment for considerable periods, for example, must be more stable than viruses that pass quickly from one host to the next. as an example of such requirements, consider the closely related polioviruses and rhinoviruses, members of two different genera of the family picornaviridae. these viruses shared a common ancestor in the not too distant past and have structures that are very similar. the polioviruses are spread by an oral-fecal route and have the ability to persist in a hostile extracellular environment for some time where they may contaminate drinking water or food. furthermore, they must pass through the stomach, where the ph is less than , to reach the intestinal tract where they begin the infection cycle. it is not surprising, therefore, that the poliovirion is stable to storage and to treatments such as exposure to mild detergents or to ph < . in contrast, rhinoviruses are spread by aerosols or contaminated mucus, and spread normally requires close contact. the rhinovirion is less stable than the poliovirion. it survives for only a limited period of time in the external environment and is sensitive to treatment with detergents or exposure to ph . general structure of viruses thin section of a herpes simplex virion (herpesviridae) in an infected hep- cell. the particle is apparently coated with an inner envelope, and is in the process of acquiring its outer envelope from the nuclear membrane. from roizman ( ). (b) machupo virus (arenaviridae) budding from a raji cell. from murphy et al. ( ). (magnification: , ×). (c) sindbis virus (togaviridae) budding from the plasma membrane of an infected chicken cell ×). (e) a portion of the cell surface with sv filaments (paramyxoviridae) in the process of budding. from compans and choppin ( ). ( , ×). (f) a row of sv virions budding from the surface of a monkey kidney cell. cross sections of the nucleocapsid can be seen within several of the particles principles of virus structure functional implications of protein-protein interactions in icosahedral viruses principles of virus structure structural biology of viruses ultrastructure of animal viruses and bacteriophages: an atlas. ultrastructure in biological systems cryoelectron microscopy adding the third dimension to virus life cycles: three-dimensional reconstruction of icosahedral viruses from cryo-electron micrographs. microbiol membrane proteins organize a symmetrical virus mapping the structure and function of the e and e glycoproteins of alphaviruses structures of immature flavivirus particles conformational changes of the flavivirus e glycoprotein visualization of membrane protein domains by cryo-electron microscopy of dengue virus the refined crystal structure of hexon, the major coat protein of adenovirus type , at . Å resolution nucleocapsid and glycoprotein organization in an enveloped virus three-dimensional structure of poliovirus at . Å resolution the fusion glycoprotein shell of semliki forest virus: an icosahedral assembly primed for fusogenic activation at endosomal ph a ligand-binding pocket in the dengue virus envelope glycoprotein x-ray crystallographic structure of the norwalk virus capsid structure of a human common cold virus and functional relationship to other picornaviruses the structure of simian virus refined at . Å resolution the canyon hypothesis in a nutshell: structure and assembly of the vaccinia virion structure of dengue virus: implications for flavivirus organization, maturation, and fusion a structural perspective of the flavivirus life cycle rotavirus proteins: structure and assembly bluetongue virus assembly and morphogenesis budding of alphaviruses key: cord- -ywz w po authors: maus, carsten title: component-based modelling of rna structure folding date: journal: computational methods in systems biology doi: . / - - - - _ sha: doc_id: cord_uid: ywz w po rna structure is fundamentally important for many biological processes. in the past decades, diverse structure prediction algorithms and tools were developed but due to missing descriptions in clearly defined modelling formalisms it’s difficult or even impossible to integrate them into larger system models. we present an rna secondary structure folding model described in ml-devs, a variant of the devs formalism, which enables the hierarchical combination with other model components like rna binding proteins. an example of transcriptional attenuation will be given where model components of rna polymerase, the folding rna molecule, and the translating ribosome play together in a composed dynamic model. single stranded ribonucleic acids (rna) are able to fold into complex threedimensional structures like polypeptide chains of proteins do. the structure of rna molecules is fundamentally important for their function, e.g. the well studied structures of trna and the different rrna variants. but also other transcripts of the dna, i.e. mostly mrnas, perform structure formation which has been shown to be essential for many regulatory processes like transcription termination and translation initiation [ , , ] . the shape of a folded rna molecule can also define binding domains for proteins or small target molecules which can be found for example within riboswitches [ ] . the enormous relevance for many biological key processes led to raised research efforts in identifying various rna structures over the past decades. unfortunately the experimental structure identification with nmr and x-ray techniques is difficult, expensive, and highly time-consuming. therefore, many in silico methods for rna structure prediction were developed which cover different requirements. diverse comparative methods exist using alignments of similar rna sequences to predict structures [ , ] , but also many single sequence prediction algorithms work very well. some of them predict the most stable rna structure in a thermodynamical equilibrium, e.g. [ , , ] , whereas some other simulate the kinetic folding pathway over time [ , , , ] . the latter is also in the focus of the presented modelling approach here. results of rna structure predictions as well as kinetic folding simulations have reached a high level of accuracy and thus in silico folding became a widely used and well established technique in the rna community. however, none of the existing tools and programs provides a flexible integration into larger system models which is also due to the fact that they are written in proprietary formalisms and do not distinguish between model description and simulation engine. to illuminate the importance of the folding processes and the possibility to integrate them into larger models, lets take a look at a concrete example of gene regulation. the tryptophan (trp) operon within bacterial genomes represents one of the best understood cases of gene regulation and has been subject to various modelling approaches [ , ] . tryptophan is an amino acid, a building block for the production of proteins. the trp operon includes five consecutive genes, coding for five proteins. the joint action of the proteins permits the synthesis of trp through a cascade of enzymatic reactions. this ability is vital since the bacterium may be unable to feed on trp from the environment. as long as trp is obtained from the surrounding medium, its costly synthesis is impaired by a threefold control mechanism: repression of transcription initiation, transcriptional attenuation, and inactivation of the cascade of enzymatic reactions actually producing trp. each of these are triggered by trp availability. transcriptional attenuation follows if transcription starts although trp was available. this has a small but non-negligible chance. as soon as rna polymerase (rnap) has transcribed the operon's leader region into an mrna molecule, a ribosome can access this. the ribosome starts translating the mrna content into a growing sequence of amino acids. the speed of the ribosome depends on trp availability. the ribosome advances quickly as long as trp is abundant, which prevents rnap from proceeding into the operon's coding region. the attenuation is caused by the formation of a certain constellation of rna hairpin loops in presence of a trp molecule at a distinct segment of the mrna molecule (figure ). attenuation depends on the synchronised advance of both rnap and ribosome, and their relative positioning with respect to mrna. in [ ] a model of the tryptophan operon was developed, in which repressors, the operon region, and the mrna were modelled individually. however, the latter in not much detail. only the repression of transcription initiation was included in the model. consequently, the simulation result showed stochastic bursts in the trp level, caused by the repressor falling off, which started the transcription. integrating a transcriptional attenuation model would have prevented this non-realistic increase in trp concentration, and mimicked the threefold regulation of the trp more realistically. however, the question is how to model the attenuation. as this regulating process depends largely on structure formation, modelling of rna folding would be a big step in the right direction for reflecting attenuation dynamics. additionally, modelling interactions between mrna and rnap as well as mrna and the ribosome are needed because both influence the kinetic folding process and rna termination structures break up gene transcription. the focus of this paper is the rna folding process, but at the end we will also give a detailed outlook how the composed model of tryptophan attenuation looks like and how the individual model components act together. the reason for rna folding is the molecules' general tendency to reach the most favourable thermodynamical state. complementary bases of rna nucleotides can form base pairs by building hydrogen bonds similar to dna double helices. adenine (a) and uracil (u) are complementary bases as well as cytosine (c) and guanine (g). in addition, the wobble base pair g-u is also frequently found in rna structure folding. each additional hydrogen bond of base pairs affords a small energy contribution to the overall thermodynamic stability, but there is another chemical interaction which is even more important for the rna structure than just the number and type of base pairs. it's called base stacking and describes the interactions between the aromatic rings of adjacent bases by van-der-waals bonds. base pair stacking is the cause why an uninterrupted long helix is thermodynamically more favourable than a structure of multiple single base pairs or short helices interrupted by loop regions, even if the number and type of base pairs are equal. since the s, significant progress has been done on identifying thermodynamic parameters of different base pair neighbourhoods and structural elements like hairpin loops, e.g. [ , ] . this was a precondition to develop rna structure prediction algorithms based on energy minimisation, i.e. finding the thermodynamical most stable structure. rna structures are hierarchically organised (see figure ). the most simple hierarchy level is the primary structure which is nothing else than the linear sequence of nucleotides. two nucleotides are linked over the ' and ' carbon atoms of their ribose sugar parts resulting in a definite strand direction. the secondary structure consists of helices formed by base pairs and intersecting loop regions. such structural elements are formed rapidly within the first milliseconds of the folding process [ ] . interacting secondary structure elements finally build the overall three-dimensional shape of rna molecules. although they are formed by simple base pairs like secondary structures, helices inside loop regions are often seen as tertiary structures. such pseudoknots and higher order tertiary interactions are, due to their complexity and analog to many other rna structure prediction methods, not covered by our model. however, it should not retain unstated here that there are some existing tools which can predict pseudoknots quite well, e.g. [ ] . primary structure secondary structure tertiary structure as already mentioned, typically kinetic rna folding simulations, as e.g. [ , , ] , are aimed at efficiently and accurately simulating the molecules structure formation in isolation rather than supporting a reuse of rna folding models and a hierarchical construction of models. for approaching such model composition, we use the modelling formalism ml-devs [ ] , a variant of the devs formalism [ ] . as devs does, it supports a modular-hierarchical modelling and allows to define composition hierarchies. ml-devs extends devs by supporting variable structures, dynamic ports, and multi-level modelling. the latter is based on two ideas. the first is to equip the coupled model with a state and a behaviour of its own, such that macro behaviour the macro level does not appear as a separate unit (an executive) of the coupled model. please recall that in traditional devs coupled models do not have an own state nor a behaviour. secondly, we have to explicitly define how the macro level affects the micro level and vice versa. both tasks are closely interrelated. we assume that models are still triggered by the flow of time and the arrival of events. obviously, one means to propagate information from macro to micro level is to exchange events between models. however, this burdens typically modelling and simulation unnecessarily, e.g. in case the dynamics of a micro model has to take the global state into consideration. therefore, we adopt the idea of value couplings. information at macro level is mapped to specific port names at micro level. each micro model may access information about macro variables by defining input ports with corresponding names. thus, downward causation (from macro to micro) is supported. in the opposite direction, the macro level needs access to crucial information at the micro level. for this purpose, we equip micro models with the ability to change their ports and to thereby signalise crucial state changes to the outside world. upward causation is supported, as the macro model has an overview of the number of micro models being in a particular state and to take this into account when updating the state at macro level. therefore, a form of invariant is defined whose violation initiates a transition at macro level. in the downward direction, the macro level can directly activate its components by sending them events -thereby, it becomes possible to synchronously let several micro models interact which is of particular interest when modelling chemical reactions. these multi-level extensions facilitate modelling, figure depicts the basic idea, see also [ ] . the central unit in composed models using rna structure information is an rna folding model. therefore, we first developed a model component which describes the folding kinetics of single stranded rna molecules. it consists of a coupled ml-devs model representing the whole rna molecule and several atomic models. each nucleotide (nt) of the rna strand is represented by an instance of the atomic model nucleotide which is either of the type a, c, g, or u meaning its base. they are connected via ports in the same order as the input sequence (primary structure) and have knowledge about their direct neighbours. for example, the nt at sequence position is connected with the nt number on its ' side and on the ' location it is connected with the nt at position (see figure ). state variables hold rudimentary information about the neighbours, to be exact their base type and current binding partners. "binding partner" means a secondary structure defining base pair and the term is used only in this context here and does not mean the primary backbone connections. if a partner of a nucleotide changes, an output message will be generated and the receiving (neighboured) nucleotides will update their state variables. holding information about other atomic model states is normally not the case in devs models as they are typically seen as black boxes. however, here it is quite useful because of some dependencies concerning base pair stability. base pairs are modelled by wide range connections of nucleotides via additional interfaces. whereas the rna backbone bonds of adjacent nucleotides are fixed after model initialisation, the connections between base pairing nucleotides are dynamically added and removed during simulation (figure ). therefore, two different major states (phases) of nucleotides exist: they can be either unpaired or paired. as already stated in section . , base pair stability depends on the involved bases and their neighbourhood, especially stacking energies of adjacent base pairs provide crucial contributions for structure stabilisation. in our kinetic folding model, base pair stability is reflected by binding duration, i.e. the time advance function of the paired phase. thus, pairing time depends on thermodynamic parameters for nucleic acids base stacking which were taken from [ ] and are also be used by mfold version . [ ] . this thermodynamic data set not only provides the free energy (gibbs energy) for a given temperature of • c, but also the enthalpy change Δh of various stacking situations. the enthalpy together with the free energy and the absolute temperature allows us to calculate the entropy change Δs which allows us further to calculate the activation energy Δe a for base pair dissociation at any temperature t between and • c: Δe a is directly used as one parameter for base pair opening time, i.e. the duration of a paired phase is directly dependent on the activation energy for base pair disruption. to allow rna structures to escape from local energy minima and refold to more stable structures, the base pair dissociation time will be randomised, which leads to very short bonding times in some cases although the activation energy needed for opening the base pair is quite large. for base pair closing an arbitrary short random time is assigned with the unpaired phase of nucleotides assuming that rna base pair formation is a very fast and random process. after unpaired time has expired, the nucleotide model tries to build a base pair with another nucleotide randomly chosen from within a set of possible pairing partner positions. this set is determined by sterically available rna strand regions and thus an abstraction of spatial constraints. for example, a hairpin loop smaller than nucleotides is sterically impossible, but also many other nucleotide positions with larger distance can be excluded for secondary structure folding (see figure ). an unpaired nucleotide is not able to choose another nt for base pairing by its own. it has no global information about the rna shape which is strongly needed here. therefore, an implicit request by adding a new input port will be made to the coupled model that holds such macro knowledge and can therefore choose a valid position with which the requesting nt will try to pair next. for choosing this position at macro level two different model variants exist. the first and more simple one picks a position totally random from within the set of possible partners, whereas the second variant takes the entropy change into account when a pairing partner will be selected. the latter method prefers helix elongation in contrast to introducing new interior loops by single base pair formations and small loops will be more favourable than large ones [ ] . correct rna folding with the first method is dependent on the base pair stabilities and the random folding nuclei which are the first appearing base pairs of helical regions. this last point is less important for rna folding with model variant because the chosen binding partners are more deterministic due to loop entropy consideration. a comparison of both approaches with respect to simulation results is given in section . once a nucleotide received an input message by the macro model containing the number of a potential pairing partner, it tries to form a base pair with this nt by adding a coupling and sending a request. for a successful pairing, the partners must be of complementary base type and they must be able to pair in principle, e.g. bases can be modified so that they can not bind to others. figure illustrates the whole state flow for base pair formation and disruption of the nucleotide model component. the role of the macro model and its interactions with the micro level (nucleotides) shows the schematic organisation of the whole rna folding model in figure . already mentioned in the previous section, high level information about the whole rna molecule is needed to take sterical restrictions for base pairing into account. therefore, the coupled model holds the overall rna secondary structure which will be updated every time the state of a nucleotide changes from unpaired to paired and vice versa. this will be triggered by nucleotide port adding and removal recognised by the macro level. the same functionality is used to signalise the macro level the wish to try pairing with another nucleotide. the macro model detects a port adding, calculates the sterically possible partner set, chooses a position from within the set, and after all sends this position to the just now added nucleotide input port ( figure , nt ) . the coupled macro model is further responsible for sequence initialisation on the micro level by adding and connecting nucleotide models, i.e. it generates the primary rna structure. another task of the macro model is to observe the current folding structure for special structural patterns. this could be for example a specific binding domain for a protein or small ligand. also transcription termination or pausing structures can be of interest for observation. if observed structures are present during folding simulation, the macro level model can signalise this information and thus trigger dynamics to other components by adding new ports to itself (representing docking sites) or send messages over existing ports. a composed example model which uses this capability can be found in section . for evaluating the model's validity we simulated the folding of different rna molecules with known structure and analysed the results. three different types of experiments were done: native structure-correlates the majority of formed structures with the native structure after sufficient long simulation time? structure distribution-is the equilibrium ratio between minimum free energy (mfe) and suboptimal structures as expected? structure refolding-are molecules able to refold from suboptimal to more stable structural conformations? unfortunately, only few time-resolved folding pathways are experimentally derived and most of them treat pseudoknots [ ] and higher order tertiary structure elements [ , , ] which can not be handled by our folding model and are therefore out of the question for a simulation study. hence, some comparisons with other in silico tools were also made, although we know that one has to be careful with comparing different models for validating a model as it is often unclear how valid the other models are. because the folding model is highly stochastic, every simulation experiment was executed multiple times. typically replications were made. structural analysis of the cis-acting replication element from hepatitis c virus revealed a stem hairpin loop conformation where the helix is interrupted by an internal or bulge loop region [ ] . figure shows simulation results of its structure formation. the three-dimensional base pair lifetime plots indicate correct folding of both helical regions and only few misfolded base pairs. only small differences can be seen between simulations with the two base pair formation variants described in section . . without taking entropy into account for pairing, a bit more noise of misfolded base pairs can be observed which is not surprising due to the absolutely random partner choice. another well known rna structure is the cloverleaf secondary structure of trnas [ , ] consisting of four helical stems: the amino acid arm, d arm, anticodon arm, and the t arm. some base modifications and unusual nucleotides exist in trna which stabilise its structure formation, e.g. dihydrouridine and pseudouridine. such special conditions are not considered by our folding model as well as tertiary interactions leading to the final l-shaped form. however, folding simulations result in significant cloverleaf secondary structure formation (figure ). although there is much misfolded noise, the four distinct helix peaks are the most stable structural elements introduced during simulation, especially the amino acid arm. no fundamental difference can be seen between base pair formation model and . a third native structure validation experiment treats the corona virus s m motif which is a relatively long hairpin structure with some intersecting internal and bulge loops [ , ] . simulation of the sars virus s m rna folding indicates only for one of the native helix regions a conspicuous occurrence ( figure ). the other helices closer to the hairpin loop show no significant stabilisation. competing misfolded structural elements can be observed equally frequent or even more often. base pair formation model provides a slightly better result than the first one, but it is unsatisfying too. a reason for the result can be the multiple internal and bulge loops, which destabilise the stem and thus allow locally more stable structure elements to form. in [ ] a quantitative analysis of different rna secondary structures by comparative imino proton nmr spectroscopy is described. the results indicate that a small -nt rna has two equally stable structures in thermodynamic equilibrium, one with short helices and the other with a single hairpin. folding simulations of the same rna strand show an equal ratio of the probed structures as well ( figure ) . however, both are representing just % of all present structures which was not detected in the nmr experiments. many base pairs were introduced during simulation which are competing with base pairs of the two stable structures and thus reduce their appearance. this can be easily seen in the d matrix of figure where some additional peaks show high misfolded base pair lifetimes. simulating the rna folding with kinfold [ ] results in a five times higher amount of the -helix conformation than the single hairpin, but their total sum is about % of all molecules and thus less misfolded structures can be observed. real-time nmr spectroscopy was used by wenter et al. to determine refolding rates of a short -nt rna molecule [ ] . the formation of its most stable helix was temporarily inhibited by a modified guanosine at position . after photolytic removal of this modification a structure refolding was observed. to map such forced structure formation to relatively unstable folds at the beginning of an experiment, most rna folding tools have the capability to initialise simulations with specified structures. we used, much closer to the original wetlab experiment, a different strategy, i.e. at first g was not capable to bind any other base. the time course after removing this prohibition during simulation is shown in figure . wenter et al. detected structure refolding by measuring the imino proton signal intensity of u and u , which show high signal intensity if they are paired with other bases. accordingly we observed the state of both uracils over simulation time as well. after removal of g unpaired locking, a logarithmic decrease of structures with paired u and uniform increase of folds fig. . refolding of an deliberately misfolded small rna molecule [ ] . wetlab measurements are drawn by a x. simulation parameters: time seconds, temperature . k, base pair formation model , replications. simulations with kinfold were made with the same temperature and replication number but over a time period of seconds. with paired u can be observed reaching a complete shift of the conformational equilibrium after seconds. a very similar refolding progression was experimentally measured (single spots in figure ), but with a strong deviating time scale of factor . this could be a remaining model parameter inaccuracy or due to special experimental conditions which are not covered by our model, e.g. unusual salt concentrations. however, our model allows a quite realistic refolding from a suboptimal to a more stable rna structure. identical in silico experiments with kinfold [ ] by contrast, do not show any significant refolding (figure , nonchanging curves). the same counts for seqfold [ ] . with both approaches the energy barrier seems to be too high to escape from the misfolded suboptimal structure. local optima can more easily be overcome in comparison to other traditional pure macro methods (see figure ). we assume that even "stable" base pairs might be subject to changes, and let the nucleotides "searching" for a stable structure at micro level. this proved beneficial and emphasised the role of the micro level. however, the simulation revealed the importance of macro constraints for the folding process, and the implications of a lack of those. macro constraints that have been considered are for example the relative positioning of the nucleotides, particularly within spatial structures like hairpin or internal loops. the interplay between macro and micro level allowed us to reproduce many of the expected structure elements, e.g. figures and , although macro constraints have been significantly relaxed. these simplifications lead to "wrongly" formed structures and maybe could have been prevented by integrating terminal base stacking for pairing stability as well as less abstract base pair closing rules as macro constraints. a comparison of the two implemented base pair formation methods indicate only few differences. without taking entropy into account the noise of unstable single base pairs and short helices increases, but not dramatically. the same stable structures are formed based on both rules. having a working folding model we are now able to combine it with other model components that are influenced by or are influencing the rna structure formation and come back to the motivation, the attenuation of tryptophan synthesis. at least two further models are needed to reflect transcription attenuation: the rna polymerase and the ribosome (figure ). rna molecules are products of the transcription process which is the fundamental step in gene expression. once the rna polymerase enzyme complex (rnap) has successfully bound to dna (transcription initiation), it transcribes the template sequence into an rna strand by sequentially adding nucleotides to the ' end and thus elongates the molecule. to reflect this synthesising process, in the rna model, new nucleotide models and their backbone connections are added dynamically during simulation. this process is triggered by the rnap model component which interacts with rna. this dynamic rna elongation allows the simulation of sequential folding, where early synthesised parts of the rna molecule can already fold whereas other parts still have to be added. please note that this is not a unique feature of the model presented here, as kinetic folding tools typically realise sequential folding by just adding a new nt after a certain time delay. however, a component design allows to combine the rnap with further models (e.g. the dna template), or to model it in more detail (e.g. diverse rnap subunits), and to exchange model components on demand. the pattern observation function of the rna folding model, which is realised at macro level, allows us to look for an intrinsic transcription termination structure [ ] during simulation. if such structure is formed, the folding model removes its elongation input port meaning the release of the rna from the polymerase enzyme. at this time point the elongation stops, but structure folding and interactions with other components proceed. the ribosome enzyme complex translates rna sequences into protein determining amino acid sequences (peptides). translation starts at the ribosome binding site of mrna molecules which is reflected by a pair of input and output ports of the rna models. the translation begins after connecting it with a ribosome model. the current ribosome position with respect to the rna sequence is known by the rna model. a triplet of three rna bases (codon) encodes for one amino acid. the ribosome requests for the next codon ' of its current rna location when peptide chain elongation has proceeded. this is the case when the correct amino acid of the last codon entered the enzyme. the speed of the translation process depends strongly on the availability of needed amino acids. if an amino acid type is not sufficiently available, the ribosome stalls at the corresponding codon and thus pauses translation. a ribosome is quite big and thus - nucleotides are covered by its shape [ ] . therefore, a region upstream and downstream of the ribosome location is not able to form base pairs. as the rna model knows the ribosome location, this is handled by the rna macro level model which sends corresponding events to its nucleotide micro model components. the same counts for the helicase activity of the ribosome [ ] . for sequence translation, the macro level model will disrupt a base paired structure element when it is reached by the enzyme. whether those additional models are realised as atomic models, or coupled models depends on the objective of the simulation study. referring to the operon model presented in [ ] , the rnap, the mrna, and the ribosome would replace the simplistic mrna model, to integrate the attenuation process into the model. we presented a component-based model of rna folding processes. unlike traditional approaches which focus on the results of the folding process, e.g. stable structures in thermodynamical equilibrium, our objective has been different. the idea was to develop an approach that allows to integrate the folding processes into larger models and to take the dynamics into account, that has shown to be crucial in many regulation processes. therefore, the formalism ml-devs was used. at macro level, certain constraints referring to space and individual locations were introduced, whereas at micro level, the nucleotides were responsible for a successful base pairing and for the stability of the structure. a model component for the nucleotides and one model component for the entire rna molecule have been defined. the simulation results have been compared to wetlab experiments. therefore, the model components can be parametrised for different rna sequences (base types) as well as environmental conditions (e.g temperature). the evaluation revealed an overall acceptable performance, and in addition, insights into the role of micro level dynamics and macro level constraints. the integration of the rna folding model into a model of transcription attenuation has been sketched. next steps will be to realise this integration and to execute simulation experiments to analyse the impact of this more detailed regulation model on the synthesis of tryptophan. translation initiation and the fate of bacterial mrnas the mechanism of intrinsic transcription termination transcription attenuation: once viewed as a novel regulatory strategy genetic control by a metabolite binding mrna multiple structural alignment and clustering of rna sequences secondary structure prediction for aligned rna sequences mfold web server for nucleic acid folding and hybridization prediction fast folding and comparison of rna secondary structures a dynamic programming algorithm for rna structure prediction including pseudoknots prediction and statistics of pseudoknots in rna structures using exactly clustered stochastic simulations description of rna folding by "simulated annealing rna folding at elementary step resolution beyond energy minimization: approaches to the kinetic folding of rna dynamic regulation of the tryptophan operon: a modeling study and comparison with experimental data a variable structure model -the tryptophan operon rna hairpin loop stability depends on closing base pair coaxial stacking of helixes enhances binding of oligoribonucleotides and improves predictions of rna folding rapid compaction during rna folding combining micro and macro-modeling in devs for computational biology theory of modeling and simulation one modelling formalism & simulator is not enough! a perspective for computational biology based on james ii single-molecule rna folding rna folding at millisecond intervals by synchrotron hydroxyl radical footprinting a single-molecule study of rna catalysis and folding a cis-acting replication element in the sequence encoding the ns b rna-dependent rna polymerase is required for hepatitis c virus rna replication transfer rna: molecular structure, sequence, and properties the crystal structure of trna a common rna motif in the ' end of the genomes of astroviruses, avian infectious bronchitis virus and an equine rhinovirus the structure of a rigorously conserved rna element within the sars virus genome bistable secondary structures of small rnas and their structural probing by comparative imino proton nmr spectroscopy kinetics of photoinduced rna refolding by real-time nmr spectroscopy mrna helicase activity of the ribosome acknowledgments. many thanks to adelinde m. uhrmacher for her helpful comments and advice on this work. i will also thank roland ewald and jan himmelspach for their instructions for using james ii. the research has been funded by the german research foundation (dfg). key: cord- -vbchjexm authors: sarikavak-lisesivdin, b.; lisesivdin, s.b.; ozbay, e.; jelezko, f. title: structural parameters and electronic properties of d carbon allotrope: graphene with a kagome lattice structure date: - - journal: chem phys lett doi: . /j.cplett. . sha: doc_id: cord_uid: vbchjexm in this paper, the electronic properties of a carbon allotrope, graphene with a kagome lattice structure, are investigated. spin-polarized density functional theory (dft) calculations with grimme dispersion corrections were done. bond lengths, electronic band structure, and projected density of states were calculated. electronic band structure calculations show kagome flat-band formation with higher d-orbital contributed bonding behavior than the pristine graphene structure. the structural parameters and electronic band results of this d carbon allotrope show wider possible usage in many applications from desalination membranes to possible high-temperature superconductors. because of its unique properties and possible promising applications, graphene has attracted an important amount of attention both experimentally and theoretically since its first experimental isolation and characterization reported by novoselov et al. [ ] . with the triggered interest in graphene, searching for different carbon nanomaterials, graphene-derivatives and allotropes have also gained huge interest [ , ]. these new carbon materials have superior mechanical, electronic, and thermal features, which makes them implemented in many applications of semiconductor devices, such as electronic components [ ] , and gas sensors [ ] . in addition to these small device applications, new carbon materials, especially graphene and graphene oxide with pores, are proposed to be used as a nanofiltration membrane for desalination, drinking water production, water reuse and wastewater treatment applications [ , ] . also, on reusable or single-use personal protective equipment (ppe) or filters in air purification and air-conditioning devices, coated with modified nanomaterials like graphene or graphene-oxide with pores can enhance the repellent sars-cov- or similar viruses [ ] . with the covid- outbreak, it is well understood that the usage of enhanced filters is necessarily required to prevent aerosol transmission even in daily life. [ ] , alpha-graphyne [ ] , twin-graphene [ ] , thd-graphene [ ] , dhp-graphene [ ] , tetrahexcarbon [ ] , net y carbon [ ] and d-carbon [ ] can be listed with respect to their date of report. in addition to these theoretical studies, t-carbon was experimentally observed as t-carbon nanowires in zhang et al.'s study in [ ] . and in , d-carbon was suggested to be experimentally possible due to satisfactorily match of previously unexplained x-ray diffraction (xrd) peak of a measured chimney or detonation soot [ , ] powder to calculated d-carbon xrd peaks [ ] . with these recent signs of progress and possible future developments in experimental studies of new carbon allotropes, the electronic properties of the carbon allotrope with a kagome lattice [ ] [ ] [ ] , and kagome-like d metamaterials [ ] took attention recently. in this study, the electronic properties and the structural parameters of graphene with a kagome lattice structure are investigated with implementing the density functional theory (dft) method. our dft calculations are carried out using the atomistic toolkit (atk) software v. . [ ] . spin-polarized generalized gradient approximation (sgga) was used for exchange-correlation (xc) functional and the double-ζ (dz) orbital basis set is adopted. for sgga calculations, perdew, burke, and ernzerhof (pbe) [ ] functionals were used purely and also with grimme d dispersion correction. for further calculations, rpbe functional [ ] was used with grimme d dispersion correction. are separated with a Å vacuum space; therefore the whole structure is accepted to be a twodimensional structure. for the geometrical optimization, the maximum force on each atom is set to be smaller than . ev/Å. the optimized structural parameters for the investigated structure with sgga methods are summarized in table . the c-c bond length between cp-like structures (d ~ . Å) exhibits a featured characteristic of the double bond. the c-c bonds of cp-like structure (d ~ . Å) represent similarity with sp -sp or sp bonding. however, unconventional bent bond angles of o and o raise questions for explaining the bonds with known hybridizations (trigonal sp or sp -sp doublets) [ ] and needs a better explanation. also, bent bonds are known to result in the possibility of high chemical reactivity [ ] . the calculated bond angles are not given in table , because of obtaining perfect o and o for all three methods. the diameter of the dodecagon pores is also calculated and listed in table . as can be seen from table , the pore diameter is found smaller with the sgga-pbe method. all methods result ~ . Å of diameter, which is a suitable value for a salt filtration application. the salt rejection ability of a graphene-like membrane is known to decrease with an increase in pore size [ ] . the study of cohen-tanugi and grossman shows that most salt ions can pass through graphene with pores of diameter above . Å [ ] . therefore, the pore diameter of graphene-like d carbon allotropes should be below this value to successfully filter na + and cl − ions from water, where graphene with a kagome lattice structure fulfills this requirement and also have a very high porosity value of ~ %. with the possible experimental proof in the future, graphene membranes with a kagome lattice structure having a very high porosity value may open the possibility of the low pressure or passive desalination applications and also can be used on reusable or single-use ppe or on filters in air purification and air-conditioning devices to prevent virus transmission. graphene is already suggested as single flakes and sheet with defects in these applications and with the possible experimental confirmation of graphene with kagome lattice structure, there will be no need for introducing defects in the structure for increasing selective permeability. figure shows the spin-dependent electronic band structure of the graphene with a kagome lattice structure. band structure in the figure is calculated with sgga-pbe. however, other methods gave similar results and will be discussed shortly after. as seen in figure , the band structure has a similar behavior for spin-up and spin-down electrons. therefore, it is seen that the non-interacting pristine graphene with a kagome lattice structure shows a very small magnetic nature as the pristine graphene sheet. however, it is already shown that the dft calculations of graphene with a kagome lattice structure with doping by reducing the density of valence electrons (hole doping) makes the flat-band partially filled [ ] . this type of doping can be done with the electrostatic gating, and it is highly known that graphene has a similar behavior [ ] . this type of doping results in a ferromagnetic flat-band formation where it is possible to explore new exotic phases by tuning the filling factor of the flat-band. there is a graphene-like linear band formation at k-point. however, it is not located at the fermi level (e f ). also, it is clear that the kagome flat-bands emerge around the e f [ e ]. this kagome flat-bands are important because the electrons are confined in a very narrow energy interval with a huge number of density of states (dos) which may be related with wignercrystallization [ , ] , flat-band ferromagnetism [ ] , fractional quantum hall states [ ] and a possibility of a high-temperature-superconductivity [ ] . in figure to understand the nature of the bonds in the graphene with a kagome lattice structure, dos is projected onto orbitals of carbon atoms for sgga-pbe calculations as shown in figure (a). the main peak at the e f is mostly consisting of electronic states of the p z orbital (l= , m l = ). the main peak at the e f is also consisting of electronic states of d xz and d yz orbitals (l= , m l =± ). p z do hybridize with d xz and d yz orbitals, forming together the π band [ ] . the contribution of d xz and d yz orbitals to the total electronic states at e f is more significant in graphene with a kagome lattice structure than the pristine graphene structure. with the same calculation parameters, the total d-orbital states near to dirac point in pristine graphene structure are found to be % of total p z + d xz + d yz states. however, in graphene with a kagome lattice structure, the total d-orbital states at e f is found to be % as can be seen in figure (b). from figure (b) the long-range unvarying π bonding (p z hybrid with d xz and d yz orbitals) behavior can be observed between - ev and also can be observed in the near vicinity of dirac point which is located at . ev. it is important to point that the density of states with d xz and d yz orbitals follows in shape that of pz, which results the long-range unvarying π bonding mentioned above. the similarity of the shape of density of states of d xz and d yz orbitals to the pz, is found to in agreement with gmitra et al.'s study for the pristine graphene [ ] . in this study, structural parameters and the electronic band structure of graphene with kagome lattice structure is theoretically studied. spin-polarized dft calculations were done with sgga-pbe, sgga-pbe with grimme d correction, and sgga-rpbe with grimme d correction methods. all methods resulted in similar bond lengths and similar electronic band structure and density of state distributions. a kagome flat-band structure is observed near the e f and high number of electronic states at this kagome flat-band is mostly consisting of p z orbital and also d xz and d yz orbital contributions with a π bonding behavior. also, d-orbital contribution near k-point in graphene with a kagome lattice structure is found to be larger than the related contribution observed in the pristine graphene. the reason for the high contribution of d-orbital states between - ev needs more quantitative investigation that the plane-wave approach can be utilized in addition to the already implemented linear combination of the atomic orbitals approach. this d carbon allotrope, which has a novel electronic structure, can be a promising structure for many possible electronic device applications or it may be used mechanically in desalination applications because of suitable pore size and density of dodecagons in its structure.  structure parameters and electronic properties of graphene with kagome lattice structure is calculated,  various xc and dispersion correction methods are used and similar results are obtained,  pristine graphene with kagome lattice structure shows very small magnetic properties,  a kagome flat-band structure is observed near the e f .  high number of electronic states at this kagome flat-band is mostly consisting of p z orbital and also d xz and d yz orbital contributions with a π bonding behavior. we wish to confirm that there is no known conflict of interest associated with its publication and there has been no significant financial support for this work that could have influenced its outcome. sincerely, on behalf of all authors prof. sefer bora lisesivdin, phd corresponding author penta-graphene: a new carbon allotrope origins of dirac cones and parity dependent electronic structures of α-graphyne derivatives and silagraphynes twin graphene: a novel two-dimensional semiconducting carbon allotrope thd-graphene used for a selective gas detector encapsulation of cathode in lithium-sulfur batteries with a novel two-dimensional carbon allotrope: dhp-graphene tetrahexcarbon: a two-dimensional allotrope of carbon planar metallic carbon allotrope from graphene-like nanoribbons d-carbon: ab initio study of a novel carbon allotrope pseudo-topotactic conversion of carbon nanotubes to t-carbon nanowires under picosecond laser irradiation in methanol characterization of the condensed carbon in detonation soot a morphological investigation of soot produced by the detonation of munitions ferromagnetism and wigner crystallization in kagome graphene and related structures three-dimensional kagome graphene networks and their topological properties critical topological nodal points and nodal lines/rings in kagome graphene strain-controlled magnetic ordering in d carbon metamaterials quantumatk: an integrated platform of electronic and atomic-scale modelling tools generalized gradient approximation made simple improved adsorption energetics within density-functional theory using revised perdew-burke-ernzerhof functionals g ab initio calculations of the enthalpies of formation of c hydrocarbons a unified theory of bonding for cyclopropanes structure and reactivity of cyclopropane and its derivatives, rus water desalination across nanoporous graphene electrostatic confinement of electrons in graphene nanoribbons p x,y -orbital counterpart of graphene: cold atoms in the honeycomb optical lattice from nagaoka's ferromagnetism to flat-band ferromagnetism and beyond: an introduction to ferromagnetism in the hubbard model fractional quantum hall states at zero magnetic field bcs theory on a flat band lattice tight-binding theory of the spin-orbit coupling in graphene band-structure topologies of graphene: spin-orbit coupling effects from first principles credit author statement beyza sarikavak-lisesivdin: conceptualization; formal analysis; methodology; writingoriginal draft sefer bora lisesivdin: visualization. writing -original draft. ekmel ozbay: software; project administration; supervision. fedor jelezko: resources; supervision key: cord- -df luh v authors: dos santos-silva, carlos andré; zupin, luisa; oliveira-lima, marx; vilela, lívia maria batista; bezerra-neto, joão pacifico; ferreira-neto, josé ribamar; ferreira, josé diogo cavalcanti; de oliveira-silva, roberta lane; pires, carolline de jesús; aburjaile, flavia figueira; de oliveira, marianne firmino; kido, ederson akio; crovella, sergio; benko-iseppon, ana maria title: plant antimicrobial peptides: state of the art, in silico prediction and perspectives in the omics era date: - - journal: bioinform biol insights doi: . / sha: doc_id: cord_uid: df luh v even before the perception or interaction with pathogens, plants rely on constitutively guardian molecules, often specific to tissue or stage, with further expression after contact with the pathogen. these guardians include small molecules as antimicrobial peptides (amps), generally cysteine-rich, functioning to prevent pathogen establishment. some of these amps are shared among eukaryotes (eg, defensins and cyclotides), others are plant specific (eg, snakins), while some are specific to certain plant families (such as heveins). when compared with other organisms, plants tend to present a higher amount of amp isoforms due to gene duplications or polyploidy, an occurrence possibly also associated with the sessile habit of plants, which prevents them from evading biotic and environmental stresses. therefore, plants arise as a rich resource for new amps. as these molecules are difficult to retrieve from databases using simple sequence alignments, a description of their characteristics and in silico (bioinformatics) approaches used to retrieve them is provided, considering resources and databases available. the possibilities and applications based on tools versus database approaches are considerable and have been so far underestimated. proteins and peptides play different roles depending on their amino acids (aa) constitution, which may vary from tens to thousands residues. peptides are conventionally understood as having less than aa. proteins, on the contrary, would be any molecule presenting higher amino acid content and bothproteins and peptides-present a plethora of variations in plants. despite that, plant proteomes have been much more studied than peptidomes. it is well-known that the biochemical machinery necessary for the synthesis and metabolism of peptides is present in every living organism. from the variations of this machinery, a wide structural and functional diversity of peptides was generated, justifying the growing interest in their study. in eukaryotes, peptides are prevalent in intercellular communication, performing as hormones, growth factors, and neuropeptides, but they are also present in the defense system. besides plants and animals, several pathogenic microorganisms, peptides can serve as classical virulence factors, which disrupt the epithelial barrier, damage cells, and activate or modulate host immune responses. an example of this performance is represented by candidalysin, a fungal cytolytic peptide toxin found in the pathogenic fungus candida albicans that damages epithelial membranes, triggers a response signaling pathway, and activates epithelial immunity. there are also reports of defense-related fungal peptides. for example, the copsin, a peptide-based fungal antibiotic recently identified in the fungus coprinopsis cinerea kills bacteria by inhibiting their cell wall synthesis. regarding bacterial peptides, certain species from the gastrointestinal microbial community can release low-molecular-weight peptides, able to trigger immune responses. there are additionally peptides that act like bacterial "hormones" that allow bacterial communities to organize multicellular behavior such as biofilm formation. some peptides are known for their medical importance, as defensins that bioinformatics and biology insights present antibacterial, antiviral, and antifungal activities. for example, human alpha-and beta-defensins present in the saliva may potentially impede virus replication, including sars-cov- , besides other roles as protection against intestinal inflammation (colitis). considering the roles of plant peptides, they can also be multifunctional, and have been classified into main categories (supplementary figure s ) : ( ) peptides with no bioactivity, primarily resulting from the degradation of proteins by proteolytic enzymes, aiming at their recycling, and ( ) bioactive peptides, which are encrypted in the structure of the parent proteins and are released mainly by enzymatic processes. the first group is innocuous regarding signaling, regulatory functions, and bioactivity. so far, it has been reported that some of them may play a significant role in nitrogen mobilization across cellular membranes. the second group of bioactive peptides has a substantial impact on the plant cell physiology. some peptides of this group can act in the plant growth regulation (through cell-to-cell signaling), endurance against pathogens and pests by acting as toxins or elicitors, or even detoxification of heavy metals by ion-sequestration. comprising bioactive peptides, additional subcategorization has been proposed regarding their function. tavormina et al figure s ) based on the type of precursor: • • derived from functional precursors: originated from a functional precursor protein; • • derived from nonfunctional precursors: originated from a longer precursor that has no known biological function (as a preprotein, proprotein, or preproprotein); • • not derived from a precursor protein: some sorfs (small open read frames; usually < codons) are considered to represent a potential new source of functional peptides (known as "short peptides encoded by sorfs"). a more intuitive classification of bioactive peptides was further proposed by farrokhi et al receptors in leaves. another example is the pls (polaris) peptide that acts during early embryogenesis but later activates auxin synthesis, also affecting cytokine synthesis and ethylene response. regarding the second group, it includes peptides with signaling roles in plant defense, comprising at least subgroups, including syst (systemin) (supplementary figure s ). the syst peptides were identified in solanaceae members, like tomato and potato (acting on the signaling response to herbivory). the syst leads to the production of a plant protease inhibitor that suppresses insect's proteases. stratmann suggested that in plants, systs act to stimulate the jasmonic acid signaling cascade within vascular tissues to induce a systemic wound response. • • defense peptides or antimicrobial peptides (amps): to be fitted into this class, a plant peptide must fulfill some specific biochemical and genetic prerequisites. regarding biochemical features, in vitro antimicrobial activity is required. concerning the genetic condition, the gene encoding the peptide should be inducted in the presence of infectious agents. in practice, this last requirement is not ever fulfilled as some amps are tissue-specific and are considered as part of the plant innate immunity, while other isoforms of the same class appear induced after pathogen inoculation. plant amps are the central focus of the present review, comprising information on their structural features (at genomic, gene, and protein levels), resources, and bioinformatic tools available, besides the proposition of an annotation routine. their biotechnological potential is also highlighted in the generation of both transgenic plants resistant to pathogens, and new drugs or bioactive compounds. antimicrobial peptides are ubiquitous host defense weapons against microbial pathogens. the overall plant amp characterization regards the following variables ( figure ): electrical charge, hydrophilicity, secondary and -dimensional ( d) structures, and the abundance or spatial pattern of cysteine residues. these features are primarily related to their defensive role(s) as membrane-active antifungal, antibacterial, or antiviral peptides. regarding the nucleotide sequence, plant amps are hypervariable and this genetic variability is considered crucial to provide diversity and the ability to recognize different targets. for their charges, amps can be classified as cationic or anionic ( figure ). most plant amps have positive charges, which is a fundamental feature for the interaction with the membrane lipids of pathogens. concerning hydrophilicity, amps are generally amphipathic, that is, they exhibit molecular conformation with both hydrophilic and hydrophobic domains. silva et al with respect to their d structure, amps can be either linear or cyclic ( figure ). some linear amps adopt an amphipathic α-helical conformation, whereas non-α-helical linear peptides generally show or predominant amino acids. in turn, cyclic amps, including cysteine-containing peptides, can be divided into subgroups based on the presence of a single or multiple disulfide bonds. a peculiar feature of these peptides is a cationic and amphipathic character, what improves their functioning as membrane-permeabilizing agents. considering the secondary structures, amps may exhibit α-helices, β-chains, β-pleated sheets, and loops ( figure ). wang classified plant amps into families (α, β, αβ, and non-αβ), based on the protein classification of murzin et al, with some modifications. antimicrobial peptides of the "α" family present α-helical structures, whereas amps from the "β" family contains β-sheet structures usually stabilized by disulfide bonds. , some plant amps show an α-hairpinin motif formed by antiparallel α-helices that are stabilized by disulfide bridges. such amps present a higher resistance to enzymatic, chemical, or thermal degradation. antimicrobial peptides from the "αβ" family having both "α" and "β" structures are also stabilized by disulfide bridges. an example of amp presenting "αβ" structures are defensins, usually with a cysteine-stabilized αβ motif (csαβ), an α-helix, and a triplestranded antiparallel β sheet stabilized mostly by disulfide bonds. finally, amps that do not belong to the "αβ" group exhibit no clearly defined "α" or "β" structures. plant amps are also classified into families considering protein sequence similarity, cysteine motifs, and distinctive patterns of disulfide bonds, which determine the folding of the tertiary structure. therefore, plant amps are commonly grouped as thionins, defensins, heveins, knottins (linear and cyclic), lipid transfer proteins (ltp), snakins, and cyclotides. , these amp categories will be detailed in the next sections, together with other groups here considered (impatienlike, macadamia [β-barrelins], puroindoline (pin), and thaumatin-like protein [tlp]) and the recently described αhairpinin amps. the description includes comments on their structure, pattern for regular expression (regex) analysis (when available), functions, tissue-specificity, and scientific data availability. thionins are composed by to amino acid residues with a molecular weight around kda, considering the mature peptide. they are synthesized with a signal peptide together with the mature thionin and the so-called acidic domain. to date, there is no experimental information available about possible functions of the acidic domain, even though it is clearly not dispensable as shown by the high conservation of the cysteine residues. the thionin superfamily comprises distinct groups of plant peptides α/β-thionins and γ-thionins with distinguished structural features. the α/β thionins have homologous amino acid sequences and similar structures. besides, they are rich in arginine, lysine, and cysteine. in turn, γ-thionins have a greater similarity with defensins, and some authors classify them within this group. however, compared with the defensins, they present a longer conserved amino acid sequence. regarding the cysteine motif, it can be divided into subgroups, one with residues connected by disulfide bonds called c and the other with residues connected by disulfide bonds called c. the general designation of thionins has been proposed as a family of homologous peptides that includes purothionins. the first plant thionin was isolated in from wheat flour and labeled as purothionin. since then, homologues from various taxa have been also identified, like bioinformatics and biology insights viscotoxins (viscum album) and crambins (crambe abyssinica). they have also been isolated from different plant tissues like seeds, leaves, and roots. , thionins have been tested for different elicitors: grampositive , or gram-negative bacteria, , yeast, , insect larvae, nematode, and inhibitory proteinase. thionins are hydrophobic in nature, interact with hydrophobic residues, and lyse bacteria cell membrane. their toxicity is due to an electrostatic interaction with the negatively charged membrane phospholipids, followed by either pore formation or a specific interaction with a membrane. it has been reported that they are able to inhibit other enzymes possibly through covalent attachment mediated by the formation of disulfide bonds, as previously observed for other thionin/enzyme combinations. thionin representatives with known d structures determined by x-ray crystallography are crambin (pdb id: crn), α and β-purothionins (pdb id: phn and bhp), β-hordothionin (pdb id: wuw), and viscotoxin-a (pdb id: okh). the first to be determined was the mixed form of crambin. , it showed a distinct capital Γ shape with the n terminus forming the first strand in a βsheet. the architecture of this sheet is additionally strengthened by disulfide bonds. after a short stretch of extended conformation, there is a helix-turn-helix motif. in crambin, there is a single disulfide involved in stabilizing the helix-tohelix contacts. at the center of this motif, there is a crucial arg that forms hydrogen bonds to tie together the first strand, the first helix, and the c terminus. the first plant defensins were isolated from wheat and barley grains, initially called γ-hordothionins. due to some similarities in cysteine content and molecular weight, they were classified as γ-thionins. later, the term "γ-thionin" was replaced by "defensin" based on the higher number of primary and tertiary structures of these proteins and also on their antifungal activities more related to insect and mammalian defensins than to plant thionins. plant defensins belong to a diverse protein superfamily called cis-defensin and exhibit cationic charge, consisting of to aa with to disulfide bonds. , plant defensins share similar tertiary structures and typically exhibit a triple-stranded antiparallel β sheet, enveloped by an α-helix and confined by intramolecular disulfide bonds (figure a ). this motif is called cysteine-stabilized αβ (csαβ). the csαβ defensins were classified into groups based on their sequence, structure, and functional similarity. defensins are known for their antimicrobial activity at low micromolar concentrations against gram-positive and gramnegative bacteria, fungi, viruses, and protozoa. in addition, they present protein inhibition, insecticidal, and antiproliferative activity, acting as an ion-channel blocker, being also associated with the inhibition of pathogen protein synthesis. instead, plant defensins act in the regulation of signal transduction pathways and induce inflammatory processes, in addition to wound healing, proliferation control, and chemotaxis. in general, plant defensins do not present high toxicity to human cells, having in vivo efficacy records, with relevant therapeutic potential, and can be applied in treatments associated with traditional medicine. cools et al reported that a peptide derived from a plant defensin (hsafp ) acted synergistically with caspofungin (an antimycotic) (in vivo and in vitro) against the formation of candida albicans biofilm on polystyrene and catheter substrate, indicating that the hsafp variant presented a strong antifungal potential in the proposed treatment. other biotechnological applications of defensins are described, as in the case of ecgdf , which was isolated from a legume (erythrina crista-galli), heterologously expressed in escherichia coli and purified. ecgdf inhibited the growth of various plant and human pathogens (such as candida albicans and aspergillus niger and the plant pathogens clavibacter michiganensis ssp. michiganensis, penicillium expansum, botrytis cinerea and alternaria alternate). due to these features, ecgdf is a candidate for the development of antimicrobial products for both agriculture and medicine. non-specific lipid transfer proteins (ns-ltps) were first isolated from potato tubers and are actually identified in diverse terrestrial plant species. they comprise a large gene family, are abundantly expressed in most tissues, but absent in most basal plant groups as chlorophyte and charophyte green algae. they generally include an n-terminal signal peptide that directs the protein to the apoplastic space. some ltps have a c-terminal sequence that allows their post-translational modification with a glycosylphosphatidylinositol molecule, facilitating the integration of ltp on the extracellular side of the plasma membrane. the ns-ltps are small proteins which were thus named because of their function of transferring lipids between the different membranes carrying lipids (non-specifically, the list includes phospholipids, fatty acids, their acylcoas, or sterols). they consist of approximately aa and are relatively larger in size than other amps, such as defensins. depending on their sizes, ltps may be classified into subfamilies: ltp and ltp , with relative molecular weight of and kda, respectively. , the limited sequence conservation turned this classification inadequate. thus, a modified and expanded classification system was proposed, presenting main types (ltp , ltp , ltpc, ltpd, and ltpg) and additional types with a smaller number of members (ltpe, ltpf, ltph, ltpj, and ltpk). the new classification system is not based on molecular size but rather on ( ) the position of a conserved intron, ( ) the identity of the amino acid sequence, and figure s ) . although this latter classification system is the most recent, the conventional classification of ltp and ltp types has been maintained by most working groups. lipid transfer protein nomenclature has been confusing and without consistent guidelines or standards. there are several examples where specific ltps receive different names in different scientific articles. the lack of a robust terminology sometimes turns it quite difficult, extremely time-consuming, and frustrating to compare ltps with different roles/functions. therefore, an additional nomenclature was also proposed by salminen et al, naming ltps as follows: atltp . , osltp . , hvltpc , ppltpd , and taltpg , with the first letters indicating the plant species (eg, at = arabidopsis thaliana, pp = physcomitrella patens); ltp , ltp , and ltpc indicating the type; while the last digit (here - ) regards the specific number given to each gene or protein within a given ltp type. for the sake of clarity, the authors recommend the inclusion of a point between the type specification (ltp and ltp ) and the gene number. for ltpc, ltpd, ltpg, and other types of ltp defined with a letter, the punctuation mark was not recommended. this latter classification system is currently recommended as it comprises several features of ltps and is more robust than the previous classification systems. lipid transfer proteins are small cysteine-rich proteins, having to helices in their tertiary structure ( figure b ), which is stabilized by several hydrogen bonds. such a folding gives ltps a hydrophobic cavity to bind the lipids through hydrophobic interactions. this structure is stabilized by disulfide bridges formed by conserved cysteines, similar to defensins, although bound by cysteines in different positions. the disulfide bridges promote ltp folding into a very compact structure, which is extremely stable at different temperatures and denaturing agents. [ ] [ ] [ ] these foldings provide a different specificity of lipid binding at the ltp binding site, where the ltp structure is relatively more flexible and present a lower lipid specificity when compared with ltp . the first d structure of an ltp was established for taltp . based on d and d data of h-nmr, purified from wheat (triticum aestivum) seeds in aqueous solution. , currently, several d structures of ltps have been determined, either by nuclear magnetic resonance (nmr) or x-ray crystallography; in their free, unbound form or in a complex with ligands. the heveins were first identified in in the rubber-tree (hevea brasiliensis), but its sequence was determined later, whereas a similarity was detected to the chitin-binding domain of an agglutinin isolated from urtica dioica (l.) with cysteine residues forming a typical cys motif. the primary structure of the hevein consists of to aa, positively charged, with abundant glycine ( ) and cysteine ( - ) residues, and aromatic residues. , the chitin-binding domain is a determinant component in the identification of hevein-like peptides whose binding site is represented by the amino acid sequence sxfgy/sxygy, where x regards any amino acid. , most heveins have a coil-β -β -coil-β structure that occurs by variations with the secondary structural motif in the presence of turns in long coils in the β chain. antiparallel β chains form the central β sheet of the hevein motif with long coils stabilized by disulfide bonds ( figure c ). although the presence of chitin has not been identified in plants, there are chitin-like structures present in proteins that exhibit a strong affinity to this polysaccharide isolated from different plant sources. the presence of aromatic amino acids in the chitinbinding domain favors chitin binding by providing stability to the hydrophobic group c-h and the π electron system through van der waals forces, as well as the hydrogen bonds between serine and n-acetylglucosamine (glcnac) present in the chitin structure. , this domain is commonly found in chitinases of classes i to v, in addition to other plant antimicrobial proteins, such as lectins and pr- (pathogenesis-related protein ) members. , it may also occur in other proteins that bind to polysaccharide chitin, such as the antimicrobial proteins ac-amp and ac-amp of amaranthus caudatus (amaranthaceae) seeds which are homologous to hevein but lack the c-terminal glycosylated region. plant chitinases (class i) have the hevein-like domains, called hlds. due to the similar structural epitopes between chitinases and heveins, they are responsible for the cross reactive syndrome (latex-fruit syndrome). , among the several classes of proteins mentioned, the proteins with a high degree of similarity to hevein are chitinases i and iv. chitinases are known to play an essential role in plant defense against pathogens, also inhibiting in vitro fungal growth, especially when combined with β- , -glucanases. it also interferes with the growth of hyphae, resulting in abnormal ramification, delay, and swelling in their stretching. however, it has been shown that heveins have a higher inhibitory potential than chitinases and that their antifungal effect is not related only to the presence of chitinases ; pn-amp and pn-amp amps with hevein domains have potent antifungal activities against a broad spectrum of fungi, including those without chitin in their cell walls. , modes of action of chitinases usually include degradation and disruption of the fungal cell wall and plasma membrane due to its hydrolytic action, causing extravasation of plasma particles. , therefore, heveins have good antifungal activity, and only a few are active against bacteria, most of them with low activity. another role of hevein chitinases regards the antagonistic effect in triggering the aggregation of rubber particles in the latex extraction process in rubber trees. unlike heveins, other chitinases inhibit rubber particle aggregation. however, its action in conjunction with other proteins (β- , -glucanase) increases the effect of β- , -glucanase on rubber particle aggregation. a study by shi et al found that the interaction of the protein network related to the antipathogenic activity released by lutoids (lysosomal microvacuole in latex) is essential in closing laticiferous cells (cells that produce and store latex), not only providing a physical barrier, but a biochemical barrier used by laticiferous cells affected by pathogen invasion. knottins are part of the cysteine-rich peptides (crps) superfamily, sharing the cysteine-knot motif and therefore resembling other families as defensins, heveins, and cyclotides. their structure was initially identified by crystallography of carboxypeptides isolated from potato, showing the cysteine-knot motif with aa and cysteine residues. they are also called "cysteine-knot peptides," "inhibitor cysteine-knot peptides," or even "cysteine-knot miniproteins" because their mature peptide presents less than aa, forming interconnected disulfide bonds in the cysteine-knot motif, characterizing a particular scaffold. this conformation confers thermal stability at high temperatures. for example, the cysteine-stabilized β-sheet (csb) motif derived from knottins presents stability at approximately °c with only disulfide bonds. the knottins may have linear or cyclic conformation. however, both exhibit connectivity between the cysteines at positions - c, - c, and - c, forming a ring at the last bridge ( figure d ). knottins have different functions, such as signaling molecules, response against biotic and abiotic stresses, root growth, symbiotic interactions as well as antimicrobial activity against bacteria, fungi, virus, and insecticidal activity, among others. knottins antimicrobial activity has been attributed to the action of functional components of the plasma membrane, leading to alterations of lipids, ion flux, and exposed charge. the accumulation of peptides on the surface of the membrane results in the weakening of the pathogen membrane, resulting in transient and toroidal perforations. in the course of a large-scale survey to identify novel amps from australian plants, , an amp with no sequence homology was purified. its complementary dna (cdna) was cloned from macadamia integrifolia (proteaceae) seeds, containing the complete peptide coding region. the peptide was named miamp , being highly basic with an estimated isoelectric point (pi) of and a mass of kda. the miamp is aa long, including a aa signal peptide in the n-terminal region, bound to a aa mature region with cysteine residues. its d structure was determined using nmr spectroscopy, revealing a unique conformation among plant amps, with beta-strands arranged in greek key motifs, forming a greek key beta-barrel ( figure e ). due to its particularities, miamp was classified as a new structural family of plant amps, and the name β-barrelins was proposed for this class. this structural fold resembles a superfamily of proteins called γ-crystallin-like characterized by the precursors βγ-crystallin. this family includes amps from other organisms, for example, wmkt, a toxin produced by the wild yeast williopsis mraki. the miamp exhibited in vitro antimicrobial activity against various phytopathogenic fungi, oomycetes, and grampositive bacteria with a concentration range of . to μm generally required for a % growth inhibition (ic ). in addition, the transient expression of miamp in canola (brassica napus) provided resistance against blackleg disease caused by the fungus leptosphaeria maculans, turning miamp potentially useful for genetic engineering aiming at disease resistance in crop plants. there are few scientific publications with macadamia-like peptides, maybe because they prevail in primitive plant groups (eg, lycophytes, gymnosperms to early angiosperms as amborella and papaver), being apparently absent in derived angiosperms (eg, asteridae, including brassicaceae as arabidopsis thaliana). on the contrary, they have been identified in some monocots (as zantedeschia, zea, and sorghum). in fact, peptides similar to miamp appear to play a role in the defense against pathogens in gymnosperms, including species of economic importance (as pinus and picea) thus deserving attention for their biotechnological potential. four closely related amps (ib-amp , ib-amp , ib-amp , and ib-amp ) were isolated from seeds of impatiens balsamina (balsaminaceae) with antimicrobial activity against a variety of fungi and bacteria, and low toxicity to human cells in culture. these amps are the smallest isolated from plants to date, consisting of only aa in length. the ib-amps are highly basic and contain cysteine residues that form disulfide bonds. interestingly, they have no significant homology with other amps available in public databases. sequencing of cdnas isolated from i. balsamina revealed that all peptides are encoded within a single transcript. concerning the predicted precursor of ib-amp protein, it consists of a pre-peptide followed by mature peptide domains, each one of them flanked by propeptide domains ranging from to aa in length (supplementary figure s ) . this primary structure with repeated domains of alternating basic peptides and acid propeptide domains has, to date, not been reported in other plant species. patel et al conducted an experiment to purify ib-amp from seeds of impatiens balsamina. after purification, this peptide had its secondary structure tested by circular dichroism (cd). the results revealed a peptide that may include a β-turn but do not show evidences for either helical or β-sheet structure over a range of temperature and ph. structural information from d h-nmr was obtained in the form of proton-proton internuclear distances inferred from nuclear overhauser enhancements (noes) and dihedral angle restraints from spinspin coupling constants, which were used for distance geometry calculations. owing to the difficulty in obtaining the correct disulfide connectivity by chemical methods, the authors had built and performed separate calculations: ( ) a model with no disulfides; ( ) another with predicted disulfide bonds; and ( ) a model with alternative connectivity disulfide, as assigned from the nuclear overhauser effect spectroscopy (noesy) nmr spectra. as a result, hydrophilic patches were observed at opposite ends and opposite sides of the models, whereas in between them a large hydrophobic patch was identified. however, the study did not conclude which of the models would be the most likely representative of ib-amp , reporting only that cysteines are necessary for maintaining the structure. based on the experiment performed by patel et al, the present work built different models: model : without disulfide bonds, and the other models with different disulfide connections-model : nmr prediction by patel et al -cys; -cys and -cys; -cys, and model : disulfide bond partner prediction by dianna -cys; -cys and -cys; -cys. calculations have shown that although the peptide is small, the cysteines constrain part of it to adopt a well-defined main chain conformation. from residue to (except ), the main chain is well-defined, whereas residues to in the n-terminal region present few restrictions and appear to be more flexible (supplementary figure s ) . analyzing the rmsd (root mean square deviation), we observed that all the models lost the initial conformation and, among them, model was the most stable. models and showed a similar pattern (supplementary figure s ) , as in the models of patel et al, although model was the most flexible. little is known about impatiens-like amps mode of action. lee et al investigated the antifungal mechanism of ib-amp noting that when oxidized (bound by disulfide bridges), there occurs a -fold increase in antifungal activity against aspergillus flavus and candida albicans, as compared with reduced ib-amp (without disulfide bridges). confocal microscopy analyses have shown that ib-amp can either bind to the cell bioinformatics and biology insights surface or penetrate cell membranes, indicating an antifungal activity by inhibiting a distinct cellular process, rather than ion channel or membrane pore formation. fan et al reported the ib-amp antimicrobial activity dependent of β-sheet configuration to enable insertion into the lipid membrane, thus killing the bacteria through a non-lytic mechanism. current approaches aim to make changes in ib-amp to improve its antimicrobial activity. as an example, synthetic variants of ib-amp were fully active against yeasts and fungi, where the replacement of amino acid residues by arginine or tryptophan improved more than twice the antifungal activity. another study involving amp modification generated a synthetic peptide without the disulfide bridges (ie, a linear analog of ib-amp ), which showed an antimicrobial specificity . to . times higher than the wild-type ib-amp . puroindoline puroindolines are small basic proteins that contain a single domain rich in tryptophan. these proteins were isolated from wheat endosperm, have a molecular mass around kda, and a calculated isoelectric point higher than . at least main isoforms (called pin-a and pin-b) are known, which are encoded by pina-d and pinb-d genes, respectively. these genes share . % identical coding regions but exhibit only % identity in the ′ untranslated region. both pin-a and pin-b contain a structure with conserved cysteine residues and a tertiary structure similar to ltps, consisting of α-helices separated by loops of varying lengths, with the tertiary structure joined by disulfide bonds, of which identical to ns-ltps. the conformation of the pin isoforms was studied by infrared and raman spectroscopy. both pin-a and pin-b have similar secondary structures comprising approximately % helices, % β-sheets, and % non-ordered structures at ph . it has been proposed that the folding of both pins is highly dependent on the ph of the medium. the reduction of the disulfide bridges results in a decrease of pins solubility in water and to an increment of the β-sheet content by about % at the expense of the α-helix content. no high-resolution structure for any of the pin isoforms is available, bringing challenges to understanding the function of their hydrophobic regions, with some evidence coming only from partially homolog peptides. however, wilkinson et al proposed a theoretical model for several sequences of this amp. puroindolines are proposed to be functional components of wheat grain hardness loci, control core texture, besides antifungal activity. [ ] [ ] [ ] [ ] although the biological function of pins is unknown, their involvement in lipid binding has been proposed. while ltps bind to hydrophobic molecules in a large cavity, pins interact only with lipid aggregates, that is, micelles or liposomes, through a single stretch of tryptophan residues. this stretch of tryptophan residues is especially significant in the main form, pin-a (wrwwkwwk), while it is truncated in the smaller form, pin-b (wptwwk). [ ] [ ] [ ] puroindolines form protein aggregates in the presence of membrane lipids, and the organization of such aggregates is controlled by the lipid structure. in the absence of lipids, these proteins may aggregate, but there is no accurate information on the relationship between aggregation and interaction with lipids. the antimicrobial activity of pins is targeted to cell membranes. charnet et al indicated that pin is capable of forming ion channels in artificial and biological membranes that exhibit some selectivity over monovalent cations. the stress and ca + ions modulate the formation and/or opening of channels. puroindolines may also be membranotoxins, which may play a role in the plant defense mechanism against microbial pathogens. morris reported that the pin-a and pin-b act through similar but somewhat different modes, which may involve "membrane binding, membrane disruption and ion channel formation" or "intracellular nucleic acid binding and metabolic disruption." natural and synthetic mutants have allowed the identification of pins as key elements for antimicrobial activity. snakins are crps first identified in potato (solanum tuberosum). , due to their sequence similarity to gasa (gibberellic acid stimulated in arabidopsis) proteins, the snakins were classified as members of the snakin/gasa family. the genes that encode these peptides have ( ) a signal sequence of approximately aa, ( ) a variable region, and ( ) a mature peptide of approximately residues, with highly conserved cysteine residues. these cysteine residues maintain the d structure of the peptide through disulfide bonds, besides providing stability to the molecule when the plant is under stress , , , (figure f; supplementary figure s ). snakins may be expressed in different parts of the plant, like stem, leaves, flowers, seeds, and roots, - both constitutive or induced by biotic or abiotic stresses. in vitro activity was observed against a variety of fungi, bacteria, and nematodes, acting as a destabilizer of the plasma membrane. , , moreover, they were reported as essential agents in biological processes such as cell division, elongation, cell growth, flowering, embryogenesis, and signaling pathways. [ ] [ ] [ ] [ ] alpha-hairpinins as reported by nolde et al, alpha-hairpin emerged as a new amp with unusual motif configuration. these peptides prevail in plants and their structure was resolved based on nmr data obtained from the ecamp- peptide isolated from barnyard grass seeds (echinoa crus-galli). some α-hairpinins comprise trypsin inhibitors with helical hairpin structure and this group silva et al was recently proposed as a new plant amp family. similar to other amps, the amino acid sequences of α-hairpinins are variable. they share the conserved cysteine motif (cx cx - cx c) that form a helix-loop-helix fold and may have disulfide bridges c -c and c -c . its structural stability is maintained by forming hydrogen bonds, so that the side chains have a relatively stable spatial orientation. as reviewed by slavokhotova et al, members of alphahairpin family have been described in both mono and dicot groups, including species as echinochloa crus-galli and zea mays (both poaceae, monocot), fagopyrum esculentum (polygonaceae, eudicot), and stellaria media (caryophyllaceae, eudicot). several transcripts with α-hairpinin motif exhibit similarities to snakin/gasa genes and are sometimes positioned within this family. although the α-hairpinins structure has been published, its mechanism of action is still not resolved ( figure j , pdb id: l r). however, studies indicate they present a potential dna binding capacity. the term cyclotide was created at the end of the past century to designate a family of plant peptides with approximately aa in size and a structural motif called cyclic cysteine knot (cck). this motif is composed by a head-to-tail cyclization that is stabilized by a knotted arrangement of disulfide bridges, with conserved cysteines, connected as follows: c - , c - , c - . cyclotides are generally divided into subfamilies, mӧbius and bracelets, based on structural aspects. in addition to ccks, loops (between c - and c - ) have high similarity between both subfamilies, while the other loops (between c - and c - ) exhibit some conservation within the subfamilies , (supplementary figure s ) . to date, several cyclotides were identified in eudicot families such as rubiaceae, violaceae, fabaceae, and solanaceae, in addition to some monocots of poaceae family. in general, cyclotides may act in defense against a range of agents like insects, helminths, or mollusks. in addition, they can also act as ecbolic (inducer of uterus contractions), antibacterial, anti-hiv, and anticancer factors. all these characteristics added to the stability conferred by the cck motif turn these peptides into excellent candidates for drug development. , thaumatin-like protein thaumatins or tlps belong to the pr- (pathogen-related protein) family and received this name due to its first isolation from the fruit of thaumatococcus daniellii (maranthaceae) from west africa. thaumatin-like proteins are abundant in the plant kingdom, being found in angiosperms, gymnosperms, and bryophytes, being also identified in other organisms, including fungi, , insects, and nematodes. thaumatin-like proteins are known for their antifungal activity, either by permeating fungal membranes or by binding and hydrolyzing β- , -glucans. , in addition, they may act by inhibiting fungal enzymes, such as xylanases, α-amylases, or trypsin. besides, the expression of tlps is regulated in response to some stress factors, such as drought, injuries, freezing, and infection by fungi , viruses, and bacteria. as to the tlp structure, this protein presents characteristic thaumatin signature (ps ): , most of the tlps have molecular mass ranging from to kda, possessing conserved cysteine residues (supplementary figure s ) involved in the formation of disulfide bonds, which help in the stability of the molecule, allowing a correct folding even under extreme conditions of temperature and ph. thaumatin-like proteins also contain a signal peptide at the n-terminal, which is responsible for targeting the mature protein to a particular secretory pathway. the tertiary structure presents distinct domains, which are conserved and form the central cleft, responsible for the enzymatic activity of the protein, being located between domains i and ii. this central cleft may be of an acidic, neutral, or basic nature depending on the binding of the different linkers/receptors. all plant tlps with antifungal activity have an acidic cleft known as motif reddd due to highly conserved amino acid residues (arginine, glutamic acid, and aspartic acid; supplementary figure s ), being very relevant for specific receptor binding and antifungal activity. , , crystallized structures were determined for some plant tlps, such as thaumatin (figure g ), zeamatin ( figure h ), tobacco pr- d and osmotin, the cherry allergen pruav , and banana allergen ba-tlp, among other tlps. some tlps are known as small tlps (stlps) due to the deletion of peptides in one of their domains, culminating in the absence of the typical central cleft. these stlps exhibit only -conserved cysteine residues, forming disulfide bonds, resulting in a molecular weight of approximately to kda. they have been described in monocots, conifers, and fungi, so far. , , other tlps exhibit an extracellular tlp domain and an intracellular kinase domain, being known as pr k (pr -like receptor kinases) and are present in both monocots and dicots. for example, arabidopsis contains pr k genes, while rice has only . with the rapid growth in the number of available sequences, it is unfeasible to handle such amount of data manually. thus, amp sequences (as well as their biological information) have been deposited in large general databases, such as uniprot and trembl, which contain sequences of multiple origins. , in this sense, the construction of databases that deal specifically with amps was an important step to organize the data. during the past decade, several databases were built to support the deposition, consultation, and mining of amps. thus, these databases can be classified into groups: general and specific. the specific databases can be divided into subgroups: those containing only specific group (defensins or cyclotides) and those containing data from a supergroup of peptides (plant, animal, or cyclic peptides) (supplementary table ). in general, both types of databases share some characteristics such as the way that the data are available or the tools to analyze amps. the collection of antimicrobial peptides (campr ) is a database that comprises experimentally validated peptides, sequences experimentally deduced and still those with patent data, besides putative data based on similarity. [ ] [ ] [ ] the current version includes structures and signatures specific to families of prokaryotic and eukaryotic amps. the platform also includes some tools for amp prediction. the antimicrobial peptide database (apd) collects mature amps from natural sources, ranging from protozoa to bacteria, archaea, fungi, plants, and animals, including humans. amps encoded by genes that undergo post-translational modifications are also part of the scope, besides some peptides synthesized by multienzyme systems. the apd provides interactive interfaces for peptide research, prediction, and design, statistical data for a specific group, or for all peptides available in the database. the lamp (database linking antimicrobial peptides) comprises natural and synthetic amps, which can be separated into groups: experimentally validated, predicted, and patented. their data were primarily collected from the scientific literature, including uniprot and other amp-related databases. the database of antimicrobial activity and structure of peptides (dbaasp) contains information about amps from different origins (synthetic or non-synthetic) and complexity levels (monomers and dimers) that were retrieved from pubmed using the following keywords: antimicrobial, antibacterial, antifungal, antiviral, antitumor, anticancer, and antiparasitic peptides. this database is manually curated and provides information about peptides that have specific targets validated experimentally. it also includes information on chemical structure, post-translational modifications, modifications in the n/c terminal amino acids, antimicrobial activities, cell target and experimental conditions in which a given activity was observed, besides information about the hemolytic and cytotoxic activities of the peptides. due to the diversity of amps and the need to accommodate the most representative subclasses, several databases were established, focusing on specific types, sources, or features. there are several ways to classify amps, and they can range from biological sources such as bacterial amps (bacteriocins), plants, animals, and so on; biological activity: antibacterial, antiviral, antifungal, and insecticide; and based on molecular properties, pattern of covalent bonds, d structure and molecular targets. , the "defensins knowledgebase" is a database with manual curation and focused exclusively on defensins. this database contains information about sequence, structure, and activity, with a web-based interface providing access to information and enabling text-based search. in addition, the site presents information on patents, grants, laboratories, researchers, clinical studies, and commercial entities. , the cybase is a database dedicated to the study of sequences and d structures of cyclized proteins and their synthetic variants, including tools for the analysis of mass spectral fingerprints of cyclic peptides, also assisting in the discovery of new circular proteins. the phytamp is a database designed to be solely dedicated to plant amps based on information collected from the uniprot database and from the scientific literature through pubmed. plantpepdb is a database with manual curation of plantderived peptides, mostly experimentally validated at the protein level. it includes data on the physical-chemical properties and tertiary structure of amps, also useful to identify their therapeutic potential. different search options for simple and advanced compositing are provided for users to perform dynamic search and retrieve the desired data. overall, plantpepdb is the first database that comprises detailed analysis and comprehensive information on phyto-peptides from a wide functional range. biological data banks (dbs) are organized collections of data of diverse nature that can be retrieved using different inputs. the management of this information is done through various software and hardware resources, whose retrieval and organization can be performed in a quick and efficient way. considering biological data, information can be classified into ( ) primary (sequences), ( ) secondary (structure, expression, metabolic pathways, types of drugs, etc), and ( ) specialized, for example, containing information on a species or on a class of protein. within this third group, some references to amps can be mentioned, such as campr and apd that compile sequence data and structure retrieved from diverse sources, and also the defensin knowledgebase and the cybase which are dedicated to specific classes of peptides (defensins and cyclotides, respectively), in addition to phytamp, a specific database of plant amps (supplementary table ). the first step to infer the function of a given sequence (annotation) is to retrieve it in databases. for this purpose, approaches have been used mostly: ( ) local alignments, especially by using basic local alignment search tool (blast) and fasta ; by searching for specific patterns using ( ) regex or ( ) hidden markov model (hmm). the first approach has been widely used, since most of the information is available in databases as sequences, together with tools to align them, whereas the blast is the primary tool for doing so. this tool splits the sequence into small pieces (words), comparing it with the database. however, this approach has a limitation. small motifs may not be significantly aligned as they comprise small portions of the sequences that can be smaller than % of the total size. , due to the high variability of amps, only few highly conserved sequences can be identified using this type of inference. to reduce the effects of local alignment limitations, other strategies based on the search for specific patterns were introduced, such as regex (supplementary table ) and hmm. the regex is a precise way of describing a pattern in a string where each regex position must be set, although ambiguous characters (or wildcards) can also be used. for example, if we want to find a match for both amino acid sequences caiessk and waiesk, we can use the following expression: [cw]aies{ , }k, this expression would find a pattern starting with the letter "c" or "w," followed by an "a," an "i," and an "e," or "s," and ending with a "k." the hmms are well-known for their effectiveness in modeling the correlations between adjacent symbols, domains, or events, and they have been extensively used in various fields of biological analysis, including pairwise and multiple sequence alignment, base-calling, gene prediction, modeling dna sequencing errors, protein secondary structure prediction, noncoding rna (ncrna) identification, protein and rna structural alignments, acceleration of rna folding and alignment, fast noncoding rna annotation, and many others. using hmm, a statistic profile is included in the model, which is calculated from a sequence alignment, and a score is determined site-to-site, with conserved and variable positions defined a priori. , predicting antimicrobial activity the design of new amps led to the development of methods for the discovery of new peptides, thus allowing new experiments to be done by researchers. in this sense, the new challenge lies in the construction of new prediction models capable of discovering peptides with desired activities. the apd db has established a prediction interface based on some parameters defined by the entire set of peptides available in this database. these values are calculated from natural amps to consider features like length, net charge, hydrophobicity, amino acid composition, and so on. if we take as an example the net load, the amps deposited in the apd range from - to + . this is the first parameter incorporated into the prediction algorithm. however, most amps have a net load ranging from - to + , which then becomes the alternative prediction condition. therefore, the same method is applied to the remaining parameters. the prediction in apd is performed in main steps. first, the sequence parameters will be calculated and compared. if defined as an amp, the peptide can then be classified into groups: ( ) rich in given amino acids, ( ) stabilized by disulfide, and ( ) linear bridges. finally, sequence alignments will be conducted to find peptides of higher similarity. , , the advent of machine learning (ml) methods has promoted new possibilities for drug discovery. in ml inferences, both a positive and a negative dataset are usually required to train the predictive models. the positive data, in this case, regard preferably experimentally validated amps that can be collected in databases, whereas negative data are randomly selected protein sequences that do not have amp characteristics. , machine learning methods based on support vector machine (svm), random forest (rf), and neural networks (nn) have been the most widely used. svm is a specific type of supervised method of ml, aiming to classify data points by maximizing the margin between classes in a high-dimensional space. , random forest is a non-parametric tree-based approach that combines the ideas of adaptive neighbors with bagging for efficient adaptive data inference. neural networks is an information processing paradigm inspired by how a biological nerve system process information. it is composed of highly interconnected processing elements (neurons or nodes) working together to solve specific problems. [ ] [ ] [ ] evaluating proteomic data regarding the use of amps in peptide therapeutics, as an alternative to antimicrobial treatment, new efficient and specific antimicrobials are demanded. as aforementioned, amps are naturally occurring across all classes of life, presenting high active potential as therapeutic agents against various kinds of bacteria. the identification of novel amps in databases is primarily dependent on knowing about specific amps together with a sufficient sequence similarity. however, orthologs may be divergent in sequence, mainly because they are under strong positive selection for variation in many taxa, leading to remarkably lower similarity, even in closely related species. in this scenario, where alignment tools present limited use, strategy to identify amps is related to proteomic approaches. proteins and peptides are biomolecules responsible for various biochemical events in living organisms, from formation and composition to regulation and functioning. thus, understanding of the expression, function, and regulation of the proteins encoded by an organism is fundamental, leading to the so-called "proteomic era." the term "proteome" was first used by marc wilkins in and it represents the set of proteins encoded by the genome of a biological system (cell, tissue, organ, biological fluid, or organism) at a specific time under certain conditions. protein extraction, purification, and identification methods have significantly advanced our capacity to elucidate many biological questions using proteomic approaches. , due to the wide diversity of proteomic analysis, methods makes the choice of the correct approach dependent on the type of material and compounds to be analyzed. , two main tools are used to isolate proteins: ( ) the -dimensional electrophoresis ( -de) associated with mass spectrometry (ms) and ( ) liquid chromatography associated with ms, each one with its own limitations. [ ] [ ] [ ] obtaining native proteins is a challenge in proteomics or peptidomics, due to high protein complexity in samples, as the occurrence of post-translational modifications. alternative strategies applied to extraction, purification, biochemical, and functional analyses of these molecules have been proposed, favoring access to structural and functional information of hard-to-reach proteins and peptides. based on d gel, al akeel et al evaluated spots obtained from seeds of foeniculum vulgare (apiaceae) aiming at proteomic analyses and isolation of small peptides. extracted proteins were subjected to kda dialysis, and separation was carried out by deae-ion exchange chromatography while further proteins were identified by d gel electrophoresis. one of its spots showed high antibacterial activity against pseudomonas aeruginosa, pointing to promising antibacterial effects, but requiring further research to authenticate the role of the anticipated proteins. for amps, de is challenging due to the low concentration of the peptide molecules captured by this approach, their small sizes, and their ionic features (strongly cationic). in addition, the limited number of available specific databases and high variability turn their identification through proteolysis techniques and mass spectrometry, matrix-assisted laser desorption/ionization (maldi-ms) difficult. in addition, the partial hydrophobicity characteristics and surface charges facilitate peptide molecular associations, making analysis difficult by any known proteomic approaches. in addition, peptides are most often cleaved from larger precursors by various releasing or processing enzymes. furthermore, profiles generated do not represent integral proteome, as de has limitations to detect proteins with low concentration, values of extreme molecular masses, pis, and hydrophobic proteins, including those of membranes. due to these limitations, multidimensional liquid chromatography-high-performance liquid chromatography (mdlc-hplc) has been successfully employed as an alternative to d gels. techniques and equipments for the newly developed separation and detection of proteins and peptides, such as nano-hplc and multidimensional hplc, have improved proteomics evaluation. molecular mass values obtained are used in computational searches in which they are compared with in silico digestion results of proteins in databases. in silico approaches, usually by the action of trypsin as a proteolytic agent, may generate a set of unique peptides whose masses are determined by ms. , these methodologies are widely adopted for large-scale identification of peptide from ms/ms spectra. theoretical spectra are generated using fragmentation patterns known for specific series of amino acids. the first widely used search engines in database searching were sequest and mascot (matrix science, boston, ma; www.matrixscience.com). they rank peptide matches based on a cross-correlation to match the hypothetical spectra to the experimental one. mascot is widely used for peptidomics and proteomics analysis, including amp identification in many organisms, or to evaluate the antibacterial efficacy of new amps. evaluating new amp against multidrug-resistant (mdr) salmonella enterica, tsai et al used d gel electrophoresis and liquid chromatography-electrospray ionization-quadrupole-time-offlight tandem ms to determine the protein profiles. the protein identification was performed using the mascot with trypsin as cutting enzyme, whereas ncbi nr protein was set as a reference database. the methodology used in this study indicated that the novel amp might serve as a potential candidate for drug development against mdr strains, confirming the usability of mascot. in a similar way, umadevi et al described the amp profile of black pepper (piper nigrum l.) and their expression on phytophthora infection using label-free quantitative proteomics strategy. for protein/peptide identification, ms/ms data were searched against the apd database using an in-house mascot server, established full tryptic peptides with a maximum of missed cleavage sites and carbamidomethyl on cysteine, besides an oxidized methionine included as variable modifications. the apd database was used for amp signature identification, together with phytamp and campr . to enrich the characterization parameters, isoelectric point, aliphatic index, and grand average of hydropathy were also used (gravy) (using protparam tool), besides the net charge from phytamp database. based on label-free proteomics strategy, they established for the first time the black pepper peptidomics associated with the innate immunity against phytophthora, evidencing the usability of proteomics/ peptidomics data for amp characterization in any taxa, including plant amps, aiming the exploitation of these peptides as next-generation molecules against pathogens. other tools use database searching algorithms, such as x!tandem, open mass spectrometry search algorithm (omssa), probid, radars, and so on. these search engines are based on database search but use different scoring schemes to determine the top hit for a peptide match. general information on database search engines, their algorithms, and scoring schemes were reviewed by nesvizhskii et al. despite its efficient ability to identify peptides, database searching presents several drawbacks, like false positive identifications due to overly noisy spectra and lower quality peptides score (related to the short size of peptides). so, the identification is strongly influenced by the amount of protein in the sample, the degree of post-translational modification, the quality of automatic searches, and the presence of the protein in the databases. , in this scenario, the knowledge about the genome from a specific organism is important to allow the identification of the exact pattern of a given peptide. if an organism has no sequenced genome, it is not searchable using these methods. , once the sequences are obtained, bioinformatic tools can be used to predict peptides structure and estimate bioactive peptides. more recently, an interactive and free web software platform, mixprotool, was developed, aiming to process multigroup proteomics data sets. this tool is compiled in r (www.r-project. org), providing integrated data analysis workflow for quality control assessment, statistics, gene ontology enrichment, and other facilities. the mixprotool is compatible with identification and quantification outputs from other programs, such as maxquant and mascot, where results may be visualized as vector graphs and tables for further analysis, in contrast to existing softwares, such as giapronto. according to the authors, the web tool can be conveniently operated, even by users without bioinformatics expertise, and it is beneficial for mining the most relevant features among different samples. the central tenet of structural biology is that structure determines function. for proteins, it is often said the "function follows form" and "form defines function." therefore, to understand protein function in detail at the molecular level, it is mandatory to know its tertiary structure. experimental techniques for determining structures, such as x-ray crystallography, nmr, electron paramagnetic resonance, and electron microscopy, require significant effort and investments. all methods mentioned have their own limitations, and the gap between the number of known proteins and the number of known structures is still substantial. thus, there is a need for computational framework methods to predict protein structures based on the knowledge of the sequence. in addition, in recent years, there has been impressive progress in the development of algorithms for protein folding that may aid in the prediction of protein structures from amino acid sequence information. historically, the prediction of a protein structure has been classified into categories: comparative modeling, threading, and ab initio. the first approaches construct protein models by aligning the query sequences with already solved model structures. if the models are absent in the protein data bank (pdb), the models must be constructed from scratch, that is, by ab initio modeling, considered the most challenging way to predict protein structures. in the case of comparative modeling methods, when inserting a target sequence, the programs identify evolutionarily related models of solved structures based on their sequence or profile comparison, thus constructing structure models supported by these previously resolved models. this approach comprises main steps: ( ) fold assignment, which identifies similarity between the target and the structure of the solved model; ( ) alignment of the target sequence to the model; ( ) generation of a model based on alignment with the chosen template; and ( ) analysis of errors considering the generated model. there are several servers and computer models that automate the comparative modeling process, with swiss-model and modeler figuring as the most used. , although automation makes comparative modeling accessible to experts and beginners, some adjustments are still needed in most cases to maximize model accuracy, especially in the case of more complex proteins. therefore, some caution must be taken regarding the generated models, considering the resolution and quality of the model used, as well as homology between the model and the protein of interest. threading modeling methods are based on the observation that known protein structures appear to comprise a limited set of stable folds, and those similarity elements are often found in evolutionarily distant or unrelated proteins. the most used servers based on this approach are muster, sparks-x, raptorx, prosa-web, and most notably the i-tasser. in some cases, the incorporation of structural information to combine the sequence used in the search with possible models allows the detection of similarity in the fold, even in the absence of an explicit evolutionary relation. the prediction of structures from known protein models is, at first sight, a more straightforward task than the prediction of protein structures from available sequences. therefore, when no solved model is available, another approach is recommended, namely, the ab initio modeling. this method is intended to predict the structure only from the sequence information, without any direct assistance from previously known structures. the ab initio modeling aims to predict the best model, based on the minimum energy for a potential energy function by sampling the potential energy surface using various searchable information. , such approaches turn it challenging to produce high-resolution modeling, essential for determining the native protein folding and its biochemical interpretation. on the contrary, later resolved structures and comparisons with previously predicted proteins point to a higher successful modeling generated by ab initio methods than those generated by pure energy minimization methods, classical or even pure methods. among the most used servers and programs for ab initio modeling, we highlight the rosetta, quark, and touchstone ii. the accuracy of the models calculated by many of these methods is evaluated by cameo (continuous automated model evaluation) and by casp (critical assessment of protein structure prediction). probably the first reasonably accurate ab initio model was built in casp . since then, sustained progress was achieved in ab initio prediction, but mainly for small proteins ( residues or less). in casp , for the first time, a novel -residue protein with a sequence identity with known structures lower than % was constructed with high precision for sequences of this size. in casp , a significant improvement was reported in areas: contact prediction, free modeling, template-based modeling, and estimating the accuracy of models. the authors report that this improvement is due to the accuracy of modeling and alignment methods, as well as increased data availability for both sequence and structure. due to the number of amps deposited in the pdb (to date approximately structures), comparative modeling is the most used. however, when it comes to de novo peptide design, the most recommended choice would be ab initio or a hybrid approach that uses more than modeling method. after the generation of a model, the amp stability should be evaluated using molecular dynamics (md). molecular dynamics comprises the application of computational simulations that predict the changes in the positions and velocities of the constituent atoms of a system under given time and condition. this calculation is done through a classical approximation of empirical parameters, called "force field." if, on one hand, this approximation makes the dynamics of a system containing thousands of atoms numerically accessible, it obviously limits the nature of the processes that can be observed during the simulations. no quantum effect is visualized in a md simulation; just as no chemical bond is broken, no interactions occur between orbitals, resonance, polarization, or charge transfer effects. however, the molecules go beyond a static system. thus, md is a computational technique that can be used for predicting or refining structures, dynamics of molecular complexes, drug development, and action of molecular biological systems. molecular dynamics simulation is widely used for protein research, aiming to extract information about the physical properties of individual proteins. the results of such simulations are then compared with experimental results. as these experiments are generally carried out in solvents, it is necessary to simulate molecular systems of protein in water. these simulations have a variety of applications, such as determining the folding of a structure to a native structure and analyzing the dynamic stability of this structure. the use of md to simulate protein folding processes is one of the most challenging applications and should be relatively long (in the order of microseconds to milliseconds) to allow observing a single fold event. in addition, the force field used must correctly describe the relative energies of a wide variety of shapes, including unfolding and poorly folded shapes that may occur during the simulation. the considerable application potential led to the implementation of md simulation in many software packages, including gromacs, - amber, namd, charmm, lammps, and desmond. in addition to the above mentioned, there are other simulation types available, such as the monte carlo method, stochastic dynamics, and brownian dynamics. in the last decades, md simulation has become a standard tool in theoretical studies of large biomolecular systems, including dna or proteins, in environments with near realistic solvents. indeed, simulations have proven valuable in deciphering functional mechanisms of proteins and other biomolecules, in uncovering the structural basis for disease, and in the design and optimization of small molecules, peptides, and proteins. historically, the computational complexity of this type of computation has been extremely high, and much research has focused on algorithms to achieve unique simulations that are as long or as large as possible. the interplay between a given pathogen (eg, virus, bacteria, fungus) must be studied through a holistic approach. hostpathogen relationships are very complex and occur at diverse conceivable levels, including the cellular/molecular level of both, pathogen and host, under given environmental conditions. a most approximate understanding of these interactions at every level is the ultimate goal of "systems biology" (sb). it comprises a holistic approach, integrating distinct disciplines, as biology, computer science, engineering, bioinformatics, physics, and others to predict how a given system behaves under given conditions and what is the role of its parts. systems biology stands out because it is capable of correlating omics data for the understanding of plant-pathogen interaction. the construction of a plant-pathogen interaction network includes the reconstruction of metabolic pathways of these organisms, identification of the degree of pathogenicity, besides the expression of genes and proteins from both plant and pathogen. the networks can be classified into types: ( ) regulatory; ( ) metabolic; ( ) protein-protein interaction; ( ) signaling and regulatory; and ( ) signaling, regulatory, and metabolic. each of these networks can be plotted according to computational approaches. also, further studies are required to contemplate the construction of evolutionary in silico models and the characterization of these molecular targets in vitro. , studies of protein-protein interactions to understand the regulatory process are essential and new computational methods are necessary for this purpose with more optimized algorithms, also to remove potential false positives. thus, in-depth studies on the orientation of molecules and their linkages to the formation of a stable complex are of great importance for understanding plant-pathogen studies and also to develop new drugs. the understanding of the regulatory principles by which protein receptors recognize, interact, and associate with molecular substrates or inhibitors is of paramount importance to generate new therapeutic strategies. in modern drug discovery, docking plays an important role in predicting the orientation of the binder when it is attached to a protein receptor or enzyme, using forms and electrostatic interactions, van der walls, coulombic, and hydrogen bond as parameters to quantify or predict a given interaction. , molecular docking aims at exploring the predominant mode(s) of binding of a molecule (protein or ligand) when it binds to a protein with a known d structure based on a scoring function that has main functions: the first is to determine the binding mode and the binding site of a protein, the second is to predict the absolute binding affinity between protein and ligand (or other protein) in lead optimization, and the third is virtual screening, which can identify potential drug leads for a given protein target by searching a large ligand or protein in database. protein-protein interactions are essential for cellular and immune function. in many cases, due to the absence of an experimentally determined structure of the complex, these interactions must be modeled to obtain an understanding about their structure and molecular basis. few studies on plant-pathogen interactions include docking approaches and most studies focus on drug development for medical purposes. drug research based on structure is a powerful technique for the rapid identification of small molecules against the d structure of available macromolecular targets, usually by x-ray crystallography, nmr structures, or homology models. due to abundant information on protein sequences and structures, the structural information on specific proteins and their interactions have become crucial for current pharmacological research. even in the absence of knowledge about the binding site and limited backbone movements, a variety of algorithms have been developed for docking over the past decades. although the zdock, the rdock, and the hex have provided results with high coupling precision, the complexes provided are not very useful for designing inhibitors for protein interfaces due to constraints on rigid body docking. in this context, more flexible approaches have been developed which generally examine very limited conformations compared with rigid body methods. these docking methods predict that binding is more likely to occur in broad surface regions and then defines the sites in complex structures of high affinity. the best example is the haddock software, which has been successful in solving a large number of precise models for protein-protein complexes. a good example of its use is the study of the complex formed between plectasin, a member of the innate immune system, and a precursor lipid of bacterial cell wall ii. the study identified the residues involved in the binding site between the proteins, providing valuable information for planning new antibiotics. however, the absolute energies associated with intermolecular interaction are not estimated with satisfactory accuracy by the current algorithms. some significant issues as solvent effects, entropic effects, and receptor flexibility still need to be addressed. however, some methods, such as moe-dock, gold, glide, flexx, and surflex which deal with lateral chain flexibility, have proven to be effective and adequate in most cases. realistic interactions between small molecules and receptors still depend on experimental wet-lab validation. , despite the current difficulties, there is a growing interest in the mechanisms and prediction of small molecules such as peptides, as they bind to proteins in a highly selective and conserved manner, being promising as new medicinal and biological agents. while both "small molecule docking methods" and "custom protocols" can be used, short peptides are challenging targets because of their high torsional flexibility. proteinpeptide docking is generally more challenging than those related to other small molecules, and a variety of methods have been applied so far. however, few of these approaches have been published in a way that can be reproduced with ease. [ ] [ ] [ ] although it is difficult to use peptide docking, a recent focus of basic and pharmacological research has used computational tools with modified peptides to predict the selective disruption of proteinprotein interactions. these studies are based on the involvement of some critical amino acid residues that contribute most to the binding affinity of a given interaction, also called hot-spots. , despite the number of docking programs, existing algorithms still demand improvements. however, approaches are being developed to improve all issues related to punctuation, protein flexibility, interaction with plain water, among other issues. in this context, the capri (critical assessment of predicted interactions) is a community that provides a quality assessment of different docking approaches. it started in and since then has aided the development and improvement of the methodologies applied for docking. an evaluation was carried out for capri in , resulting in an improvement in the integration of different modeling tools with docking procedures, as well as the use of more sophisticated evolutionary information to classify models. however, adequate modeling of conformational flexibility in interacting proteins remains an essential demand with a crucial need for improvement. different docking programs are currently available, and new alternatives continue to appear. some of these alternatives will disappear, just as others will become the top choices among field users. molecular docking technique is not often used for amps, due to its standard mechanism of action based on the classical association with the external membrane of the pathogen. despite that, some amps have the ability to bind other proteins and/or enzymes, a feature still scarcely studied. in such cases, molecular docking can be useful. an example of success is the study performed by melo et al, where they showed the specific binding of a trypsin to a cowpea (vigna unguiculata) thionine, revealing that this interaction occurs in a canonical manner with lys , located in an extended exposed loop. therefore, further application of docking may bring new evidences about the antimicrobial mechanisms revealing other molecular targets of interest. it is clear that the combination of data bank information with bioinformatic tools (especially those allowing the identification of patterns, rather than sequence order) is able to revolutionize the identification of amps and prediction of their activity. the data may come from genomic, transcriptomic, or proteomic databases, or a combination of different information sources (eg, genomic and transcriptomics, transcriptomics and proteomics). supplementary figure s brings a schematic flowchart describing the steps for mining, annotation, and structural/ functional analysis of amps, in addition to some wet-lab analyses that can be integrated to assess/confirm candidate amps. similar bioinformatic approaches have been actually used to identify potential peptide candidates with anti-sars-cov- activity, especially those potentially able to interact with the spike protein and proteases involved in viral penetration. , as emphasized, plant amps show greater diversity and abundance, when compared with other kingdoms. it can be speculated that plants shelter many yet undescribed amp classes, given their vast abundance and isoform diversity. the genomic and peptidic structure of amps can be variable, with few key residues conserved, which turns their identification, classification, and comparison challenging even in the omics age. nevertheless, advances in the generation of new bioinformatics tools and specialized databases have led to new and more efficient approaches for both the identification of primary sequences and molecular modeling, besides the analysis of the stability of the generated models. despite the large availability of omics data and bioinformatics tools, most new plant peptides have been discovered by wet-lab approaches regarding single candidates. high throughput in silico methods have the potential to transform this scenario, revealing many new candidates, including some new or "non-canonical" peptides. it may be also speculated that a myriad of new peptides may exist considering even smaller peptides, still less considered and more difficult to identify. finally, in silico approaches shall in future studies be mandatory to define the design of wet-lab studies, turning the identification more efficient and requiring reasonably less time to track, identify, and confirm new candidate amps. considering the actual pandemic scenario of covid- , plant amps may be regarded as an important source of antiviral drug candidates, especially considering that some amp categories present not only antiviral effects but also a wide spectrum antimicrobial activity, act as anti-inflammatory, and also induce the immune response. of higher education personnel, biocomputational program), cnpq (brazilian national council for scientific and technological development), and facepe (fundação de amparo à ciência e tecnologia de pernambuco) for fellowships. the project is supported by the interreg italia-slovenia, ise-emh / and rc / from irccs burlo garofolo/ italian ministry of health cass performed the literature review and whore the manuscript. lz, mol, lmbv, jpbn, jrfn, jdcf, rlos, cjp, ffa, and mfo wrote specific chapters, eak and sc critically revised the text and included relevant suggestions. ambi conceived the review, wrote the introduction and concluding remarks, besides critically revising the manuscript. all authors have read the manuscript and agree to its content. supplemental material for this article is available online. prediction of protein function from protein sequence and structure plant peptides in defense and signaling plant bioactive peptides: an expanding class of signaling molecules candidalysin is a fungal peptide toxin critical for mucosal infection copsin, a 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development and application of protein-peptide docking peptide docking and structurebased characterization of peptide binding: from knowledge to know-how protein-ligand docking in the new millennium-a retrospective of years in the field unal eb, gursoy a, erman b. vital: viterbi algorithm for de novo peptide design modeling protein-protein and proteinpeptide complexes: capri th edition docking, scoring, and affinity prediction in capri potential chimeric peptides to block the sars-cov- spike receptor-binding domain peptide-like and small-molecule inhibitors against covid- the authors are very grateful to capes (coordination for the improvement key: cord- -rzda x authors: wells, stephen a. title: rigidity, normal modes and flexible motion of a sars-cov- (covid- ) protease structure date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: rzda x the rigidity and flexibility of two recently reported crystal structures (pdb entries y e and lu ) of a protease from the sars-cov- virus, the infectious agent of the covid- respiratory disease, has been investigated using pebble-game rigidity analysis, elastic network model normal mode analysis, and all-atom geometric simulations. this computational investigation of the viral protease follows protocols that have been effective in studying other homodimeric enzymes. the protease is predicted to display flexible motions in vivo which directly affect the geometry of a known inhibitor binding site and which open new potential binding sites elsewhere in the structure. a database of generated pdb files representing natural flexible variations on the crystal structures has been produced and made available for download from an institutional data archive. this information may inform structure-based drug design and fragment screening efforts aimed at identifying specific antiviral therapies for the treatment of covid- . as of late , there has been global concern over a novel respiratory disease (designated covid- by the world health organisation (who)) which originated in the wuhan region of china and has subsequently spread to multiple other countries worldwide. the virus causing covid- is a coronavirus with the taxonomic identifier sars-cov- [ ] . thanks to the diligent efforts of structural biologists, several crystal structures of proteins from sars-cov- have already been obtained and made available through the protein data bank (pdb) even in advance of journal publication. crystal structures provide us with detailed structural knowledge of the arrangement of atoms making up a protein or protein complex. a crystal structure is a static snapshot of the protein "frozen" into one of its many possible conformations. this static snapshot is rich in implicit information on the natural dynamics which the protein will explore in vivo. these natural dynamics, described by kern [ ] as a protein's "dynamic personality", can be critical to a protein's function, and can directly affect the geometry of, for example, an enzyme active site. a judicious combination of simplified methodologiesrigidity analysis, elastic network modelling, and geometric simulation of flexible motioncan work together to extract valuable information on the large-amplitude, low-frequency motions of a protein structure, at a fraction of the computational cost (in resources and time) of molecular dynamics (md) approaches [ ] . these simplified methods are strongly complementary to both computational md investigations and experimental biophysical/biochemical studies of protein behaviour. enzyme active site geometry [ ] [ ] [ ] [ ] ; and, crucially, that a structure generated from geometric simulation of a large amplitude motion can be used as input for further md investigation [ ] , as the geometric simulation retains the local bonding geometry and constraint network of the input crystal structure and forbids major bonding distortions and steric clashes. the information obtained here regarding the flexibility of the enzyme should be of use and interest in structure-based drug design and computational screening of drugs and drug-like molecules capable of binding to and inhibiting the action of the viral protease, such as the pioneering work of walsh et al. reported online within the past few days [ ] . the simulations reported here suggest that the protease structure is capable of substantial flexible motions which alter domain orientations, open and close clefts, and affect the geometry of an inhibitor-binding site. the entirety of the computational work described here was carried out in less than one working day on a laptop computer. the inputs and outputs of the simulations, principally consisting of pdb files representing flexible variations on the protease crystal structures, are available for download from the university of bath research data archive at https://researchdata.bath.ac.uk/ / [ ] with doi https://doi.org/ . /bath- methods, software and data input data: crystal structures of a viral protease were downloaded from pdb entries y e (free protease) and lu (inhibitor bound). visualisations: visualisation and some minor structural editing, viz. generation of symmetry copies, removal of water and other heterogroups, removal of alternate sidechain conformations, and renumbering of entries after addition of hydrogens, were carried out with the pymol viewer, version . [ ] . all structures were aligned to the y e crystal structure for consistency of view. addition of hydrogens, sidechain flipping: the molprobity [ ] web server hosted at duke university provides online tools carrying out these functions. rigidity analysis: carried out using the pebble-game constraint counting algorithm implemented in the software first [ ] . first should be available from arizona state university via flexweb.asu.edu; academic readers should contact the present author if there is difficulty in obtaining the software, as the site appears currently to be down. recent research has shown that the assignment of energies to polar interactions in first suffers from a bug that leads to some salt-bridge interactions with short donoracceptor distances being treated as weak rather than strong. therefore, interactions were identified and energies assigned using a recently developed corrected function, sbfirst [ ] . a copy of the sbfirst software for constraint identification is included with the deposited dataset for this study. normal mode analysis: carried out using the elnemo elastic network modelling software [ ] . this method builds an elastic network model of the protein with one site per residue (using the alpha carbon _ca_ atom coordinates as the node positions), placing springs of uniform strength between each pair of residues lying less than Å apart, and diagonalising the resulting hessian matrix to identify eigenvectors. geometric simulations of flexible motion: carried out using the froda geometric simulation engine [ ] incorporated into first. the froda engine iteratively projects an all-atom model of the protein structure along a normal mode direction [ ] while constraining the local geometry to match that of the original protein structure. frames along each such trajectory are written out as numbered pdb files. workflow and protocol: for each protein structure, the complete workflow for this study is as follows. download structure in pdb or mmcif format. in this case, the downloaded structure contains coordinates for a single protein chain, and symmetry operations to generate copies making up the crystal structure. visualise structure in pymol. generate symmetry mates. select the appropriate symmetry mate to make up the homodimer indicated in the pdb entry (under global stoichiometry) as the biological entity of interest. delete other symmetry copies. alter chain id(s) in the symmetry copy to give each chain a unique chain id. process structure through the molprobity website, adding hydrogens at electron-cloud positions, accepting all recommendations for side chain flips. download the hydrogenated structure (in which all hydrogens have a serial number of ). visualise structure in pymol for final cleaning and preparation. remove alternate conformations (i.e. those with an altloc entry which is not a blank or an a). remove water molecules and extraneous heterogroups such as glycerol, acetyl etc. save the cleaned structure; in this step, pymol renumbers the atoms sequentially, so hydrogens now have appropriate serial numbers. identify covalent and noncovalent interactions from the atomic geometry of the protein structure using sbfirst. this generates lists of covalent bonds, tagged as being either rotatable or non-rotatable (such as the c-n bond in the peptide main chain); hydrophobic tether interactions, wherever two nonpolar sidechains are closely adjacent; and polar interactions, including salt bridges and hydrogen bonds. polar interactions are assigned energies between and - kcal/mol based on the donor-hydrogen-acceptor geometry. analyse rigidity with first as a function of hydrogen-bond energy cutoff. this analysis groups atoms into rigid clusters by matching degrees of freedom against constraints. first can carry out a dilution, gradually lowering the cutoff to exclude polar interactions one by one from weakest to strongest. it is also possible to simply carry out "snapshots" of the rigidity at a series of cutoff values, which in this study were - . , - . , - . … - . , - . kcal/mol. review constraints based on rigidity information. at this stage, a small number of noncovalent constraints (one in y e, three in lu ) were identifiable as artefacts of crystal packing, and were excluded from the network when carrying out the geometric simulations. based on the rigidity analysis, a cutoff of - . kcal/mol appeared appropriate for the geometric simulations, in line with previous studies [ , ] . normal mode analysis. extract only the alpha carbon positions and run elnemo pdbmat (generation of matrix) and diagstd (diagonalise matrix) functions. the result is a series of normal modes of motion, reported as eigenvector/eigenvalue pairs. when these are sorted by eigenvalue from lowest to highest, the first six modes are trivial rigid-body motions with near-zero eigenvalues (frequencies), while the low-frequency modes from upwards represent flexible motions of the protein itself. this study examines modes to , that is, the ten lowest-frequency nontrivial modes, with modes to being shown in the figures. geometric simulation of flexible motion. for each normal mode of interest ( to ), run first with a chosen energy cutoff (- . kcal/mol) and the previously identified lists of covalent and noncovalent bonding constraints, providing the normal mode eigenvector and a direction of motion (parallel or antiparallel to the vector) along with a directed step size (in this case . Å), and invoking the froda engine implemented in first. froda carries out a series of steps. in each step, all atom positions are moved along the normal mode direction. then, the local atomic geometry defined by the bonding constraints and steric exclusion is restored by a few cycles of iterative relaxation. after a userdefined number of steps (in this case ), a numbered "frame" is written out as a pdb file. a series of such frames describes the trajectory of flexible motion. froda continues each run until a user-defined maximum number of steps (in this case ) or until "jamming" occurs where the constraints can no longer be satisfied, typically when the motion has led to severe steric clashes through the collision of two domains. output data: the inputs, scripts and outputs of the rigidity analysis, normal mode analysis, and geometric simulations for each protein have been deposited in the university of bath's data repository and are accessible from https://researchdata.bath.ac.uk/ / [ ] results and discussion the y e and lu crystal structures each contain explicit coordinates for one chain (a) of the protease, and in the case of lu , a chain (c) representing a bound inhibitor. the input structures for analysis and simulation consist of chain a and a symmetry copy (b), making up the biological homodimer. chain c is likewise duplicated to chain d. the x-ray structure does not contain explicit hydrogens, which are added using molprobity [ ] . with hydrogens added, it is then possible to detect the covalent bonds, polar noncovalent interactions (hydrogen bonds and salt bridges) and hydrophobictether interactions using sbfirst [ ] (see methods). the polar interactions are rated with an effective energy between and - kcal/mol based on the donor-hydrogen-acceptor geometry. pebble-game rigidity analysis [ , ] divides the structure into rigid clusters and flexible regions based on the distribution of degrees of freedom and constraints. this rigid cluster decomposition (rcd) depends on the set of polar interactions that are included, based on an energy cutoff that excludes weaker interactions. information on the relative stability of different portions of the structure is typically obtained at cutoffs around a "room temperature" value of - kcal/mol. rcds are shown in figure . with the inclusion of weaker polar constraints, a single large rigid cluster extends across both chains of the protease dimer. as weaker constraints are eliminated, the large cluster extends across the n-terminal domains (roughly residues - ) of both chains, which consist largely of beta-sheet structure, while the largely alpha-helical c-terminal domain breaks up into several smaller rigid clusters, each one a single helix. with further elimination of constraints, the beta-sheet regions become fully flexible. flexible motion of a protein structure is best carried out at with a rigidity cutoff such that the structure is fully flexible. the mobility studies that follow are carried out with a cutoff of - . kcal/mol, similar to previous studies on enzymes [ , ] . it is important to appreciate that even at this lower cutoff the structure is still thoroughly constrained by noncovalent interactions, as illustrated in figure . the core of each folded domain is rich in hydrophobic interactions, as is the interface region between the two beta-sheet domains as the two chains form the homodimer, and arrays of strong polar interactions constrain secondary structure (for example, the backbone hydrogen bonds within alpha helices and beta sheets). noncovalent constraints in the lu structure, shown as green (hydrophobic) and red (polar) lines. the protein mainchain is shown in cartoon representation. the circled area highlights a small set of interdomain hydrophobic constraints, one between the ala residues of the two chains, and one each between residue thr of one chain and leu of the other. in the y e structure only the ala interaction is present. these interactions may be considered an artefact of crystal packing. also highlighted in figure are a small set of hydrophobic interactions assigned between the n-terminal domains of chains a and b. in the y e structure, a single tether connects the sidechains of the ala residues of both chains. in the lu structure illustrated, there are two additional tethers nearby, each connecting the sidechains of the thr residue of one chain and the leu of the other. since the two n-terminal domains are otherwise not connected by noncovalent interactions, and these tethers are not involved in forming a rigid cluster, it seems reasonable to consider these tethers an artefact of crystal packing, and eliminate them from further consideration. their deletion does not affect the rigid cluster decompositions of the structures. if these tethers were retained, they would somewhat limit (but not entirely prevent) the central cleft-opening motion discussed below. normal modes, representing directions of motion intrinsic to the structure, are easily obtained from an elastic network model [ ] (see methods) in which the protein is represented as one site per residue and springs of uniform strength are placed between every pair of sites with a separation less than Å. in the lu structure, the amino acid residues of the bound inhibitor are included in the elastic network. of the n normal modes of a structure with n residues, six modes of near-zero frequency represent the trivial rigid body motions of the structure, while the low-frequency modes from upwards represent modes of flexible motion. when the structure moves along a low-frequency mode direction, the global geometry changes with minimal change in the local geometry, making these directions of "easy" motion for the protein. the sign of a normal mode eigenvector is arbitrary; motions parallel and antiparallel to each bias direction must be investigated equally. linear projection of a protein structure along a normal mode direction would rapidly introduce unphysical distortions into the structure. the geometric simulation approach implemented in froda [ , ] instead applies a bias, moving the structure along a normal mode direction, while maintaining steric exclusion and the bonding geometry and noncovalent constraints of the input structure. this approach rapidly generates physically realistic flexible variations on the input structure which retain all-atom steric and bonding detail. geometric simulations of the lowest-frequency nontrivial modes of motion ( upwards) show that the protease structure is capable of substantial amplitudes of easy motion covering several Ångstroms distance in global root-mean-square displacement (rmsd). in particular, the orientations of the alphahelical domains relative to the beta-sheet domains can change through flexing and rotation about the interdomain "hinges", and the beta-sheet domains themselves are large enough to display bending and twisting motions within themselves. these variations are illustrated for the y e structure in figure and for lu in figure . , shown in cartoon representation and coloured from blue to red along each chain. each flexible variation is the th frame of a geometric simulation, lying at an all-atom rmsd of to Ångstroms from the starting crystal structure. note the substantial variations of domain orientation achieved. each variation is labelled with the normal mode used to bias the geometric simulation, from to , and with the direction of bias, parallel (+) or antiparallel (-) to the mode eigenvector, whose sign is arbitrary. : flexible variations (outer circle) of the lu structure (centre), shown in cartoon representation and coloured from blue to red along each chain. the bound inhibitor is shown as spheres. each flexible variation is the th frame of a geometric simulation, lying at an all-atom rmsd of to Ångstroms from the starting crystal structure. note the substantial variations of domain orientation achieved. each variation is labelled with the normal mode used to bias the geometric simulation, from to , and with the direction of bias, parallel (+) or antiparallel (-) to the mode eigenvector, whose sign is arbitrary. flexible variations on a protein crystal structure are potentially relevant to structure-based drug design in at least two ways. firstly, flexible motion can open or expose clefts and potential binding sites not directly visible in the static crystal structure. since the protein in vivo will be exploring its flexible motion thanks to the brownian-motion driving force of its solvent (e.g. cytosol), such latent sites may constitute valid target areas for inhibitors. secondly, the global low-frequency motion couples to variations in the binding site/active site geometry [ , ] . knowledge of the range of flexible variation here is potentially useful for structurebased drug design and/or fragment screening, since attention can be focussed on candidate molecules that interact robustly with the binding site and tolerate its flexibility, in preference to molecules that interact well only with the crystal structure and not with its flexible variations. y e crystal structure (a) and cleft opening motion along modes -(b) and +(c). bottom row: lu crystal structure (d) and cleft opening motion along modes + (e) and + (f). protein structure is shown as spheres and coloured by amino acid type. green, aliphatic or aromatic residues; yellow, cysteine or methionine; red, hydroxyl (serine and threonine); magenta, acidic; purple, amidic; blue, basic. a more detailed view of this opening cleft is shown in figure . the residues are coloured to bring out the chemical character of the amino acid side chains. the portions of the alpha-helical domain surfaces that move aside to open the cleft display largely hydrophobic residues (aliphatic or aromatic side chains). intriguingly, the exposed surface area within the cleft is richly lined with a series of basic residues (lysine and arginine), and is flanked by acidic (aspartic and glutamic acids) and polar (threonine) residues. in the crystal structures the basic and acidic residues appear to be involved in a network of inter-and intra-domain salt bridge interactions which stabilise the core of the homodimer. an antagonist capable of targeting this zone of strongly polar surface geometry exposed by flexible motion could potentially disrupt the dynamics of the enzyme and interfere with its function. detail view of interdomain cleft opening (structure lu , mode +, th frame of geometric simulation). protein structure is shown as spheres and coloured by amino acid type. green, aliphatic or aromatic residues; yellow, cysteine or methionine; red, hydroxyl (serine and threonine); magenta, acidic; purple, amidic; blue, basic. one end of the bound inhibitor is visible as grey spheres in the lower left. the exposed cleft is rich in basic, acidic and polar residues. in the lu structure, a binding site on the flank of each beta-sheet domain is occupied by the inhibitor n-(( -methylisoxazol- -yl)carbonyl)alanyl-l-valyl-n~ ~-(( r, z)- -(benzyloxy)- -oxo- -((( r)- oxopyrrolidin- -yl)methyl)but- -enyl)-l-leucinamide (chain c of the structure), hereinafter "n ". this inhibitor is listed in the pdb as prd_ ; it is a peptide-like inhibitor, with a central core of amino acids modified at either end with heterogroups, and was previously known to inhibit a protease of a feline coronavirus [ ] (see pdb entry eu ). in the y e structure the same binding cleft is empty, which means that its geometry is likely to change in the course of flexible motion of the beta-sheet domain. figure shows the n binding site of the lu structure and the corresponding region of the y e structure. as noted, all structures have been aligned to the y e crystal structure for ease of viewing and comparison. figure also shows flexible variations of the y e site in the course of flexible motion. a set of residues forming a rough square around the binding site (glu , gln , asn and thr ) are highlighted in white as a guide to the eye. it is immediately visible that openings, closings and flexible distortions of the site are an intrinsic feature of the flexible motion of the protease. note in particular the effect of motion along the lowest-frequency nontrivial mode direction, mode . motion biased antiparallel to the mode eigenvector ( -) includes an opening of the cleft, while motion biased parallel to it ( +) closes it. since the n inhibitor is quite deeply embedded in the binding site of the lu structure, opening and closing of this cleft in the course of the protein's natural motion may be necessary for molecules to access the cleft. on visual inspection, the site in the y e structure is clearly slightly more closed that in lu , consistent with the proposition that such opening/closing motions are intrinsic to the protein. - + - + - + - + figure : top row: the n inhibitor binding site of the lu structure, and the corresponding empty site of the y e structure. rows below: flexible variations of the y e site from geometric simulations along normal modes as indicated. residues - of a single chain of the protease are shown as spheres and coloured by amino acid type. green, aliphatic or aromatic residues; yellow, cysteine or methionine; red, hydroxyl (serine and threonine); magenta, acidic; purple, amidic; blue, basic. to highlight the binding site, residues glu , gln , asn and thr are coloured white. substantial opening and closing of the binding site cleft occurs during the flexible motion. the rigidity and flexibility of two recently reported crystal structures (pdb entries y e and lu ) of a protease from the sars-cov- virus, the infectious agent of the covid- respiratory disease, has been investigated using pebble-game rigidity analysis, elastic network model normal mode analysis, and all-atom geometric simulations. this computational investigation of the viral protease follows protocols that have been effective in studying other homodimeric enzymes. the protease is predicted to display flexible motions in vivo which directly affect the geometry of a known inhibitor binding site, e.g. through an opening/closing motion, and which open new potential binding sites elsewhere in the structure. a database of generated pdb files representing natural flexible variations on the crystal structures has been produced and made available for download from an institutional data archive. this information may inform structure-based drug design and fragment screening efforts aimed at identifying specific antiviral therapies for the treatment of covid- . naming the coronavirus disease (covid- ) and the virus that causes it dynamic personalities of proteins rapid simulation of protein motion: merging flexibility, rigidity and normal mode analyses structure and function in homodimeric enzymes: simulations of cooperative and independent functional motions a complete thermodynamic analysis of enzyme turnover links the free energy landscape to enzyme catalysis exposing the interplay between enzyme turnover, protein dynamics, and the membrane environment in monoamine oxidase b. biochemistry protein flexibility is key to cisplatin crosslinking in calmodulin structure and function of l-threonine- -dehydrogenase from the parasitic protozoan trypanosoma brucei revealed by x-ray crystallography and geometric simulations the flexibility and dynamics of protein disulfide isomerase main protease structure and xchem fragment screen dataset for "rigidity, normal modes and flexible motion of a sars-cov- (covid ) protease structure the pymol molecular graphics system molprobity: more and better reference data for improved all-atom structure validation protein flexibility predictions using graph theory. proteins-structure function and genetics salt bridge impact on global rigidity and thermostability in thermophilic citrate synthase elnemo: a normal mode web server for protein movement analysis and the generation of templates for molecular replacement constrained geometric simulation of diffusive motion in proteins comparative analysis of rigidity across protein families crystal structure of feline infectious peritonitis virus main protease in complex with synergetic dual inhibitors key: cord- -w gc nx authors: nan title: poster presentation abstracts date: - - journal: j pept sci doi: . /psc. sha: doc_id: cord_uid: w gc nx nan background and aims: homodimerization of myd adapter protein is essential for nf-kb activation in the inflammatory pathway triggered by il- and tlr [ ] . we designed a peptidomimetic of the myd tir domain consensus peptide arg-asp-val-leu-pro-gly-thr [ ] , named st . here, we report its synthesis and biological activity. we also report the synthesis and biological activity of its enantiomer, st , and its diastereoisomers, st and st . methods: the structure of the myd tir domain consensus peptide is subdivided into three distinct portions, the most important of which is a b-turn. in the peptidomimetic design we changed the b-turn with a tricyclic spirolactam [ ] , already known [ ] . we synthesized this building block, its enantiomer and two of possible diastereoisomers by "in solution" synthesis. based on semiempirical calculation of heat of formation [ ] , we could predict the right stereochemistry of the products selectively obtained in the last cyclization step. results: these four compounds were tested for their biological activity by reporter gene assay (rga). some coimmunoprecipitation experiments were also carried out and we report their results. conclusions: the results show the activity of st and its isomers on our target, with limited specificity towards their stereostructure. introduction of a methylene bridge between the cα(i+ ) and the n(i+ ) atoms in an open peptide (i) to mimic simultaneously the cαh(i+ ) and hn(i+ ) protons (β-lactam scaffold assisted design -β-lsad) has proven to be a practical tool for the preparation of monotopic β-turn peptidomimetics (ii, r = r = h), according to the principle of separation of constraint and recognition elements . in this work we report a short, general, and stereocontrolled synthesis of multitopic β-lactam scaffolds of type vi. α-alkyl serinates iii are converted into the corresponding enantiopure nnosyl-aziridines iv which undergo "in situ" ring-opening with amino acids v. subsequent base-promoted cyclization affords the n-protected α-alkyl-α-amino-β-lactams vii. incorporation of the novel scaffolds into linear and cyclic peptides and their conformational features are also presented, most of them showing stabilized β-and γ-turn conformations. poly(amino acids) are emerging as promising therapeutic carriers finding widespread application in the field of drug delivery. in this context, polyproline polymers have been used to solubilize poorly water-soluble proteins, in affinity chromatography for the purification of platelet profilin, and more recently, in the design of dendrimers. poly(amino acids) are most conveniently synthesized by polymerization of the corresponding amino acid n-carboxyanhydride (nca). in spite of the interest of polyproline, the preparation of proline n-carboxyanhydride (pro-nca) renders poor synthetic yields. in this work a new method for the preparation of pro-nca in high yields and purities is described. amino acid n-carboxyanhydrides are obtained by the method described by fuchs. but, in the case of proline, the n-carbamoyl chloride does not cyclise spontaneously as it takes place with other amino acids, and the use of a non-nucleophilic base is required for the cyclisation. a tertiary amine, such as triethylamine, is commonly used but it renders a low conversion of the n-carbamoyl chloride to the expected pro-nca, together with the presence of the pro-pro diketopiperazine byproduct. in the present work, polymer-supported bases have been used instead of triethylamine. higher yields of pro-nca, and very low percentages of diketopiperazine have been obtained. in addition, no tertiary amine contamination was observed. polymer-supported bases could also be recycled and pro-nca yields were reproducible. in conclusion, we have developed an efficient method for pro-nca preparation with polymer-supported bases. the introduction of novel nonproteinaceous heterocyclic amino acids into peptides results in new compounds with interesting structural, physicochemical and biological properties. the transformation of amino acid side chains after the peptide assembly is a convenient method of generating such modified peptides. taking into account the biological activity and complexing abilities of nitrogen-containing heterocycles, we investigated the formation of imidazole, benzimidazole and quinoxaline moieties using condensation with various aldehydes and α-dicarbonyl compounds after classical peptide synthesis on solid support. the imidazole synthesis utilizes the n-terminal or side chain amino group of amino acids, whereas a derivative of phenylalanine, β-( -amino- -nitrophenyl)alanine, was developed for benzimidazole and quinoxaline synthesis. the modified peptides were purified by preparative hplc and characterized by esi-ms, uv and nmr. in conclusion, we developed a straightforward method of synthesis of peptides with specific ion affinity and spectral characteristic. the broad range of commercially available aromatic aldehydes and dicarbonyl compounds makes possible the synthesis of combinatorial libraries of modified amino acids and peptides. part of this work was supported by a grant no. t a from the ministry of education and science. nmda receptors belong to the ionotropic group of glutamate receptors. the activity of the receptor can be altered by compounds acting at binding sites. the (r,s)-(tetrazol- -yl)glycine (tg) has been shown to be a highly potent nmda (n-methyl-d-aspartic acid) receptor agonist with exitotoxic effects [ ] . the aim of our studies was to investigate the chelating ability of tg towards copper(ii) ions. copper is widely distributed throughout the body with a distinct concentration in the brain. copper enters cells as complex and seeks out targets requiring it to function. for these reasons it was interesting to evaluate stability and structure of tg -copper(ii) complexes. the equilibrium and structural properties of complex species were characterized by ph-metric and spectroscopic (uv-vis and epr) methods. in the system, polymeric species are dominant at acidic ph range having { nh , coo-} coordination with possible ntetr bridging elements. monomeric complexes were found at physiological ph. the two tg molecules are bound to copper ion via four nitrogen donors. the formation of two {nh , ntetr} donor sets results in very strong metal-ligand interactions and the complex species are very stable over a wide ph region. we have also performed an investigation on similar tetrazole compounds in order to compare the chelating ability of the tetrazole moiety . the targets of our studies were , -diamino- h- , , , -tetrazole [ ] and tetrazole aspartic acid. references continuing work in that field, we synthesized oxytocins containing tetrazole analogues of amino acids. the -tetrazolyl group is widely used in medicinal chemistry as an isostere of the carboxyl group. compounds containing tetrazole ring appear to be metabolically more stable than their carboxylic analogues and have comparable acidity. we synthesized derivatives of aspartic, glutamic, and alpha-aminoadipic acids containing h-tetrazole ring in side chains. these derivatives were then used for syntheses of oxytocin analogues substituted in position . apart from above we also obtained two analogues with tetrazole analogue of glycine in position . the first one contains h-tetrazole ring, the second one has tetrazole ring substituted with methyl group in position . oxytocin analogues possessing amino acids with tetrazole ring in side chains were synthesized on amide resin using fmoc methodology. in the case of analogues with c-terminal tetrazole ring, fragments - were synthesized on resin and then coupled with suitable dipeptides in solution. all obtained peptides show no pressor and rather low uteronic activity. however, for some analogues the uterotonic activity when measured in the presence of magnesium ions was several times higher. in humans, two classes of defensins, α-defensin and β-defensin, have been identified on the basis of tissue specificities and structural features including their modes of disulfide pairing. in general, particular combinations with disulfide bonding in cysteine-containing peptides are critical for expressing their intrinsic biological activities. in the case of human α-and β-defensins, however, disulfide isomers without the native pairing were demonstrated to exhibit similar antimicrobial activity to that of the native defensins. therefore, to assess the biological activities of defensins as well as defensin-based therapeutics, extreme care is required in the chemical synthesis of these peptides to avoid ambiguity in quality. in the present study, we synthesized human α-defensin- , - and - , and human β-defensin- , - , - and - by employing boc chemistry, and determined the optimal conditions for folding the respective reduced peptides preferentially into a native conformation. among the factors affecting the oxidative folding in the presence of reduced and oxidized glutathione, the buffer concentration and reaction temperature were essential. all the synthetic human α-and β-defensins were confirmed to have the respective native disulfide pairing by sequential analyses and mass measurements with cystine segments obtained by enzymatic digestion. all the human α-and β-defensins could be efficiently oxidized to the α-and β-defensin-type disulfide structure, respectively, under several conditions determined in the present study. these synthetic peptides of high homogeneity were used to accurately assess the antimicrobial activity. native chemical ligation is based on the reaction of a peptide bearing a c-terminal thioester group with an n-terminal cysteinyl peptide, leading to the formation of an amide bond at the aa-cys junction. the key starting materials for native chemical ligation are unprotected c-terminal thioester peptides. thioester peptides are often prepared using boc/benzyl solidphase peptide chemistry. however, the widespread use of the fmoc/tert-butyl chemistry for peptide synthesis, over the boc/benzyl method, has stimulated the development of methods allowing the preparation of thioester peptides that are compatible with the basic treatments used to remove the fmoc alpha-amino protecting group. we report here a novel method for thioester peptide synthesis that is based on the use of the sulfonamide safety-catch linker. once the peptidyl chain is assembled by fmoc/tert-butyl chemistry, the thioester function is generated on the solid-phase through an intramolecular n,s-acyl shift. the procedure seems to be insensitive to the bulkiness of the amino acid directly attached to the sulfonamide linker. the thioesters were successfully used for native chemical ligations in solution or on the solid support. we optimized the recognition sequence of the substrate and the reaction conditions with respect to the yield. the sortase-mediated ligation was successfully applied to the synthesis of cellpenetrating peptide-pna conjugates which showed enhanced activity in antisense experiments compared to pna alone. this ligation strategy was also employed for the coupling of a chemically synthesized construct of the extracellular loops of the crf-receptor with the corresponding n-terminal receptor domain, which was expressed in e. coli. this kda protein behaves like an artificial receptor, binding specifically natural ligands. linear gramicidins represent the most investigated family of antibiotic peptides forming ionic channels. gramicidins produced by bacillus brevis are hydrophobic peptides composed of amino-acids with d and l configuration strictly alternate. the presence of d-amino acids in the sequence of gramicidin a (hco-val-gly-ala-dleu-ala-dval-val-dval-trp-dleu-trp-dleu-trp-dleu-trp-nhch ch oh) should possible make the peptide highly resistant to proteolysis [ ] . striking features like ethanolamine group in c-terminus, the n-terminal n-formylated valine and the high hydrophobicity of the peptide sequence, make the solid-phase synthesis of gramicidin a very tricky. therefore, we followed a new synthetic strategy for peptide chain elongation assisted by microwave energy. in fact, microwave energy has been demonstrated to produce highpurity compounds with more rapid reaction times, enhancing coupling rates and efficiency in difficult syntheses [ ] . however, microwave-assisted solid phase peptide synthesis (mw-spps) has not been yet extensively investigated. in this context, we synthesized gramicidin a by mw-spps in high yield and purity, enhancing reaction rate compared to the traditional spps. thermal disruption of peptide aggregation, induced by microwaves, is possible favorable for obtaining this particularly difficult sequence. gramicidin a was incorporated in synthetic lipid bilayers, self-assembled on mercury electrodes, characterized by hydrophilic spacers interposed between the metal and the lipid bilayer. we tested the behaviour of gramicidin a in biomimetic membranes using electrochemical impedance spectroscopy (eis), ac voltammetry and other electrochemical techniques [ ] . [ csf (glc) is an n-glucosylated peptide to be produced in large scale by peptlab because it is the active molecule of the first specific diagnostic/prognostic test for monitoring disease activity and guiding therapeutic treatments of multiple sclerosis patients [ ] . in order to develop a synthetic protocol by an automated instrumentation, increasing yield, purity of the crude, and reaction time, a microwave-assisted solid phase peptide synthesis was validated comparing the use of the new generation of triazine-based coupling reagents (tbcrs) with a series of commonly used ones. activation of carboxylic acids by tbcrs is particularly effective because of formation of triazine "superactive esters". the usefulness of tbcrs as coupling reagents has been recently confirmed in the synthesis of z-, boc-, and fmoc-protected dipeptides, sterically hindered amino acids, in the synthesis of esters, in manual and automated spps of difficult peptide sequences, and head-to-tail constrained cyclopeptide analogues [ ] . moreover, we also demonstrated tbcrs efficient in a microwave-assisted solution synthesis of the n-glucosylated building block fmoc-asn(glcoac )-oh using a manual monomode microwave instrument [ ] . this building block was used to obtain csf (glc) comparing the efficacy of a monomode microwave automatic instrument with the traditional solid-phase peptide synthesizers such as the manual and automatic in batch systems, as well as the continuous-flow one. it is known that enzymatic peptide synthesis is more advantageous than chemical synthesis in many aspects; it is highly stereoselective, racemization-free and requires minimal side-chain protection. the method is, however, limited to the use of amino acid derivatives which meet the enzymatic specificity as a coupling component. this problem may be solved using enzymes which have wide specificity of substrate. but in this case, secondary hydrolysis of the resulting peptide may arise from the inherent nature of the protease. in this matter, ficin and ficin-like enzymes were used as cysteine protease to analyze the diminishment of specificity for the substrate. the cysteine protease-catalyzed peptide coupling reaction has been studied by using synthetic fourteen boc-amino acid phenyl and naphthyl esters as acyl donor. the reaction conditions were optimized for organic solvent, ph, and concentration of acceptor. the coupling reaction was carried out by incubating an acyl donor ( mm) with an acyl acceptor (ala p-nitroanilide, mm) and enzyme ( . u) in a mixture of gta buffer ( mm, ph . ) and dmso ( : ) at ْc. the progress of the coupling reaction was monitored by rp-hplc. the products were obtained in satisfactory yields. non-enzymatic glycosylation, also called glycation, is a common modification in living organisms formed by the reaction of carbohydrates with free amino groups of peptides and proteins. it is a slow chemical reaction yielding amadori products undergoing further oxidation and degradation reactions finally leading to advanced glycation end-products (age). amadori products are early markers for ageing, diabetes mellitus and alzheimer's disease. despite the clinical importance of these amadori products, universal protocols to synthesize amadori modified peptides are still missing. here we describe a solid phase strategy for the glycation of specific amino groups on partially protected resin bound peptides using a global post synthetic approach. the peptides were synthesized by standard fmoc/tbu-chemistry using carbodiimide activation. the lysine position to be modified was incorporated with a methyltrityl protected ε-amino group, which can be selectively cleaved after completion of the peptide synthesis with % tfa in dichloromethane. the partly deprotected peptide was glycated in methanol using a ten-fold molar excess of , - , -di-o-isopropylidene-aldehydo-β-d-arabino-hexos- -ulo- , -pyranose and nabh cn for h at °c. after cleavage the overall yields were in the range of - % for the tested octapeptides. all byproducts were well separated by rp-hplc allowing a simple purification strategy even for medium-sized peptides. thus the general strategy presented here allows routine synthesis of amadori peptides at reasonable yields and purities using standard protocols established in most laboratories synthesizing peptides. -chlorotrityl chloride resins are recommended for the synthesis of c-terminal proline peptide acids to overcome diketopiperazine formation during chain assembly. however, we have found these (and similar) resins to be unsuitable for the synthesis of peptides greater than residues. for example, the chemokine guinea pig eotaxin, ( residues c-terminal proline) assembles poorly if not at all on a -chlorotrityl resin. we sought to circumvent these problems in the chemical synthesis of peptides and proteins, through the development of a resin-swap procedure. whereby the initial c-terminal protected tripeptide is assembled on a -chlorotrityl resin, liberated from the solid-support, then reattached to a resin that is suited for long chain peptide / protein synthesis. using this approach, the synthesis of guinea pig eotaxin is reported. the tripeptide fmoc-thr(but)-lys(boc)-pro-oh was assemble on -chlorotrityl resin, cleaved with % tfe in dcm and attached to wang resin using standard protocols. peptide assembly gave the gp eotaxin in % overall yield (as determined by uv monitoring). fmoc-on cleavage, purification and tag removal followed by folding gave the native chemokine in good yield. choice of resin is one of the most critical factors in ensuring a successful peptide synthesis, we have shown the superiority of wang resin over chlorotrityl resin in the synthesis of medium and long peptides and developed a method for the synthesis of c-terminal proline containing peptides which overcomes the problem of diketopiperazine formation. the technique is being applied to the synthesis of other c-terminal proline peptides e.g. human eotaxin and ip . dimerization of cell receptors, involved in antigen presentation, is an essential step in several cellular signal transduction processes, therefore substances that are able to modulate such a process are of potential therapeutic value. dimeric peptide ligands could represent useful tools to cause dimerization of such receptors. a similar strategy applies dimerization of ligands, interacting with dimeric proteins or proteins with multiple binding sites, to design molecules with enhanced affinity. dimeric analogs of the immunosuppressory hla class ii fragments were synthesized using suitably modified, standard fmoc solid-phase protocols and mbha-resin. the dimerization was achieved by crosslinking n-terminal amino groups of the peptides with the commercially available mixture of poly(ethyleneglycol)biscarboxylic acid (average mw , length range - Å), activated by esterification with pentafluorophenol. the same procedure was applied to synthesize a series of dimeric analogs of c-terminal fragments of plexin-b, consisting of two undecapeptides, linked by the polyethyleneglycol spacers. other biand polyvalent linkers were also investigated. our results demonstrated that the amino-terminal dimerizations of the tested hla-fragments resulted in enhanced immunosuppressive activities, whereas interaction of pdz dimer with the plexin fragments led to about -fold increase in affinity, as compared to their monomeric counterparts. [background and aims] elucidation of alzheimer's disease (ad)-related aß - dynamic events is a difficult issue due to uncontrolled polymerization. [methods] based on the "o-acyl isopeptide method" (chem. commun. , ; j. am. chem. soc. , , ), we have developed a novel photo-triggered "click peptide" of aß - ( ), e.g., " -n-nvoc- -aiaß ( )", in which a -nitroveratryloxycarbonyl (nvoc) group was introduced at ser in -o-acyl isoaß - ( -aiaß , ). [results] i) the click peptide did not exhibit the self-assembling nature under physiological conditions due to one single modified ester; ii) photo-irradiation of the click peptide and subsequent o-n intramolecular acyl migration afforded the intact aß - ( ) with a quick and one-way conversion (so-called "click"); and iii) no additional fibril inhibitory auxiliaries were released during conversion to aß - ( ). [conclusions] this method provides a novel system useful for investigating the dynamic biological functions of aß - , such as the self-assembly and aggregation processes in ad. several insulin analogues have recently been introduced clinically for improved treatment of diabetes. industrial productions of such insulins are based on microbial expression systems, which are highly efficient, but generally limited to the proteogenic amino acids. also, some sequences form inclusion bodies or fail to express. the total chemical synthesis of insulin in research scale was a landmark achievement in peptide science. however, the most commonly used method relies on recombination of a-and b-chains under "random" folding and pairing of the three disulfide bridges. this folding/oxidation step is difficult and low yielding. a general approach using a removable auxiliary which can direct correct formation of disulfide bridges is highly desirable. in the pancreas as well as in microbial expression systems, insulins are prepared and folded as single chain precursors, with a c-peptide connecting the a and bchains. the c-peptide helps direct the orientation of a and b-chains in obtaining the correct disulfide pairing and overall peptide folding. upon folding, the c-peptide is removed enzymatically. we report here a new method for total chemical synthesis of insulin by use of fmoc-based step-wise solid-phase synthesis of single-chain precursors followed by cpeptide directed folding and cleavage of c-peptide, thereby allowing total chemical synthesis of novel insulins with unnatural substitutions. -chloro- -methoxy- , , -triazines a-c anchored on cellulose, silica or wang resin were prepared by the treatment of , -dichloro- -methoxy- , , -triazine with appropriate solid support in the presence of a base. immobilized, environmentally friendly triazine coupling reagents a-c were obtained by treatment of a-c with n-methylmorpholinium p-toluenesulfonates in the presence of hcl acceptor. the loading of the solid carriers were calculated from n, s contents, determined by microanalysis. all prepared immobilized n-triazynylammonium toluenosulfonates a-c have been found stable at room temperatures. activation of carboxylic components afforded triazine activate esters a-c connected to the support. treatment of a-c with appropriate amino components gave amides or peptides. the final products, chromatographically homogenous amides and peptides, were isolated by filtration or extraction from the solid support. mutter's pseudoproline dipeptides and sheppard's hmb derivatives are powerful tools for enhancing synthetic efficiency in fmoc spps. they work by exploiting the natural propensity of n-alkyl amino acids to disrupt the formation of the secondary structures during peptide assembly. their use results in better and more predictable acylation and deprotection kinetics, enhanced reaction rates, and improved yields of crude products. however, these approaches have certain limitations: pseudoproline dipeptides can only be used for sequences containing serine or threonine, and the coupling of the amino acid following the hmb residue can be extremely difficult. to alleviate some of these shortcomings, we have prepared fmoc-ala-(dmb)gly-oh and fmoc-gly-(dmb)gly-oh. these dmb-dipeptides can be incorporated into peptides in place of ala-gly and gly-gly, resulting in peptides containing structure breaking (dmb)gly residues. by introducing the (dmb)gly residue as part of a dipeptide unit, the need to acylate the highly hindered secondary amino group of (dmb)gly is avoided. on treatment with tfa the dmb group is cleaved regenerating gly. to test the efficacy of our new derivatives in expediting the synthesis of hydrophobic peptides, we undertook the preparation of the challenging neurotoxic prion peptide - <b> </b>; this peptide reportedly can not be made using fmoc spps methods. the dipeptides marked in bold were systematically substituted with the appropriate dmb peptides. the effects of the substitution were evaluated using conductivity monitoring and lc-ms analysis of the crude peptides. h-lys-thr-asn-met-lys-his-met-<b>ala-gly</b>-ala-ala-ala-<b>ala-gly</b>-ala-val-val-<b>gly-gly</b>-leu-gly-oh <b> </b> th efficient dipeptide production form unprotected l-amino acids with the novel enzyme l-amino acid α-ligase. k. tabata , h. ikeda , m. yagasaki , s. hashimoto background and aims: application of α-dipeptides has been limited due to the lack of cost-effective manufacturing methods. the known methods require the protection of amino acid(s) to fix the order of the amino acids ( fig. ) . furthermore, they usually accompany the formation of longer peptides. to establish the costeffective manufacturing method, a novel activity which synthesizes α-dipeptides from two unprotected l-amino acids was screened. methods and results: a gene was found in the genome of bacillus subtilis by in silico screening based on a putative reaction mechanism. the purified protein coded on the gene, i) catalyses α-dipeptide formation from unmodified l-amino acids with a specific order in an atp-dependent manner, ii) never forms tri-or longer peptides, and iii) takes a wide variety of l-amino acids but no d-amino acids. the enzyme was tentatively named l-amino acid α-ligase (lal). the whole cell reaction of a recombinant e. coli strain expressing lal and polyphosphate kinase (ppk) with two l-amino acids and polyphosphate (polyp) enable the efficient production of many dipeptides with a certain order of the constituent amino acids through the coupling reaction of lal and ppk (fig. ) . conclusion: a novel enzyme, lal, enables to synthesize dipeptides cost-effectively directly from unmodified l-amino acids. t. ye marine organisms continue to provide rich sources of structurally unique and pharmaceutically active compounds. due to the difficulties in the isolation of significant quantities of these natural products, synthetic chemistry serves an important role in their structural assignment and biological evaluation. antifungal agents have received considerable attention recently since the spread of hiv has left many people open to fungal infections, and there is a rapidly growing number of drug resistant strains of fungus emerging. ll- g gamma is a cyclodepsipeptide isolated from the marine fungus hypoxylon oceanicum and structurally assigned in by schlingmann. the structure of ll- g gamma was determined by a combination of chemical degradation, chiral chromatography and spectroscopic analysis. ll- g gamma uniquely combines a beta-ketotryptophan and a polyketide portion within a macrolactone ring. ll- g gamma has exhibited potent activity against fungal strains and as such, is an attractive compound to develop as a future therapeutic agent. to date, there have been no reported studies towards the synthesis of ll- g gamma. we have completed the total synthesis of ll- g gamma by employing the macrolactamization followed by a c-h oxidation as the key step. aspartimide (aminosuccinimide, asu) formation is the first step in the degradation of asp/asn containing peptides and proteins. the reaction is especially prevalent at asx-gly sites and results in a variety of rearranged and racemized products. the bases used in fmoc-tert-based spps promote the formation of asu and related products. we recently found that the dmb backbone protection efficiently prevents secondary structure formation at gg sites and is orthogonal with respect to standard fmoc spps. here we explore the use of dmb, tmb and nbzl groups (z) for the synthesis of "difficult"/asu-prone peptides, in three different schemes: a) fmoc-asx-(z)gly-oh dipeptide building blocks; b) fmoc-(z)gly-oh monomer building blocks, and c) two steps "submonomeric" approach for synthesis of substituted n-benzyl glycines on the resin. we tested the new methods on two model peptides vkd/ngyi and ha - hiv-tat - (h-g lfgaiagfi engwegmidg grkkrrqrrr -oh) fusion peptide. the yield and purity of the products reach and even exceed the level in control experiments obtained with hmb protection and the peptides were found free of asu/piperidides. the acid removal of the dmb protection is ~ % faster than that of hmb. the submonomeric route (strategy c) is especially simple, efficient, cost effective and it allows the use of different amines for halogen-displacement. the backbone protecting groups used were in many respects superior to the commercial reagents and applicable for synthesis of both peptide acids and peptide amides. the use of nbzl-nh for halogen displacement represents a new method for preparation of backbone-caged peptides. alkyl bonded silica gels historically have been the standard in reversed phase (rp) purification of biomolecules such as synthetic peptides, small proteins, and oligonucleotides. silica gels provided the resolving power needed for challenging separations and the mechanical stability required to be operated industrially under high pressure conditions. the chief disadvantage of silica gels is poor chemical stability under alkaline conditions, which limits their capability to withstand rigorous clean and sanitization -in-place (cip/sip) protocols. as a result, polymeric media have gained recent market attention because of their excellent chemical stability, which enables full compatibility with modern cip/sip protocols. however, first generation polymeric gels lacked both the resolving power and the mechanical stability to be compatible with industrial high pressure dynamic axial compression (dac) hardware. rohm and haas' advanced biosciences division recently introduced a new, monospheric, micron, high performance polymeric rp material. unlike existing softer polymeric gels, this product has higher mechanical stability which enables it to be used effectively with industrial dac / hplc hardware. in addition, this material provides high resolving power for the most challenging industrial separations, because of its unique and selective pore structure, as well as its small monospheric particle size. finally, because of its excellent chemical stability, the media is not limited in the range of ph that can be used. the combination of mechanical stability for high throughput, chemical stability for long lifetime in use, and high resolution for high yield, together translate to an effective cost-in-use solution for industrial polishing processes. we have developed new types of peptide nucleic acids with improved water solubility by introducing ether linkages and pyrrolidine rings in the main chain; pyrrolidine-based oxy-pnas (popnas). in this work, cellular uptake and endosomal release of the trans-l-popna oligomers, one of stereoisomes of the popna, were investigated. the cellular uptake was achieved by combining the popna oligomer with an n-terminal -mer peptide of an influenza virus hemagglutinin protein (ha ) that is labeled with a rhodamine fluorophore at the n-terminal and covalently linked with a hepta-arginine unit at the c-terminal (rho-ha -r ). the fluorescence images of the cho cells after incubation with fam-po( ) [fam-o-cag tta ggg tta g-gly-nh ] in the absence and presence of rho-ha -r were observed with confocal laser-scanning microscopy. incubation with fam-po( ) alone, no internalization of the oligomer was observed. in the presence of rho-ha -r , however, fam-po( ) was successfully internalized into cho cells and, more importantly, the fluorescence spread over the whole cell. the fluorescence image indicates that the popna oligomer in combination with the ha -r peptide was transferred into cytoplasm within h. since both the red (rho) and green (fam) fluorescence spread over the cytoplasm, the popna oligomers that were taken up into endosomes together with the rho-ha -r were released into cytoplasm as the disruption of the endosomes by the ha peptide. in summary, the popna oligomers were readily taken up into cytoplasm of cho cells, when combined with a ha -r peptide. most of functional rnas have post-transcriptional modifications, some of which are quite important for their structure and function. thus, for studying such rnas, it is necessary to use purified raw rnas obtained from living organisms. isolation of native rna is necessary also in the case of analyzing the sequence and modifications of mature rna, which may be different from simple transcript of its gene. therefore, rna isolation method is required. many previous reports demonstrated isolation of rnas, especially trnas. most common and traditional purification methods are based on successive column chromatographies. it seems difficult to apply such method to every trna because effective combination of columns varies among individual trnas. to overcome the difficulty, a sequence-specific selection method using a solid-phase dna has been devised. in this method, a trna can be purified from rna mixture by a single step. however, this method needs high temperature treatment, which might assist hydrolysis of rna strand and might impair heat labile modifications. pna-rna hybrid has been known to be much more stable than dna-rna hybrid. thus pna-based rna purification method seems to be possible for wider variety of rnas in lower temperature, in comparison with dna-based method. in this study, we attempted to purify a single rna, such as a trna and a noncoding rna, from rna mixture by using immobilized pna. r. pipkorn , w. waldeck , h. spring , j. jenne , k. braun background and aims: safe drug delivery technologies are pivotal for genetic interventions, but viral vectors baer the risk of inflammatory reaction. questions concerning the efficacy of delivery of the genetic substances, the desired topical gene activation and targeting must be answered. therefore we attempted to develop a membrane non-perturbing delivery system for transport of inactive functional genes into cells and tissues. genes can be subsequently activated at the target site. our concept bases on the use of peptide-nucleic-acids (pnas) resistant against proteases and nucleases, oligonucleotide derivatives, in which the phosphate-backbone has been replaced with ethylen-amin connected alpha-amino-ethylglycine-units. methods: peptides conjugates were composed and synthesized according to the solid phase synthesis and protecting group chemistry strategies. pna sequences were conjugated covalently, non cleavable, with a capronic acid spacer to the nls, pkkkrkv. pnas have gained broad attention in antisense/antigene experiments and as diagnostic tools. in principal, they can be synthesised with several activating reagents known from peptide synthesis. namely, hatu or pybop are often used. synthesis with hatu is more laborious, because preactivation is needed in order to avoid guadinylation of the n-terminus of the growing pna-chain. we wanted to use pybop, because preactivation should not be needed in this case, which is especially useful in automated synthesis. surprisingly, in the pybop-mediated syntheses of mer pnas we obtained products showing molecular masses approx. da above the expected ones. detailed analysis revealed, that the modification occurred at the only guanine residue in the sequence. in order to further characterise the side reaction, a short pna fragment was synthesised using hatu and pybop activation, respectively, and cleaved from the resin with and without the n-terminal fmoc-group. while synthesis with hatu gave the desired products, pybop partly activates the aromatic carboxy group of the guanine residue, which is substituted by piperidine during subsequent fmoc cleavage. the modified sequences could be further characterised by ms/ms-fragmentation. our results show that care must be taken when synthesising pnas with pybop activation. on the other hand, this reaction possibly opens an opportunity to synthesise guanine derivatives. the opioid receptor system in the central nervous system (cns) controls a number of physiological processes including pain, reward, gastrointestinal and cardiovascular functions. as a consequence, most pain modulating compounds currently available cause a variety of side-effects. the endogenous ligands for the opioid receptors are a series of peptides that includes endomorphin- . endomorphin- has been shown to elicit potent anti-nociception through the highly selective activation of µ-opioid receptors. it is this receptor that mediates supraspinal analgesia and thus, selectivity for this receptor results in analgesia without affecting other processes. therefore, endomorphin- is considered a promising lead compound for the development of a new, safer pain medication. we have synthesized a large number of lipid-and carbohydrate-modified endomorphin- analogues and screened these compounds for their binding and activation of µ-and δ-opioid receptors in sh-sy y cells as well as caco- cell monolayer permeability and plasma stability. compounds conjugated with either a lipoamino acid or sugar moiety on the c-terminus lost binding affinity by several orders of magnitude, whilst n-terminal conjugations resulted in minimal loss of binding affinity. a number of analogues showed pm binding affinity and high apparent permeability, and of these compounds, one has been selected for assessment in nociceptive and neuropathic pain models. in addition to these pre-clinical studies, internalization and tolerance formation of these compounds has also been measured in an effort to synthesise a non-tolerant opioid agonist. endomorphin- analogues with a high degree of amphiphilicity cause increased receptor internalization and subsequently less tolerance formation. a. marcinkowska , l. borovičkova , j. slaninowá , z. grzonka carbohydrate moieties of glycopeptides and glycoproteins play different decisive roles in various biological phenomena. conformation and solubility of proteins are influenced by the oligosaccharide chains, which can also inhibit the proteolytic degradation. as a result, the synthesis of glycopeptides is an attractive field that contributes to understanding of mutual interactions between both moieties and for their biological interest. the synthesis of glycopeptides requires a combination of synthetic methods from both carbohydrate and peptide chemistry. moreover, this synthesis needs stereoselective formation of the glycosyl bond between a carbohydrate and a peptide (amino acid) part, and also an appropriate protecting group methodology that allows selective deblocking of only one functional group in these polyfunctional molecules. in the present work we modified the oxytocin and vasopressin structure with glycoamino acids. transformations of fmoc-protected serine and threonine derivatives into appropriate o-glycosylated precursors suitable for solid phase peptide synthesis were worked out. the -and -o-glycosides were synthesized from fmocserine and fmoc-threonine allyl esters and appropriate glycosyl bromide using hanessian's modification of the koenigs -knorr reaction. these n--fmoc-protected glycosides were used in synthesis of glycopeptides. eight analogues of oxytocin modified in position were obtained. we have also prepared two types of lysin-vasopressin analogues modified with glycoamino acid, in which the glucuronic acid was attached to the ω-amino group of lysine in position through the amide bond. glycosylated analogues of oxytocin and vasopressin display an increased stability towards enzymatic degradation, and retain some hormonal activities. supported by grants: ds/ - - - (zg) and z (js) according to many authors the formation of amadori products is a key stage in the glycation process. glycated proteins may show allergenic properties and potentially initiate autoimmunological processes. they may also serve as the markers of diabetes. to our best knowledge, all procedures concerning the synthesis of peptide-derived amadori products reported in literature are based on "in solution" approach which makes them tedious and time consuming. a modified method of the solid phase synthesis of peptide-derived amadori products based on direct alkylation of the deprotected ε-amino groups with , : , -di-oisopropylidene-β-d-arabino-hexos- -ulo- , -pyranose in the presence of sodium cyanoborohydride was proposed. isopropylidene groups, protecting the sugar moiety in the obtained conjugate, were removed with trifluoroacetic acid containing % water. studies on optimization of the reaction performed on the model peptide attached to a wang resin, fmoc-lys-leu-leu-phe-(resin), showed that the best yield of the product is attained with a two-fold excess of , : , -di-oisopropylidene-β-d-arabino-hexos- -ulo- , -pyranose and a five-fold excess of sodium cyanoborohydride. the identity of the product was confirmed by high resolution ms. the several side products were isolated and their structures will be discussed. our results prove that the synthesis of glycated peptides in the solid phase is feasible. the lack of homogeneous glycoproteins in sufficient quantities is an ongoing challenge in glycobiology. in order to solve this problem researchers have turned to a variety of approaches ranging from mutant eukaryotic strains to the highly demanding total synthesis of glycoproteins. [ ] using rnase b as a model nglycoprotein [ ] we have searched a path to assemble this enzyme employing a combination of chemical and recombinant methods. native chemical ligation [ ] allows the coupling of protein segments of unrestricted size in a chemoselective manner. we have developed solid phase methods to produce the required thioester building blocks - -sr (a) and glycopeptide thioester - -sr (b) containing an n-glycan at asn on a dual linker pega resin. [ ] the remaining segment - (c) was expressed in e. coli as a fusion protein and released by intein mediated protein cleavage. [ ] sequential coupling of the three rnase segments requires the use of a protective group at the n-terminus of segment b compatible with the oligosaccharide part. dysfunctional mutations of antitrypsin can result in a loss of elastase inhibitory activity or allow self-aggregation to occur and cause emphysema and cirrhosis, respectively. insights of the mechanism of disease provide strategy to cope with the aberrant protein aggregation and may bring potential therapeutic agents. in the present work, we describe our effort to identify effective anti-protein polymerization ligands by the employment of combinatorial technology. antitrypsin from human plasma was purified by glutathione sepharose and mono q-sepharose column chromatography. both ala-scanning and peptide shortening were carried out systematically to explore the structural requirements necessary for binding. combinatorial chemistry was then employed to conduct the library screening experiments. assessment of peptide binding was achieved through an unique gel electrophoresis assay. the structural requirements and the minimal peptide length required for binding were revealed by our systematic approach. this information was critical for the design of combinatorial library and the discovery of antitrypsin binding peptides with much improved affinity and specificity. there is currently no effective cure for z antitrypsin related cirrhosis and emphysema. the synthesis and screening of combinatorial libraries offer avenues to increase throughput and ultimately lead to the discovery of inhibitory peptides to the polymerization of pathogenic antitrypsin. with the rapidly increasing number of biopharmaceuticals in the industrial pipeline the need for efficient and expedient purification procedures is growing ever greater. affinity chromatography is one of the most promising technologies in this regard, as it offers very high selectivity and can often replace lengthy and expensive traditional chromatographic procedures. the use of combinatorial split-and-mix libraries is a powerful tool for discovering new affinity ligands but the technique has been limited by the laborious spectroscopic and chemical analysis needed to identify the binding ligand. we have previously introduced a novel bead encoding technology based on a -dimensional image recognition of patterns made by fluorescent particles randomly distributed inside larger beads. [ ] the beads are read prior to each chemical transformation by an instrument featuring three fluorescence microscopes at a rate of , beads per hour. we here present the development of small peptidomimetic affinity ligands for the human growth hormone (hgh) by the use of this technology. the library was sought enriched prior to synthesis by in silico screening of a virtual combinatorial library using a large number of diverse building blocks. binding ligands were identified by incubation with fluorescence tagged hgh. [ the cinnamic acids and their derivatives have been found to possess a variety of biological effects, including antiviral, antimicrobial, antitumor and antioxidant activity. for example, several hydroxycinnamic acid conjugates with amino acids, isolated from plant sources showed enhanced antioxidant activity. the synthesis of cinnamic acid amides and their opioid activity was also cited in the literature. however the synthesis and pharmacological properties of sinapoyl-peptide amides continues to be virtually unexplored. on the other hand, the synthesis and opioid activity of analogs of tyr-mif- has been well documented by us. herein we present a synthesis of a series of sinapoyl -peptide amides where sinapic acid were attached consecutively to both c-and n-end of the tyr-mif- peptide chain: sa-pro-xaa-gly-nh ; sa-tyr-pro-xaa-gly-nh ; pro-leu-gly-nh(ch )nnh-sa sa=sinapic acid; xaa=leu, unusual aminoacid; n= , to obtain the sinapoyl-peptide-amides, both fmoc-and boc-based spps approach were used. analgesic activity was determined by the randall-sellitto paw-pressure test. the antioxidant effects were examined by dpph test as well. studies to establish the importance of introducing the sinapoyl moiety in the tyr-mif- molecule for the antioxidant and opioid activities are underway. several proteins are involved in the transcription of dna to mrna, among which the basic leucine zipper (bzip) proteins. these transcription factors bind specific dna sequences by dimerization and inserting short alpha-helices into the dna major groove. because the dimerization domain is only required to obtain the correct geometrical positioning of the alpha-helices, we will replace it by a dipodal steroid scaffold with defined stereochemistry. due to orthogonal protecting groups, a unique feature of this scaffold is the possibility to design not only homodimers, but also heterodimers. therefore this strategy allows for the construction of both major/major groove and major/minor groove binding peptides, either mimicking naturally occurring proteins or designing peptides with new binding properties. native chemical ligation and staudinger ligation are both suitable for the construction of these peptide dimers. moreover, a combination of solution-and solid-phase chemistry allows for the generation of combinatorial libraries. the increasing number of antibiotic-resistant bacteria is a global health problem. therefore the development of new highly efficient drugs is one of the major tasks of this century. as an example of peptides, which inhibit the growth of e. coli, we demonstrate an easy and rapid method for finding peptides with optimized antimicrobial properties. as a first step we built a modular construct. this construct consists of a constant cationic and a variable module. the cationic module was choosen to achieve cellpenetrating properties. the variable module was expected to act as the virtual active part of the peptide. to increase the proteolytic stability of the peptide we synthesized them in cyclic form. in the first step we used the combinatorial approach to screen approximately . . peptide sequences in the variable region in order to find highly active peptides against e. coli. to optimize the identified sequence, we substituted all amino acids of the sequence with other amino acids and building blocks. additionally, in order to increase stability we modified the bridging. in this way we were able to uncover peptides with high antimicrobial activity as well as proteolytic stability and reasonable solubility. a series of melanocortin active core tetrapeptide hfrw nonpeptide imitations has been prepared using a combination of solution and solid phase synthesis. most of them included residue of -( -imidazolyl) propylamine or histamine as substitutes of histidine. phenylalanine residue, which is included in melanocortins was replaced by residues of derivatives of , '-disubstituted isopropylidenedicyclohexane, , '-disubstituted bicyclohexane, , -disubstituted cyclohexane, , -disubstituted cyclooctane, and , -, , -or , disubstituted benzenes. instead of arginine, residues of oligomethylene diamines, -butyl- -ethyl- , -pentanediamine, , '-methylene-bis(cyclohexylamine), and , '-diaminodiphenylmethane were introduced. -naphtyloxyacetyl-, ( - h-indol- -yl)-butyryl-, -phenyl-ethanesulfonyl-and naphthalene- -sulfonyl-groups served as replacement of tryptophan residue. tested on binding assay on melanocortin receptors, active core imitations exhibited a micromolar affinity to them. isopropylidenedicyclohexane and bicyclohexane derivatives showed about fold higher affinity compared with corresponding derivatives of cyclohexane, cyclooctane or disubstituted benzene. obestatin is a novel endogenous ghrelin-associate peptide, which is involved in the regulation of food intake and weight gain. it was shown to be anorexigenic, able to decrease food intake, gastric emptying and jejunal motility. although obestatin and ghrelin originate from a common prepropeptide of residues, they are reported to exert opposing physiological roles, by binding distinct receptors belonging to the subgroup of type a gpcrs [ ] . obestatin was found to be the natural ligand of the orphan gpr receptor, a gpcr, expressed in jejunum, duodenum, stomach, pituitary, ileum, liver and hypothalamus. as many other peptides involved in the obesity process, it is a new and interesting drug target for the discovery of new anti-obesity molecules. in particular, the first step for the design of new molecules with potential improved anti-obesity activity, is the elucidation of the obestatin conformational features. here, we present the synthesis and the conformational analysis by nmr and cd spectroscopies of obestatin and its related -mer c-terminal sub-fragment, in aqueous solution and in membrane mimicking environment. the data outline the obestatin c-terminal portion as the region characterized by significant conformational features potentially opened to interesting future developments. [ a total of isolates of rhizobium were collected from root nodules of medicago sativa and melilotus officialis plants in different regions of isfahan province .all of isolates on ty medium formed white ,slimy colonies with smooth margins and their inoculation on to roots of young alfalfa plants produced spindly nodules . the nodules developed with some of the isolates were big and pinkish ,although the rest of isolates produced small and white nodules .the speed of nodulation for all the isolates was almost similar and the related nodules were appeared within two weeks . the production of brown pigments on aged colonies of some isolates on ty or ty supplemented with l_tyrosine and copper sulfate revealed that these isolates of s. meliloti are melanin-producing rhizobia.based on the motility and sensitivity to antibiotics tests ,all of the isolates formed a reasonably homogenous group .however a few of them were able to produce an anti-microbial compound which was found to inhibit a number of isolates of s. meliloti .the compound did not suppress the growth of other bacteria . partial purification and spectrophotometery of the compound suggested that it likely belong to the antimicrobial polypeptides .considering on their physiological and biochemical properties ,none of the isolates were selected as a superior and competitive strain ,although based on nodulation efficiency , melanin and antimicrobial compounds production capability the isolate s. meliloti sm and sa were nominated to investigate in details. cyclotides are a fascinating family of plant-derived peptides characterized by their head-to-tail cyclized backbone and knotted arrangement of three disulfide bonds. this conserved structural architecture, termed the cyclic cystine knot, is responsible for their exceptional resistance to thermal, chemical and enzymatic degradation. cyclotides have a variety of biological activities but their insecticidal activities suggest that their primary function is in plant defense. in this study we determined the cyclotide content of the sweet violet viola odorata, a member of the violaceae family. we identified cyclotides from the aerial parts and roots of this plant, of which are novel sequences. the new sequences provide information about the natural diversity of cyclotides and the role of particular residues in defining structure and function. as many of the biological activities of cyclotides appear to be associated with membrane interactions, we used hemolytic activity as a marker of bioactivity for a selection of the new cyclotides. the new cyclotides were tested for their ability to resist proteolysis by a range of enzymes and, in common with other cyclotides, were completely resistant to trypsin, pepsin and thermolysin. the results show that while biological activity varies with the sequence the proteolytic stability of the framework does not, and appears to be an inherent feature of the cyclotide framework. the structure of one of the new cyclotides, cycloviolacin o , was determined and shown to contain the cyclic cystine knot motif. this study confirms that cyclotides may be regarded as a natural combinatorial template that displays a variety of peptide epitopes most likely targeted to a range of plant pests and pathogens. furthermore, the inherent stability of the framework makes it an excellent scaffold for protein engineering applications. warfarin is the most widely prescribed anticoagulant drug for the prevention and treatment of arterial and venous thromboembolic disorders.because of large interpatient variability in the dose-anticoagulant effect relationship and a narrow therapeutic index careful dosage adjustment based on inr is essential. warfarin is available as a racemic mixture of two enantiomers,(s)-and (r)-warfarin. in contrast to (r)-warfarin, which is metabolized by multiple cytochrome p s(cyps), including cyp a and cyp a ,(s)-warfarin, is predominantly metabolized to -hydroxywarfarin by polymorphic cyp c . since the potency of (s)-warfarin is much higher than that of (r)-warfarin, about -to -fold,any change in the activity of cyp c gene is likely to have a significant influence on the anticoagulant response. previous in vitro findings revealed that certain variants in the cyp c gene are associated with large interindividual differences in the pharmacokinetic and pharmacodynamic outcomes of warfarin therapy. three major alleles have been found to date in humans:arg /ile , and cys /ile and arg /leu , and arg /leu , which have been designated cyp c * (wild-type), cyp c * , and cyp c * , respectively. we have investigated this polymorphism in iranians that has not been described previously. genomic dna was isolated from whole blood. for detection of cyp c * , and cyp c * variants, a protocol based on pcr technique and endonuclease digestion with kpni, ava ii was used. in this research work, we have studied a group of patients, in which warfarin therapy was initiated. recently new -residue antimicrobial peptides -arenicins were isolated from coelomocytes of marine polychaeta arenicola marina and their sequences were determined [ ] . there are two isoforms of arenicins which differ only with single amino acid. these peptides have no structure similarity to any previously identified antimicrobial peptides. we have synthesized and estimated the antibacterial properties of arenicin- : rwcvyayvrvrgvlvryrrcw. the linear peptide was prepared by solid phase method using boc-technology without any problem. however the cyclization caused the appreciable difficulties. the following methods of oxidation were used: oxygen of air, k fe(cn) and hydrogen peroxide in aqueous or organic media. the best results were obtained by using hydrogen peroxide in methanol, but and in this case the yield of the aim peptide did not exceed %. synthetic arenicin had the same hplc profile and maldi-tof spectra as a natural molecule. the peptide showed an antimicrobial activity against gram-positive bacteria: peptidergic hormones and neurotransmitters are known to be produced by the specific cleavage of their precursor proteins that per se have no biological functions. the neutrophil-activating peptides we recently identified, however, are the peptides cleaved from mitochondrial proteins by proteolysis. therefore, we named them "functional cryptic peptides" because they are hidden in protein sequences. some of these peptides activate gi type of g proteins directly, and neutrophils are suggested to be stimulated by the direct (i.e., not via gpcrs) activation of g proteins. these peptides had features, in common, in their distributions of charged and hydrophobic amino acid residues, but homologies in their primary structures were not apparent. in the present study, we predicted functional cryptic peptides that activate g proteins, based on the distribution of charged and hydrophobic residues. receptors for these peptides were also investigated by the direct cross-linking experiments between peptides and their targeted proteins. the finding of functional cryptic peptides is expected to lead to the identification of novel signaling mechanisms where such peptides are involved in the regulation of bio-functions. the fragment - of the precursor of human interleukin- alpha (pil- α) (gk-vlkkrr) appeared to have more than % homology with corticotropin fragment - (gkpvgkkrr). we have previously synthesized the octapeptide gkvlkkrr (referred to as leucocorticotropin, lct) and found its high affinity binding to corticotropin receptors on various immunocompetent cells in human and mouse. in this study we investigated the interaction of lct with rat adrenal cortex membranes and the effects of lct on the level of -oxycorticosteroids (cs) in rat adrenal glands and plasma in vivo. lct was labeled with tritium by the high-temperature solid-state catalytic isotope exchange reaction to specific activity of ci/mmol. receptor binding studies revealed that tritium-labeled lct bound with high affinity and specificity to corticotropin receptor on rat adrenal cortex membranes (kd = . nm). lct at concentrations of . - nМ was found to have no influence on the adenylate cyclase activity in adrenal cortex membranes, while intranasal injection of lct to rats at doses of - microg/kg was found to inhibit the secretion of cs from the adrenals to the bloodstream. thus, lct is an antagonist of corticotropin receptor. comarin derivatives such as warfarin are prescribed widely for treatment and prevention of thrombosis. warfarin is the widespread oral anticoagulant drug employed, but its required dose is highly variable both inter-individually and inter-ethnically. so it is desirable to develop strategies to predict the warfarin dose response in patients before initiation of anticoagulation. the vitamin k-dependent γ-carboxilation system, consists of the vitamin k-dependent γ-carboxylase, which requires the reduced hydroquinone form of vitamin k as a cofactor and the warfarin sensitive enzyme vitamin k , -epoxide reductase (vkor), which produces the cofactor. warfarin exerts its anticoagulant effect by inhibiting the vitamin k epoxid reductase enzyme complex (vkor) that recycles vitamin k , -epoxide to vitamin k hydroquinone. a component of the vkor termed vkorc , has now been identified as a therapeutic target site of warfarin. point mutations were identified within the gene encoding vkorc in individuals who required large doses of wafarin to maintain therapeutic anticoagulation. however the relationship between the primary structure of vkorc and the mechanism of action of warfarin is poorly understood. in previous works we have shown that naturally occurring functional protein fragments affect cell proliferation [ ] . their mechanisms of action involve receptors of "classical" regulatory peptides, or are non-receptoric [ ] . among protein kinases involved are pka, camkii, mapk [ ] . in organism bioactive functional protein fragments could participate in maintenance of tissue homeostasis. in present work, homeostatic potential of functional protein fragments was studied in compare with classical regulatory peptides. the panel of test substances was formed, including signal transduction modulators (pka, pkc, ca +-channel activators), classical regulatory peptides (bradykinin, somatostatin, met-enkephalin, endothelin, neurotensin) and fragments of functional proteins (β-actin fragments from ( - ) and ( - ) segments; valorphin (β-globin ( - )); neokyotorphin (α-globin ( - ); short acidic peptides from multiple precursors). their activity was tested in cultures derived from similar sources but differing in transformation degree (e.g., mouse embryonic vs. tumor fibroblasts) and/or culturing conditions. the factors most affecting cell sensitivity to the test substances were (in order of the importance decrease): ( ) cell type; ( ) transformed vs. normal phenotype; ( ) cell density; ( ) serum supply. activity of fragments of functional proteins, showing general correlation with other test substances, was more influenced by the culturing conditions (i.e., cell population status). thus, fragments of functional proteins could be regarded as partners of "classical" homeostasis regulators, playing role of finer tuners of tissue proliferative status. the study was supported by ras presidium programme "molecular and cell biology" histone-like proteins in bacteria are small basic proteins that contribute to the control of gene expression, recombination and dna replication. they are also an important factor in compressing the bacterial dna in the nucleoid. among the hlps, hu protein is attracted to dna containing structural aberrations such as four way junctions or single stranded lesions. this protein plays an important role in binding as a dimer and bending dna. it also contributes to the beginning of the dna replication. in this study we showed that a -kda protein, probably hu exists in halobacillus karajiensis which is a novel gram positive moderate halophile bacteria that was recently isolated from surface saline soil of the karaj region, iran. since hu is purified and characterized in e.coli we used this bacteria as the control in this study. the -kda protein extraction was carried out by using pca % which is normally used for extracting histones from eukaryotic cells. the results of running the protein extracts on sds-page demonstrated a band around -kda which was seen in protein extracts of this protocol. these results supported the hypothesis of the existence of a -kda protein in halobacillus karajiensis. sufficient oxygen and nutrient delivery is a necessity for tumors. when oxygen supply decreases, tumors initiate growth of new blood vessels. low grade astrocytomas, a class of malignant brain tumors, grow along the existing vessels in a process called co-option. hypoxia is induced in the progression from grade iii to grade iv astrocytomas (glioblastoma multiforme, gbm) which in turn triggers the formation of a new and distinct tumor vasculature. the new vessels formed by tumor-triggered angiogenesis differ by molecular composition from their normal vascular counterparts. we are utilizing phage displayed peptide libraries to identify peptides that specifically home to either co-opted or angiogenic brain tumor vessels. furthermore, we aim at characterizing differentially expressed endothelial markers (receptor molecules) to get a better understanding of the molecular changes in the vasculature. several rounds of in vivo biopanning was performed in mouse models of astrocytomas to isolate a phage pool that has up to a hundred-fold homing to low grade tumor lesions. out of the selected pool we discovered peptides capable of homing and accumulating to the tumor islets and co-opted vasculature. the homing potential of our newly identified peptides has shown to be highly specific for clusters formed by the tumor cells and co-opted "early" vessels within these palisades. these homing peptides represent promising candidates to selectively target co-opted vessels and tumor lesions in the brain and act as lead compounds in identification of surface molecules (receptors) differentially expressed by co-opted tumor vessels. the αvβ integrin receptors play an important role in human tumor metastasis and growth. the inhibition of these receptors by antibodies or by cyclic peptides containing the arg-gly-asp( rgd) sequence may be used as selectively treatment to suppress the disease. our research group has previously described that the formal introduction of a single carbon atom to bridge the cα (i) and n(i+ ) contiguous residues of a linear or cyclic peptide leads to α-amino-β-lactam peptidomimetics containing predictably placed β-turn and γ-turn motifs, respectively. the combination of these results with the well-known capacity of rgd tripeptide for inhibition of the biological answer in integrin led us to the design of the following cyclic peptide. the adhesion and cell-growth "in vitro" assays using human umbilical vein endothelial cells (huvec), as well as "in vivo" assays with xenograph mice revealed that the rgd peptidomimetic was active to micromolar concentrations, slightly better than the reference compound in this field: cilengitide®. whole saliva is composed of secretions from parotid, submandibular and sublingual glands, and smaller ones from saliva of minor salivary glands (e.g. palatal and labial). saliva contains a variety of proteins and polypeptides. one of them is statherin, a multifunctional -amino acid residue, phosphominiprotein. this peptide is present in human parotid and submandibular saliva . the aim of our study was to investigate the stability of statherin in extracts of the major salivary glands. submandibular, sublingual and parotid gland tissues were obtained at autopsy h after death. samples of the gland tissues were homogenized, centrifugated ( , g, min., c) and the supernatants were frozen and stored at - c prior to analysis. synthetic statherin was added to the supernatants before analysis ( microgram/ml). the samples were analysed for the presence of the peptide by the matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (maldi-tof ms}technique and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds page). statherin has been found to be decomposed in extracts of parotid and submandibular glands and also in the extract of sublingual glands. the dramatic increase in research for new anthrax therapeutic approach was prompted by potential use of the causative agent of anthrax bacillus anthracis as a biological weapon. anthrax toxin consists of three proteins, the protective antigen (pa) and the two enzymes lethal factor (lf) and edema factor (ef) that are carried through the membrane of the target cell upon binding to specific site on the membrane receptor-bound pa. lethal factor and edema factor were found to cooperate to promote immune evasion of the bacterium. here we describe the production of peptide inhibitors of pa-lf binding, obtained by selecting pa-binding peptide by a competitive panning of a phage peptide library, using recombinant lf. we selected several mer peptides, which were synthesized in tetra-branched multiple antigen peptide (map) form, inducing resistance to proteolytic degradation ( ) and maintaining biological activity of phage peptides. lead tetra-branched peptides were systematically modified by progressive shortening and residue randomization, to obtain an increase of peptide affinity and inhibitory efficiency. affinity maturation of lead sequences enabled selection of a peptide which has an ic at least one log lower that any other lethal-toxin-inhibiting peptide described so far and is effective for in vivo neutralization of anthrax toxin activity ( ) . the same peptide can also efficiently inhibit the binding of ef to pa and ef-induced camp increase in different cell lines. microtubules are dynamic polymers that have important roles in eukaryotic cellular processes such as signal transduction, cell polarity, vesicular transport and chromosomal movement. the dynamic behavior of microtubules has been studied both in vivo and in vitro. the effect of arsenic trioxide on microtubule polymerization has been studied under in vivo experimentation shown that it inhibits formation of mitotic bundles. we studied the mechanism of arsenic trioxide effect on polymerization of microtubule protein purified from sheep brain in vitro. microtubule polymerization has been conducted by adding mm gtp to purified tubuline in pem buffer at oc for minutes and simultaneously followed by measuring turbidity ( nm). the results shown that lag time of polymerization (nucleation step) is affected by increasing concentrations of arsenic trioxide from - micromolar. moreover the rate of elongation step was decreased exponentially by increasing arsenic trioxide concentration. electron micrographs also showed microtubules length decrement due to arsenic trioxide. the results have shown the inhibitory effect of arsenic trioxide on microtubule polymerization via its effect on nucleation step as well as elongation rate. background and aims: alzheimer's disease (ad) is the major cause of dementia among the elderly. the increase in life expectancy worldwide demands new therapies for ad urgently. self-association of the amyloid ß-protein (aß) into neurotoxic assemblies, a seminal event in the etiology of ad, is considered to follow interactions of the c-terminus of the -residue form of aß (aß ). we hypothesized that molecules with high affinity for the c-terminus of aß will disrupt aß oligomerization. a series of c-terminal fragments (ctfs) of aß , aß(x- ) with x = - , has been prepared to study their potential to inhibit aß oligomerization and neurotoxicity. methods: attenuation of aß assembly by ctfs was studied by quantitative analysis of oligomer size distributions using a photo-cross-linking assay followed by sds-page. biological activity of ctfs themselves and as inhibitors of aß -induced neurotoxicity was assessed by mtt reduction assay using differentiated pc- cells. the structure of the ctfs was studied by circular dichroism (cd) spectroscopy and ion mobility spectrometry-mass spectrometry (ims-ms) coupled with molecular dynamics (md) simulations. results: ctfs were found to inhibit aß oligomerization in a length dependent manner with minimal or no toxicity of the ctfs themselves. certain ctfs were found to inhibit aß -induced neurotoxicity. cd spectra indicate that increasing peptide length results in growing ß-sheet content. structures based on experimentally determined cross-sections support the existence of a previously proposed turn around residues gly -gly . the data suggest that aß ctfs can serve as lead compounds for development of peptidomimetic drugs for treatment and prevention of ad. background and aims: human kallikrein hk is a prostate specific serine protease, which expression level is elevated in aggressive human prostate cancer suggesting a possible role in a tumour growth and spreading. since hk protease is highly prostate specific, inhibition of its activity is a possible method to prevent tumour growth without interfering the function of the other proteases. we have identified hk specific linear peptide inhibitor by using phage display techniques. in order to design peptide for in vivo studies we tested the protease stability of the linear and the cyclic forms of the peptide. methods: the prerequisite of the binding was studied by using conventional ala-replacement method and the most optimal sequence was selected for further studies. the stability of the original linear form, acetylated form, peptide with cystein bridge and head-to-tail cyclic peptide was tested with modified trypsin (sequencing grade) and with human plasma. results: both linear versions and peptide with cystein bridge were unstable and were degraded during the first minutes in both stability tests. head-to-tail form of the peptide was stable in both tests during the first minutes. conclusions: since our peptide contains arginine there was a possibility that our peptide is sensitive to trypsin and other serum proteases. indeed both linear and one cyclic from degraded in our tests. only head-to-tail peptide was stable during the first hours suggesting protease resistant folding. background and aims: a large number of anticancer agents has been developed in recent years. however, these agents have very little or no specificity which leads to systemic toxicity. among them paclitaxel is considered to be one of the most important drugs in cancer chemotherapy; however, this agent has also lack of selectivity to the tumor tissue. therefore, development of tumor-targeting prodrug is highly promising. methods: to activate cytotoxic agent specifically at the tumor tissue, we developed a new prodrug strategy based on o-n intramolecular acyl migration, which is a well-known reaction in peptide chemistry, and photodynamic therapy. results: we synthesized a prodrug which has a coumarin derivative conjugated to the amino acid moiety of isotaxel (o-acyl isoform of paclitaxel). the prodrug was selectively activated by visible light irradiation ( nm) leading to cleavage of coumarin. finally, paclitaxel was released by subsequent o-n intramolecular acyl migration. conclusion: we synthesized and evaluated a novel type of paclitaxel prodrug. this prodrug showed promising kinetic data. therefore, we believe that photoactivation can be promising novel strategy for design of tumor-targeting prodrugs. the search of new immunosupressants, exhibiting the mechanism of action characteristic for cyclosporine a (csa) and fk- is an important challenge for medicinal chemistry. cyclolinopeptide a (cla) natural cyclic nonapeptide [cyclo(leu-ile-ile-leu-val-pro-pro-phe-phe)] possesses a strong immunosuppressive activity comparable with that of csa in low doses. the possibility of practical application of cla as a therapeutic agent is limited due to its high hydrophobicity. it has been suggested that the tetrapeptide sequence pro( )-pro( )-phe( )-phe( ) is responsible for the interaction of the cla molecule with the proper cellular receptor. in order to evaluate the role of this tetrapeptide unit for biological activity of native peptide, we decided to modified this fragment. in this communication we present linear and cyclic cla analogues in which phenylalanine residues in position and/or have been replaced with amphiphilic; alphahydroxmethylphenylalnine or homophenylalanine . the synthetic strategy and biological activity will be evaluated. resistance to currently used small molecule antibiotics develops at an alarming rate. while resistance to β-lactams in clinical isolates is primarily due to hydrolysis of the ring by β-lactamases, when bacteria develop resistance to fluoroquinolones or aminoglycosides, the sequences of the target biopolymers are altered. earlier we developed a family of antibacterial peptide derivatives that kill bacteria by inhibiting protein folding and are active in animal models of infection. in the current study we examined the synergy between antibiotics acting by different modes of action. inhibition of properly folded active resistance enzymes was completely efficacious to recover the activity of amoxicillin, a β-lactam antibiotic against strains that were originally resistant to this molecule. some activity of ciprofloxacin was also recovered by reducing the load of the induced self-defense dnak protein, but the synergy between the antibacterial peptide and the fluoroquinolone did not yield full bacterial killing. the mode of action of the synergy is indeed inhibition of protein folding because no such effect could be observed with kanamycin where resistance involves changes in the target protein sequence. as opposed to current β-lactamase inhibitors and combination therapies that work against only a limited number of strains, inhibition of all protein folding in bacteria is a universally applicable treatment option. elimination of resistance to β-lactams by proline-rich peptide derivatives may represent a viable avenue to give second life to these antibiotics for which large stockpiles are available for pharmaceutical companies in both patented and generic forms. the integrin αiibβ is the major integrin-adhesion receptor on platelets. in unstimulated platelets αiibβ is present in a resting conformation state. upon platelet activation by agonists, αiibβ receives intracellular signals (inside-out signaling) that allow its rapid conversion to a high-affinity state capable of binding soluble ligands, resulting in platelet aggregation. the intracellular signals include proteins that bind to the cytoplasmic tails of the two subunits α and β of the integrin, or integrin-associated membrane proteins. in vivo charge swapping mutation studies suggested that αiib and β tails have a direct site of interaction between αiib (r ) and β (d ). peptides derived from the cytoplasmic tail sequences can specifically induce or block αiibβ activation in platelets. the aim of this study is to develop peptide analogues based on the cytoplasmic tail sequences of both αiib and β subunits that could inhibit platelet thrombus formation by specifically disrupting the inside-out signaling pathway. peptide analogues of the αiib and β subunits spanning the sequences αiib- - , αiib - - , αiib - - , αiib- - and β - - , β - - , β - - were synthesized in their free state, palmitoylated and/or tagged with the tat fragment - and carboxyfluorescein-labeled, in order to investigate their membrane permeability, as well as their inhibition of the platelet aggregation. inflammatory pain begins when noxious stimuli (thermal, chemical or mechanical) excite sensorial neurons called nociceptors. the activation of nociceptors leads to the opening of some ionic channels and depolarization of the cell membrane. one of these channels is trpv , which is directly implied in thermal hyperalgesia associated to inflammation. in previous work it has been found that peptoid h-arg- - c ( fig. ) inhibits the activation of trpv by blocking the pore entrance. however, this compound showed toxicity in vivo. the aim of our work is the design and synthesis of new compounds, based on the structure of h-arg- - c, with better therapeutic properties. we synthesized some new non-competitive antagonists of trpv that exhibit notable anti-inflammatory and analgesic activity in vivo. th. skarlas, e. panou-pomonis, d. krikorian, m. sakarellos-daitsiotis, c. sakarellos erf is a transcriptional repressor with tumor suppressor activity regulated by the ras/erk signaling pathway. it has been shown that erf interacts with, and is phosphorylated by erks in vitro and in vivo. this phosphorylation determines its subcellular localization and biological function. erf exhibits a high degree of specificity and sensitivity for erks. the major objective of our study is to provide proof of principal for a specific anti-cancer approach targeting the ras-pathway, which is commonly activated in human tumors, via the stimulus of the downstream effector erf. this will be attained by modeling specific peptide inhibitors that block the erf phosphorylation and inactivation by the ras/erk signaling pathway. we present the design and synthesis of peptide inhibitors incorporating the fsf and fkf motifs, known to play a critical role in the erf/erk interaction, in their free forms or conjugated to a carrier. ubiquitinium is a well known mechanism in protein degredation of eukaryotic cells ,in which many obsolte and corrupted three dimentional structure protein ,become marked by covalent attachment of ubuquitin through a multi-step enzymatic pathway.ubiquitin is a small , . kda peptide of amino acid residues that targets such substrtes for proteolysis in proteasome .recnt studies showed that an extra cellular ubiquitination process also taking place in the epididymes of humans and other animals marks protein on the surface of the defective sperm .it appears that structurally and functionally defective sperm become surface ubiquitinated by epididymal epithelial cells. a certain portion of ubiquitin -labeled sperm is phagocytosed and the remaining is ejaculated .hence ubiquitin on the sperm surface could be a good marker of semen quality control in men. the aim of present study is to purify ubiquitin from packed blood cells , to produce and purify antiubiquitin antibodies,to design an immunofluorescence assy for detection of defective sperm, to compare the percentage of ubiquitinated sperm in oligoasthenotertozoospermia and normozoospermia and finally to determine correlations between sperm parameters and sperm ubiquitination. p. vakalopoulou, ch. anastasopoulos, g. stavropoulos, s. yiannis c-terminal analogues of substance p (sp) have been studied for their ability to prevent tumor growth or the proliferation of several cancer cell lines. the incorporation of damino acids into the sequence of sp and n-methylation of peptide bonds have shown to protect sp from the action of plasma and tissue peptidases. aiming to design and prepare more potential antagonist of cancer cells proliferation and taking into account that all the metabolites of the c-terminal hexapeptide analogue [arg , d-trp , , mephe ]sp - (antagonist g) possess the n-me group and d-trp residue, we proceeded to the synthesis of peptoid-peptide hybrids. they are oligomeric peptido-mimetics containing the residue [-Ν(bzl)-ch -co-]=(nphe). the incorporation of n-substituted glycine in peptide chains has been proved to improve their stability against proteases and give biologically active peptides. thus, the tetrapeptoid-peptide hybrids h-arg -d-trp -nphe -d-trp -oh and h-d-trp -nphe -d-trp -leu -oh, corresponding to metabolites of antagonist g and also the hexapeptoid-peptide hybrids glp -d-trp -Νphe -d-trp -leu -glu(obzl) -ΝΗ and glp -d-trp -Νphe -d-trp -leu -glu(obzl) -oh have been synthesized. the latter have incorporated the amino acid residues glp at the n-terminal and glu(obzl) at the c-terminal of the analogue, which have shown to give to the analogues increased resistance and biological activity. all the products were purified (hplc), identified (esi-ms) and set about for study their biological properties and activity against cancer cells proliferation. a chemokine receptor cxcr has multiple critical functions in normal and pathologic physiology. cxcr is a gpcr that transduces signals of its endogenous ligand, cxcl (stromal cell-derived factor- , sdf- ). the cxcl -cxcr axis plays an important role in the migration of progenitors during embryologic development of the cardiovascular, hemopoietic, central nervous systems and so on. this axis has recently been proven to be involved in several problematic diseases, including hiv infection, cancer cell metastasis, leukemia cell progression, rheumatoid arthritis (ra) and pulmonary fibrosis. thus, cxcr is a great therapeutic target to overcome the above diseases. fourteen-mer peptides, t and its analogs, were previously found to be specific cxcr antagonists that were identified as hiv-entry inhibitors, anti-cancer-metastatic agents, anti-chronic lymphocytic/acute lymphoblastic leukemia agents and anti-ra agents. cyclic pentapeptides, such as fc [cyclo(d-tyr-arg-arg-l- -( -naphthyl)alanine-gly)], were previously found as cxcr antagonist leads based on pharmacophores of t . in this symposium, we would like to report the development of low molecular weight cxcr antagonists involving fc analogs and other compounds with different scaffolds including leaner-type structures. erythropoietin (epo) controls the proliferation and differentiation of red blood cells. it activates epor by inducing dimerization and reorientation of two receptor chains. peptides mimicking the action of epo, epo mimetic peptides (emp), have been discovered by phage display, interacting with the receptor on the active site and competing with the hormone [ ] . another peptide, epor derived peptide (erp), was reported to activate the receptor through an alternative site distant from the hormone binding site, and to have synergic action with epo [ ] . we report the design of new synthetic epo-r agonists by dimerization of active peptides. pegbased polyamide linkers of precise length were used to link the molecules, using oxime chemistry [ ] . these peptides include emps that have been homo-dimerized through their n-or c-terminus. a hetero-dimer of one emp and one erp peptides was also created. biological characterization of the molecules is currently under investigation. [ the envelope spike (s) glycoprotein of the severe acute respiratory syndrome associated coronavirus (sars-cov) mediates the entry of the virus into target cells. recent studies point out to a cell entry mechanism of this virus similar to other enveloped viruses, such as hiv- . as it happens with other viruses peptidic fusion inhibitors, sars-cov s protein hr derived peptides are potential therapeutic drugs against the virus. it is believed that hr peptides block the six-helix bundle formation, a key structure in the viral fusion, by interacting with the hr region. it is a matter of discussion if the hiv- gp hr derived peptide t (enfuvirtide) could be a possible sars-cov inhibitor given the similarities between the two viruses. we used fluorescence spectroscopy techniques to test the possibility of interaction between both t (hiv- gp hr derived peptide) and t- with s protein hr and hr derived peptides. our biophysical data show a significant interaction between a sars-cov hr derived peptide and t . however the interaction is only moderate (kb = ( . ± . ) × m- ). this finding shows that the reasoning behind the hypothesis that t , already approved for clinical application in aids treatment, could inhibit the fusion of sars-cov with target cells is correct but the effect may not be strong enough for application. [ ] were used to investigate the structure, dynamics and thermodynamics of the known complex between erythropoietin mimetic peptides (emp) and erythropoietin receptor (epor). with gromacs . bioinformatics software, we have obtained from the known emps about the key functional amino acids required for effective epo mimetic action. then we systematically altered the amino acids in those peptides, and simulated the complex to observe the differences between the altered peptides with the original ones. based on these results, we designed new emps of potential significance. in order to fast identify the mimetic action of these new peptides, we synthesized these peptides and labeled the epor binding peptide (ebp) with quantum dots [ ] , to study the binding of these new emps to epor. our results illustrate a principle for fast identifying receptor-specific sites importance for receptor internalization, and for enhancing sensitivity to hormones and other agonists. blood vessel formation largely contributes to the pathogenesis of numerous diseases, including ischemia and cancer [ ] [ ] . in this regard therapeutic strategies aim to stimulate vascular growth in ischemic tissues and suppress their formation in pathologies like in tumour and diabetic retinopathy. placental growth factor (plgf), an homolog of vascular endothelial growth factor (vegf), ( % amino acid sequence identity), stimulates angiogenesis and collateral growth in ischemic heart and limb. whereas vegf exerts it biological function through the binding to both vegf receptor- (vegfr- or flt ) and vegfr- (or kdr) plgf binds specifically to flt . the complex plgf/flt constitutes a potential candidate for therapeutic modulation of angiogenesis and inflammation [ ] . the binding between plgf and flt- has multipunctual features [ ] and potential antagonist must have a sufficient molecular surface to spatially distant contact points. we have used an elisa-like screening assay to select antagonists of plgf/flt- complex from a large random library of tetrameric unnatural peptides (complexity: ^ = . molecules) identifying two active molecules with an about m ic . the relative stability of identified peptides were assessed in human serum and their inhibitory properties were tested in a capillary-like tube formation assay performed with human umbilical vein endothelial cells (huvec the αvβ integrin is a cell adhesion receptor involved in angiogenesis and tumor cell invasion. the tripeptide motif rgd is the αvβ minimal recognition sequence and many rgd-containing peptides have been investigated as radiopharmaceuticals for targeting angiogenesis and tumor metastatic phenotype. since rgd sequence binds also to other integrins, the aim of the present study was to develop and characterize a selective αvβ ligand suitable for imaging. a novel peptide containing the rgd loop covalently linked to an echistatin domain (crgdechi) was designed, synthesized and then tested for selective binding to αvβ integrin. a panel of peptides were used for comparison. adhesion assays showed that the novel peptide was able to inhibit adhesion of αvβ overexpressing cells but not αiibβ and αvβ overexpressing clones. in conclusion the novel peptide showed a high affinity and specificity for αvβ integrin. the design of new molecules, based on the lead compound presented here, is currently ongoing with the aim at developing novel anticancer drugs and/or new class of diagnostic noninvasive tracers as suitable tools for αvβ -targeted therapy and imaging. background and aims: short peptides like leu-pro-phe-phe-asp (lpffd) and leu-pro-tyr-phe-asp-amide (lpyfda) can influence the structure and aggregation of ß-amyloid peptides. soto's pentapeptide lpffd has been published as a ß-sheet breaker (bsb). it is necessary to gain more information about the nature of the interaction of aß and the pentapeptides mentioned above for the understanding of their action and for the possible development of future therapeutic agents. methods: in this study radioligand binding assay, diffusion ordered nmr spectroscopy, dynamic light scattering, circular dichroism and ft-infrared spectroscopy was used. results: it was shown by radioligand binding assay and diffusion ordered nmr spectroscopy that both pentapeptides bind to aggregated aß. dynamic light scattering, circular dichroism and ft-infrared spectroscopy revealed, that after the treatment of the aß with the pentapeptides aß fibrils are still present. conclusion: both peptide can bind to aß and can cause small conformational changes of aß, however, they cannot prevent completely the formation of aß fibrils in - micromolar concentration using : molar ratio of aß and the bsb peptide. peptide arrays are convenient tools for the analysis of antibodies, protein binding domains and to address other biological questions. here we present a new method to produce identical copies of arrays on microscope slides. the peptides are synthesized on modified cellulose-discs, using a variation of the spot-method introduced by ronald frank more than years ago [ ] . the new array format overcomes several limitations of the spot-method, e.g. the low throughput with only one copy of the library and the large sample volumes that are needed for membrane incubations. for the presented arrays modified cellulose discs with covalently bound peptides are dissolved after synthesis. the resulting solutions can be spotted onto glass slides by conventional spotting techniques. three dimensional layers of cellulosepeptide molecules are formed on the surface of the supports used for spotting. a virtually unlimited number of identical arrays can be printed and assays are performed with a sample volume of µl or less. as application example we show mapping experiments of the streptavidin recognition site with a peptide library containing histidine-proline motives. because of the much higher peptide loading compared to conventional arrays, the formed -dimensional structure might be superior for protein-interaction studies with even low binding constants. [ the interaction between the cap binding protein, eukaryotic initiation factor (eif) e, and the scaffolding protein eif g is critical for the formation of the heterotrimeric eif f translation initiation (ti) complex (eif e/eif g/eif a). elevated levels of eif e and eif g found in several human solid tumor cancers and the induction of malignant transformation in animal models by overexpression of eif e and the reversal of this phenotype by treatment with anti-sense rna, suggest the importance of the eif e/eif g interaction in the excessive translation of oncogenic proteins. eif g binds to eif e through a conserved eif e-binding motif yx l (non-specified (x) and hydrophobic ( ) amino acids) that interacts with an hydrophobic hot spot on eif e. we report here the identification of a putative eif e anionic exosite that is distinct from the hot spot and contributes to the binding of eif g-derived ligands. our strategy focuses on in situ eif e-templated click reaction-mediated assembly of hybrids comprised from an anchoring minimal eif g-derived peptide fragment, which binds to the hot spot, and a series of complementing positively charged fragments targeting the anionic exosite. we synthesized a training set of [ , , ] triazole-containing hybrid peptides that are potent inhibitors of eif e/eif g interaction. moreover, we achieved in situ eif e-templated assembly of these hybrids from the corresponding fragments via click reaction in the absence of cu(i) catalysis. as such, we demonstrate a proof-of-concept for a new paradigm in the development of inhibitors of protein-protein interaction merging click reaction with fragment-based and in situ target-templated approaches. goodpasture disease is an autoimmune pathology caused by the accumulation of reactive autoantibodies against the alpha- of collagen iv. goodpasture antigenbinding protein (gpbp) is a ser/thr protein kinase that phosphorylates the alpha- chain and might be important in human autoimmune pathogenesis [ ] . we are carrying out in our laboratories the biophysical and functional characterization of gpbp protein. in the presence of some proteins and at specific experimental conditions, gpbp participates in structurally ordered intra-and inter-protein aggregation processes. structure prediction programs identify four different domains for gpbp: an n-terminal domain showing pleckstrin homology (ph domain); a central domain with high tendency to form coiled-coils; a domain with ww features; and a c-terminal start domain ('star-related lipid-transfer'). using the tango algorithm [ ] , we have identified several aminoacid sequences in the gpbp start domain of vertebrates with high tendency to participate in protein aggregation. in this work we present the synthesis and structural characterization of a collection of peptides derived from the sequences described above. we recently developed a combinatorial library screening protocol to identify hpq-containing cyclopeptides that bind streptavidin more tightly than its linear analogues. the relative affinities in ic of these structurally constrained ligands and its linear counterparts were measured by a captured enzyme-linked immunosorbent assay. however, their intrinsic binding kinetics remained to be elucidated. in this work, surface plasmon resonance (spr) was employed to directly determine the kinetics and thermodynamics of the ligands binding to a streptavidin chip. solid-phase peptide synthesis was carried out using standard fmoc chemistry. spr experiments were carried out using biacore optical biosensor. streptavidin was immobilized onto a cm sensor chip using the standard amine coupling procedure. the equilibrium dissociation constants and kinetic on/off rates of n-to-side chain and n-to-c cyclopeptides were deduced by scatchard analysis and computational simulation, respectively. it was found that both cyclopeptides exhibited similar binding kinetics and bound streptavidin far more avidly than its linear form ( -fold). in addition, the reversed (qph) linear and cyclic peptidyl ligands were hardly recognized by streptavidin. not only the binding specificity was distinguished qualitatively, but also the entropic advantage acquired by the pre-organized conformation over its linear analogues was demonstrated quantitatively by spr in this study. the mutation of tumour suppressor genes in the progression of cancer is well characterized. for example, p is found to be mutated in approximately % of cancers and the loss of this proteins activity has been shown to lead to the deregulation of cell growth and apoptosis. the potential of peptide aptamers to inhibit protein/protein interactions in a highly specific manner makes them very attractive as research reagents or as target validation tools in anti-cancer drug discovery. more interestingly, these molecules have the potentially to inhibit the activity of proteins which are key regulators of cancer cell growth and therefore could act as synthetic tumour suppressor proteins. we used peptides based on known protein/protein interactions, as well as peptides isolated using display technologies, for the design of protein aptamers that were used to analyze pathways critical in controlling cancer cell growth. a range of scaffolds were used to present these peptides in an effort to optimize the peptides activities. data relating to the activity of these peptide aptamers in vitro as well as in cellular systems will be discussed. the cyclic undecapeptide urotensin ii (u-ii) is the endogenous agonist for the u-ii receptor (ut), a gq coupled gpcr. current views suggest that binding by agonist, but not antagonist, leads to induction of stabilization of an active receptor conformation. we have previously probed the interactions of urotensin ii with rat ut (rut) using a series of photolabile u-ii analogues containing p-benzoyl-l-phenylalanine (bpa). it was found that the c-jun n-terminal kinases (jnks) are important mitogen-activated protein kinases. these ser/thr protein kinases are activated by various growth factors, cytokines, and cellular stresses. jnks have been shown to play a key role in phosphorylation of proteins in signal transduction of different diseases including cancer, neurodegenerative, cardiovascular, and inflammatory diseases. therefore, these enzymes are considered as important therapeutic target proteins. the interactions of jnk with peptides are of special interest for development of novel specific atp-noncompetitive inhibitors. interactions of this kinase and its mutants with various substrates were demonstrated in vivo using yeast ras-recruitment system. bioinformatical tools have been developed to predict optimized binding peptides as well as to correlate sequence position and amino acid with binding effiency to extract binding determinants. biomolecular interaction analysis have been performed for selected peptide sequences using surface plasmon resonance (spr) technology. real time measurements of the binding of peptides to the different isoforms jnk and jnk resulted in the determination of affinities as well as kinetic constants for association and dissociation. experimental results and their bioinformatic analysis are discussed with respect to critical features of potential atpnoncompetitive inhibitors. the b-domain of is one of the five nearly homologous domains of staphylococcal protein a. this domain contains three alpha-helices which are assembled in an anti parallel three-helix bundle. the b-domain binds the fc region of mammalian immunoglobulins through the n-terminal fragment that contains two alpha-helices. the c-terminal helix does not interact with fc but it is necessary for the correct folding and immunoglobulin recognition of the b-domain. to search for new peptide analogues of the c-terminal helix that bind the n-terminal fragment, a ¨one-bead one-compound¨ library of peptides was designed based on the sequence of the c-terminal helix. active peptides were obtained after incubation of the library with the n-terminal fragment and rabbit immunoglobulin g labelled with fluorescein. new peptides were found and their sequences identified by maldi tof-tof mass spectrometry. the synthesis of the two most active peptides was carried out and the binding with the n-terminal fragment was confirmed by cd spectroscopy. the nterminal fragment peptide showed an increase in helicity when the c-terminal wild type peptide or some analogues were present in solution. the complete domains with the c-terminal fragment mutations were synthesized and structurally characterized by cd and nmr spectroscopy. the wild type and the new mutants adopt predominantly an alpha-helical structure. the interaction between rabbit immunoglobulin g and the wild type b-domain and the new analogues was investigated using surface plasmon resonance. although compared with the wild type, the mutants exhibited different kinetics, they were able to bind the immunoglobulin with high affinity. a. jaśkiewicz, e. bulak, h. miecznikowska, k. rolka sfti- , a strong trypsin inhibitor, was isolated in from seeds of sunflower. it is homodetic -amino-acid-residue peptide containing a disulfide bridge. because of its small size and strong trypsin inhibitory activity, this inhibitor became an interesting model for studying enzyme -inhibitor interactions. sfti- possesses one reactive site located at the lys -thr peptide bond and therefore is able to interact with the enzyme in a : stoichiometry. in this report we describe chemical synthesis and kinetic studies of a series of sfti- analogues containing double sequences of the wild inhibitor. their structures contain combinations of disulfide bridges and/or head-to-tail cyclization. each of these analogues contains two trypsin-specific reactive sites. we expect that kinetic studies should answer the question whether such dimeric analogues are able to interact simultaneously with more then one trypsin molecule and how this fact affects their inhibitory potency. in addition, we alsopresenttwo analogues in which we substituted the disulfide bridge with a carbonyl one. since carbonyl bridge has not been previously introduced into molecules proteinase inhibitors, we decided to check its impact on the activity and proteolytic stability of such modified analogues. ubiquitination, the covalent attachment of one or multiple polymerized ubiquitins is a post-translational modification of proteins, which has manifold functions. it mainly determines the protein for degradation, but also activation, deactivation or substrate alteration. due to its ubiquitinous distribution in all eucarionts no high-affinity antibodies could be originated. highaffinity ligand peptides are of interest to study ubiqutination. based on bioinformatical considerations and investigations of ubiquitin-interacting proteins short peptide sequences were selected. by using a peptide array specific ubiquitin binding was monitored and quantified with label free detection based on reflectometric interference spectroscopy (rifs). the results from rifs were confirmed by detection of binding in solution with fluorescence correlation spectroscopy (fcs) using carboxyfluorescein and s -labelled peptide amides. binding constants were determined by isothermal calorimetry (itc) and rifs. finally h, n-nmr chemical shift analyses of the peptides with the highest affinity were carried out, which allowed the localisation of the interaction site of ubiquitin with the peptide the results from all four methods correlated very good. they showed fast equilibria within s and binding constants down to the low micromolar range. nmr results revealed hints for discrimination possibilities between lys and lys polymerized ubiquitins. ( f is a potent neutralizing mab that binds the human respiratory syncytial virus (hrsv) f protein and is a promising candidate for clinical development. the majority of neutralizing antibodies to hrsv f protein map to two regions of the protein designated site ii and site iv,v,vi. to further characterize the f epitope, we employed a trypsin digestion of a hrsv f protein- f mab complex, followed by mass spectrometry analysis of the resulting recovered mab bound peptide. one peptide at m/z was captured by the f mab. sequence assignment was based upon mass and matched with the database from a virtual digest. this peptide was assigned as residues - [tkctasnknrgiiktfsngcdyvsnk] of the hrsv f protein which spans antigenic site iv,v,vi. to further delineate the epitope, the binding of f mab to a series of peptides corresponding to antigenic site iv,v,vi in the hrsv f protein was determined. based on the peptide elisa data, the f-binding region could be reduced to - sequence [ctasnknrgiiktfs] . as demonstrated by the substitution analysis, r and k significantly contribute to epitope binding, but another positively charged residue, k makes a minor contribution to the binding. both, the peptide elisa and proteolytic digestion of the mab-antigen complex approaches identified the same region of hrsv f protein as being critical for the binding of f. furthermore, these data confirm the results obtained using complementing genetic approaches using a panel of mutations in recombinantly expressed f protein and selection of antibody escape virus mutants (data not presented). the recently identified uracil-dna specific nuclease (ude) is the first representative of a new family of nucleases. the protein sequence has no detectable homology to other proteins except a group of sequences present in genomes of other pupating insects (vertessy et al, submitted). to analyze the physiological function of this protein, peptide conjugates were prepared to serve as synthetic antigens for the generation of antibodies against isoforms of dutpase, an enzyme inherently involved in preventing the synthesis of uracil-dna [ , ] . we used poly[lys(seri-dl-alam)] (sak) as a synthetic branched polypeptide [ ] or bovine serum albumin (bsa) as a natural macromolecular carrier. peptides were prepared by solid phase method utilizing syro (mulltisyntech gmbh, germany) peptide synthesizer, using fmoc chemistry and dipci/hobt-mediated coupling on rink-amid mbha resin. a c-terminal cys(acm) was added to the native sequences for incorporation of sh group into the peptide. in case of sak choroacetylated polypeptide was conjugated with sh-peptide to form thioether linkage. the maleimidobenzoyil-n-hydroxyszukcinimid (mbs) derivative of bsa was used to introduce the peptide into the macromolecule. antibodies have been developed as diagnostic tools and therapeutics for many different diseases. however, the isolation and preparation of intact specific antibodies is often very tedious or even unfeasible. recent studies have shown that single paratope peptides might be well capable to mimic corresponding antigen ligands [ , ] , suggesting that paratope peptides from a native antibody might have many advantages, e.g. for molecular vaccine design and targeting. we have developed a new method for identification of paratope-containing peptides by proteolytic affinity-proteome analysis in combination with high resolution fticr-mass spectrometry (fticr-ms) [ ] . in the present study we used hen eggwhite lysozyme (hel) and a polyclonal rabbit anti-lysozyme antibody (helpab) as a model system. the direct determination of paratope peptides was obtained by selective binding of a dtt-cleavage mixture of the anti-lysozyme antibody to immobilised hel, followed by proteolytic digestion of the antibody-antigen complex (paratope excision, parexprot). two specific paratope peptides were identified by maldi-fticr-ms, and the corresponding peptide sequences were identified by database search within a - ppm threshold. additionally, the identified paratope peptides were synthesised and characterised by affinity mass spectrometry, which ascertained their full binding specificity to lysozyme. the propeptide blocks the active site of inactive zymogen of cathepsin d and is cleaved off during maturation. we have designed a set of peptidic fragments derived from the propeptide structure and evaluated their inhibitory potency against mature cathepsin d using kinetic activity assay. the mapping localized two segments in the propeptide involved in the inhibitory interaction with the enzyme core: n-terminus of propeptide plays a major role and the active site anchor plays a minor role according to their respective ki values. in addition, a fragment derived from the mature n-terminus of cathepsin d displayed inhibition, which supports its proposed regulatory role. the mechanism of interaction of both propeptide segments was characterized by the mode of inhibition and by spatial modeling of propeptide in cathepsin d zymogen. using fluorescence polarization measurements, kd in nanomolar range was determined for the n-terminal propeptide segment. the inhibitory potency of the active site anchor segment was modulated by ala val mutation that was reported to be associated with cathepsin d pathology. . by comparing the resulting low-energy conformations using different sets of atoms, specific conformational features common only to the high/medium affinity compounds were identified. they included the spatial arrangement of the three most important pharmacophoric side chains tyr , arg , and nal as well as the orientation of the xaa -arg amide bond, which together represent a "minimalistic" d pharmacophore model for binding of the cyclopentapeptide antagonists to cxcr . this model rationalizes the data for the cyclopentapeptides as well as for the peptidomimetic cxcr antagonist krh- . automated docking of the pharmacophore model to the d structure of the tm region of cxcr revealed that the pharmacophoric groups of the cyclopentapeptide ligands were involved in favorable interactions with their counterparts in cxcr . for instance, the hydroxyl group of tyr formed a hydrogen bond with lys , the guanidino group of arg formed a salt bridge with glu , and the backbone carbonyl of xaa -arg formed a hydrogen bond with lys . this finding gives additional support for the suggested d pharmacophore model, and also provides opportunities for rational design of cxcr mutants to map potential contacts with peptide ligands. with the successful completion of the human genome project, the next challenge is to assimilate enormous amount of genetic information generated and to assign functions to a large number of proteins encoded. although the dna chip technology to detect the abundance of mrnas has been established, it is known that the abundance of mrnas and proteins does not correlate. thus, protein detection methods for reproducible and quantitative investigation of protein networks are strongly required. we attempted to establish a novel protein detection system based on a fluorescent measurement that does not require labeling of target molecules and preparation of secondary antibodies. we focused on a steric hindrance caused by the interaction between a target protein and a specific capture agent. when a target protein interacts with a specific capture agent immobilized on solid surface, we assumed that a steric hindrance in the vicinity of a capture agent increases. in order to detect the differences in the steric hindrance, we utilized a fluorescent system with the staudinger reaction. this reaction is a chemical ligation between a phosphine and an azide group. these two functionalities are unreactive with protein surfaces under biological conditions. we incorporated an azide group into an immobilized capture agent and investigated the efficiency in the staudinger reaction between the azide and an external triphenyl phosphine derivative. it was found that a target protein bound to the capture agent immobilized onto the solid support interferes with the efficiency in the staudinger reaction. the major histocompatibility complex (mhc) has a crucial role to initiate the immune response via the binding of the peptide fragments (epitopes) of foreign antigens and their presentation to the t-cell receptors (tcr). the co-receptor molecule cd enhances the binding between tcr and mhc ii. small molecules that mimic surfaces of mhc-ii may lead to blockage of the autoimmune response and the development of drugs for immunotherapy. hla-dqa * /dqb * (dq ) and hla-dqa * /dqb * (dq ) are highly correlated to autoimmune diseases as sjogren syndrome (ss) and systemic lupus erythematosus (sle). the non polymorphic β regions of the modelled hla-dq , which are exposed to the solvent and may disrupt the interaction of dq with cd + t lymphocytes were determinated using the getarea program. it was found that the regions - (arg-asn-asp-gln-glu-glu-thr-thr) and - (glu-tyr-trp-asn-ser-gln-lys-glu) display the highest solvent accessibility. peptide analogs of these regions were synthesized, by the fmoc/otbu solid phase strategy, purified by rp-hplc and characterized by mass spectrometry esi-ms. the dimeric analogs of the peptides, designed to mimic the superdimeric nature of the immunosuppressory fragments of hla class ii molecules were also synthesized and investigated. conformational studies were performed with cd spectroscopy and biological experiments are in progress. background and aims: aggregates of β-amyloid peptide (aβ) play central role in the etiopathology of alzheimer's disease (ad). short peptides like c. soto's pentapeptide lpffd and lpyfd-amide synthesized in our laboratory are neuroprotective agents against aβ assemblies both in vitro and in vivo. however, the mechanism of their neuroprotective effect has not yet been fully understood. methods: transmission electron microscopy (tem), cd, ft-ir, diffusion ordered nmr spectroscopy, dynamic light scattering, and radioligand binding assays were used. results: all the methods applied showed that the pentapeptides mentioned above do not break the fibrillar structure of aβ, that is these molecules are not real β-sheet breakers (bsb). the pentapeptides bind to aβ fibrils and cause small structural changes by intercalating into the aβ assemblies. fibrils of aβ survive one week treatment with the pentapeptides using them in to -time molar excess. conclusion: all the results in our laboratory show that the short peptides have long-term interaction on aβ-assemblies. in the first step they bind tightly to the aβ surface and prevent further interaction of aβ fibrils with the neuronal membranes. after this step the short peptides can be built into the structure of aβ-assemblies with intercalation causing a less ordered β-conformation. proteolytic enzymes (neprilisin, ide) could cleave and hydrolyze aβ peptides after this structural change, therefore the short peptides are good drug candidates for the treatment of ad. cellular processes in normal and pathogenic cell states are regulated by external stimuli via complex networks of catalytic and non-catalytic protein-protein interactions. we have developed methodology for the synthetic variation of peptides and peptidomimetics using polymer reagents including linker reagents enabling polymer-supported cacylations. [ ] in combination with the virtual screening of protein subsites, we have demonstrated the application of the novel synthetic methods to inhibitor optimization for various proteases including plasmepsin ii, hiv protease, and sars coronavirus main protease. [ , ] moreover, multivalent peptide polymers have been developed for the intracellular targeting of proteins. [ ] this methodology was now extended to the inhibition of peptide-protein interactions by small molecules. for this purpose, we have composed a library of , small molecules by algorithmic searching of a database of bioactive molecules with virtually designed substructures (fragments). high throughput assays were developed on the basis of fluorescence and fluorescence polarization detection. despite the scepticism regarding the inhibition of protein-protein interactions with small molecules, efficient hit molecules have been developed for several intracellular targets and were subjected to synthetic variation and cellular follow-up assays. the essential event in platelet adhesion to the blood vessel wall after injury or in thrombosis is the binding to sub-endothelial collagen of plasma von willebrand factor (vwf), a protein which interacts transiently with platelet glycoprotein (gp) ibalpha , slowing circulating platelets to facilitate their firm adhesion through other collagen receptors, e.g. integrin alpha beta and gpvi. to locate thevwf-binding site in collagen iii; we synthesized overlapping triple-helical peptides which comprise the whole native sequence of collagen iii . peptide # (gpogpsgprgqogvmgfogpkgnd (o is hydroxyproline)) alone bound vwf, with an affinity comparable to that of native collagen iii. immobilized peptide # supported platelet adhesion under static and flow conditions, processes blocked by an antibody which prevents the vwf a domain from binding full-length collagen. truncated and alaninesubstituted triple-helical peptides derived from # either strongly interacted with both vwf and platelets, or lacked both vwf and platelet binding. thus, we identified the sequence rgqogvmgf as the minimal vwf-binding sequence in collagen iii. the present work completes our understanding of the collagen-vwf interaction, providing information on crucial sequences in collagen that perfectly complements our existing knowledge of the collagen-binding site in vwf and may assist in targeting the collagen-vwf interaction for therapeutic purposes solid phase assay systems such as enzyme-linked immunosorbent assay (elisa), surface plasmon resonance (spr), and overlay gels are used to study processes of protein-protein and protein-peptide interactions. the common principle of all these methods is that they monitor the binding between soluble and surfaceimmobilized molecules. following the use of bovine serum albumin (bsa)-peptide conjugates or isolated synthetic peptides and the above-mentioned solid phase assay systems, we were able to demonstrate that positively charged peptides, which would be expected to repulse each other, can interact with each other. both the elisa and spr methods showed that the binding process reached saturation with kd values ranging between and nm. no interaction was observed between bsa conjugates bearing positively charged peptides and conjugates bearing negatively charged peptides or with pure bsa molecules, strengthening the view that interaction occurs only between positively charged peptides. however, interactions between the same peptides were not observed in solution when was monitored by nuclear magnetic resonance (nmr) or by native gel electrophoresis. thus, it appears that for positively charged molecules to interact one of the binding partners must be immobilized to a surface, a process that may lead to the exposure of otherwise masked groups or atoms. the relevance of our findings for the use of solid phase assay systems to study interactions between biomolecules will be discussed. the hematopoietic progenitor kinase (hpk ), a mammalian hematopoiesis-specific ste kinase, contains a cluster of four proline-rich sequences called p , p , p and p located after the kinase domain. these pro-rich regions play an important role in the interactions of this kinase with different adapter proteins. previous studies showed that p , which contains the canonical pxxpxr motif, and p and p with the canonical pxxpxk motif interact with the c-terminal sh domain of hematopoietic lineage cell-specific protein (hs ) even if with different affinity. hs protein shares a high amino acid sequence and structural similarity to cortactin although their functions differ considerably. here we report the results of our investigation on the interaction between the c-terminal sh domain of cortactin and the four proline-rich motifs of hpk . these interactions were analyzed by non-immobilized ligand interaction assay by circular dichroism (nilia-cd). upon peptide addition, the binding was monitored by the cd changes of the trp side-chains of the conjugate gst-sh cort. the dissociation constants kd were determined analyzing the cd data at nm using a nonlinear regression method. the results demonstrate that gst-sh cort displays an affinity binding higher than that found with the corresponding hs domain and that the four hpk pro-rich regions are not equivalent. p appears to bind with the highest affinity (kd= . µm), followed by p (kd= µm) and p (kd= µm), whilst p does not interact at all. the generation of a fibrin clot is mediated by the regulated activation of a series of serine proteases and their cofactors. factor viii in its activated form, fviiia, acts as a cofactor to the serine protease fixa, in the conversion of the zymogen fx to the active enzyme fxa. both fviii and fix are essential for normal coagulation, deficiencies of either are associated with the bleeding diatheses hemophilia a and b, respectively. the role of fviiia is to bind factor ixa, generating the phospholipid-dependent intrinsic factor xase complex. at least two interactive sites have been identified for the enzyme-cofactor interaction. the ser -gln region within the a subunit has been shown to be crucial for viiia-ixa interaction. in an attempt to study this interaction, we synthesized a series of peptides of - loop of the a subunit. the syntheses of these peptides were carried out by using spps and fmoc/but methodology. the synthesized compounds were purified by rp-hplc and lyophilized to give fluffy solid, identified by ft-ir, nmr and es-ms spectra. these compounds were tested for inhibitory activity on human platelet aggregation in vitro, by adding common aggregation reagents to citrated platelet rich plasma (prp). the aggregation was determined using a dual channel electronic aggregometer by recording the light transmission. and ci/mmol, respectively. both tritiated aβ peptides were used in cat brain( in vivo) experiments and it was found that the peptide aggregates enter the neurons within min (electronmicroscopic autoradiography). this transport is most probably an endocytotic pathway. aβ aggregates could interact also with cytoplasmic proteins such as -phosphoglyceraldehyde dehydrogenase etc. we suppose that aβ assemblies can interact both with membrane receptors (nmda, ampa, ach) and with cytoplasmic proteins triggering neuronal dysfunction and death. background and aims: ww domains are the shortest known protein domains and contain a stable three-stranded b-sheet, which presents the binding site for prolinerich ligands. this interaction is mediated by hydrophobic interactions between aromatic and hydrophobic residues of the domain, and the polyproline core of the ligand. as part of our ongoing efforts aimed at synthetically mimicking conformationally defined protein binding sites ( ), we have designed and synthesized linear and cyclic peptides covering the binding site of the ww domain of human yes-associated protein (hyap-ww), whose structure in complex with a proline-rich ligand had been solved by nmr spectroscopy ( ) . methods: peptides were synthesized by spps, purified by hplc, and characterized by d-nmr spectroscopy, as well as by molecular dynamics calculations. affinities of the peptides to a hyap-ww ligand were determined in direct and competitive binding assays. results and conclusions: a cyclic peptide covering the sequence stretch of hyap-ww that contains its primary contact residues for proline-rich ligands, was found to compete with the domain for binding to a hyap-ww ligand. long-ranging noes identified in the nmr spectra of this peptide indicate a conformation, in which sequentially distant residues are brought into spatial proximity, likely through formation of a beta-sheet. these result demonstrate the feasibility of functional, as well as structural, mimicry of conformationally defined protein binding sites through synthetic peptides. the rockefeller university, new york, ny, usa integrins constitute a family of transmembrane cell surface receptors. they are involved in cell-cell and cell-extracellular matrix interactions. thus, they participate in many physiological and pathophysiological processes and are of crucial importance for the living organism. integrins possess two non-covalently bound subunits, &alpha; and &beta;, that jointly participate in ligand binding. these dimeric proteins show very high specificity in recognition of natural ligands. for example, α β integrin recognizes vcam- (vascular cell adhesion molecule ) and fibronectin through binding amino acid motifs tqidspln and ldv, respectively. on the other hand, fibronectin is a classical ligand for α β integrin with the recognition motif rgd. as shown, identification of the integrin ligands occurs through small recognition amino acid sequences (mostly tripeptides). thus, small cyclic peptides possessing a recognition motif in the appropriate three-dimensional conformation are able to interfere with the integrin-ligand interactions and act as inhibitors. the aim of this investigation is the characterisation of small cyclic peptides containing the rgd motif and the determination of the selectivity and specificity of these inhibitors. two new pentapeptides with -amino-cyclopropane- , -dicarboxylic acid monomethyl ester ((+/-)acc) were synthesized and tested. peptides were characterized in biological assays with living cells (k and wm ) and in surface plasmon resonance binding studies. experiments have shown that cyclic peptide cyclo-(arg-gly-asp-(+)acc-val) is a very potent inhibitor (ic -value in nm range) of interaction between vitronectin and αvβ or αvβ integrins. when preparing biotin-labelled peptides as ligands for avidin-based assays, it is chemically most expedient to locate the biotin label on the n-terminal group of the peptide. this is done without any regard to how this may affect peptide-target interactions, biotin-avidin binding, and the solubility properties of the resultant peptide. in many instances, the products are poorly soluble, have little biological activity, and poor affinity for avidin. problems can also arise during the synthesis of such nterminally biotinylated peptides due to the poor solubility and reactivity of many of the reagents used for biotin introduction. to overcome these limitations, we have developed an extremely simple method for synthesising peptides c-terminally with biotin. peptides can now be easily prepared by standard solid phase techniques either n-or c-terminally labelled, and screened to determine the optimum presentation for the biotin. in cases studies using protein-protein interaction and kinase assays, peptides c-terminally labelled with biotin gave better sensitivity. y. yang , j. eble , n. sewald many bacterial pathogens bind and enter eukaryotic cells to establish infection. invasin is an outer membrane protein required for efficient uptake of yersinia into m cells. invasin mediates its entry into eukaryotic cells by binding to members of β integrin family that lack insertion domains (i domains), such as α β ,α β ,α β ,α β , and αvβ . this type of peptide-protein interaction is an ideal subject for the rational design of inhibitors. the integrin binding motif consists of one loop region with a conservative asp residue and two synergistic regions. the aim of this project is to synthesize cyclic peptides based on the invasin binding epitope sdms. this sequence has to be positioned in a β-turn with asp in i+ position for optimal activity of the peptide. also the arg and asp residue, which are about . Å and . Å respectively away from the asp residue of the sdms loop in invasin, should be investigated. peptides that mimic these recognition sites have been synthesized and tested as ligands for the integrin peptide-dna cross-linking is a very powerful tool for studying peptide-dna complexes. it transforms non-covalent complexes into covalent complexes, which renders characterization of the adduct by classical techniques (mass spectroscopy, nmr,…) much easier. the aim of our research is to develop a new method for peptide-dna cross-linking involving the incorporation of a furan moiety. the strategy is inspired by the naturally occurring process of oxidative furan ring opening by cytochrome p . the resulting cis-butene- , -dial has been shown to react with amino-and sulfhydryl groups of macromolecules such as proteins and dna. in our research, dna binding peptides are modified with a furan moiety and then chemically oxidized into a reactive enal. this enal can react with dna to form a covalent peptide-dna complex. previous attempts to selectively oxidize furan modified minor groove binding peptides consisting of n-methylpyrrole building blocks failed. we are now applying the same strategy on major groove binding peptides consisting of natural amino acids. currently the oxidation conditions are being optimized so that the furan moiety undergoes selective oxidation. these optimized conditions will be applied to known dna binding peptides, in order to obtain a peptide-dna cross-link. we coupled octanoyl or palmitoyl group to the n-terminus of an analogue of sv nuclear localization signal (nls) peptide, sv - (ser ) to investigate the effect of fatty acid chain length on the conformation of the lipopeptide-antisense oligodeoxynucleotide (odn) complexes and to establish the optimal peptide/odn molar ratio (rm) for the effective delivery of odn into the cells. the odns used in this study were targeted towards either the green fluorescent protein (gfp) mrna and the junction sequence between ews and fli genes. the conformational change of odn at different rm values was followed by circular dichroism (cd), attenuated total reflection-fourier transform infrared (atr-ftir) spectroscopy and atomic force microscopy (afm). the sv peptide-mediated odn transfer into nih/ t cells was studied by epifluorescence microscopy. the interaction between the hiv- regulatory protein rev and rev responsive element (rre) of hiv- mrna has emerged in the last decade as an important target in antiviral therapy. the rev-rre interaction is essential for the replication of hiv. the rev protein binds to the rre site located in the env coding region of the full length viral mrna and facilities the export of the rna from the nucleus, while protecting it from the cell's splicing machinery. in the published nmr structure of the rre/rev-derived peptide complex, an -helical segment of rev binding domain recognises a specific region of rre. an approach is described to design a new class of -hairpin peptidomimetic ligands for hiv- rev protein, which inhibit its binding to the rre rna. a model -hairpin peptide served as a scaffold to pre-organise side chains into a geometry similar to that seen in a helical peptide. a library of peptidomimetics was prepared by grafting sequences related to the rna recognition element in rev onto a hairpin-inducing d-pro-l-pro template. the electrophoretic mobility shift assay (emsa) revealed that all of the designed peptidomimetics bind to rre and the best examples show affinities (kd) in a nanomolar range. these new ligands show a novel approach to designing rev peptidomimetics, represent interesting leads for the development of more potent hiv rre/rev inhibitors and permit more detailed studies of the mechanism of binding to rna. a. napiorkowska, a. sawula, m. olkowicz, p. mucha, p. rekowski tat (trans-activator of transcription) is the protein which controls the early phase of hiv- replication cycle. it is a potent viral trans-activator containing from to amino acid residues which binds to tar rna. the fundamental role of tat is promoting effective elongation of viral mrna (vmrna). binding of tat to tar is mediated by a -amino-acid, highly basic arg -lys-lys-arg -arg-gln-arg-arg-arg sequence of the arm (arginine rich motif); the key role in these interactions is played by arg . the goal of our research was to investigate the interaction of -nucleotide tar rna with synthesized tat peptide analogues using capillary electrophoresis (ce), a powerful analytical technique of biochemical studies. changes in electrophoretic mobility of the tar peak are employed for monitoring tar-tat complex formation. ce experiments were performed using lpa-coated capillary and a physical gel containing buffer. native arm fragment tat( - )nh , its analogues ac-tat( - )nh and ac-[lys ]tat( - )nh and analogues substituted in position with alanine-, homoalanine and lysine-derived amino acids containing nucleobases (adenine, guanine, cytosine, uracil, thymine) and nucleosides (adenosine, guanosine, uridine and cytidine) in the side chain were studied. specific interactions and complex formation were observed for both the native arm peptide fragment and selected tat analogues. the research is aimed at improving our understanding of the molecular mechanism of peptide-nucleic acid interaction, as well as evaluating the usefulness of selected nucleobase-containing amino acids as point probes for investigating peptide-rna interactions. interactions between proteins and dna are important to all living organisms. the goal is to investigate the molecular recognition between dna and the transcription factor phob of e. coli on the single molecule level and to identify amino acids required for dna binding. phob is composed of a transactivation domain (amino acids - ) and a dna binding domain (amino acids - ) that binds to specific dna sequences (pho boxes) containing a tgtca sequence. [ ] chemical synthesis of peptide epitopes present in the dna binding domain of phob and isolation of the whole dna binding domain of phob was performed. the protein was purified using intein mediated protein purification. an additional cysteine residue was ligated to the protein using intein mediated ligation reactions and will be used for immobilization and labeling. in single molecule force spectroscopy (afm) experiments it has been shown that both a peptide with a native phob-sequence and the recombinant protein bind to dna. competition experiments were performed to prove specific dna binding. [ ] mutated peptides and proteins where strategic amino acids were replaced by alanine have also been examined to reveal the contributions of single residues to molecular recognition. the binding contribution of the proteins is determined by surface plasmon resonance, electrophoretic mobility shift assays and fluorescence correlation spectroscopy. we investigated the biophysical characteristics and the pore formation dynamics of naturally occurring and synthetic peptides forming membrane-spanning channels by using isolated rod outer segments (os) of reptilia and amphibia recorded in the whole-cell configuration. once blocked the two os endogenous conductances (the cgmp channels by light and the retinal exchanger by removing one of the transported ion species from both sides of the membrane, i.e. k+, na+ or ca +), the os membrane resistance (rm) could be > gΏ. therefore, any exogenous current can be studied down to the single channel level. macroscopic currents of amplitude of ~ pa were recorded in symmetric k+ or na+ (> mm) and ca + ( mm) from the commercially available alamethicin mixture, the synthetic alamethicin f / (a major component of the natural mixture), and selected analogues applied at µm concentration at - mv. once applied and removed the peptide, the current activates and deactivates with a time constant of about ms. the synthetic analogues [glu(ome) , , ] and [glu(ome) , ] produce a current of about pa at µm concentration, and they show a strong activation by hyperpolarization as alamethicin f / itself. clear single channel events were observed when the concentration of all of the alamethicin peptides is reduced to < nm.<br> these results indicate that the three gln residues at positions , and of alamethicin f / are not a key factor for pore formation and its conduction properties. in general, the pore assembly and disassembly are very fast and cooperative events. the translocation mechanism of penetratin (rqikiwfqnrrmkwkk) is not clear, but the involvement of cell membrane was supposed. recent studies with phospholipid model membranes have shown that penetratin interacts only with negatively charged liposomes. we aimed to analyse the effect of penetratin on liposomes composed of different phospholipids (dppc/dppg : - : ) by fluorescence spectroscopy. in the first set of experiments, liposomes labelled with fluorescent markers (dph, ans and tma-dph) were incubated with penetratin and the fluorescence polarisation was determined as a function of the temperature. in the range of - mol/mol phospholipid/penetratin ratio, no change in the transition temperature was observed indicating that penetratin has no influence on the membrane structure. next, we have analysed the interactions between phospholipids and penetratin through changes in the intrinsic fluorescence of the peptide due to the presence of two w residues in its sequence. comparing the emission spectra corresponding to penetratin in aqueous media or in presence of vesicles one can clearly appreciate a blue shift. this indicates that that tryptophan residues are mainly exposed to a hydrophobic environment. analysis of the main band shows low values of polarization suggesting a free motion of the peptide chain. on the contrary polarization measured for penetratin mixed with liposomes results in higher values. this indicates that hydrophobic residues, like trp, are inserted into the bilayer and their motion is restricted. these data suggest the presence of interation sensed strongly by trp properties. cyclopeptide antibiotic gramicidin s (gs) has antimicrobial activity against gram-positive and gram-negative bacteria and some fungi. but non-specific action of gs and its high lytic potential limits therapeutic application of gs. we attempted to elucidate in which way gs molecule could be modified to lose its haemolytic side effects. gs molecule interacts directly with membrane phospholipids due to electrostatic and hydrophobic interaction. naturally, changes in the state of a lipid bilayer cause changes in the gs molecule binding to a bilayer. we studied the effect of gs on human blood platelets and the effect of platelet membrane state on the gs-induced disaggregation of cells with the help of turbidimetric and microscopy techniques. we modified the membrane state by temperature, osmotic stress, ionizing irradiation, lipid oxidation. depending on concentration gs causes platelet shape change and activation. when added to preliminary aggregated (in response to physiological agonists -thrombin, epinephrine, adp) platelets, gs causes crumble of cells aggregates. the rate and extent of platelet disaggregation under the effect of gs non monotonously depends on temperature (range of - °c) and irradiation dose (up to gy). parameters of the gs interaction with membranes are determined by the mobility of membrane lipids. factors modifying the lipid bilayer change the degree and the speed of the gs interaction with platelet membranes. results obtained permit to use gs for testing the state of membrane lipids and on the other hand allow to suppose ways of gs molecule modifications to achieve its tolerance to blood cells. g. bai , p. gomes , r. seixas , m. hicks , m. prieto , m. bastos eukaryotic antibiotic peptides (eaps) have been widely studied for the past years as an alternative to conventional antibiotics due to emergence of multi-resistant microbial strains, and significant efforts targeting increasingly potent and specific antimicrobial peptides are being made. one interesting approach in peptide antibiotics is based on hybrid sequences derived from natural eaps, with ca ( resistance to conventional antibiotics has stimulated a search for alternative therapeutics for microbial infections, a possible source that has gained much interest in recent years are antimicrobial peptides. antimicrobial peptides target the cell membrane directly, which is a key feature as evolution has shown bacteria have had difficulty in altering their membrane composition and organization to mount a suitable defence against these peptides. a common theory is that peptides that bind strongly exhibit high biological activity, but our real-time quantitative binding studies via surface plasmon resonance (spr) have shown that this correlation does not always hold. as more information on the molecular details of membrane disruption is required, we have used atomic force microscopy (afm) to visualise peptide insertion and changes in membrane morphology by a range of antimicrobial peptides in situ. interaction studies were performed with a series of phospholipid mixtures that mimic either mammalian cells (high in phosphatidylcholine and cholesterol) or microbial cells (high in phosphatidylethanolamine and phosphatidylglycerol). the present study may assist in the design of new specific antimicrobial peptides with high antimicrobial activity and low host toxicity. proportions of popc and popg as models. very high molar ratio partition constants (( . +- . )x ^ and ( . +- . )x ^ ) were obtained for the bacterial models (popg:popc : and : , respectively), these being about one order of magnitude greater than the partition constants obtained for the less anionic mammalian model systems (( . +- . )x ^ for the % popc system). at low lipid:peptide ratios there were significant deviations from the usual hyperbolic-like partition behavior of peptide vesicle titration curves, especially in the case of the most anionic systems. membrane saturation was shown to be related to such observations and mathematical models were derived to further characterize the peptide-lipid interaction under these conditions. the calculated peptide-to-lipid saturation proportions, together with the determined partition constants, suggest that the minimal inhibitory concentrations of omiganan pentahydrochloride could represent the conditions required for bacterial membrane saturation to occur. the hemolytic pore-forming toxin sticholysin ii (stii) produced by the sea anemone stichodactyla heliantus belongs to the actinoporin protein family. the n-terminal domain of these proteins is required for interaction with membranes. to investigate the role of stii´s n-terminal domain in membrane binding and in the molecular mechanism of hemolysis, peptides corresponding to residues to , or shorter fragments from this region, were synthesized. in some peptides leu was replaced by trp. all peptides exhibited hemolytic activity, albeit to a lesser extent than the whole protein. moreover, peptides lacking the - hydrophobic stretch were less active. the longer peptides were also able to permeabilize phospholipid vesicles. conformational studies were performed in aqueous solution and in membranemimicking environments. cd spectra showed that, while the shorter, more hydrophilic peptides, displayed a random conformation, the longer peptides underwent aggregation with increasing concentration, ph, and ionic strength. in the presence of trifluoroethanol and upon binding to detergent micelles and phospholipid bilayers, the peptides showed a propensity to acquire -helical conformation, as expected for the sequence comprising residues to . fluorescence spectra demonstrated that the first residues of stii´s n-terminus penetrate more deeply into the bilayer, whereas residues - are located more superficially. this is in agreement with the predicted amphipathic nature of the helix formed by these residues and corroborates the existing hypotheses for the role of the n-terminal domain in the process of membrane insertion and pore formation. among a great number of antibacterial peptides a group of trp-rich peptides is of special interest. taking into consideration, that in most of proteins tryptophan is not frequently occurring amino acid, the biological meaning of a high content of tryptophan in structure of these antimicrobial peptides is particularly interesting. in the present study we carried out the investigation of antimicrobial and hemolytic activities of selected trp-rich peptides and their action on microbial membrane: ilpwkwpwwpwrr-nh - indolicidin (i) pitwpwkwwkgg-nh - b (ii) plswffprtwgkr-nh - gsp- a (iii) fpvtwrwwkwwkg-nh - puroindoline (iv) vrrfpwwwpflrr-nh - tritrypticin (v). sunflower trypsin inhibitor sfti- is the smallest and the most potent known peptidic trypsin inhibitor from the bowman-birk class of proteins [ ] . this head-to-tailcyclized -amino-acid peptide contains one disulfide bridge and a lysine residue (lys ) present in the p position, which is responsible for inhibitor specificity.as was reported by us and other groups, sfti- analogues with one cycle only retain trypsin inhibitory activity. very recently we have shown [ ] that introduction of nsubstituted glycine residues mimicking lys and phe (denoted as nlys and nphe) in the p position of monocyclic sfti- with disulfide bridge yielded potent trypsin and chymotrypsin inhibitors, respectively. in this novel class of proteinase inhibitors contains completely proteolytic resistant p -p ' reactive site. in the present communication we report chemical synthesis and determination of trypsin and chymotrypsin inhibitory activity of a series of ten sfti- analogues modified in the p position by these peptoid monomers (nlys and nphe). each of the synthesized peptomeric (peptide-peptoid hybrid polymer) sfti- analogues contains one of the following cycles: head-to-tail, disulfide bridge formed by cys, by pen and by cys/pen residues. the impact of the different cycles introduced into sfti- structure on proteinase inhibitory activity will be discussed. s-protein contains a proteolytic processing site and two interacting heptad repeat regions denoted as hr-n and hr-c. following processing of s-protein mediated by host cellular protease/s, the c-terminal s -fragment fuse with host cell membranes via its hr-n and hr-c domains that form coiled coil -helix bundle (trimeric of dimers)-crucial for its receptor-mediated viral fusion. our objective in this work is to study the proteolytic site using model peptides and also to examine the interaction of hr-n and hr-c domains using fluorescence microscopy and other techniques. thus we synthesized an intramolecularly quenched fluorogenic peptide containing the proposed cleavage site [abz-eqdrntr evfatyx, abz= -amino benzoic acid and tyx= -nitro tyrosine] and showed by kinetic measurements that this cleavage is mediated most efficiently by furin, followed pc and pc . other potential substrates were also tested and compared. above cleavage can be blocked by specific-pc-inhibitors in a dose-dependent manner. in addition using fluorescent-labeled peptides derived from hr-n and hr-c domains, circular dichroism spectra and surface assisted laser desorption mass spectral interest has grown to develop specific and potent inhibitors of this enzyme. our objectives in this study are to generate soluble recombinant human (h)ski- enzyme, design potent inhibitors and study its dmodel structure. we have successfully expressed hski- enzyme lacking its transmembrane domain in hek- cells and purified the enzyme via chromatography. in addition we developed ski- inhibitors by using pseudo-and multi-branch peptide approaches. in first approach we inserted dipeptide isosteres amino oxy acetic acid (aoaa) or -amino , dioxa octanoic acid (adoa) at scissile p -p ' position ((r l) of hski- . a typical example is gryssrrl(adoa)aip . other dipeptide isosters were also incorporated at the cleavage site of either ski- prodomain or lassa virus glycoprotein. in second approach we prepared and -branch peptides containing hski- - segment. these peptides inhibit ski- in competitive manners with varying degrees ranging from low m to high nm ic . circular dichroism spectra indicated strong interactions of inhibitors with ski- consistent with observed inhibition profile. a d-model structure of catalytic domain of ski- indicated a broad catalytic pocket cysteine proteases are of great importance in biochemical processes and these enzymes are used in biotechnology, food industry and agriculture. in this connection synthesis of high selectivity and high specificity substrates for cysteine proteases is of importance. enzymatic synthesis of peptides is a good tool for obtaining different biologically active peptides. immobilized serine proteases, subtilisin carlsberg and α-chymotrypsin immobilized on poly(vinyl alcohol) cryogel (pvacryogel), proved to be a convenient biocatalyst for such kind of syntheses. the high specific chromogenic substrate for cysteine proteases assay glp-phe-ala-pna was obtained with high product yields (up to % in h) using subtilisin and chymotrypsin immobilized on pva-cryogel. the reaction was carried out according to the following scheme: glp -the residue of pyroglutamic acid, pna -p-nitroanilide. the influence of initial concentrations of components, the reaction mixture composition, the biocatalyst content and time on product yield was studied. it was shown that the optimal conditions are: dimethylformamide-acetonitrile mixture : (v/v), initial concentrations - mm, and enzyme-to-substrate ratio - : . this approach was used in order to synthesize analogous substrates, containing different fluorogenic and chromogenic groups as well as other amino acids in p position. the obtained substrates were tested for the papain assay. peptidyl-a-ketoaldehydes represent attractive lead compounds and intermediates in the development of potent protease inhibitors due to their structural similarity with peptide aldehydes, previously known to be excellent inhibitors of serine-and cysteine protease. recently, we demonstrated the application of polymer cyano methylene-and carboxylato methylene phosphoranes in the assembly of a-hydroxy-b-amino esters (norstatines), a,b-diketoesters, and a,b-unsaturated ketones. [ , , ] we now present a further development of our reagent linker approach employing peptidyl-a-ketoaldehydes and diamino propanoles . carboxylato methylen phosphoranes derived from bromo acetic esters which are readly acylated without racemization, play the key role in our synthetic concept. herein we show the oxidative cleavage to peptidyl-a-ketoaldehydes using dimethyldioxirane (dmd) in acetone as oxidant, after saponification and decarboxylation on the solid support. diamino propanoles were furnished via the reductive amination of resin-bound peptides. over the past few years nuclear magnetic resonance has emerged as a powerful means for lead molecular identification and optimization.on the other hand, the f nmr has been used succesfully in several structural studies, protein folding studies and for the identification of active compounds, using a very similar methodology that the one used in the present work. the methodology required the labeling of the substrate with a cf moiety. the enzymatic reaction is performed with the cf substrate and quenched, using an enzyme inhibitor. f nmr is then used to monitor the evolution of both substrate and product. only two peaks are observed, the starting substrate and the cleaved substrate. this nmr method has some advantages: fluorine nmr is very sensitive, , times that of the proton. there are no spectral interference from protonated solvents, buffers or detergents typically present in the enzymatic reactions.the f isotropic chemical shift is extremely sensitive to small structural perturbations resulting in different chemical shift for the signals of the substrate and product. isotopic labeling of the protein is not required. as a model, caspase- , which play a critical role in the initiation of apoptosis process and hiv- protease were chosen. two different kind of libraries were screened: one based on natural products from plant and animal extracts used in tradicional chinese medicine and a second one corresponding of a synthetic library with two sublibraries of and compounds.with this methodology it has been possible to identify some compounds with very promising inhibitory properties. background and aims: human kallikrein (hk ), a prostate specific serine protease, regulates the activity of several factors that may participate in proteolytic cascades promoting tumor growth and metastasis. thus, inhibition of its enzymatic activity is a potential way of preventing growth and metastasis of prostate cancer. moreover, specific ligands for hk have potential use for targeting and in vivo imaging of prostate cancer and for development of novel assays. methods: to find peptide ligands we panned several phage display peptide libraries against active recombinant hk captured by a monoclonal antibody exposing the active site of the enzyme. alanine scanning and amino acid deletion analyses were performed to elucidate the motifs required for hk inhibition. results: from libraries expressing and amino acid long linear peptides we isolated six different hk -binding peptides. three of these peptides are specific inhibitors of the enzymatic activity of hk . amino acid substitution and deletion studies indicated that motifs of amino acids are necessary for the inhibitory activity. conclusions: we have developed specific hk inhibitors by phage display technology. these novel hk specific peptides are potentially useful for treatment and targeting of prostate cancer. peptidylarginine deiminase iv (padiv) catalyzes the citrullination of arg residues in various peptides and proteins, such as histone, resulting in the production of citrullinated proteins in granulocytes [ , ] . the citrullination mechanism of histone subunits and its functional effects in cells are not well known yet in detail. recently, it has been reported that the protein deimination/citrullination by pad iv plays a role in rheumatoid arthritis [ ] . this implicates that the citrullination of histone may be related to rheumatoid arthritis. in order to further study the citrullination mechanism of histone, we explored the citrullination sites of histone h a and h by pad iv using a series of synthetic peptides. recently, hagiwara et. al. reported that pad iv only citrullinates the arg of histone h a as well as the arg in histone h [ , ] . in order to investigate the citrullination mechanism, the n-terminal peptides of histone h a and h were chemically synthesized and examined the citullination by pad iv. the n-terminal acetylation effect of the n-terminal synthetic peptide was also estimated on the citrullination by padiv. the velocity of each arg residues in the n-terminal peptides were estimated in vitro. the results indicated that padiv recognizes the specific arg residues in the synthetic peptide, and that the n-terminal acetylation of the histone peptides dramatically affects on the substrate recognition of padiv. in addition, the cd spectra of the n-terminal peptides were measured to elucidate the structural specificity for the recognition of pad iv. background and aims. prolyl oligopeptidase (pop) is a serine peptidase that cleaves oligopeptides after prolyl residues. it has been associated with cognitive disorders. pop inhibitors have been shown to enhance cognition in monkeys ( ) and to improve performance in verbal memory tests in humans ( ) . in the present study, the p l-prolyl residue of pop inhibitors was replaced by two l-proline mimetics, the -t-butyl-l-prolyl group and the (r)-cyclopent- -enecarbonyl group. the effect of the mimetics on in vitro potency, lipophilicity and binding kinetics were studied. methods. the l-proline mimetics were synthesized according to the published procedures ( , ) with minor modifications. the ic and ki values and the binding kinetics were determined for porcine pop. the log p values were determined with the shake-flask method. results. the replacement of the p l-prolyl residue by the l-proline mimetics gave compounds which were equipotent with their parent structures. both l-proline mimetics increased lipophilicity but the effect of the -t-butyl-l-prolyl group was more pronounced. while the -t-butyl-l-prolyl group increased the dissociation half-life of the enzymeinhibitor complex, the (r)-cyclopent- -enecarbonyl group decreased it. conclusions. both l-proline mimetics perfectly mimicked l-proline at the p position of pop inhibitors. these mimetics can be used to modify the lipophilicity and the binding kinetics of pop inhibitors. the proteasome is an essential multicatalytic protease of the ubiquitin proteasome pathway. as a prime executor of regulated proteolysis, the proteasome controls almost all aspects of cell metabolism from signal transduction to cell cycle and differentiation. pharmacological intervention into proteasome activity leads to cell apoptosis. this observation was applied to successfully treat multiple myeloma, since the cancer cells exhibit substantially higher sensitivity to competitive inhibition of proteasome than normal cells. however, the complete shutting down of the proteasome catalyzed proteolysis leads to serious side effects resulting from the disruption of proteolytic homeostasis even in noncancerous cells. here, we show an alternative approach to control the proteasome activity using peptide based noncompetitive regulators. the cathelicidins derived peptides rich in proline and arginine (pr) residues have been found to affect activity of all the proteasome complexes both in vivo and in vitro, likely by binding to the face of the enzyme. mechanism and structural constrains of the pr peptides dictating their influence on the proteasome remain elusive. our results indicate that there are three sequence related properties of the pr peptides controlling their effectiveness as proteasome regulators: length of the peptide, distribution of a set of positive charges at the peptide n-terminus, and positioning of proline residues. far uv cd spectroscopy demonstrates that these properties also correlate with the structure of pr peptides. in particular, it seems that structural propensity of the pr peptides to form beta-turns are required to bind to proteasome as regulatory competent molecules. our work is focused on the search of selective, low-molecular cathepsin b peptide inhibitors acylated with the (e)- -(benzylsulphonyl)acroyl group (bsa). the double bond, embedded in the bsa moiety is activated by two electron-withdrawing groups and may be a good target for the michael-type addition of the catalytically active -sh group. three series of peptide derivatives possessing general structures: bsa-phe-asn(r)-oh, bsa-ile-x(oh)-n(ch ) and bsa-x-pro-oh were synthesized in solution and characterized by enzyme kinetic studies against papain, cathepsins b and k. it should be noted that all the investigated compounds were competitive and reversible inhibitors of the enzymes examined. using d h nmr (tocsy, cosy, roesy) and c nmr spectroscopy along with theoretical calculations (analyse program) we determined the conformational properties of two most potent and selective cathepsin b inhibitors. this work was supported by grant ds/ - - - . background and aims: we have developed peptides inhibiting human kallikrein- (hk ) activity. as hk is overexpressed in prostate cancer tissue, these peptides are potentially useful for treatment and diagnosis of prostate cancer. two of the potential physiological substrates for hk are proform of prostate specific antigen (propsa) and insulin-like growth factor-binding protein- (igfbp- ). both of these might participate in the regulation of prostate cancer growth: igfbp- by inhibiting igf-dependent tumor growth and psa by degrading extracellular matrix. we aimed to study whether our hk -inhibiting peptides inhibit also hk activity towards natural protein substrates, i.e. activation of propsa and degradation of igfbp- . methods: the effect of the peptides on the activation of propsa by hk was studied by preincubating the peptides with hk , followed by addition of psa and specific psa substrate. igfbp- degradation was studied by two specific immunoassays, one detecting only native igfbp- , while the other one also detected proteolytically cleaved forms of the protein. results: hk -inhibiting peptides were found to inhibit propsa activation and igfbp- degradation by hk in a dose dependent fashion. conclusions: we have developed new peptides inhibiting hk activity towards natural substrates, like propsa and igfbp- . the peptides might be useful for treatment of prostate cancer and other diseases associated with increased hk activity. from the seeds of garden four-o'clock and spinach we isolated two serine proteinase inhibitors (mjti i -mirabilis jalapa trypsin inhibitor and soti i -spinacia oleracea trypsin inhibitor), which are probably representatives of a new family of inhibitors. the purification procedures of these inhibitors included affinity chromatography on immobilized methylchymotrypsin in a presence of m nacl, ion exchange chromatography and/or preparative electrophoresis and finally rp-hplc on c column. their primary structures (fig. ) differ from those of known trypsin inhibitors, but showed significant similarity to one another, as well as to the antimicrobial peptides isolated from the seeds of mirabilis jalapa (mj-amp , mj-amp ), mesembryanthemum crystallinum (amp ) and phytolacca americana (amp- and pafs-s) and from hemolymph of acrocinus longimanus (alo- , and ). the equilibrium association constants (ka) of mjti i and soti i with bovine -trypsin were found to be about - m- . mjti i and soti i have been synthesized using solid-phase method. the synthesized inhibitors and inhibitors isolated from plants have similar properties. the disulfide bridge pattern in both inhibitors was established after digestion with thermolysine, followed by the maldi-tof: cys -cys- , cys -cys and cys -cys . s. cosgrove, l. rogers, c. hewage, j.p. malthouse aspartyl proteases are required for the multiplication of the aids virus and for producing the amyloid protein which causes alzheimer's disease. hiv protease inhibitors have been highly effective in treating aids patients and it is hoped that potent inhibitors of the beta secretases will also prove effective in treating alzheimer's disease. therefore inhibitors of the aspartyl proteases have great therapeutic potential. we have shown that the peptide glyoxals are potent inhibitors of the thiol protease papain and of the serine proteases subtilisin and chymotrypsin. using c-nmr we have been able to show that glyoxal inhibitors react reversibly with an active site nucleophile in these enzymes to form a tetrahedral adduct which is tightly bound by the enzyme. in the present work we synthesise c-enriched peptide glyoxals, we assess their inhibitor potency, and use c-nmr to examine how the inhibitors interact with the aspartyl protease pepsin. z-ala-ala-[ - c]phe-glyoxal was synthesised from [ - c]phenylalanine which was converted to its methyl ester. this was then coupled with z-ala-ala to give z-ala-ala-[ - c]phe-ome which was hydrolysed to the free acid. this was converted to the diazoketone and transformed into z-ala-ala-[ - c]phe-glyoxal using dimethyldioxirane. nmr spectra at . t were recorded with a bruker avance drx standard-bore spectrometer. we show that peptide glyoxal inhibitors can be potent inhibitors of pepsin and that pepsin only binds one of the four glyoxal forms (one non-hydrated, one fully hydrated and two partially hydrated forms). alzheimer`s disease (ad) is the most common cause of dementia in older people. a major factor in the pathogenesis of ad is the cerebral deposition of amyloid fibrils, consisting of amyloid β peptides (aβ), as senile plaque. the -to amino acid long aβ is generated by the proteolysis of β-amyloid precursor protein (app) by β-and γ-secretases. since bace , a unique member of the pepsin family of aspartyl proteases initiates the pathogenic processing of app by cleaving at the n-terminus it is a molecular target for therapeutic intervention in ad proteolytic activity was found to occur, to a variable degree, in digestive organs of all studied organisms over the entire ph range. the common feature was the existence of two activity peaks, in the acid (ph . - . ) and alkaline (ph . - . ) zones, as well as a similar protease set containing e and d cathepsins, a trypsin-like enzyme, elastase, and collagenolytic proteases. proteolytic activity in the hepatopancreas of crab and sea star was found to be an order higher than in other study objects. high protease activity in crab hepatopancreas is an evolutionary mechanism compensating for a poor differentiation of digestive system, low substrate specificity of enzymes, and cold environment. trypsin activity in digestive organs of invertebrates suggests that a trypsin-like enzyme is a genetically old one, an evolutionary origin of all serine proteases. a difference of kind between vertebrates and invertebrates is that the latter have cathepsine activity (absent in vertebrates) and no pepsin activity. it is of interest to develop enzyme inhibitors containing a light activated switch that can be used to control binding and inactivation of an enzyme. several inhibitors containing the azobenzene photoswitch group have previously been developed and have shown changes in activity of around two times on photoswitching. this study aimed to improve this switching by more extensive derivatisation of azobenzene to closer resemble the peptide substrates of proteases. a series of peptidomimetics containing the azobenzene photoswitch group were synthesized and assayed against the protease alpha-chymotrypsin. these compounds contained azobenzene, linked to a known chymotrypsin inhibitory group (either a trifluoromethylketone or boronate ester), and otherwise designed to be peptide-like. in some cases both ends of the azobenzene moiety were derivatized in order to increase the impact of photoswitching on the shape of the compound and thus its enzyme binding strength. assays showed that most compounds were reversible inhibitors of chymotrypsin, with low micromolar inhibition constants (ki or ic ). up to four times increase in enzyme inhibition on light activated switching of the azobenzene group conformation was obtained. a number of peptidyl derivatives structurally based upon the inhibitory sites of cystatins has been synthesized. these compounds are prone to proteolytic degradation, are rapidly excreted and poorly bioavailable. the majority of this problems might be overcome by use of peptidomimetics with structures resembling those of previously synthesized peptidyl derivatives. among the peptidomimetics are azapeptides, in which alpha-ch group of amino-acid residue is replaced by a nitrogen atom. the azapeptides have recently been demonstrated as potent and selective inhibitors of cathepsins b and k. it was shown that azapeptide inhibitors bind along the active site cleft of cathepsin b in a bent conformation. this bent structure is likely to result from the mobility of the bonds in the vicinity of the inserted azaamino acid residue as well as from the interaction with enzyme. in our present work we have studied the peptide of a sequence: z-arg-leu-arg-gly-ile-val-ome, which is characterized by one major and three minor conformations. the replacement of alpha-ch group in the gly residue of peptide chain of z-arg-leu-arg-gly-ile-val-ome by the nitrogen atom likely results in rigid conformation. our aim was a comparison of structure of the parent peptide z-arg-leu-arg-gly-ile-val-ome and a selective cathepsin b inhibitor z-arg-leu-arg-agly-ile-val-ome by using h-nmr. severe acute respiratory syndrome corona virus associated main protease (sars cov mpro protease), alternatively known as chymotrypsin-like protease ( clpro), is a mediator of virus infection cyclus and from there a therapeutic target. a peptide aldehyde library targeting the sars corona virus main protease (sars-cov mpro, alternatively known as clpro) was designed on the basis of three different reported binding modes and supported by virtual screening. a set of peptide aldehydes were prepared by a newly developed methodology and investigated in an inhibition assay against sars-cov mpro. [ ] protected amino acid aldehydes furnished by the racemization-free oxidation of amino alcohols with dess-martin periodinane were immobilized on threonyl resins as oxazolidines. following boc-protection of the ring nitrogen yielding the n-protected oxazolidine linker, peptide synthesis was performed efficiently on this resin releasing deprotected products under mild hydrolysis conditions. the library was tested in a new fluorimetric enzyme assay for sars cov mpro. via immobilization of the fluorophor, -( -amino- -methyl- -coumarinyl)-acetic acid, the substrate actsavlq-amca was prepared, surprisingly displaying a higher affinity than the native substrate. several potent inhibitors were found with ic values in the low micromolar range. interestingly, the most potent inhibitors seem to bind sars-cov mpro in a non-canonical binding mode. currently, the initial screen is extended towards the discovery of small molecule inhibitors of sars corona virus main protease. literature: a method of bromelain cleavage of surface glycoprotein hemagglutinin (ha) from the influenza a virions was initially employed for ha ectodomains crystallographic study [ ] . the remaining spikeless subviral particles were used by us earlier for ha c-terminal fragment extraction and mass spectrometric (ms) investigation [ ] . now sds-page analysis of the subviral particle preparations revealed several additional bands in a range of - kda together with major viral proteins comparing to intact virions (figure, m matrix protein, f -f -m protein fragments, np-nucleoprotein). maldi-tof ms analysis of the in-gel trypsin hydrolyzates has shown that the additional bands are fragments of М protein. this was confirmed by n-terminal sequencing of the protein fragments electroblotted from the bands. concentration of sh-reducing reagent in bromelain digestion reaction influenced on the m fragment bands intensity. we conclude that due to membrane destabilization during ha spikes removing, m protein localized under viral membrane inside intact virions becomes accessible to limited proteolysis by bromelain. [ dipeptidyl peptidases (dpp's) sequentially release dipeptides from polypeptides. among those enzymes, dppiv, fapα, dpp , dpp and dppii cause the release of n-terminal dipeptides containing proline or alanine at the penultimate position. they are all members of clan sc, a group of serine proteases that contains proline-specific peptidases. dipeptidyl-peptidase iv (dppiv) is the best studied member of this group of enzymes and has become a validated target for the treatment of type diabetes over the last years. the development of inhibitors for the related enzymes (i.a. dppii) has only started recently. this poster presents selected products synthesised to further elaborate the structure-activity relationship for dpp ii inhibitors with a , -diaminobutyrylpiperidine basic structure. this class of compounds was described earlier by our group as the hitherto most potent and selective inhibitors of dpp ii. starting from n -p-chlorobenzyl-substituted uamc , our lead compound, two types of modifications were proposed: • the synthesis of n -(di)alkyl-and arylalkyl analogues; • the synthesis of -methyl analogues. in our previous study, we reported potent and small-sized bace inhibitors containing phenylnorstatine [( r, s)- -amino- -hydroxy- -phenylbutyric acid; pns] at p position as a transition-state mimic. in developing more active compounds, we focused our attempts on the p position, where we replaced the pns by its thioderivative. herein, we present the synthesis of a novel phenylthionorstatine [( r, r)- -amino- -hydroxy- -(phenylthio)butyric acid; ptns] as a p moiety with hydroxymethylcarbonyl (hmc) isostere, and then an application to the bace inhibitors design. we have synthesized ptns starting from readily available n-benzyloxycarbonyl-serine and after multistep reaction (including weinreb amide formation, thiophenyl group introduction, through cyanohydrin derivative the transformation into the -hydroxy ester and then acid). purification was done by column chromatography and rp-hplc. peptides were synthesized by the fmoc based solid phase method and characterized by maldi-tof ms. the peptide inhibitors were adopted to enzyme assay using a recombinant human bace and a fluorescence-quenching substrate. bace inhibitory activity was determined based on the decrease% of the cleaved substrate by the enzyme.we have synthesized ptns and then the ( r, r)-enantiomer was applied to spps (solid phase peptide synthesis). we synthesized octa-and pentapeptide-type inhibitors of bace containing pns or ptns at the p position. these compounds were enzymatically tested and showed high bace inhibitory activity. a novel derivative of pns, ptns, was synthesized, and evaluated in comparison to corresponding pns. the inhibitors with ptns exhibited a slightly higher inhibitory activity against bace comparing to those with pns. this study suggests possibilities of the application of ptns to design other aspartyl proteases inhibitors. the αν β integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis, mainly by interacting with matrix proteins through recognition of an arg-gly-asp (rgd) motif. inhibition of the αν β integrins with a cyclic rgd peptide impairs angiogenesis, growth and metastasis of solid tumours in vivo. the aim of this study was to investigate the effects of replacement of amino acids by aza-β -amino acid analogs in cyclic rgdpeptides as αν β -integrin antagonist on angiogenesis, microcirculation, growth and metastasis formation of a solid tumour in vivo. the selectivity profile of these antiadhesive cyclopeptide is rationalized by a special presentation of the pharmacophoric groups. thr rgd motif resides in position i to i+ of a regular γ-turn. we synthetized linear and cyclic aza-β rgd-peptide with the purpose to examine the effect on the conformation and the activity. are aza-β amino acids γ-turn mimetics? the preferred conformations were determined by nmr. prostaglandins are involved in a large number of biological activities mediated by their g-protein coupled receptors (gpcrs). the prostaglandins pgf alpha receptors are found specifically in uterine muscle, where they initiate parturition and labor. the pgf alpha receptor plays a key role in preterm labor, for which medical and social costs are estimated at $ billion per year in the usa (the highest per patient cost of any disorder). peptide mimics have been developed in our laboratory ( , ) , that serve as allosteric antagonists of the pgf alpha receptor. the importance of the turn geometry of the central residue in these peptide mimics has been investigated using enantiomeric indolizidin- -one beta-turn mimics which can respectively induce type ii and ii' geometry. our presentation will discuss the synthesis and biology of these novel allosteric modulators of prostaglandin pgf alpha receptor activity. it was shown that luteinising hormone -releasing hormone (lhrh) receptors are overexpressed in the most of adenocarcinoma cells in contrast to their low content in normal tissues. these data create the basis for lhrh analogues application in therapy of breast, ovary, prostate, lung, intestine, liver and kidney cancers. both agonists and antagonists utility for the targeting of cytotoxic moiety to the tumor cells is well documented. however, the number of lhrh analogues possessed their own cytotoxic activity is still very limited. we nicotianamine (na) that was first isolated from the leaves of nicotiana tabacum l [ ] , is known as a key biosynthetic precursor of phytosiderophores. various studies have proved that nicotianamine plays a significant role in plants as an iron, nickel, zinc ... transporter [ ] . the aim of our study was to synthesize unnatural analogues of na via peptide intermediates, to investigate the mechanisms of metal transport and accumulation within the plant. we found that the strategy developed for na synthesis could not be applied when the azetidine ring was changed for pyrrolidine ring and we investigated a new route to synthesize such analogue. these synthetic pathways will be discussed. the primary physiological roles of arginine vasopressin (avp), [cycle - (h-cys -tyr -phe -gln -asn -cys -pro -arg -gly -nh )], involve vasoconstriction of vascular smooth muscles, via v a receptor, and antidiuretic action in kidney (blood osmolality regulation) via v receptor. binding of avp to the v a receptor subtype also stimulates glycogenolysis in the liver and promotes platelet aggregation. in addition, activation of the v b (also known as v ) receptor causes adrenocorticotropic hormone release from the anterior pituitary. v b receptors are also present in the brain where avp functions as a neurotransmitter. in the recent years by the salivary glands of several bloodsucking animals like, teaks, leeches, vampire bats and so forth are isolated plenty of proteins and peptides with different molecular weight and well established anticoagulant activity. many of the strongest anticoagulants isolated by bloodsucking animals are found in the extract of salivary glands of different kinds of leeches. such leech is the haementeria officinalis, from which is isolated the most active inhibitor of factor xa -ats. in order to study the role of some amino acids in the process of interaction among peptides mimetics and the active site of serine proteinases, some fragment analogues of ats's active site by replacement of some amino acids with the other with similar structure or with unnatural amino acids were synthesized. in the present work the synthesis and the anticoagulant activity according to the aptt and ic of the newly synthesized peptides and structure-activity relationship will be discussed. rational design of peptides is a challenge which would benefit from a better knowledge of their rules of sequence-structure-function relationships. peptide structures can be approached by spectroscopy and nmr techniques but data from these approaches too frequently diverge. structures can also be calculated in silico from primary sequence information using three algorithms: pepstr, robetta and peplook. the most recent algorithm, peplook introduces indexes for evaluating structural polymorphism and stability. the method uses a de novo search of energy minima by an iterative boltzmann-stochastic procedure and using a combination of phi/psi couples derived from the structural alphabet for protein structures proposed by etchebest et al. for peptides with converging experimental data, calculated structures from peplook and, to a lesser extent from pepstr are close to nmr models. the peplook index for polymorphism is low and the index for stability points out possible binding sites. for peptides with divergent experimental data, calculated and nmr structures can be similar or, can be different. these differences are apparently due to polymorphism and to different conditions of structure assays and calculations. the peplook index for polymorphism maps the fragments encoding disorder and the mean force potential score indicates which residues will be most available for interactions with partners. this should provide new means for the rational design of peptides. several diseases like cancer metastasis, rheumatoid arthritis and chronic lymphocytic b-cell leukemia are linked to the interaction of the cxcr chemokine receptor to its natural ligand, the amino acid protein stromal cell-derived factor- α (sdf- α). [ ] one strategy for the treatment of these diseases could be to block the interaction between cxcr and sdf- α with small cxcr antagonists. furthermore, radiolabeling of suitable compounds with appropriate radioisotopes could provide agents for imaging of cxcr expression in vivo via pet. previous studies by fujii et al. on cxcr antagonists led to a high affinity cyclic pentapeptide with the sequence cyclo[gly-d-tyr-arg-arg-nal]. [ ] to further improve this structure, different approaches have been chosen with respect to metabolic stability, bioavailability, conformational rigidity and chemical versatility for radiolabeling. first, an n-methyl scan of the backbone amides was performed to influence conformational freedom and to increase metabolic stability and bioavailability. n-methylation of arginine residues yielded peptides with moderate affinity (ic<sub> </sub>-values: nm (n-me)arg<sup> </sup> and nm (n-me)arg<sup> </sup>, resp.) whereas n-methylation of other amino acids significantly decreased the affinity (ic<sub> </sub>> nm). by substitution of arg<sup> </sup> by ornithine, the affinity was mostly retained. [ ] the amino group of orn can be alkylated or acylated via radiolabeled groups containing short lived isotopes. moreover, the bioavailability should be improved as the high basicity of the two guanidino groups could be reduced. first ornithine-acylated derivatives showed ic<sub> </sub> values between - nm enabling for the first time <sup> </sup>f-radiolabeling of small cxcr antagonists for pet imaging in vivo. binding of ligands to integrins plays a major role in cell adhesion, migration, and signal transduction of cells. these interactions are important not only for normal cell functions, but also in pathogenic processes. the v integrin for example is involved in tumor cell adhesion and osteoporosis. the association of ligands is specific and requires minimal recognition sequences. therefore, suppression of integrin activity using competitive inhibitors bears great pharmacological potential. the tri-peptide sequence rgd is a prominent recognition sequence of integrin ligands. two new cyclic pentapeptides were synthesized containing the tripeptide sequence rgd as well as -amino-cyclopropane- , -dicarboxylic acid monomethyl ester (acc) and valine varying only with respect to the stereochemistry of acc. both the (+) (all r) and (-) (all s) isomers of acc were incorporated. acc is a cyclic -amino acid as well as a cyclopropyl analogue of aspartic acid. biological tests with cell lines expressing mainly v and v integrin show a higher inhibitory activity of cyclo-(-arg-gly-asp-(+)acc-val-). in order to derive a structure-activity relationship of these two isomers, solution structures in dmso-d were investigated by nmr spectroscopy. subsequently, structural information was obtained by applying distance restraints derived from the nmr spectra in distance geometry/simulated annealing and molecular dynamics calculations. due to the rigidity of the cyclopropyl unit in acc, the structure of the cyclopeptide is significantly influenced by the integrated propane ring, thus explaining the different biological properties. integrins are an important family of cell adhesion molecules. currently, members are known. among other functions, integrin α β is involved in inflammatory processes, leukocyte migration and tumor angiogenesis. the structure of its natural ligand vcam- , including the binding loop sequence tqidspln, has been determined by x-ray crystallography. therefore, it is possible to apply the concept of spatial screening: using small cyclic peptides with structure inducing building blocks, the binding motif is presented in different well-defined structural arrangements. for this study, a series of cyclic penta-and hexapeptides based on the tqidspln sequence has been synthesized. β-homoamino acids, i.e. β -amino acids with proteinogenic side-chains, have been incorporated as structure inducers for spatial screening. although β -amino acids are supposed to prefer the central position of Ψγ-turns, less data exist than for e.g. d-amino acids. apart from the structural characterization of potential high affinity ligands for integrin α β , a major goal of this work is to provide a better understanding of the influence of β -amino acids on the structure of cyclic peptides. the structures of the peptide library have been investigated by nmr spectroscopy, followed by dg/sa and md calculations. the results substantiate the γ-turn inducing capability of β-homoamino acids, but also prove the formation of different turn structures in certain cases. a comparison to the x-ray structure of vcam- shows that the structure of the binding sequence has been successfully approximated by some of the peptides. biological activity tests should lead to meaningful structure-affinity relationships. neuropeptide y (npy) is a -amino acid peptide amide and binds to the so-called y receptors. its most dominant element is the c-terminal alpha-helix spanning amino acid residues - . residues - form a polyproline helix with highly conserved proline residues at positions , and , followed by a loop structure. the importance of the polyproline helix strongly varies between different receptor subtypes. it obviously plays no role in ligand binding at the y receptor subtype, whereas the n-terminal segment is of importance for ligand binding at y and y . in order to further study the importance of the polyproline helix we introduced a conformationally constrained pyridone dipeptide mimetic at different single positions by solid phase peptide synthesis using fmoc/tbu strategy. the resulting peptides have been investigated in cell lines that selectively express y and y receptor, respectively. different methods including radioactive competitive binding assay, cd and nmr have been applied to investigate conformation and interaction of receptor and ligand. loss of affinity at the y receptor is independent of the position and about -, -and -fold, respectively, when introduced once, twice and thrice. introduction of the building block in position / leads to the most reduced affinity at the y receptor subtype but, surprisingly, affinity can partially be regained by introduction of the dipeptide at two additional positions. the position of the dipeptide is of greater importance at y . these novel peptides clearly indicate the importance of proline residues and the structure of the n-terminus for ligand binding. interactions of src homology (sh ) domains with phosphotyrosine (py) containing ligands is critical for regulating cellular processes. the cytosolic protein tyrosine phosphatase shp- contains two sh domains. an intramolecular interaction of the n-terminal sh domain with the catalytic (ptp) domain renders the enzyme inactive in the native state. binding of a py-ligand to shp- n-sh leads to a conformational shift and the dissociation of the sh -ptp complex [ ] . in previous studies we investigated the topographical and conformational preferences of the n-sh domain of shp- using conformationally restricted linear and cyclic peptides derived from the natural interaction partner ros py [ ] . we identified peptides that showed an increased binding affinity for the n-sh domain and partially inhibited ros-mediated shp- activity. on the basis of these results we hypothesized that an imperfect fit of the py+ and py+ side chains might be responsible for the inhibitory effect. in order to confirm this hypothesis we synthesized a new series of peptides and evaluated their biological activity. to better understand the role of each individual sh domain in the activation process we also determined the binding affinity against the c-sh domain and the activation profile of different shp- mutants. pull-down assays of the interactions of the py-ligands with full length shp- confirmed the results obtained for the binding to the individual sh domains. proteins are targets for photo-destruction due to absorption of incident light by endogenous chromophores. mass spectroscopic data presented evidence that structural modification observed upon irradiation of goat alpha-lactalbumin at nm results from tryptophan (trp) mediated cleavage of disulfide bonds [ ] . the aim of the recent studies is to define structural elements that direct the destructive influence of near-uv light on the disulfide bridges of proteins. most of the proteins of the immunoglobulin superfamily contain a so called triad, consisting of two s atoms, forming a disulfide bridge, and a single trp in their close vicinity [ ] . we have indications that this arrangement gives rise to a photolytic degradation similar to that described in our earlier studies for goat alpha-lactalbumin [ ] . we therefore investigated the influence of uv light on the single chain variable fragment (scfv) of a monoclonal antibody ( d a ) [ ] which contains two triads. the results showed that after irradiation of the wild type scfv (i) new bands (degradation products) appeared in electrophoresis experiments and (ii) the affinity for its antigen, von willebrand factor decreased. by site-directed mutagenesis, we modified the critical trp-residues to perform a parallel study on these mutants. background and aims: it is known that thrombomodulin has important function which prevents thrombus. we found kmylcvckn (m, n >= ) peptides derived from thrombomodulin had strong anti-thrombus activity in our recent studies. these peptides formed two structures, parallel and anti-parallel, as dimers, we examined the relation between structure and activity. methods: two peptides of kkkylc(acm)vckkk and kkkkylcvc(acm)kkkk were synthesized by fmoc chemistry. dimer peptides were made by removing acm with iodine, after dissolving in . m tris hcl buffer (ph . ) and oxidizing the mixture of these synthesized peptides spontaneously. then three peptides shown in figure were separated using rp-hplc. the peptide concentration in normal human pooled plasma was micro moles / l when measuring aptt (activated partial thromboplastin time). results: the anti-parallel formed peptide, peptide b, was prolonged aptt approximately . times, although two parallel formed peptide, peptide a and c, were not significantly different from the aptt of normal plasma. conclusions: these peptides have structure-activity relationship, we observed that the anti-parallel formed peptide had strong anti-thrombus activity. insect kinins share a highly conserved c-terminal pentapeptide sequence phe-xaa-xbb-trp-gly-nh , where xaa can be tyr, his, ser or asn and xbb can be ala but is generally ser or pro. they are potent diuretic peptides that stimulate the secretion of primary urine by malpighian tubules, organs involved in the regulation of salt and water balance ( ). the insect kinins preferentially form a cis-pro, type vi β-turn. insect kinin analogs containing tetrazole ( ) and -aminopyroglutamate ( ), both cis-peptide bond, type vi β-turn motifs, demonstrate significant activity in the in a cricket diuretic assay. in this study, we compare the diuretic activity of insect kinin analogs incorporating the four stereochemical variants of the -aminoglutamate (apy) motif. three of the insect kinin analogs incorporating the stereochemical variants, ( the need for new effective and to mammalian cells non-toxic antifungal agents increases in parallel with the expanding number of immunocompromised patients at risk for invasive fungal infections. in our laboratory we have produced a serie of low-molecular peptide derivatives of the general structure: x-arg-leu-nh-ch(ipr)-ch -nh-y (where x and y were acyl groups with aromatic carbocyclic system). we have found and earlier reported that some of these display high antimicrobial activities against several clinically important gram-positive pathogenic bacteria. in this study we have by solution methods synthesized a group of low-molecular compounds and investigated their antifungal activity. the study included both candida and aspergillus species. we have found that some of the compounds were highly fungicidal. we also made a conformational study in which the residues were separately replaced by selected hydrophobic amino acids and their equivalents. the conformational study showed that the desirable stable intramolecular structure could only be formed in the presence of some vital components. this work was supported by grant ds/ - - - . increased resistance of bacterial pathogens to currently employed antibiotics has resulted in efforts to develop antimicrobial compounds with new mechanisms of action. previously, we have synthesized some high potent antimicrobial compounds based upon the n-terminal binding fragment of human cystatin c. some derivatives of the general structure: x-arg-leu-nh-ch(ipr)-ch -nh-y ( ) (where x and y were acyl groups with aromatic carbocyclic system) have displayed the broad antibacterial spectrum and high activity against several clinically important gram-positive pathogens, including multi-resistant staphylococci. herein, the synthesis and structure -antibacterial properties relationship for two series of analogues of are presented. the x and y groups in were replaced by selected substituents with various geometry and distance between aromatic moieties and carbonyl. we have established the general structural features which the discussed class of peptide derivatives should possess in order to displaying the particular antimicrobial activity. this work was supported by grant ds/ - - - . we have synthesized beta-endorphine-like decapeptide immunorphin sltclvkgfy which corresponds to the - sequence of the heavy chain of human igg. immunorphin was found to be a selective agonist of non-opioid (naloxone-insensitive) beta-endorphin receptor. the purpose of this study was to prepare [ h]immunorphin and characterize by its using the non-opioid beta-endorphin receptor on mouse peritoneal macrophages and membranes isolated from various rat organs. by use of tritium-labeled immunorphin ([ h]sltclvkgfy) with specific activity of ci/mmol, non-opioid beta-endorphin receptors were revealed and characterized on mouse peritoneal macrophages and rat myocardium, spleen, adrenal, and brain membranes. since dehydroamino acids are quite reactive and various thiol nucleophiles are known to add to their double bonds [ , ] , we hoped that these compounds might act as alkylating inhibitors of cathepsin c (dipeptidyl-peptidase i). its main function is protein degradation in lysosymes, but it is also found to participate in the activation of neuraminidase and proenzymes of serine proteinases (leukocyte elastase, cathepsin g, granzyme a) [ , ] . it is well known, that phosphonodipeptides structural analogues of synthetic substrates of cathepsin c are the model substances in designing the new inhibitors of this enzyme. for that reason we have undertook the synthesis, theoretical and structural investigations of phosphonic analogues of dehydropeptides. gly-∆zphe-abupo(ome) gly-∆zphe-alapo(oet) gly-∆zphe-leupo(ome) gly-∆zphe-valpo(oet) gly-∆zphe-glypo(ome) gly-∆zphe-nbupo(oet) the structure and conformational preferences in this group of peptides had been investigated by mean of nmr techniques. in order to find the interactions between compounds-enzyme (cathepsin c) and interpret the results of biological test, the molecular modelling methods had been used. the interaction of v integrin receptor with its ligands is selectively implicated in various processes, like angiogenesis, bone-formation, tumor genesis and tissue-genetic migration of embryonic cells. several cyclic rgd pentapeptides are known as selective ligands for v integrin receptor. the aim of this study was to prepare a new conjugate, composed of the cyclo[rgdfc] derivative and a branched chain polycationic polypeptide, poly[lys(dl-alam)] (ak). the cyclopeptide was prepared on -cl-trityl chloride resin by fmoc/tbu strategy. the "head-to-tail" cyclisation was achieved in a diluted solution of dmf in the presence of bop and hobt coupling reagents and diea base. coupling of the cyclopeptide to ak polymer was carried out by thioether linkage. adhesion properties of soluble cyclic rgd peptides and their plate-immobilized forms were studied. free cyclopeptides evoke aggregation of cultured primary neural and cloned neural stem cells, while their plate-immobilized forms fail to support cell adhesion. on the contrary, in case the newly synthesized ak-c(rgdfc) conjugate such induction of cell aggregation was not observed. whereas immobilizing this derivative to either glass, or plastic was found to support cell-attachment in case of various cell types. in addition, all cell lines investigated -including also the primary neural cells -attached to ak-c(rgdfc) coated surface and survived, grew or differentiated even in the absence of serum. our data suggest that cyclic rgd -polypeptide conjugates represent a new tool to investigate selective cell adhesion and may provide a novel scaffold-material for directed cell-seeding. in the ph-induced channel closure in combination with the pip interactions. however, their detailed regulations are still remaining unclear. therefore, in the present study, we investigate these crucial residues with electrophysiological recordings and rationally designed mutagenesis based upon our structural analysis of kir . tetramer. lys- is located fairly close to the intracellular channel gate and protrudes its long side chain positive charge into the pore. this may interfere with the potassium flow by providing repulsion charge while ph is lowered, which pushes the channel towards its closed state. mutation to met- therefore reduces such ph-sensitivity. on the other hand, arg- is supposed to be responsible for the maintenance of channel opening in the presence of pip . loss of positive charge at this site may lead to the enhanced ph-sensitivity due to an abolished or reduced pip interaction. more interestingly, the double mutant for both sites reveals a compensation scenario. in combination with the discussion for the role of previously known r-k-r triad, our data provide very clear structural explanation for the exact functional roles of these basic residues in the regulation of ph-sensitive channel gating. mouse obese cart peptides are neurotransmitters involved in feeding, stress and endocrine regulation. leptin, a long-term adiposity signal, upregulates expression of cart in the hypothalamus. recent findings of co-localization of cart and cholecystokinin (cck)-a receptor (responsible for satiety effect of cck) in brain and gastrointestinal tract suggest a neurochemical link between cart peptides and cck. in normal fasted mice, cart( - ) peptide decreased food intake after intracerebroventricular (icv) administration in a dose-dependent manner. anorectic effect of cart peptide was enhanced by peripherally administered cck- , while cck-a receptor antagonist, devazepide blocked the effect of cart peptide on food intake. we used two mouse obesity models in this study: monosodium glutamate (msg) and diet-induced obese (dio) c bl mice. both dio and msg mice had substantially increased fat to body mass ratio compared to their controls and were hyperleptinemic. msg mice were hypophagic and neither cart peptide nor cck- and devazepide had any effect on food intake of these mice. dio mice fed high-fat diet showed slightly decreased sensitivity to central administration of cart peptide, effect of cck- on food intake was preserved. in conclusion, cart peptide and cck- showed a synergistic effect on feeding in control mice that pointed to their probably integrated action in the central nervous system. analogously, devazepide suppressed cart anorectic effect. in msg obese mice, effects of both cart peptide and cck- on food intake were diminished due to disrupted signaling in hypothalamus. in dio mice, additive effects of cart and cck- were partly preserved inspite of hyperleptinemia and increased adiposity. b. chini , s. stoev , l.l. cheng , m. manning , were subsequently shown not be selective for the rat v b receptor [ ] . peptides a-d served as excellent leads to the design of selective agonists for the rat vp v b receptor [ ] . replacement of the arg residue in a-d by lys, orn, dap and dab, led to the first potent and selective agonists for the rat v b receptor [ ] . we now report that three of these; d the aim of the de novo peptide synthesis and the incorporation of cofactors is the construction of artificial protein models. these model systems can be used for understanding the structure-function relationship of native proteins and might open a way for possible applications. protegrin- (pg- ) is an -amino acid peptide with an amidated c-terminus, which forms an antiparallel beta-sheet, constrained by two disulfide bridges. the native sequence of pg- is highly cationic, containing six positively charged arginine residues. it was found that the structural features such as amphiphilicity, charge and shape are important for the cytolitic activity of pg- . in this study we investigate the sar (structure activity relationship) of two pg- analogues: rglcycrgrfcvcvg-nh (bm- ) and rglcyrprfvcvg-nh (bm- ). our antimicrobial activity studies of these peptides show that the bm- peptide is active against microbe species as well as the native pg- , whereas the bm- is completely inactive. the bm- analoque is shorter than native pg- and contains only three arginine residues, therefore is much cheaper in the chemical synthesis, what could be an advantage of this antimicrobial peptide. the conformational studies of both analogues were performed by using d h-nmr technique (in dmso-d ) and molecular dynamics studies. the d solution structure of both analogues was established using interproton distances and torsion angles. for simulated annealing calculations the xplor program was used. our conformational studies show that the bm- forms a regular beta-hairpin structure, which is very similar to that of the native pg- peptide, whereas the bm- analogue is very flexible, what could be a reason of the antimicrobial inactivity. copper amine oxidases (ec . . . ) catalyze the oxidative deamination of primary amines to the corresponding aldehydes, ammonia and hydrogen peroxidase. these enzymes are ubiquitous, occurring in micro-organisms, plant and animals. activity of this enzyme increases under various stress conditions including thermal and water stresses. although lsao is not a thermostable enzyme, it is in maximum stability and activity above physiological temperatures. in this study we have investigated the kinetics of thermal denaturation of lentil seedling amine oxidase (lsao) by measuring its denaturation constant (kden) at various temperatures from to degrees centigrade in mm phosphate buffer, ph . . the results of thermal inactivation curves as well as measuring of a at various temperatures were used to calculate kden. moreover, activation energy (ea) for denaturation reaction was obtained from corresponding arrhenius plot. our results showed that unfolding process started to occur at degree centigrade and ea of denaturation was changed at degree centigrade proving a dominant conformational change of the enzyme at this temperature. the results of the kinetic study are coincident with previously reported equilibrium studies denoting the optimum and melting temperature of the enzyme are and degree centigrade, respectively. development and advancing of enzymatic processes used for production and modification of natural polysaccharides are now major biochemistry challenges. the paper investigates enzymatic systems in invertebrates, in particular, an enzymatic complex obtained from the hepatopancreas of red king crab paralithodes camtschaticus, and clarifies its effect on the mechanism of chitin and chitosan hydrolysis. chitinolytic activity was estimated with spectrophotometer using -(dimemylamine)-behzaldehyde method by the concentration of n-acetyl-d-glucosamine which is educed under chitinolysis. total glycolytic activity was defined by the sum of n-acetyl-d-glucosamine and d(+)-glucosamine in the reaction with potassium hexaferricyanide (iii). content of d(+)glucosamine in the hydrolysates of chitin and chitosan was estimated by highly effective reverse-phase liquid chromatography (helc) of aminosaccharides with ortho-phthalaldehyde. the paper studies the process of chitin and chitosan glycolysis and the effects of different factors (ph, temperature and time of incubation, enzyme/substrate ratio) on the total glycolytic activity of the enzymatic complex from crab hepatopancreas, which is compared with a previously studied proteolytic and exochitinase activities. a mechanism of enzymatic hydrolysis of chitin and chitosan is suggested. study results allowed the following conclusions concerning glycolytic and deacetylase activity of ep: ) ep induces the formation of a monomer (n-acetyl-d-glucosamine) and oligomers (chitin and chitosan) with low deacetylation. thus, ep is characterised by a marked endochitinase (endochitosanase) activity; ) n-acetylglucosamine deacetylase and, apparently, exochitosanase activity was not revealed; ) it was found that chitinase and protease activities of ep are associated with different enzymes. [background] in opioids, the n-terminal amino acid ', '-dimethyl-l-tyrosine (dmt) enhances bioactivity by orders of magnitude. c-terminal modification of the dmt by a methyl group, h-dmt-nh-ch , exhibited µ-opioid receptor affinity (kiµ = . nm) equivalent to that of morphine; however, antinociception was only . - . % [ ] . dmt plays an important role in the message domain to anchor opioid ligands into the active site of opioid receptors, specifically to trigger biological activity by the µ-opioid receptor. [methods] dimerization of dmt through diaminoalkanes [ ] or , -bis-(aminoalkyl)- ( h)-pyrazinone produced potent opioidmimetics with high affinity for µ-opioid receptors (kiµ = . - . nm), agonism (gpi, ic = . - . nm), and antinociception in mice after systemic and oral administration, which verified passage through the epithelial membranes of the gastrointestinal tract and blood-brain barrier [ ] . ( -aminocycloalkane- -carboxylic acid) . in this case the ring consists of five atoms. knowing that acylation of the nterminus of several known b blockers with a variety of bulky groups has consistently improved their antagonistic potency in the rat blood pressure assay, the apc substituted analogues were also synthesized in n-acylated form (with -adamantaneacetic acid (aaa)). the activity of eight new analogues was assayed in isolated rat uterus using a modified holton method in munsick solution and in rat blood pressure tests. the results clearly demonstrated the importance of the position in the peptide chain into which the sterically restricted apc residue was inserted. apc at positions led to preservation or reduction of antagonistic qualities, respectively. acc at position enhanced antagonistic qualities in blood pressure test and led to preservation of activity in antiuterotonic test.. in most cases acylation of the n-terminus led to enhancement of antagonistic potencies. our findings offer new possibilities for designing new potent and selective b blockers. background: during the course of developing opioidmimetic analgesics, data revealed that the n-terminal residue ', '-dimethyl-l-tyrosine (dmt) plays an important role in anchoring opioid ligands in the active site of opioid receptors. as a single residue c-terminally extended with an aminomethyl group exhibited µ-opioid receptor affinity (kiµ = . nm) similar to morphine; however, antinociception was only . - . % [ ] . in order to develop potent µ-opioid agonists, the dimerization of tyr or dmt through diaminoalkanes [ ] or , -bis-(aminoalkyl)- ( h)-pyrazinones [ ] resulted in production of unique opioidmimetics with high receptor affinities and potent biological activities [ ] . methods: the synthesis of opioids and opioidmimetics and the determination of their receptor binding characteristics were performed as described previously [ ] [ ] [ ] . results and conclusion: newly synthesized -(tyr-nh-butyl)- -(tyr-nh-propyl)- ( h) pyrazinone and -(tyr-nh-propyl)- -(tyr-nh-butyl)- ( h) pyrazinones (i and ii) exhibited fairly high binding affinity towards µ-opioid receptor (kiµ = . and . nm, respectively). replacement of tyr with dmt in i and ii gave opioidmimetics iii and iv (kiµ = . and . nm, respectively); they exhibited -and -fold higher binding affinity than the tyr derivatives. while iii is a dual µ-/δ-opioid agonist, iv is only a µ-opioid agonist. these findings pave the way to design additional µ-opioid receptor agonists and antagonists for therapeutic application. divalent cations have been known for a long time to influence significantly binding to receptors and biological activity of the peptide oxytocin (ot). there is very low binding of h-ot to the receptors in the absence of these ions. it has been speculated where the divalent cations work. recently an article appeared showing formation of a complex divalent cation-ot and stressing the importance of n-terminal amino group for binding and activity [ ] . however deamino analogues of ot are also very active and their binding is also influenced by divalent cations. we have studied ot, deaminooxytocin (dot) and an ot antagonist (antag) by means of electrospray ms and we have observed that all these compounds form molecular adducts with zn +, mg +, mn + and ca +. in binding experiments using -i antag, the quantity of tracer bound to membranes of hek cells having stable expressed human ot receptor strongly depends on the character and concentration of divalent ions. displacement curves using unlabelled antag do not change in the absence or presence of mm of tested divalent ions. on the other hand, displacement curves using unlabelled ot and dot are shifted to the left in the presence of mg + and mn +, and to much lesser extent by zn + and ca +. all this points to the idea that the divalent ions do work on the site of membrane receptors. biologically active peptides exhibit multiple conformations in solution. thus, the synthesis of conformationally restricted analogues is a valuable approach for determining structure -activity relationships. restrictions can be imposed e.g. through the formation of cyclic structures within the peptide framework by disulfide bridges, or by substitution of chosen amino acid residues that limit conformational freedom, thus forcing the peptide backbone and/or side chains to adopt specific orientations. in recent years, conformationally constrained analogues of bioactive peptides seem to be a feasible approach to providing useful informations concerning threedimensional structure of such compounds which, in turn, could rationalize our knowledge about structure-biological activity relationships and thus help to design peptides with desired pharmacological properties. steric restrictions can be introduced by the formation of cyclic structures within the peptide backbone or by incorporation of amino acids with limited conformational freedom, which in turn results in specific orientations of the peptide backbone and its side chains. another approach to reduce the flexibility of the analogue is substitution of chosen amino acids with various types of pseudopeptides prepared trough short-range cyclizations. the present work is a part of our studies aimed at clarifying the influence of sterical constraints in the n-terminal part of arginine vasopressin (avp) and its analogues on the pharmacological activity of the resulting peptides. we describe the synthesis of four new analogues of avp substituted at positions and or and with two diastereomers of -amino-pyroglutamic acid and four peptides in which we combined the above modification with the placement of mercaptopropionic acid (mpa) at position . all the peptides were tested for their in vitro uterotonic, pressor and antidiuretic activities in the rat. different strategies to modulate shp- activity protein tyrosine phosphatase shp- consists of two sh domains n-terminal to the catalytic (ptp) domain and a short c-terminal tail. the binding of a py-ligand to the n-sh domain is required for an efficient activation of shp- phosphatase activity. the specificity of the shp- sh domains is determined by the py-residue (position ) and residues at positions - , + and + . combinatorial peptide library methods revealed different classes of consensus sequences for both sh domains [ , ] . in addition, the importance of residues c-terminal to py+ (+ to + ), in particular for binding to the n-sh domain, has been demonstrated [ ] . together with investigations of the determinants for optimal sh domain binding and stimulation/inhibition of shp- activity [ ] , these informations were useful for the generation of different strategies for effectors of shp- activity. peptides cyclized between different positions of the general consensus py- to py+ were synthesized and evaluated with respect to n-sh domain binding and stimulation of phosphatase activity. structure-activity studies have revealed that the specificity of an integrin towards its rgd-containing ligands can be evaluated through the distances between the cβ atoms and/or the distance between the charged centers of arginine and aspartic acid as well as, the pseudo-dihedral angle (pdo), composed by the r-cζ, r-cα, d-cα and d-cγ atoms, which defines the relative orientation of the arg and asp side chains. in a previous study [ ] , the antiaggregatory activity of rgd peptide analogues, i.e. their ability to act as fibrinogen receptor αiibβ antagonists, was correlated with the above structural criteria. our results suggested that the fulfillment of the criterion - ο < pdo < + ο is a prerequisite for an analogue to exhibit activity. in the present study, we examine the above criteria to rgd-containing peptides, derived from the active sites of the ecm proteins fibrinogen, fibronectin and vitronectin, as well as, from the cryptic rgd site of von willebrand factor. the correlation of the structural data with the biological activity of compounds, are in good agreement with the previously mentioned - ο < pdo < + ο criterion. furthermore, our results show that the differences in activity of compounds, which display similar distances between the charged centers of arg and asp, can be better evaluated by the pdo structural criterion. acknwolegments : this work was supported by grants from eu and the hellenic ministry of education ( heraklitos). references : the gpiib/iiia receptor, which is a member of the integrin family, is the most abundant receptor in the surface of platelets and can interact with a variety of adhesive proteins including fibrinogen, fibronectin and von willebrand factor. fibrinogen binding on gpiib/iiia is an event essential for platelet aggregation and thrombus formation. mapping of the fibrinogen binding domains on gpiib subunit suggested the sequence - as a putative binding site [ ] . this region was restricted to sequence gpiib - (ymesradr) using synthetic octapeptides overlapping by six residues [ ] . the ymesradr octapeptide inhibits adp stimulated human platelets aggregation and binds to immobilized fibrinogen. in this study we present the conformational analysis of three synthetic analogues yaesradr (a ) ymesaadr (a ), and ymesraar (a ), using nmr spectroscopy and distance geometry calculations. common structural characteristic of peptides a and a is the interaction between the side chains of arg and glu , however in a the guanidino group of arg seems to form salt bridges with both glu and asp . peptide a is stabilized only by a week interaction between arg and glu side chains. the interactions between the residue side chains provoke different overall shape of the three molecules. the most populated structural family of a exhibits a π backbone shape, a a turn around -s a -, while a an almost extended shape. background and aims: endomorphin- (em- : h-tyr-pro-phe-phe-nh ), endogenous opioid peptide isolated from bovine and human brain, has high affinity and selectivity for the mu receptor and produces potent and prolonged analgesia in mice [ ] . in this presentation, the incorporation of ethylene-bridged phe-phe unit (eb[phe-phe]) or piperidine carboxylic aid (pic) in position was carried out to obtain more potent agonist or antagonist with stability against dipeptidyl peptidase iv (dpp iv). methods: the synthesis of eb[phe-phe] unit was achieved according to the procedure of lammek b. et al. [ ] protected peptides were synthesized by a solution method using boc-chemistry. the final products were identified by maldi-tof mass spectrometry and elemental analyses. the receptor binding affinity of peptides was assessed by radio-ligand receptor binding assay using mu and delta opioid receptors from rat brain membranes or cos- cell membranes expressing each opioid receptors. muc glycoprotein, produced by the epithelium of the colon, built up mainly of repeat units of ptttpitttttvtptptptgtqt , can be underglycosylated in colon carcinoma. we have been studying the epitope structure of the muc repeat unit with the mucin peptide specific mab monoclonal antibody. this antibody recognizes the ptgtq sequence as minimal, and ptptgtq as optimal epitope. our interest lies in the modification of this epitope with maintained or enhanced specificity, and we aim to clarify the effect of different epitope modifications on mab antibody binding: a) amino acid changes in the flanking region, b) glycosylation in the epitope core and in the flank. for this we have prepared a) libraries of ax( )ptgtqaa and atptgtqx( )a peptides, and x( )ptgtqx( ) heptapeptides based on the antibody binding properties of the libraries; and b) glycopeptides pt(galnac)ptgtq, ptpt(galnac)gtq and ptptgt(galnac)q. the peptides were prepared by solid phase synthesis; after purification, esi-ms and amino acid analysis characterisation their antibody binding properties were studied by competitive elisa. our results show that a) although all amino acids in positions x( ) and x( ) resulted in antibody binding; in position x( ) hydrophobic, in x( ) aromatic residues provided stronger binding than that of the native peptide; b) glycosylation on thr( ) did not influence the binding of mab , but on thr( ) the presence of n-acetyl-galactosamine, interestingly, slightly increased the antibody recognition. these findings could be useful in designing synthetic peptide vaccines for tumour therapy. histidines play essential role in binding of biological metal ions, either in small or macromolecular chelating molecules, e.g. in metalloenzymes. therefore the low molecular weight polyhistidine type ligands are of potential importance as model substances. continuing our investigations on a novel branched oligopeptide type ligand -(his) (lys) lys-nh -prepared by solid phase peptide synthesis, we investigated the metal ion binding properties with zinc(ii) and copper(ii). the eight primary metal-binding sites are the four imidazole and four ammine groups on the ligand. phpotentiometric titrations revealed, that up to ph all these donor atoms loose their protons on increasing ph. the competition between the protons and the metal ions results the decrease of pka values to about - in the case of copper(ii) and to about - in case of zinc(ii) ion. this reflects the higher stability of the complexes formed with copper(ii) in spite of the weak axial coordination that seems to occur in zinc(ii) complexes. combined potentiometric, spectrophotometric, cd and nmr spectroscopic methods were utilized to investigate the speciation and the structure of the complexes formed in aqueous solution. the prepared cu(ii) complexes cleaved dna, but it is not known whether in oxidative or in hydrolytic manner. because of this ambiguity further studies with zn(ii) complexes will be undertaken. this work has received support through sapstclg nato collaborative linkage grant and from the hungarian science foundation (otka t ). lgr . further studies have shown that in both male and female gonads, insl and lgr represent a paracrine system important for meiosis induction in the ovary and male germ cell survival in the testis. thus insl may have clinical applications in fertility management. we undertook to determine the key structural elements responsible for its unique actions. methods: alanine-scanned analogues of human insl and mimetics of the b-chain alone were prepared by solid phase peptide synthesis. each was subjected to cd spectroscopy for secondary structure analysis and assayed for in vitro lgr binding and activation activity. the tetrapeptide h-dmt-d-arg-phe-cbp was found to be a selective µ agonist [ic (gpi) = . ± nm] with -fold lower potency than the corresponding, highly potent tiq -tetrapeptide, but with still -fold higher potency than leu-enkephalin. in conclusion, we developed selective, cbp-containing δ antagonists and µ agonists with significant potency. recently, we described the syntheses and biological activities of several opioid peptide analogues that contained the n-terminal sequence - , common to dermorphin and deltorphin. some of them showed very high agonist potency both in the gpi assay and in the mvd assay [ , ] . in this work, we designed new analogues in which the sequences were elongated at the c-terminal to obtained the full sequences of dermorphin (a) and deltorphin (b). the syntheses of compounds and their biological activity profiles will be discussed. background and aims: endomorphin- (em- : tyr-pro-phe-phe-nh ) is very potent endogenous opioid peptide, which exhibits high affinity and selectivity for the mu-opioid receptor [ ] . previously, we had reported that [ac c ]-em- containing -aminocyclopropane- -carboxylic acid (ac c) exhibited higher affinities than em- for the mu-opioid receptor [ ] . in order to clarify that the substitution of -aminocycloalkane- -carboxylic acids (acnc: n indicates the number of carbon atoms in a ring) for pro in position of em- is efficient to obtain higher affinity for the mu-receptor, we synthesized . therefore, the replacement of pro to ac c and ac c will be efficient to make these analogs adopt bioactive conformation and exhibit high affinity for mu-receptor. in the past few years, many attempts have been made to prepare a synthetic insulin. the biological activity of insulin is known to be closely related to the c-terminal octapeptide fragment of its b-chain.it was found that b gly and b phe were present in all insulins so far obtained from various animal species indicating the significance of these two residues.it would therefore seem desirable to study the effect of each of these two amino acid residues or both on biological activity of the octapeptide fragment of the b-chain. a heptapeptide arg-phe-tyr-thr-pro-lys-ala-och , corresponding to (b -b ) insulin des gly -phe , and an octapeptide arg-phe-phe-tyr-thr-pro-lys-ala-och , des gly were synthesized using the solid phase method. the c-terminal ends of both peptide were converted to methyl ester by transesterification cleavage from the resin. the side chain protecting groups were removed by hf. manual counter current distribution method was used for purification of the free peptides. the way to solve the evaluation of tyrosine containing peptide was studied. the free methyl ester peptides were administered for insulin-like activity test by glucose metabolism in the rat fat cells technique in vitro. aim of this study is to develop peptides as useful tools for degradation of synthetic dyes, which are often pollutants. we focused our interest in peroxidases, a class of enzymes reported to efficiently degrade azo and anthraquinonic dyes. in particular, the fungus versatile peroxidase (vp) of pleurotus eryngii can perform this degradation. therefore, our goal is the synthesis of a peptide based on this peroxidase able to emulate its biological function. the linear and cyclic peptide sequences were derived by the theoretical model of vp [pdb: a ], which determined the amino acids fundamental for the desired function of the active sites. in particular, the residues instrumental for the coordination of the heme, the mn binding site, and the long range electron transfer pathway [ ] , were pin-pointed. moreover, we calculated the radius of the heme cavity. the next step was the synthesis of these peptides in order to verify the coordination of the heme and optimize their sequences. the syntheses were carried out by solid-phase following the fmoc/tbu strategy. because of purification difficulties of the fully-protected peptide, we undertook an alternative synthetic pathway, based on a solid phase head-to-tail cyclisation strategy, following the fmoc/tbu/allyl three-dimensional protection scheme [ ] . next steps will be to test the coordination properties of the synthetic peptides, with respect to the heme, and further computational studies based on the new model of pleurotus the calcium plays an important role in biochemical pathways. it binds to enzymes and proteins in a different process. aspartic (asp, d) and glutamic (glu, e) acid side chains are the main ligands of calcium, but the contribution of the backbone carbonyl groups in the binding is also important. generally the binding places in the proteins are an unstructured loop between two helixes ( -or alpha-helix). the common sequence is the so-called ef-hand motif, which contains amino acids [ ] . it is already known that some proteins also bind calcium with a non-ef-hand loop. for example alpha-lactalbumins have a ten amino acid long sequence for binding [ ] . it is an asp rich sequence where asps are closer to each other than in ef-hand motif (-k fldddltdd -) but only asps side chains take part in calcium coordination. we constructed a series of cyclopeptides to mimic the loop structure of alpha-lactalbumin [ ] . in this study we focus on determining the importance of conservative amino acids within the ca + binding loop of this protein, using microcalorimetry (itc). the itc measurements were performed in different organic solvents and at different temperature. the synthesis of fatty acids in adipose tissue. in this article, we present the solution structure of gip in water and tfe/water determined by nmr spectroscopy. the calculated structures are characterised by the presence of an -helical motif between residues ser -gln and phe -gln respectively. the helical conformation of gip is further supported by cd spectroscopic studies. six gip( - )ala - analogues were synthesised by replacing individual n-terminal residues with alanine. alanine scan studies of these n-terminal residues showed that the gip( - )ala was the only analogue to show insulin secreting activity similar to that of the native gip. however, when compared with glucose its insulinotropic ability was reduced. for the first time, these nmr and modelling results contribute to the understanding of the structural requirements for the biological activity of gip. a knowledge of the solution structure of gip and of the role of its individual residues will be essential in the understanding of how they interact with the gip receptor. efrapeptins are pentadecapeptides produced as a mixture of six closely related analogues (efrapeptin c-g) by the fungus tolypocladium niveum and other members of this species. they consist predominantly of the nonproteinogenic amino acids -aminoisobutyric acid (aib), isovaline (iva), -alanine ( ala) and pipecolic acid (pip), have an acetylated n-terminus and bear an unusual cationic c-terminal headgroup derived from leucinol and , -diazabicyclo[ . . ]non- -ene. efrapeptin c is a competitive inhibitor of the f -atpase and active against the malaria pathogen plasmodium falciparum. an anti-proliferative effect was also reported. conformational analysis of efrapeptin c in trifluoroethanol and dimethylsulfoxide was conducted to obtain structure-affinity relationships. the absence of amide-and -protons resulted in an imperfect assignment and unsatisfying conformational study. specific deuteration of methyl groups in aib did not simplify the assignment. cd and ft/ir spectra hint to helical or beta-turn secondary structures as main structure elements. residual dipolar couplings (rdc) were measured in a stretched cross-linked poly(dimethylsiloxane) gel in dichloromethane. the impact of the rdc on the conformational analysis led to an improved high resolution structure from simulated annealing protocols and consolidated the formation of a helical structure of efrapeptin c in nonpolar solution which is comparable with the binding pocket of the f -atpase. finally, the dynamics of the resulting structures was studied using the gromos force field in explicit solvent. serotonin selective reuptake inhibitors (ssris) are currently among the most frequently prescribed therapeutic agents of depression. their therapeutic use includes also obsessive-compulsive disorder, panic disorder, bulimia. the serotonin transporter (sert) is the target of serotonin selective reuptake inhibitors (ssris). altough the inhibition is the proximal event in antidepressant action, the clinical benefit of antidepressant medications requires weeks of continuous dosing, indicating that their mechanism of action involves events downstream from acute transporter blockade. long-term effects of ssri treatment may be due to changes in intrinsic properties of sert structure, function, or regulation. thus, understanding the mechanism of action of sert remains a primary goal in the search for developing novel treatments for diseases associated with serotonergic dysfunction. in the present study experimentally determined ligand selectivity of the buspirone analogues toward the serotonin transporter was theoretically investigated on the molecular level. the model of serotonin transporter based on the crystal structure of bacterial homologue from aquifex aeolicus (leutaa) was constructed using the traditional homology modelling approach. a series of docking experiments with ssri's were conducted, using interactive molecular graphics techniques combined with energy calculations and analysis of the transporter-ligand complexes. structural information about the serotonin transporter and its molecular interactions with ssri's is important for understanding the mechanism of action of these drugs and for development of drugs with improved potency and selectivity. the protein kinase c (prkc) is a member of a super-family of the eukaryotic receptor protein kinases. it forms dimers and is anchored in the membrane, with a cytoplasmic kinase domain and an external domain, presumably acting as a sensor. prkc enables formation of biofilms of bacillus subtilis which show a high degree of spatial organization. they colonize various surfaces and produce complex antibiotic resistant communities. prkc acts as a ser/thr kinase with features of the receptor kinase family of eukaryotic hanks kinases. our current study involved theoretical modeling of the protein kinaze prkc complexes with the modified atp. the ligands were selected from a set of molecular probes developed by k. shah and coworkers [ ] . each modified atp molecule was docked to the active site of the kinase molecule using autodock genetic algorithm procedure. the optimized structures of the complexes were submitted to the molecular dynamics simulations in the amber force field. we obtained four optimized structures of prkcc complexes in water. the results suggest the great similarity of our complexes with human cyclin-dependent kinase [ ] complexes. background and aims. indolicidin is a -residue antimicrobial peptide, which was isolated from bovine neutrophils. this molecule possesses a wide spectrum of antibacterial, antifungal and antiviral activity, furthermore it has also haemolytic effect. data derived from structural investigations led to considerably diverse conclusions regarding the secondary structure of this peptide, therefore the aim of this study was to examine the effect of cis-trans isomerization on the conformational properties of this antimicrobial peptide. methods. the conformational analysis of indolicidin containing cis or trans xxx-pro peptide bonds was performed by simulated annealing calculations with the use of amber force field. results. for the conformers of indolicidin with cis or trans xxx-pro peptide bonds, the evolving secondary structural elements were examined and poly-proline ii helix and type vi beta-turn were identified. in the case of this peptide, various intramolecular interactions may play an important role in stabilizing the structure of conformers. therefore the presence of the h-bonds between backbone atoms, the aromatic-aromatic interactions between the side-chains of trp amino acids and the proline-aromatic interactions between the side-chains of trp and the pyrrolidine rings of pro amino acids was investigated. conclusions. the conformational comparison of the peptides possessing cis or trans xxx-pro peptide bonds resulted in different secondary structural elements for both isomers, which are the poly-proline ii helix and type vi beta-turn for the trans and cis isomers of indolicidin, respectively. the occurrences of various intramolecular interactions are in agreement with the observed secondary structures. we have shown the monte carlo conformational search using macromodel is useful for conformational study of oligopeptides prepared from alpha, alphadisubstituted alpha-amino acids. moreover, we have studied conformational analysis of oligopeptides containing chiral alpha, alpha-disubstituted alphaamino acids to predict the helical screw sense of helical structures. here we report computational study on conformation of oligopeptides containing cyclic alpha, alpha-disubstituted alpha-amino acids with side-chain chiral centers. background and aims. the homopolymeric amino acids (hpaas) are polypeptides consisting of the same amino acids. some of them play a relevant role in the formation of several neurodegenerative diseases. most probably the poly-(ala) and poly-(gln) are the best representatives of these peptides because of their important biological effects. our aim was to perform conformational analysis and structural investigation of these two hpaas. methods. to explore the conformational spaces of the peptides, simulated annealing (sa) and random search (rs) calculations were carried out using amber force field. two different forms of the hpaas were modelled: either with charged n-terminal amino group and c-terminal carboxyl group, or with the n-and c-termini blocked by acetyl and n-methyl amide groups, respectively. results. for the conformers obtained by sa and rs calculations, the occurrences of various secondary structural elements like different types of beta-turns, gammaand inverse gamma-turns, alpha-helix, -helix, poly-proline ii helix and beta-strand were investigated. in the cases of various helices and beta-strand, segments with different lengths characterized by these secondary structures were determined along the entire sequence of peptides. for the conformers of the hpaas, the intramolecular h-bonds formed between the backbone atoms as well as between the backbone and side-chain atoms were identified. the vasopressin and oxytocin receptors (v ar, v r and otr) are membrane-embedded proteins belonging to the large family a g protein-coupled receptors (gpcrs). they are involved in crucial physiological functions as the regulation of water metabolism, control of blood pressure and stimulation of labor and lactation, mediated via v r, v ar and otr, respectively. as such, they are involved in a number of pathological conditions and are important drug targets. understanding their inhibition and activation mechanisms may improve design of ligands capable of selective stimulation or blockade of the respective receptors presenting the therapeutic targets. to investigate the otr, v ar and v r interactions with agonists and antagonists thirty computer models of receptor-ligand complexes have been modeled via docking and molecular dynamics (md) and analyzed in details. the receptor models were built on rd crystal structure template or using the coordinates of mii-gtα( - ), for non-active and activated models, respectively. the ligands (arginine vasopressin, oxytocin, desmopressin, atosiban ([mpa ,d-tyr(et) ,thr ,orn ]ot) and barusiban (mpa ,d-trp ,ile ,allo-ile ,asn ,abu ,mol ) were docked into the receptors. the complexes have been embedded into the hydrated popc bilayer and submitted to ns unconstrained md in the amber force field. the relaxed systems have been obtained and analyzed in details. the receptor residues responsible for agonists/antagonists binding have been identified and mechanism of binding involving the highly conserved residues has been proposed. a three-dimensional models of the neurohypophyseal hormone receptors were constructed using a multiple sequence alignment and either the crystal structure of bovine rhodopsin or the complex of activated rhodopsin with gta c-terminal peptide of transducin rd*-gt( - ) prototype to obtain nonactive or activated receptor models, respectively. analogs were docked to v ar, v r and otr, both non-active and activated models. the low-energy receptor-ligand complexes, with properly docked analogs were submitted to the constrained simulated annealing (csa), in vacuo. the relaxed receptoranalog models were obtained. the residues responsible for analogs binding to v ar, v r and otr have been identified and presumable biological activity of these compounds was determined. n-methyldehydroamino acids belong to non-standard amino acids found in nature. n-methyl-(z)-dehydrophenylalanine was found in tentoxin, a selective weed killer, having been produced by several phytopathogenic fungi of the alternaria genus. n-methyl-(z/e)-dehydrobutyrine and n-methyldehydroalanine are components of nodularins and microcystins, families of hepatoxins produced by species of freshwater cyanobacteria, primarily nodularia spumingena and microcystis aeruginosa. the simplest n-methyl dehydropeptides, ac-delta(me)xaa-nhme (where xaa = ala, (z/e)-abu, (z/e)-phe, and val) and, for comparison, the saturated ac-l-(me)ala-nhme analogue were investigated using computational methods. cis-trans b lyp/ - +g**//hf/ - g ramachandran potential energy surfaces were created. the conformers found were optimised at the b lyp/ - +g** level. the effect of the electrostatic solute/solvent (water) interaction on the solute energies was investigated within the scrf method using the polarisable continuum model (pcm) on the geometries of solutes in vacuo. it was found that for all the studied dehydropeptide molecules the lowest conformer (phi, psi = ~ - °, °) has the cis n-methyl amide bond. this feature seems to be independent of the dehydroamino acid moieties, the c-beta substituent and the z/e configuration. the pi-electron conjugation as well as the n-h···n hydrogen bond play the dominant role in the stability of this conformer (see figure) . the preliminary nmr investigations into the conformational preferences of the studied molecules in solution confirm the theoretical results obtained. the strong tendency of the n-methyl amide bond to adopt the cis configuration seems to be the reason why n-methyldehydroamino acids are found in small natural cyclic peptides, where they ensure the conformational flexibility necessary for biological action. the purpose of this study was to determine the potentials of mean force (pmf) of the interactions between models of nonpolar amino acid side chains in water. the potentials of mean force (pmf's) dependent on orientation were determined for systems forming hydrophobic and diagonal complexes composed of side-chain models of alanine, valine, leucine, proline and iso-leucine, respectively, in water. for each hydrophobic pair in water a series of umbrellasampling molecular dynamics simulations with the amber force field and explicit solvent (tip p water model) were carried out and the pmfs were calculated by using the weighted histogram analysis method (wham). in all cases a characteristic shape of pmf plots for hydrophobic association were found, which was manifested as the presence of contact minima and solvent separated minima. depths of contact minima for all systems studied were about kcal/mol. in this work we compared the ability of two theoretical methods of ph-dependent conformational calculations to reproduce experimental potentiometric-titration curves of two models of peptides: ac-k -nhme in % methanol (meoh)/ % water (h o) mixture and ac-xx(a) oo-nh (xao) (where x is diaminobutyric acid, a is alanine, and o is ornithine) in water, methanol (meoh) and dimethylsulfoxide (dmso), respectively. in theory, in all three solvents, the first pka of xao is strongly downshifted compared to the value for the reference compounds, the water and methanol curves have one, and the dmso curve has two jumps characteristic of remarkable differences in the dissociation constants of acidic groups. the predicted titration curves of ac-k -nhme are in good agreement with the experimental ones; better agreement is achieved with the md-based method. the titration curves of xao in methanol and dmso, calculated using the md-based approach, trace the shape of the experimental curves, reproducing the ph jump, while those calculated with the edmc-based approach, and the titration curve in water calculated using the md-based approach, have smooth shapes characteristic of the titration of weak multifunctional acids with small differences between the dissociation constants. quantitative agreement between theoretically predicted and experimental titration curves is not achieved in all three solvents. the poorer agreement obtained for water than for the nonaqueous solvents suggests a significant role of specific solvation in water, which cannot be accounted for by the mean-field solvation models m a. papakyriakou , g.f. vlachopoulos , g.a. spyroulias , e. manessi-zoupa , p. cordopatis angiotensin-i converting enzyme (ace) belongs to the m family of the ma clan of zinc metallopeptidases and can act either as a dipeptidyl carboxypeptidase, which catalyses the proteolytic cleavage of dipeptides from the carboxy terminus of a wide variety of peptides, or as an endopeptidase, which hydrolyses peptides bearing amidated c-termini. among the former category of ace peptide substrates, the most distinguished are those involved in blood pressure regulation, such as angiotensin i (angi) and bradykinin (bk). in the latter category falls the gonadotropin-releasing hormone (gnrh) in an attempt to analyze molecular interactions at atomic level we simulated the ace-substrate complexes, using the recently determined d crystal structure of ace testis isoform and a knowledge-based docking method in order to insert the peptide substrate (angi, bk and gnrh) of ace into its catalytic cleft. in order to introduce the effect of protein mobility and gain information about enzyme-substrate recognition and interaction we have sampled the conformational space of these complexes via molecular dynamics simulations with explicit solvent representation. we have also performed molecular dynamics calculations with tace-inhibitor complexes, such as lisinopril, as well as with tace mutated at specific sites, such as the ligands of the two buried chloride ions that have been shown to affect substrate activity. our results provide new insights into the role of specific domains of tace and their implication in the enzyme activity, which is not readily apparent from the available crystal structures. two main mechanisms for the propagation of action potential in myocytes are: ) the free flow of local circuit current through gap junctions and ) the effect of electrical field. here we study effect of each mechanism and their importance during action potential propagation. method: we simulated the cardiac myocyte by the orcad software, then used the model of sinoatrial node to stimulate the myocytes model and studied the propagation of action potential with and without gap junction. result: our results show that, although gap junction solely is not able to mimic physiological condition, but it is necessary for normal cardiac functioning. on the other hand, electric field is not sufficient for successful propagation of action potential and the existence of gap junction is necessary. anthrax is a disease of animals and humans, caused by the bacterium bacillus anthracis. anthrax toxin (at) consists of three proteins, one of which is the anthrax lethal factor (alf). alf is a gluzincin zn-dependent highly specific metalloprotease (~ . kda), which belongs to the m family of the ma clan of zinc metalloproteases. alf cleaves most isoforms of mitogen-activated protein kinase (mapk)-kinases (meks) close to their amino termini, leading to the inhibition of one or more signaling pathways. no data are available on the enzyme-substrate interaction at the molecular level. therefore, we performed classical molecular dynamics simulations on the alf-mkk/mek complexes in order to probe protein-substrate interactions. the simulations pinpointed specific hydrophobic as well as electrostatic alf-peptide substrate interactions and these data were exploited in the building of virtual combinatorial libraries of di-and tri-peptides using the twenty native aminoacids. by applying docking simulations to anthrax zn-metalloprotease around . peptide substrates were virtually screened according to their binding affinity. data suggest that complexes of alf with peptides substrates bearing arg, trp, lys and phe aminoacids, exhibit the highest binding affinity providing evidence for electrostatic interactions between negatively charged residues of alf's active site and positively charged side-chains of di/tri-peptides. new libraries of substrates were built incorporating non-protein residues, organic moieties and chelating groups. alf-substrate complexes with the best score (in terms of binding energy) are further analysed. in the present studies we designed and synthesised seven new bradykinin (bk) analogues and evaluated them in the in vivo rat uterotonic assay using a modified holton method in munsick solution on a strip of rat uterus and in blood pressure test. we used [arg , hyp , thi , , d-phe ]bk, the b antagonist of vavrek and stewart as a model, when designing our analogues. in all cases, the n-terminus of our peptides is acylated with bulky substituent. we previously reported that acylation of the n-terminus of several known b antagonists with various kinds of bulky acyl groups has consistently improved their antagonistic potency in rat blood pressure assay. on the other hand, our earlier results seem to suggest that effects of acylation on the contractility of isolated rat uterus depend substantially on the chemical character and size of the acyl group, as we observed that this modification may either change the range of antagonism or even transform it into agonism. the peptides were synthesized by the solid-phase method using the fmoc-strategy the modifications proposed either preserved or increased the antagonistic potency in the rat blood pressure test. on the other hand, the seven substituents, differently influencend the interaction with the rat uterine receptors and except one led to decrease of antiuterotonic activity. in both cases acylation of the n-terminus led to enhancement of antagonistic potencies. our results may be of value in the design of new b agonists and antagonists. the formations of amyloid fibrils have been reported as for various amyloidosis. several structural models of fibrils are proposed for respective proteins so far. however, their common basic structures and universal features to induce amyloid fibril formations are not known in detail. previously, we examined intermolecular interactions among the several amino acid residues in barnase, which is known to form amyloid-like fibril. based on the experimental results using a series of mutant barnase, we discovered that the interactions between hydrophobic side-chains are the most essential driving force to form the fibrils and that both intermolecular and inter-sheet interactions in the fibril maintain highly ordered molecular packing. in the present paper, we describe a novel prediction method for core regions of various fibril-forming proteins and show the verification of the above possible structural principle. at first, we calculated the interaction's score between side-chains in the antiparallel orientation of beta-strands. next, the peptides with predicted sequences of fibril cores, a couple of high-scored regions with a designed turn moiety to induce a hairpin-like form, were chemically synthesized by spps. as a result, the formation of amyloid fibrils was confirmed for most of high-scored sequences. in addition, we also applied this method to prion protein, we could predict possible beta-strands with hetero-paired orientation. some synthetic peptides involving these strands were proved to have fibril-forming ability. thus, we have developed the novel method to predict the core regions that induce amyloid fibrils. a principal factor analysis (pfa) is a very efficient way of identifying patterns in the data sets even if the patterns are hard to find (e.g. in the high dimensional data sets). this is the reason why the pfa method can be powerful tool for analyzing molecular dynamics (md) trajectories. it is possible to reduce dramatically the trajectory size without loosing significant structural information by applying the pfa procedure. we used this tool for interpretation of results from the molecular dynamics simulations of the model of the transcription factor nf-kb. nf-kb is a protein involved in the numerous biological processes such as regulation of immune response, inflammation, various autoimmune diseases and is used by many viruses, including human immunodeficiency virus (hiv), to activate transcription of their own genes. only the trajectory of the backbone atoms of the nf-kb were subjected to the further analysis. peptides contain many basic sites such as side chains of basic amino acid residues, oxygen and nitrogen atoms of amide groups, and terminal amino groups. these parts can interact with protons. this interaction can change conformational behaviour of peptides and, consequently, their biological functions. the interaction becomes even stronger in the gas phase. in that case, the stability of the peptide chain is influenced, which may have impact on peptide fragmentation during mass spectroscopy analysis of peptide structures. in this study, we will present the interaction of proton with carbonyl oxygens in the model of alanine tripeptide. quantum chemical calculations employing density functional theory using hybrid b lyp functional and - ++g** basis set were used to describe this interaction and also to find possible pathways of proton transfer among interaction sites. two different mechanisms of proton transfer were found. the first mechanism is represented by an isomerization of the proton around the double bond of the carbonyl group. the second mechanism is based on the large conformational flexibility of the tripeptide model where all carbonyl oxygens cooperate. the later mechanism exhibits nearly half energy barrier of the rate-determining step compared to the first one. we focus our attention on situation, in which methyl groups attached to alpha atoms in tripepetide model influence the conformational behavior. results will be presented for all four possible stereochemical configurations. a. papakyriakou , p. galanakis , p. gazonis , g.a. spyroulias p protein is one of the most effective defensive weapons of human body against carcinogenesis, due to its tumor suppression properties. it has been noticed, in many types of cancer, that the functions of p are being downgraded or even suppressed and this fact is ought to the presence of mutated forms of p or to the complete absence of the protein. the suppression of p levels is being indirectly regulated by the protein itself, which activates the expression of a gene, the oncogene mdm (murine double minute ), which expresses the mdm protein, known as human-mdm or just hdm . hdmx protein is a homologue protein to hdm and is being implicated, through various biological processes, in the suppression of p . however, recent experimental evidence suggests that hdm and hdmx proteins are not the only ubiquitin ligases that negatively regulate p through ubiquitin pathway. two recently discovered e ligases, cop and pirh , are also proposed to promote p for degradation. all these proteins function as e ligases bearing a ring finger domain. these domains are characterized by their high content in cysteines and the binding of two zn(ii) ions while they catalyze the latter stage of protein signaling for proteolysis by the s proteasome, through the ubiquitin pathway. the structure variation and the stability of these ring fingers is studied through molecular dynamics simulations of - ns and structure variations are analyzed in a structure-function correlation basis. semax is a synthetic analogue of adrenocorticotropic hormone acth - . it is a nootropic agent containing seven amino acids met-glu -his-phe-pro-gly-pro without hormonal (adrenocorticotrophic) activity. semax is neuroprotective via a mechanism involving the regulation nitric oxide (no) and lipid peroxidation. semax proved to be highly effective in abating the rise in no and restoring neurologic functioning [ ] . it was found to improve intellect and memory in healthy human. it is effective in rehabilitation of people with memory and motor disorders, parkinson's and hantington's diseases, after cerebral stroke and head trauma [ ] . to study conformation dynamics in connection with in vivo activity of semax the molecular dynamics method of standard protocol was applied [ ] . semax and about twenty its analogs were studied. using cluster analysis method semax was found to be more labile among various synthesized analogous (met-gln -his-phe-pro-gly-pro; gly-glu-his-phe-pro-gly-pro; lys-glu-his-phe-pro-gly-pro; glu-his-phe-pro-gly-pro; his-phe-pro-gly-pro). because of collective degree of freedom it has one more stable configuration that is unreachable in analogs. singularities of semax and analogous were studied using -d, -d poincare maps, auto and crosscorrelation functions of special type in terms of topological structure of energy hypersurface. this work was supported by rfbr (pr. - - ), russian ministry of education and science, moscow government and crdf. rhodopsin (rd) is the only representative of g-protein coupled receptors (gpcrs) whose structure has been described with high resolution. thus, it has become the structural prototype for other gpcr. these receptors are involved in transduction of various signals into the cell and actions of many hormones and neurotransmitters. about % of all drugs act through gpcr. growing evidence that rd and related gpcrs form functional dimers/oligomers, followed by direct proof (using atomic force microscopy -afm) that in the retina rd associates into a paracristalline network of rows of dimers, need models of rd-transducin (g t -heterotrimeric protein) complex that would envision an optimal rd dimer/oligomer amenable to satisfy all well documented interactions with gt. current model includes tetramer built of two activated (metaii) and two inactive rd molecules, ligands stabilising metaii: gtα(ile -phe ) and gtγ(asp -cys )farnezy, lipid bilayer built of pc (phosphatidylocholin head groups) , ps (phosphatidyloserine) and pe (phosphatidyloetanolamine) (all three types of phospholipids contain the polyunsaturated docosahexaenoyl chain -dha) and water. experimental data concerning shape of oligomer, conformational changes in metaii, proper interactions and distances among residues have been looked upon. the poster shows results of the molecular dynamic carried in amber force field for ~ ps in the periodic box. conformational changes which took place during simulation caused proper adaptation one another monomers in tetramer and ligands to activated receptors. the human cystatin c (hcc) is a one of known domain swapping proteins. during this process, one of the hcc β-hairpins (β -l -β ) changes its conformation forming long β-strand. this conformational transition destabilizes the monomer structure and leads to domain-swapped dimer. the causative force for changing the βhairpin conformation is assumed to be the alleviation of distortions of the l -loop val amino acid residue's backbone. following the above assumption and our previous conformational studies of the hcc β-hairpin peptide we investigated the influence of the point mutations, v d, v p and v n of the val residue, on the β-hairpin peptide structure. the conformational studies by means of cd spectroscopy and molecular dynamics studies were performed. the study revealed that the hcc peptide with the wild-type sequence has the strongest tendency from all studied peptides to form a β-hairpin structure. on the basis of these results we conclude that the presence of distortions in the val residue of l -loop is unlikely to cause the d domain swapping of the human cystatin c. acknowledgments: this work was financially supported by the ministry of scientific research and information technology of poland under grant t a . temporin a (ta) (flpligrvlsgil-nh ) and temporin l (tl) (fvqwfskflgril-nh ) are small, basic, hydrophobic, linear antimicrobial peptides amide found in the skin of the european red frog, rana temporaria. these peptides have variable antibiotic activities against a broad spectrum of microorganisms, including clinically important methicillin-sensitive and resistant staphylococcus aureus as well as vancomycin-resistant enterococcus faecium strains. to gain further insight into the mechanism of action of these small antimicrobial peptides, we have investigated their conformational behaviour in different environmental conditions. more specifically, we deeply investigated by solution nmr spectroscopy in water and water/dmso ( : ) solutions as isotropic solutions and mm aqueous solution of dpc (dodecylphosphocholine) was used as membrane mimetic environment. understanding the basis of the interactions of temporins with membranes could be crucial for the design and synthesis of potent antimicrobial agents. cripto is the founding member of a family of soluble and cell bound growth factors known as egf-cfc [ ] distinguished by the presence of an n-terminal signal peptide, two distinct cysteine-rich domains (crd) and a c-terminal hydrophobic region involved in cell surface attachment by a post-translational gpi modification. the characteristic crds, known as egf-like and cfc domains (from the first members cripto, frl and cryptic), both span about residues with disulfide bridges [ ] each, which, presumably, beside a possible functional modularity, confer them also a structural independence. in this work we have focused our attention on the cfc domain of mouse cripto. the domain has been produced by ssps, along with variants bearing mutation on h and w , that have been described as crucial for alk receptor recognition. the two variants have been purified and refolded, achieving the correct disulfide bridges, and then comparatively analyzed by cd spectroscopy under different ph conditions; thus obtaining experimental insights on the structural arrangements of this new class of protein domains. furthermore, the binding properties of wild type and mutants cfc domains to alk receptor have been determined by using an elisa-based assay. our results demonstrated that the cfc domain alone can directly bind alk in the absence of additional ligands and, furthermore, confirmed a role of h /w in cripto/alk interaction. there is considerable interest in the pharmacology of the two cholecystokinin (cck) receptors ccka-r (or cck- ) and cckb-r (or cck- ) that mediate the biological action of the cck hormone. they are membrane receptors belonging to the superfamily of g-protein coupled receptors (gcpr) and are predominantly located in the gastrointestinal tract and in the central nervous system, respectively. a library of cyclic peptide analogues derived from the octapeptide c-terminus sequence of the human cholecystokinin hormone [cck( - ), or cck ] has been designed, synthesised and characterised. the peptide analogues have been rationally designed to specifically interact with the cck type b receptor (cckb-r) on the basis of the structure [ ] of the bimolecular complex between cck and the third extracellular loop of cckb-r [namely, cckb-r( - )]. the new ligands showed binding affinities generally lower than that of parent cck . anyway, structure activity relationship data underline that preservation of the trp -met motif is essential, and that the phe side chain and a carboxylic group close to the c-terminal end must both be present. the nmr conformational study in dpc micelles of the compound endowed with maximal binding affinity (cyclo-b , ic = m) shows that this compound presents the turn-like conformation, centred at the trp -met segment, as planned by rational design, and that such conformation is stabilised both by the cyclic constrain and interaction with the micelle. cripto is the founding member of a family of extracellular growth factors called egf-cfc found in mouse, human, chicken, xenopous and zebrafish [ ] . these proteins are characterized by the presence of an n-terminal signal peptide, a c-terminal hydrophobic region and two highly conserved cysteine-rich domains, the egf-like (epidermal growth factor) and the cfc (cripto/frl /cryptic). cripto is strictly required in the early embryonic development and contributes to deregulated growth of cancer cells in adults, since it is highly over-expressed in many solid carcinomas. it has been proposed that each single domain of cripto could bind different protein partners, playing different functional roles [ ] . on this grounds, investigation of the single domains d-structures can have also strong functional implications. we present here an extensive conformational analysis of the mouse cfc domain ( - sequence) and of the w a mutant based on nmr data. sequences have been synthesized by spps and refolded reconstituting the correct disulfide bridges [ ] . the molecular models have been built by computational methods using the nmr data collected under both acidic (ph ) and nearly physiological (ph ) conditions. both domains show a globally extended folding with three strands linked by the three disulfide bridges and two connecting loops, in which h and w , key residues in receptor binding, are exposed to the solvent urantide, a selective antagonist. thus, we carried out a study aiming at the characterization of conformational arrangement and affinity properties of ut extracellular segments.we measured by surface plasmon resonance (spr) technology the binding affinities of the three ligands, u-ii, urp and urantide towards the three extracellular loops of ut. furthermore, the secondary structures of the synthetic receptor fragments in presence of dodecylphosphocholine micelles and interaction with ut ligands were analysed using nmr spectroscopy. spr data showed that the ec loop ii was able to recognize the ligands u-ii, urp and urantide with similar affinities while none of these two ligands were able to interact with the extracellular loop i. furthermore, the absence of binding of urantide, a peptide antagonist, suggested strongly that loop iii would be involved in the signal transduction process and implies that u-ii and urp, but not urantide, would bind to ut according to a common pattern. moreover, the results indicate that potent ut antagonists could be designed by producing highaffinity ligand targeting the extracellular loop ii. also, the spr and nmr studies revealed that the synthetic structural ut domains contained some of the conformational and chemical features essential for the binding of hu-ii, urp and urantide to hut. synthetic cysteine-rich replicates of naturally occurring peptides such as hormones, neurotransmitters, enzyme inhibitors, defensins and toxins often can be oxidatively folded in high yields to their native structure. the presence of identical cysteine patterns in the sequence were found to lead to identical disulfide connectivities and homologous spatial structures despite significant variability in the non-cysteine positions. therefore, it is generally accepted to attribute the disulfide connectivities based on the homology of their cysteine pattern. minicollagen- from the nematocysts of hydra is a trimeric protein containing n-and c-terminal cysteine-rich domains involved in the assembly of an intermolecular disulfide network. examination of three-dimensional structures of peptides corresponding to these folded domains by nmr spectroscopy revealed a remarkable exception from the general admitted rule [ ] . despite an identical cysteine pattern, they form different disulfide bridges and exhibit distinctly different folds. additionally, comparative analysis of the oxidative folding revealed for the c-terminal domain a fast and highly cooperative formation of a single disulfide isomer, the n-terminal domain proceeding mainly via an intermediate that results from the fast quasi-stochastic disulfide formation according to the proximity rule. to our knowledge, this is the first case where two short peptides with identical cysteine pattern fold uniquely and with high yields into defined, but differing, structures. therefore, these cysteine-rich domains may well represent ideal targets for structure calculations to learn more about the elementary information encoded in such primordial molecules. the conformational change of the cellular prion protein, prpc, to its virulent "scrapie" form, prpsc, is believed to be responsible for prion infectivity. and several studies suggest that the prion disulfide bond is important for the stability, structure, and propagation of prion oligomers. to test this hypothesis, we selected two conserved peptides flanking the disulfide bond in the sheep prion protein, and measured the secondary structure of these peptides with circular dichroism, hydrogen/deuterium exchange, and molecular dynamics simulations. our preliminary data suggests that the two peptides do not adopt stable secondary structure, native or otherwise. thus, the folding intermediate of a prion protein seems unlikely to comprise local structure around the disulfide bond. the conformationally labile cα-tetrasubstituted α-amino acid residue bip possesses non isolable (r) and (s) atropoisomers. we have previously reported that in the linear dipeptides boc-bip-α-xaa*-ome with α-xaa* = ala, val, leu, phe, (αme)val and (αme)leu residues at the c-terminal position of bip, the onset of an equilibrium between diastereomeric conformers with unequal populations could be observed by cd and h nmr. the phenomenon of induced circular dichroism (icd) represents the basis for the "bip method", an easy and fast configurational assignment for chiral α-amino acids. in search for an extension of the bip method, we investigated the boc-bip-β-xaa*-ome dipeptide series with β-xaa* = β -hala, β -hval, β -hleu, β -hpro, β -hphe, or the cyclic β , -amino acids ( s, s)/( r, r)-achc and ( s, s)/( r, r)-acpc. low-temperature ( k) h nmr spectra in cd od revealed the presence of two conformers. significant d.r. (diastereomeric ratio) values were observed for all combinations of bip with both β -and cyclic β , -amino acids. cd analysis in meoh solution of the boc-bip-β-xaa*-ome dipeptides allowed us to conclude that the cd resulting from the induced axial chirality in the biphenyl core of the bip residue gives clear information on the β-xaa* configuration for both β -and cyclic β , -amino acids (except the aromatic β -hphe), with a p torsion of the biphenyl axial bond of bip being preferentially induced by (l)-β -xaa* as well as cyclic ( s, s)-β , -xaa* c-terminal residues. we have recently reported that the induced circular dichroism (icd) of the biphenyl core of boc-bip-xaa*-ome dipeptides based on the conformationally labile cαtetrasubstituted α-amino acid residue bip could allow an easy and fast configurational assignment for both α-and β-xaa* amino acid residues. in search for other biphenyl/xaa* architectures in which a transfer of central to axial chirality could result in a potentially useful icd, we considered n-substituted , -dihydro- hdibenz[c,e]azepine (daz) derivatives from α-and β-amino acids as interesting candidates. in the present communication, we report the syntheses, and the h nmr and cd analyses of a series of (daz)xaa*-ome amino esters derived from α-, β -, and cyclic β , -xaa* residues, namely d- β-peptide molecules possess interesting conformational characteristics and biological properties. they may represent a new class of rigid foldamers potentially useful as templates or spacers. d-structures of β-peptides have been experimentally investigated using x-ray diffraction and various spectroscopic techniques, but they have never been doubly spin labelled and studied by epr. a terminally protected β-hexapeptide, based on trans-( r, s)-β-toac and trans-( s, s)-achc, synthesized using classical solution methods, was found by ft-ir absorption and cd techniques to adopt the - -helical conformation. a set of four, terminally blocked, hexapeptide sequences, each characterized by four strongly helicogenic aib residues and all combinations of the two isomeric ile/allo-ile residues at positions and was synthesized by solution methods and fully characterized. a detailed solution (by ft-ir absorption, nmr, and cd) and solid (crystalline)-state (by cd and x-ray diffraction) conformational investigation allowed us to validate our assumption that all four peptides are folded in well developed - -helical structures. however, the most relevant conformational conclusion extracted from this d-analysis is that the handedness of the - -helical structures formed does not seem to be sensitive to the configurational change at the β-carbon atom of the constituent ile versus the diastereomeric allo-ile residues (in other words, the dominant control on this important structural parameter appears to be exerted by the chirality of the amino acid α-carbon atom). these results complement published findings on the diverging relative stabilities of the intermolecularly h-bonded β-sheet structures generated by ile versus allo-ile homo-oligopeptides. taken together, these data represent an experimental proof for the intuitive view that potentially different conformational properties are magnified in a strongly self-aggregated homo-peptide system (as compared to weakly self-aggregated, helical, host-guest peptides such as those investigated in this work). in a first approach to β-sandwich proteins the hydrophobic core between two symmetrical sheets each with four antiparallel β-strands was computationally designed by packing of amino acid side chain conformations (rotamers) in an initially given backbone structure. the proteins were synthesized by coupling four peptides with β-hairpin structure to a cyclic decapeptide template (tasp). an aggregation observed by equilibrium ultracentrifugation with the first designed proteins was decreased to a dimer by increasing the surface charge in two further variants of this protein from - to + and + . replacement of l-pro by d-pro in the loops and the template proved to stabilize the β-structure. these results led us to an improved design of an asymmetric core with algorithms for selection of proteins with a minimal number of atom clashes and cavities in the core, and a maximum number of hydrogen bonds after energy minimization. this protein termed beta-mop (modular organized protein) was synthesized in amounts to allow a characterization by cd, ftir, tryptophan fluorescence during reversible unfolding, and by high resolution nmr. nmr measurements of diffusion indicate a dimeric structure. the β-structure is stable up to °c ( k) as determined by d h nmr showing sharp resonance lines. the d h, h dqf-cosy spectrum at mhz shows a typical βsheet distribution extending well into the characteristic regions > . ppm (for amide protons) and > . ppm (for hα signals). all data indicate a well folded protein with β-structure. a.s. galanis , z. spyranti , n. tsami , g.a. spyroulias , e. manessi-zoupa , g. pairas , i.p. gerothanassis , p. cordopatis angiotensin-i converting enzyme (ace) belongs to the m family of the ma clan of zinc metallopeptidases and can act either as a dipeptidyl carboxypeptidase, or as an endopeptidase. among the ace peptide substrates, the most distinguished are angiotensin i (angi) and bradykinin (bk) due to their role in blood pressure regulation. despite the fact that biological data strongly suggest that the two active sites exhibit different selectivity and activity towards physiological and exogeneous substrates none experimental evidence for the interaction of angi and bk with ace catalytic sites, is available so far. a dual approach for studying the structure and physicochemical determinants of ace-angi/bk interaction has been performed. the first involves the application of molecular dynamics simulations (presented elsewhere in this book) and the second is making use of the solid-phase synthesized - aa ace catalytic site maquettes (csm) bearing the native sequence and the application of the nmr spectroscopy, and presented herein. therefore, high-resolution multinuclear nmr spectroscopy was applied to analyze the conformational features of ace substrates angi and bk in dmso or aqueous mixtures. then titration experiments were conducted and ace csms were titrated by angi/bk peptides, while monitored by nmr. d h- h tocsy and noesy experiments were used in order to map the interaction site of both substrates and csm through chemical shift perturbation and comparison of noe signal differentiation. competitive binding studies were also carried out through titration studies of csm-angi/bk and known ace inhibitors. a. carotenuto , p. grieco , l. auriemma , e. novellino , v.j. hruby the melanocortine receptors are involved in many physiological functions, including pigmentation, sexual function, feeding behavior, and energy homeostasis, making them potential targets to treat obesity, sexual dysfunction, etc. understanding the conformational basis of the receptor-ligand interactions is crucial for the design of potent and selective ligands for these receptors. the conformational preferences of the cyclic melanocortin agonists and antagonists mtii, shu , [pro ]mtii, and pg (table ) when two chromophores are chirally oriented and close enough to one another in space, their excited states couple and become non-degenerate. this phenomenon, termed exciton coupling, produces a typical bisignate cd curve. the intensity of the cd couplet is dependent on the molar extinction coefficient and the distance between the interacting chromophoric moieties, while the sign is governed by the angle between the effective electron transition moments. in particular, exciton coupling over a long distance can be observed only with strongly absorbing chromophores, e. g. porphyrin derivatives, characterized by their extremely intense and sharp soret band near nm. in this work we examined by the exciton coupled cd method the combined distance and angular dependencies, generated by the seven conformationally restricted β-turn and - -helical spacer peptides -l-ala-[l-(αme)val]n-(n = - ) on a system formed by two intramolecularly interacting -carbamido- , , , -tetraphenylporphyrin chromophores. these porphyrin derivatives are confirmed to be excellent reporter groups. we find that not only the centerto-center separation (from to Å) between the two chromophores, but the orientation (roughly parallel or perpendicular) between the directions of their effective transition moments as well, are responsible for the onset or even for the modulation of the intensity of the exciton coupling phenomenon. in particular, the porphyrin…porphyrin interaction is still clearly detectable over the long distance of ca. Å when the two chromophores are about perpendicularly oriented. a. hetényi , g.k. tóth , c. somlai , t.a. martinek , f. fülöp β-peptides are probably the most thoroughly investigated peptidomimetic oligomers. to extend the field of β-peptides towards the construction of possible new secondary structures, the replacement of the cα and cβ atoms of the β-amino acid with heteroatoms could be an attractive modification, for example cβ-atom of β-peptides by an nr moiety, leading to hydrazine peptides. in the literature, there are only a few studies [ ] [ ] [ ] about hydrazine peptides, and hydrazine peptides with cyclic side-chain have not been studied yet. in order to determine the secondary structure preference of -amino-pyrrolidine- s-carboxylic acid homo-oligomers (figure ), their potential energy hypersurface were probed at the ab initio b lyp/ - g** level. the calculations predicted the -strand to be the most stable structure. the hydrazino-peptides in question were synthetized on solid support, and their structures were characterized by nmr and cd methods. the results were found to be in good accordance with the -strand structure. cathepsin c [ec . . . ]( ), which belongs to family of cysteine proteases, catalyzes hydrolysis of n-terminal peptide, preferential glyphe. this enzyme may play a part in chronic airway diseases ( ) . also increaser level of enzyme was found in case of cancer, rheumatism and muscle's distrophy ( ) ( ) ( ) . for this reason we have undertook investigations of peptides containing two dehydroamino acid residues, which could act as alkylating inhibitors of this enzyme. to define structure and conformation of investigated peptides we were used different methods of nmr spectroscopy, including standard d experiments, protonproton correlations, proton-carbon correlations, and d noe experiments. to complete structural research computational chemistry methods had been used. in order to predict the biological activity of investigated peptides, the simulation of docking process of these peptides to enzyme active site had been made and after that correlated with results of enzymatic test.. the obtained results suggest, that investigated peptides containing two ∆phe residues (z and e isomers respectively) in solution have bent conformation, which is stabilized by intermolecular hydrogen bonds. these results are confirmed by the results of theoretical calculations. also simulation of docking process have showed two possible peptide's orientation in active site of cathepsin c and allowed the rational interpretation of biological test's results. turns are important elements of secondary structure in peptides and proteins. different types of turns are distinguished according to the number of residues involved. the most abundant is the β-turn, which involves four consecutive amino acids with the co at position i hydrogen-bonded to the i+ nh. the γ-turn is centred at a single residue and is generally stabilized by a hydrogen bond between the i co and the i+ nh. model dipeptides rco-l-pro-xaa-nhr' are the smallest systems able to adopt the β-turn conformation, which is favoured by the presence of proline at i+ . a peptide of this series, incorporating a cyclopropane amino acid (xaa), has been shown to accommodate two consecutive γ-turns in the solid state [ ] , instead of the expected β-turn conformation. the double γ-turn encountered is unique among crystalline short linear peptides. in fact, the γ-turn is observed almost exclusively in low-polarity solvents, and only a few oligopeptides of cyclic structure exhibit a γ-turn in the crystal. this is the first time that the strong tendency of pro-xaa dipeptides to adopt a β-turn in the solid state has been switched to the γ-turn. theoretical calculations [ ] also show the high preference of this cyclopropane amino acid for the γ-turn conformation. [ oxidative stress plays an important part in the development of cardiovascular disease (cvd). haptoglobin is a hemoglobin-binding protein that has a major role in providing protection against heme-driven oxidative stress. there are two common alleles for haptoglobin ( and ), and the three phenotypes, haptoglobin - , haptoglobin - , and haptoglobin - , differ in their ability to function as antioxidants. we determined whether there was a relation between the haptoglobin phenotype and the development of coronary artery diseases. haptoglobin (hp) phenotypes were determined in iranian patients with coronary artery diseases. we performed haptoglobin (hp) genotyping by polymerase chain reaction (pcr) using allele-specific primer-pairs. in multivariate analyses controlling for conventional cvd risk factors, haptoglobin phenotype was a highly statistically significant, independent predictor of cvd. the odds ratio of having cvd in patients with the haptoglobin - phenotype was . times greater than in patients with the haptoglobin - phenotype. an intermediate risk of cvd was associated with the haptoglobin - phenotype. these results suggest that haptoglobin phenotype is an important risk factor in determining susceptibility to cardiovascular disease which may be mediated by the decreased antioxidant and antiinflammatory actions of the haptoglobin allelic protein product. the special feature of proteins involved in alzheimer's or prion diseases is their ability to adopt at least two different stable conformations. the conformational transition that shifts the equilibrium from the functional to the pathological isoform can happen sporadically. it can also be triggered by mutations in the primary structure, changes of different environmental conditions, or the action of chaperones. elucidation of the molecular interactions that occur during the transformation from α-helix to β-sheet and the consecutive formation of amyloids on a molecular level is still a challenge. therefore, the development of small peptide models that can serve as tools for such studies is of paramount importance. we succeeded in generating model peptides that, without changes in their primary structure, predictably react on changes of diverse environmental parameters by adopting different defined secondary structures. these de novo designed peptides strictly follow the characteristic heptad repeat of the α-helical coiled coil structural motif. furthermore, domains that favour β-sheet formation and aggregation can be generated. alternatively, those peptides can be equipped with functionalities that allow either the binding of metal ions or the interaction with membranes. as proof of our concept we showed that the resulting secondary structure of such peptides will strongly depend on environmental parameters. thus, this system allows to systematically study the interplay between peptide / protein primary structure and environmental factors for peptide and protein folding on a molecular level. the pathogenesis of alzheimer's disease (ad) is strongly linked to neurotoxic assemblies of the amyloid β protein (aβ). aβ is a soluble component of human plasma which by an unknown mechanism becomes aggregated and neurotoxic. some genetic mutations within the aβ sequence cause very early onset of ad-like diseases, probably by facilitation of aβ assembly into neurotoxic species. recently, it was found that not amyloid fibrils, but smaller aβ assemblies initiate a pathogenic cascade resulting in ad. therefore, preventing the folding of nascent aβ monomers would have therapeutic benefit. to uncover details of structural changes accompanying the aggregation process, especially its initial stage, we have decided to study the aβ( - ) fragment and its mutation-related variants. our recent studies on this aβ fragment using the cd method and the aggregation test have proved it a good model for structural studies. the obtained results confirmed that the aggregation process follows the scheme with an α-helical intermediate and pointed out differences in the behaviour of aβ variants. to further confirm the scheme of the structural changes accompanying aggregation we have applied ftir spectroscopy and analysed aggregation-induced changes of the amide i band which is directly related to peptide backbone conformations. the ftir spectra analysis indicate that water addition provoked conformational changes are strongly dependent on the aβ( - ) variant and in some cases the formation of α-helical intermediate seems to be preceded by helix formation. to verify this hypothesis the temperature dependent atr ftir spectra will be analysed. supported by ug bw grant. amino acid octarepeats present in the prion protein bind to cu + and are considered as a potential periplasmic copper ion transporters. this octarepeat is located in the unstructured region of the prion protein, which is supposedly not intricately involved in prion aggregation. our group is involved in exploring the function of octarepeats with a special emphasis on their possible role in amyloid fibril formation and aggregation. in this context, we have prepared truncated peptide constructs derived from the prion protein octarepeat phgggwgq and have reported their fibrillation activity. we will present aggregative behavior of a truncated bis-pentapeptide, containing gggwg segment, when tethered with a flexible linker diaminobutane. fibrillar architectures were observed by this bis conjugate after incubation in water which was probed by different microscopic and steady state fluorescence techniques. further investigations with ki revealed a homogeneous environment of the two tryptophan moieties in the conjugate. in the absence of other side-chains, it is likely that fibril formation involves hydrophobic interaction between tryptophan indole moieties and main chain backbone interactions. interestingly, a facilitator role for aromatic-glycine motifs for amyloid aggregation has been proposed based on bioinformatics search of the swiss-prot and trembl databases. collagens are known to fold into a highly ordered rode-shaped triple helix with stretches of lower and higher suprastructural stability and even disruptions to modulate recognition by other proteins that interact with the extracellular matrix [ ] . to increase understanding of folding and stability of the collagen triple helix, we have adressed the design of photocontrolled collagenous peptides. our aim was to crosslink two side chains of the repetitive (xaa-yaa-gly) sequence motifs of collagen model compounds via an azobenzene chromophore in analogy to our previous studies on photomodulation of the conformational preferences of cyclic peptides and more recently of hairpin-peptide model systems [ ] . molecular modeling experiments suggested appropriate sequence positions that could result in triple-helical peptides with conformational stabilities that can be modulated by cis/trans isomerization of the azobenzene moieties. as light switchable crosslinker azobenzene- , '-n-( -iodo- -butynenyl)carboxyamide was synthesized for reaction with two ( s)-mercaptoproline residues placed in suitable xaa and yaa positions, respectively. by this approach a fully folded triple helix was obtained upon thermal relaxation, and unfolding was induced by irradiation at nm. the favorable optical properties of the azobenzene derivative together with the regular suprastructure resulted in a valuable model system that allows for ultrafast time-resolved studies of collagen folding and unfolding. amyloid formation is connected with alzheimer's disease, parkinson's disease, finnish familial amyloidosis. after protein misfolding short peptide sequences act as "hot spots" providing the driving force for protein aggregation in amyloid fibrils. we have identified one of these sequence stretches in the abl-sh domain of drosophila (dlsfmkge) whereas the human homologous region (dlsfkkge) is predicted to be less amyloidogenic. the possible reason for the difference of amyloid formation propensities of the two peptides was investigated by molecular dynamics (md) of β-sheet structures. the antiparallel alanine β-sheets consisting of two and ten strands were constructed, minimized, and mutated to the sequences dlsfmkge and dlsfkkge. all four systems: ) dlsfmkge -two strands, ) dlsfkkge -two strands, ) dlsfmkge -ten strands, ) dlsfkkge -ten strands, were surrounded by Å layer of water molecules over the solute and subjected to md, amber . force field, ntp protocol. the md runs were started at the temperature of k and the temperature was elevated stepwise by degrees till k. the results show considerably higher hydrogen bond percentage for dlsfmkge than that one for dlsfkkge during the course of the simulation, thus suggesting that dlsfmkge is a potential fibril-maker, but dlsfkkge is not. two strand β-sheet systems were stable until k. the ten strand β-sheets are more stable. angiotensin-i converting enzyme (ace) has a critical role in cardiovascular function, which consists of cleaving the carboxy terminal his-leu dipeptide from angiotensin-i producing a potent vasopressor octapeptide, angiotensin-ii. there are two isoforms of ace. the somatic isoform is present in all human cells except the testis cells, where the testicular isoform is produced. the major difference between these two types is that, the somatic form has two active sites, at the n-and c-end respectively while the testicular has only one, which is almost identical to the somatic c-terminal active site. here we report the structural study of a aa peptide (previously expressed in bacteria), which corresponds to an extended domain of the human somatic n-terminal active site of ace (ala -gly ) by circular dichroism experiments, and the overexpression in bacteria, purification and structural study, using circular dichroism techniques, of a aa peptide which corresponds to an extended domain of the human somatic c-terminal active site of ace (ala -gly ). following the subclonning into an appropriate expression vector and the expression, the peptide was isolated from the inclusion bodies using chromatography techniques. the recombinant protein fragment had a molecular weight, measured by esi-ms, of kda which was in consistence with the theoretical calculation based on the dna sequence. the recombinant peptides acquired their theoretically calculated secondary structure only when , , -trifluoroethanol is present at a concentration of ~ %. in order to elucidate their structures, solutions of these peptides, labeled with n and/or c, will be studied by nmr spectroscopy. aggregation of peptides is believed to trigger various degenerative diseases but it also plays an important role for the preparation of peptide fibres and peptide-based biomaterials. it is therefore extremely important to understand the mechanisms involved in peptide aggregation and be able to control them. studies were performed on a library of amphiphilic peptides, designed around the sequence of a model antimicrobial peptide rich in leucine and lysine. the library also included peptide hybrids in which natural amino acids were replaced by non-proteinogenic omega-amino acids, such as -aminohexanoic acid and -aminononanoic acid. the aim was to estimate the aggregation and its correlation with the biological activity by using a fluorescence technique commonly employed to calculate the cmc (critical micelle concentration) of surfactants. peptides and peptide hybrids were synthesized on solid support using the fmoc polyamide protocol. they were purified by semi-preparative rp-hplc and characterized by esi-ms and analytical rp-hplc. the aggregation behaviour of the synthesized molecules was investigated in water by steady-state fluorescence measurements using pyrene as fluorescent probe. peptides were dissolved in water/pyrene or water/pyrene/ . fluorescence spectroscopy has become an extremely valuable technique for conformational studies of biopolymers, the development of peptide-based chemosensors, and biochemical research in general. in this connection, synthetic amino acids as fluorescent probes to be incorporated into a peptide chain may exhibit significant advantages over the related protein (trp and tyr) residues in terms of potentially different and ameliorated properties. we recently designed and prepared a new fluorescent amino acid, antaib, based on a planar anthracene core and belonging to the class of achiral, ciα↔ciα cyclized, cα-tetrasubstituted α-amino acids (strong β-turn and helix inducers in peptides). peptides based on antaib combined with (l)-ala residues were synthesized and subjected to a conformational analysis. more specifically, the protected derivatives boc-antaib-oet (oh) and fmoc-antaib-otbu (oh) were prepared in seven steps from , , conformational transitions in peptides and proteins emerge to play the major role in the genesis and evolution of prion related diseases and alzheimer's disease (ad). in this context, conditions influencing this transition and the following aggregation process are of paramount interest. peptides and proteins that are involved in aggregation processes contain potential metal binding sites. the concentration of metal ions in the brain tissue is naturally high and zn in the mm range has been found in ad amyloid plaques. thus, it is widely accepted that metal complexation is one of the key incidents that lead to conformational transitions and aggregation. we present here a coiled coil based model peptide system with an intrinsic amyloid forming tendency which can be used to study the impact of different metal ions on secondary structure and aggregation. metal complexing histidine residues were incorporated to create potential binding sites which, depending on their position and the nature of the metal ion, dictate folding and aggregation. the time dependent conformational transition was monitored by cd-spectroscopy. aggregates were characterized by cryo tem. high resolution fticr-ms experiments revealed information on the stoichiometry of the peptide-metal complexes. in the absence of metal ions the presented peptides formed amyloids in a time range of weeks. depending on the his positions and milieu conditions, the nature of the metal ion determines folding and aggregate morphology. furthermore, metal binding was shown to inhibit the amyloid formation. a challenge to our understanding of protein folding is the design of a protein from first principle, i.e. starting from geometric restraints and applying properties of amino acids expected to be essential for folding to a defined structure. we developed a program to calculate the backbone coordinates of antiparallel strands to match the surface of an elliptical cylinder. various parameters like the number and shearing of the strands and the ellipticity of the structure can be varied. the relative orientation of the β-strands and the geometrical features of the hydrogen-bonds were derived from statistical analyzes of natural β-sheet structures. iterative cycles of core-packing with amino acid rotamers, molecular dynamics simulation and energy minimization with the charmm forcefield are used to include backbone movements and to minimize the risk of trapping an energetically unfavorable structure. the quality of residue packing is assessed with the help of criteria which proved to be successful in our design of β-sandwiches. at the protein surface, a network of salt-bridges with an excess of positive charges has been designed to increase the stability and the solubility of the protein. the final sequence is synthesized by standard solid phase fmoc-chemistry. insights gained from the analysis of the synthesized structure with ftir and cd spectrometry should help us to refine the parameters for subsequent designs. with this strategy, we hope to contribute to a better understanding of protein folding. the immunoglobulin binding protein g ( igd) from streptococcus species consists of amino acids residues, which form two antiparalell-packed beta-hairpins and an alpha-helix in the middle of the sequence packed to the beta-sheet. the second hairpin was found to be stable in isolation. this fragment is therefore likely to be the first folding initiation site of the protein which could provide an adequate nucleation center on which the rest of the polypeptide chain would find a favorable environment to fold. thus, among the two beta-hairpins, the - fragment of igd corresponding to the c-terminal beta-hairpin was synthesized. in our studies, we investigated different environmental and temperature conditions for formation of the - beta-hairpin structure. its structure was examined by means of cd spectroscopy in water, buffer solutions (ph = to ) and in aqueous solutions of trifluoroethanol. additionally, its structure was investigated in the solid state by ftir spectroscopy. the cd studies revealed that the - fragment of igd in water forms mainly a statistical-coil structure, whereas the ftir technique shows formation of a regular beta-sheet structure. nmr spectroscopy and calorimetric measurements were carried out at various temperatures. our studies show that the - fragment at low temperature exists in an equilibrium between two conformations -a regular beta-hairpin and a statistical-coil. although increasing temperature resulted in shifting the equilibrium in the direction of the statistical-coil structure, the overall beta-hairpin shape of the - fragment was maintained. prion diseases are characterized by the conversion of the physiological cellular form of the prion protein (prpc) into an insoluble, protease-resistant abnormal scrapie form (prpsc) with an highly beta-sheet content [ ] . the analyses of intrinsic structural propensities of the prp c-terminal domain showed an high conformational flexibility for the αhelix fragment which indicates that this region may be particularly important in the prpc→prpsc transition [ ] . therefore conformation-based approaches focalized on helix region appear to be the most promising for the study of prion protein misfolding. recent studies on tetracycline properties showed that this molecule binds and disrupts prp peptide aggregates and inactivates the pathogenic forms of prp [ , ] . a fluorometric titration of the fluoresceinated peptide corresponding to prion protein helix- with tetracycline has been carried out to determine the value of the apparent dissociation constant of this interaction (estimated to be ± nm). remarkably, the fluoresceinated peptide exhibits in water a canonical α-helical cd spectrum, that is maintained even in presence of tetracycline. accordingly, docking calculations and molecular dynamics simulations suggest that tetracycline interacts preferentially with the c-terminal end (residues - ) of helix- with a significant involvement of the treonine rich region. in the last decades, a series of discoveries have shed light on the role played by the carbohydrate moiety in glycoproteins. it has been shown that covalently linked sugar moieties influence peptide/protein properties such as hydrophobicity, conformation, biostability and bioactivity. the design of carbohydrate-peptide analogs with increased, retained or modified biological activity requires an understanding of their conformational preferences both in solution and in the receptor-bound state. in our recent work we have created two classes of well-structured linear and cyclic carbohydrate-modified analogs of opioid peptides, leu-enkephalin and leuenkephalin amide. the first class represents a group of compounds in which the linear peptide is alkylated at the n-terminal position by -deoxy-d-fructose unit, while its cyclic analog possesses an ester bond between the c- hydroxy group of the sugar moiety and the c-terminal carboxy group of peptide, -deoxy-dfructofuranose acting as a bridge between the leu-enkephalin terminal parts. the rigid -membered imidazolidinone ring is characteristic for the second class of compounds. in these adducts an imidazolidinone moiety connects the acyclic sugar residue with the linear peptide chain. in the corresponding bicyclic imidazolidinone analogs -membered ring is formed through an ester bond between the primary hydroxyl group of the d-gluco-pentitolyl residue and the cterminus of peptide. this work reports the comparative cd and ftir spectroscopic properties of the prepared glycopeptides in comparison with data on the non-modified flexible parent peptides performed in different solvents in order to expose the structural and conformational differences caused by a keto-sugar, rigid membered imidazolidinone ring and/or cyclization. the α-helical coiled coil structural motif consists of two to five α-helices which are wrapped around each other with a slight superhelical twist. the simplicity and regularity of this motif have made it an attractive system to study the role of complementary interactions for protein folding. here we present a systematic study showing that intermolecular electrostatic interactions between positions e and g of the helices are in competition with the intramolecular interactions between positions e/b and g/c. those competitive interactions affect folding and stability of the motif which were monitored by temperature dependent cd-spectroscopy. incorporation of oppositely charged amino acids in positions e/b and g/c reduced considerably electrostatic repulsion between equally charged amino acids in positions e and g. in addition coiled coil stability can be increased by the alkyl part of the amino acid side chains in positions e and g. studies with natural and unnatural amino acids showed that the longer this alkyl part the better is the hydrophobic core protected from solvent. therefore the repulsion of equally charged amino acids in positions e and g can be overruled by involving them either into attractive intrahelical electrostatic interactions or into hydrophobic core formation. human cystatin c (hcc) is a amino acid residues protein that reversibly inhibits papain-like cysteine proteases. this inhibitor belongs to the amyloidogenic proteins shown to oligomerize through d domain swapping mechanism. the crystal structure of hcc reveals the way the protein refolds to produce symmetric dimer while retaining the secondary structure of the monomer. the monomeric form of hcc consists of a core with a five-stranded antiparallel β-sheet wrapped around a central α-helix. the hcc dimerization is preceded by an opening movement of l loop from β -l -β hairpin and separation of the β -helix-β fragment from the remaining part of the molecule. the amino acid sequence of β -helix region suggests additional possible partial unfolding in the n-terminal part of helix. in order to investigate the structural stability of β -helix region the peptide corresponding to the helix and its n-terminal truncated analogs was synthesized along with the peptide analogs of helix containing point mutations that could stabilize helical structure of the n-terminus. the peptides were synthesized by the solid-phase method using fmoc/tbu tactics. purified products were identified by maldi-tof. the secondary structure content was calculated from the cd spectra using selcon . the random coil was the predominant structure of the peptide corresponding to the α-helical fragment of hcc and its n-truncated analogs. however, an increase of αhelix content was observed in some of the peptides corresponding to the helix containing point mutations. we expect that these mutations could stabilize the hcc monomer and suppress dimerization. the tumor suppressor protein p is a trarnscription factor that triggers cell-cycle arrest and apoptosis in response to genotoxic stress signals. the tetramereric structure of the p , which is essential for its activity as a transcription factor, is formed as a dimer of dimers. while the primary dimer is constructed from inter molecular formation of a two-stranded anti-parallel b-sheet and a two anti-parallel a-helix bundle, the secondary dimer is stabilized through interactions between residues on the surface of the primary dimer. from various substitution experiments on p , it has been shown that hydrophobicity of phe is critical for the tetramer formation of p . also we have substituted three phenyl groups of p with cyclohexylalanine (cha) and showed that phe cha is dramatically stabilized against temperature, chemical denaturant, and organic solvent by cd measurements. here, to clarify the mechanism of the stabilization of phe cha, we analyzed its three dimensional structure using x-ray crystallography. we obtained two kinds of crystals, one is a hexagonal bipyramid crystal in the space group of p<i> <sub> </sub> </i> with <i>a</i>= . Å, <i>b</i>= . Å, <i>c</i>= . Å diffracted to about . Å, and another is tabular crystal in the space group c<i> </i> with <i>a</i>= . Å, <i>b</i>= . Å, <i>c</i>= . Å diffracted to about . Å. in these crystals, the peptides formed tetramers which are very similar to those observed in the wild-type. the structure of the pocket where the side chain of cha is incorporated was defined to elucidate the hydrophobic interaction to determine the stability. helices shown in proteins, as a secondary structure, almost always form right-handed screw sense. this right-handedness is believed to result from the chiral center at the αposition of proteinogenic l-α-amino acids. among proteinogenic amino acids, l-isoleucine and l-threonine possess an additional chiral center at the side-chain β-carbon besides the α-carbon. however, no attention has be paid how the side-chain chiral centers affect the secondary structures of their peptides. recently, we have reported that sidechain chiral centers of chiral cyclic α,α-disubstituted amino acid (s,s)-ac( )c(dom) affected the helical secondary structure of its peptides, and the helical-screw direction could be controlled without a chiral center at the α-carbon atom. herein, we synthesized a chiral bicyclic α,α-disubstituted amino acid (r,r)-ab( , )c and its homopeptides, and studied the relationship between the chiral centers and the helical-screw handedness of peptides. contrary to the left-handed helices of (s,s)-ac ( ) four threefinger-toxins (tfs) have been purified from the pooled venom of golden krait (bungarus fasciatus, i.e. bf) from thailand and studied previously. these peptide toxins contain - residues and or pairs of disulfide bonds, and are rich in β-structure. we herein analyzed the tf-isoforms in bf venoms from kolkata (eastern india), hunan province (eastern china) and indonesia to study the geographic variations and structure-function relationships of the venom polypeptide family. a total of five or six tfs of low lethality were purified from each of the geographic venom samples, the n-terminal sequences and accurate masses of the peptides were determined. the cdnas encoding some of these tfs were also cloned and sequenced. full peptide sequences were deduced and match with those of the tfs purified from crude venom. intra-species variations of the venom tfs were found to be surprisingly high since sequence-identities between the majorities of orthologous toxins in different geographic samples are only - %. most of the bf proteins were not neurotoxic by electrophysiological assays using chick biventer-cervicis and mouse diaphragm neuromuscular tissues. the toxins appear to be associated with weak toxins or non-conventional snake venom tfs as analyzed by a phylogenetic tree. the reason behind their lack of neurotoxicity would be discussed. v. moussis, e. panou-pomonis, c. sakarellos, v. tsikaris peptides involved in neurodegenerative diseases can adopt at least two different stable secondary structures. amyloid-forming proteins can experience a conformational transition from the native, mostly α-helical structure, into a ß-sheet rich isoform. the latter conformation is probably present in intermediates for the formation of amyloids. the conformational change can be triggered by protein concentration or environmental changes. therefore, our aim was to generate a de novo designed peptide that contains structural elements for both, stable α-helical as well as ß-sheet formation. this model peptide can be used to elucidate the conformational changes dependent on concentration and ph. the design is based on the well studied α-helical coiled coil folding motif. the conformation and structure of the resulting aggregates were characterized by cd-spectroscopy and cryo transmission electron microscopy. as a result, three distinct secondary structures can be induced at will by adjustment of ph or peptide concentration. low concentrations at ph . yield globular particles of the unfolded peptide whereas, at the same ph but higher concentration, defined ß-sheet ribbons are formed. in contrast, at high concentrations and ph . , the peptide prefers highly ordered α-helical fibers. in conclusion, we successfully generated a model peptide that, without changes in its primary structure, predictably reacts on environmental changes by adopting different defined secondary structures. thus, this system allows to systematically study now the consequences of the interplay between peptide primary structure and environmental factors for conformation on a molecular level. cells respond simultaneously to a multitude of different signals. inside a cell signals from activated receptors are integrated by networks of enzymatic reactions and molecular interactions, leading to a spectrum of cellular responses. in order to understand the relationship between a specific cellular stimulus and a cellular response, methods are required to detect in parallel the pattern of molecular interactions. a large number of molecular interactions is mediated by protein domains, binding to linear peptide motifs. lysates of activated and resting cells were incubated on peptide microarrays carrying peptides corresponding to such binding motifs of signalling domains. binding of proteins to a spot of the array was probed by immunofluorescence. jurkat t cells were stimulated either with the phosphatase inhibitor pervanadate or with antibodies directed against cell surface receptors. upon activation of t cells, numerous changes in the pattern of molecular interactions were detected for a total of proteins and peptides. these changes were caused either (i) by masking or unmasking of a binding domain which resulted in a reduced or increased binding of a protein to the microarray or (ii) by recruitment of a protein into a complex that in turn bound to the microarray. the changes were dependent on the nature of the stimulus. the human melanocortin receptor (hmc r) was constructed to contain a flag epitope and a hexahistidine tag at the amino-terminus as well as at the carboxyl terminus to facilitate purification. stably transfected hmc r in human embryonic kidney (hek ) cell lines that expressed the receptor resulted in a kd value of . and . nm respectively in each case when the super potent agonist mtii was competed with [ i]ndp-α-msh. treatment of the tagged receptors in the hek cells with agonist resulted in down-regulation which indicates that these tagged receptors retain their biological functions. the hmc r was solubilized from cell membranes with n-dodecyl-β-d-maltoside and purified at a nickel chelating resin and a newly constructed affinity column. the purified hmc r was a glycoprotein that migrated on sds/page with a molecular mass of kda. the results from matrix-assisted laser desorption ionization-time of flight (maldi-tof) mass spectrometry was used to identify and characterize peptides derived from the hmc r following in-gel digestion with chymotrypsin. the phosphorylation sites were identified on the purified human melanocortin receptor with agonists (peptide vs small molecule) treatment. the discovery of antibiotics in the s has been one of the most revolutionary events in the history of medicine. however, during last decades, the increase of antibiotic resistance has significantly hampered the application of antibiotics. therefore, further scientific effort to find new antibiotics with novel mechanisms of action is of high importance. insects are the largest and the most diverse group of living animals on earth. they have potentially been confronting high variety of microorganisms. as a result, they have evolved powerful defense system, thus representing vast source of novel potential therapeutics. we chose larvae of fleshfly neobellieria bullata for identification and characterization of new promising molecules, peptides or proteins, which participate in immunity response against microbial infections. the hemolymph of the third-instar larvae of neobellieria bullata was used for isolation. the larvae were injected with bacterial suspension of escherichia coli or staphylococcus aureus to induce antimicrobial response. the hemolymph was separated into crude fractions, which were subdivided by rp-hplc. isolated fractions were characterized by uv-vis spectroscopy, amino-acid analysis, mass spectroscopy, -d and -d sds electrophoresis, capillary zone electrophoresis, ion-exchange hplc, tryptic digests and n-terminal sequencing. we found out significant antimicrobial activities against escherichia coli, staphylococus aureus or pseudomonas aeruginosa in several fractions. using real-time pcr, we followed and compared levels of mrna of different proteins and peptides in induced and non-induced larvae. despite the fact that many technological advances are currently involved in proteome analysis, like two-dimensional gel electrophoresis and mass spectrometry, there is still a great need for the development of novel engineered chemical probes for proteomics and interactomics. here, we describe our approach concerning the study of proteome and interactome of proteins involved in cell-matrix interactions. it relies on the use of a small synthetic inhibitor chemically modified to allow for its immobilisation to magnetic beads or affinity chromatography materials. proteins will be detected together with their native interaction partners because of nondenaturing conditions. this general procedure is applied for the enrichment of metalloproteinases, especially matrix metalloproteinases, which are potential target in tumour therapy. hydroxamic acids are known to be potent inhibitors of metalloproteinases. marimastat is a reversible inhibitor with a good potency and shows activity towards a wide range of metalloproteinases. the synthesis of new marimastat derivatives will be reported. the parent compound is modified with a linker to allow immobilisation on a solid surface. binding studies were performed using surface plasmon resonance. this approach is not only appropriate for the generation of metalloproteinase proteome subsets by affinity column or using magnetic beads, but also to enrich and isolate interaction partners of the target proteins. the present report summarizes the latest data devoted to theoretical and experimental investigation of the high temperature solid state catalytic isotope exchange reaction (hscie) that takes place in peptides and proteins by the action of deuterium and tritium [ ] . the available ms-procedures, designed to estimate the amount of protein, are aimed at derivatization at different stages of sample preparation, and as the best result, it is only possible to achieve quality comparison of the objects involved. the hscie reaction allows the production of evenly deuterium labelled proteins and peptides, and their application makes it possible to create a qualitative mass spectrometry method for protein analysis. introduction of definite amounts of these deuterium-labeled proteins into biological objects, prior to isolation, separation and trypsinolysis, will generate quantitative information concerning the composition of the proteins under study. tritium labelled proteins produced at a temperature of - oc carry the isotopic label in all the peptide fragments and completely retain their enzymatic activity. the proteins' reactivity is dependent on their three-dimensional structure. the hscie reaction has been shown to be used both in the production of tritium labelled proteins and in the investigation of spatial interactions in protein complexes. in addition, evenly deuterium and tritium labelled peptides can be used in studies of the kinetics and transformation paths of peptides in the organism's tissues. immunization of mice with type ii collagen (cii) from rat leads to development of collagen-induced arthritis (cia). susceptibility to cia is associated with the major histacompatibility class ii protein h- aq that binds the glycopeptide epitope cii - ( ) and presents it to helper t cells. [ ] to explore the interactions in the system and to stabilize towards in vivo degradation, amide bond isosteres have been introduced in its backbone.glycopeptide was virtually docked into the binding site of a comparative model of h- aq. based on the hydrogen bonding network between the peptide backbone and h- aq, the amide bond between ala -gly was chosen for isosteric replacement. to vary the geometric and hydrogen bonding properties, mimetics of the dipeptide were synthesized with the amide bond replaced by ψ[ch nh], ψ[coch ] and ψ[(e)-ch=ch], respectively. these were introduced in using solid-phase synthesis to give glycopeptidomimetics that were biologically tested for their ability to bind to the h- aq protein and for recognition by t cells. shown to be a potent neuroprotective factor in various pathophysiological models. despite its therapeutic potential in diverse neurodegenerative diseases, its short in vivo half-life limits its utility as a useful clinical agent. moreover, the development of a peptidomimetic that reproduces the pharmacological activity of pacap is unlikely since the pharmacophores are spreaded throughout the entire peptide chain. therefore, the development of pacap analogues with lower susceptibility to proteolysis represents a first step toward clinical applications. in the present study, derivatives of both pacap and pacap with particular chemical modifications were developed targeting specific peptidase sites of action. results indicate that the incorporation of an acetyl or a hexanoyl group at the n-terminus and modifications at the ser residue contributed to increase stability against dipeptidyl peptidase iv, the major enzyme involved in pacap degradation. moreover, after determination of pacap metabolites in human plasma, the amide bond between residues and was substituted by a ch nh surrogate and this derivative showed increased plasma stability. all modified peptides were tested for their ability to induce pc differentiation. the effects of pacap analogs on pc cells are mediated through the pac receptor which is the major receptor involved in the neuroprotective effects of pacap. this study exposes interesting data concerning pacap metabolism in isolated human plasma and demonstrates the possibility of increasing the metabolic stability of pacap without significantly reducing its biological activity. species of the fungal genus trichoderma are commercially used as bioprotective agents against fungal plant diseases. more than strains were collected from their natural habitats and evaluated for biocontrol properties. seven of the most active isolates exhibiting strong biological activity towards eutypa dieback and esca diesease of grapevine were classified as trichoderma brevicompactum, or shown to be closely related to that species. these strains were screened for production of peptaibiotics. the formation and synergistic action of hydrolytic enzymes and peptaibiotics were to play an important role in mycoparasitism. after background and aims: conotoxins are short, disulfide-rich neurotoxins that target various ion channels and receptors. these peptides have desirable pharmacological properties to become therapeutics for neurological disorders; several conotoxins have already reached clinical development stage. our long-term goal is to improve bioavailability, metabolic stability and pharmacokinetics of conotoxins using a variety of chemical modifications. methods: we designed and chemically synthesized conotoxin analogs containing two distinct types of backbone modifications: ( ) peptide-peptoid chimeras (conopeptoids) of alpha-conotoxin imi and ( ) peptide chimeras of mu-conotoxin kiiia containing non-peptidic "backbone spacers". results: conopeptoid-imi, containing ala replaced by n-methyl glycine potently blocked activity of nicotinic acetylcholine receptors. in mu-conotoxin kiiia, aminohexanoic acid or amino- -oxapemtanoic acid were inserted to be a part of the peptide backbone. the two oxidized analogs containing "backbone prosthesis" differed in their hydrophibicity profile, but they both potently inhibited neuronal sodium channels. conclusion: our results suggest that backbone engineering may become an effective method of producing conotoxin analogs with modified bioavailability. to increase the stability and the therapeutic efficacy of peptide sequences from myelin oligodendrocyte protein (mog) that act as multiple sclerosis (ms) antigens, we grafted them onto a framework of a particularly stable class of peptides, the cyclotides. they are a recently discovered family of cyclic plant peptides with superb intrinsic stability. the limitations of linear peptides as drugs due to their instability and poor bio-availability can be overcome by using the cyclotide scaffold as a framework for novel drug design. peptide epitopes from mog protein were incorporated onto the framework of the model cyclotide kalata b by means of boc-spps approach. after successful backbone cyclisation and oxidation of the cysteine residues, the peptides were purified to high purity with rp-hplc. nmr chemical shift analysis was used to assess whether the grafted analogues have a stable scaffold, similar to that of kalata b . a structure of a representative peptide was determined and it shows remarkable resemblance to the native scaffold of kalata b . the activity of the bioengineered peptides has been tested in vivo. a group of mice injected with one of the peptides have shown a depression in the clinical score and have not fallen ill. this is an exciting result that shows the first active bioengineered cyclotide in an animal model of disease. the structural information from nmr studies will be used in conjunction with the results from the activity studies in a feedback loop to design second-generation lead molecules. a.d. de araujo, p.f. alewood conotoxins are small, disulfide-rich peptide neurotoxins produced in the venom of marine cone snails that enable these molluscs to capture their prey. these compounds exhibit a high degree of selectivity and potency for different ion channels and their respective subtypes, making them useful tools in the investigation of the nervous system. due to the key role of ion channels in many physiological processes, conotoxins are also excellent drug lead candidates in drug discovery, with some examples currently undergoing clinical trials and one recently approved drug (prialt®). like other peptide drugs, the use of conotoxins in drug development is limited by the low oral bioavailability of these compounds due to pre-systemic enzymatic degradation and poor penetration through the intestinal mucosa. peptide analogs that mimic the native structure and incorporate rationally designed non-natural modifications in the disulfide framework or peptide backbone may exhibit increased resistance to proteolytic degradation. in biological environments, disulfide linkages are susceptible to attack by enzymes and reducing agents (such as glutathione). therefore we carried out the synthesis of redox-stable conotoxin analogs in which intrinsic disulfide bonds were replaced by thioether linkages. in this work, wehave explored two solid-phase synthetic routes in the preparation of thioether conotoxin mimetics: the first route based on peptide assembly using a tetra-orthogonally protected lanthionine building block, and the second route based on intramolecular on-bead cyclisation between cysteine and betabromoalanine residues. thyrotropin releasing hormone (trh, l-pglu-l-his-l-pro-nh ), a tripeptide synthesized in the hypothalamus, operates in the anterior pituitary to control levels of tsh (thyroid-stimulating hormone) and prolactin. the thyrotropin-releasing hormone (trh) receptor (trh-r) belongs to the rhodopsin/β-adrenergic receptor subfamily of seven transmembrane (tm)-spanning, g protein-coupled receptors (gpcrs). the two g-protein-coupled receptors for trh, trh receptor type (trh-r ) and trh receptor type (trh-r ), have been cloned from mammals and are distributed differently in the brain and peripheral tissues. the trh receptor subtype- appears to mediate the hormonal and visceral effects, whereas trh receptor subtype- has been implicated in its central stimulatory actions. identification of critical features of the trh, separation of its multiple activities through design of selective analogues and affinity labels have been elusive and unfulfilled goals for more then years. this presentation will highlight our studies on effect of the biological activity of trh with the introduction of alkyl groups of varying sizes at the n- and c- position of the centrally placed histidine residue of trh peptide. the requisite n--boc-dialkyl-l-histidines as building scaffolds have been synthesized in six steps from l-histidine methyl ester dihydrochloride and used for the synthesis of various trh analogues. ghrelin, the natural ligand for the growth hormone secretagogue receptor (ghsr- a), has received a great deal of attention due to its ability to stimulate growth hormone secretion and to control food intake. during these last years, ghrelin analogues or mimetics gained interest for their implication in food intake regulation. in the course of our studies concerning ghrelin analogs, we developed new ligands of the ghsr- a based on heterocyclic structures. interestingly, one heterocyclic family presented high affinity for the ghrelin receptor. a structure activity study was performed within this family and led to potent ghsr- a agonists and antagonists. the binding affinities were determined by displacement of radiolabelled ghrelin and the agonist or antagonist character was measured by intracellular calcium mobilisation. the first in vivo results revealed that in vitro activities and in vivo responses were not correlated. particularly, binding to ghsr- a and in vivo gh release and food intake control were not fully correlated. these results strongly suggest the presence of receptor subtypes that modulate ghrelin actions. some examples will be reported. further investigations are going on in the laboratory. background and aims: alzheimer's disease (ad) related beta-amyloid (aβ) peptides possess high propensity towards aggregation. nowadays one of the major directions of the drug-design against ad is the synthesis of putative amyloid aggregation inhibitor molecules (aai) which are able to hinder the formation of these toxic amyloid aggregates. in the present work we report the design, synthesis and investigation of putative aais derived form the peptide sequence aiigl identical to aβ( - ). methods: aβ( - ) peptide and putative aggregation inhibitors were synthesized manually using fmoc-chemistry and dcc/hobt activation. studies on both the aβ aggregation and the effect of the aais were performed with several instrumental techniques. the total amount of the aggregates was determined by thioflavine-t binding assay, while their size distribution was characterized with dynamic light scattering (dls). aggregated aβ forms were visualized with transmission electron microscopy (tem). the binding affinity of the aais to aβ fibrils was studied in saturation transfer nmr experiments, while in vitro viability assays were performed on cultured human sh-sy y neuroblastoma cells to monitor the neuroprotective effect of the amyloid aggregation inhibitors. results and conclusions: derivatives of aβ( - ) were synthesized and tested concerning their neuroprotective effect against aβ-mediated toxicity. the most promising candidates were examined with physico-chemical methods to characterize their aggregation altering ability. the pentapeptide riigl-amide proved to be the most powerful neuroprotective compound, and it was also able to alter considerably the aggregation kinetics of aβ( - ) . this molecule might serve as a lead compound in the drug-design against ad in the future. m. mattiuzzo, a. bandiera, m. benincasa, i. samborska, m. scocchi, r. gennaro antimicrobial peptides (amps) are ancient molecules of the innate immune defence system. most of them kill bacteria by lysing their membranes. the proline-rich group of amps represents an exception, as they act via a permeabilization-independent mechanism that is likely based on recognition of molecular targets/transporters. however, specific internal targets have not yet been identified for most of these amps. bac is a pro-rich amp isolated from bovine neutrophils that is capable to translocate across membranes to target hypothetical intracellular proteins. in this study, we used a molecular approach to identify putative targets for bac by selection of peptide-resistant mutants followed by identification of the genes responsible of this resistance. to this aim, an e. coli strain susceptible to the fully active fragment bac ( - ) was subjected to chemical mutagenesis and a number of peptide-resistant mutants was obtained. a pool of genomic dna from these mutants was used to construct a plasmid library that was used to transform a susceptible strain. this approach allowed the identification of different clones that provided a high level of resistance to bac . sequence analysis revealed the presence of genes originating from five different regions of the e. coli chromosome. among them, one clone contained ptrb, a gene coding for a serine peptidase broadly distributed among gram -bacteria, which could be involved in resistance by degrading the peptide. other resistanceconferring clones, which encode for membrane proteins that may be involved in peptide translocation across the memmbrane, are currently under investigation. lycotoxins are peptides from the venom of the wolf spider that were predicted to have an amphipatic alpha-helical structure and confirmed to possess significant antimicrobial and pore-forming activities. [ ] we became interested in these peptides as potential leishmanicidal agents against leishmania donovani promastigotes and leishmania pifanoi amastigotes. thus, lycotoxin i (lyi) and lycotoxin ii (lyii), [ ] and shortened analogues lyi - , lyi - , lyii - and lyii - , were synthesized as c-terminal carboxamides. short-and long-term parasite proliferation measurements showed all peptides except lyi - to be active against both promastigotes and amastigotes at the micromolar range, and suggest peptide effects on parasites to be irreversible. lyii, that showed the lower activity, became more leishmanicidal upon residue trimming, whereas the most active lyi displayed the opposite behaviour. a set of complementary techniques showed that lycotoxin peptides are membrane-disruptive to promastigotes. electron microscopy showed that two populations of promastigotes, one with intact parasites and the other extensively damaged, are formed upon addition of the peptides at their ec . all peptides were non-hemolytic for sheep erythrocytes below micromolar. tissue transglutaminase (tg ) is an enzyme that plays a key role in the pathogenesis of the celiac disease. tg is the main autoantigen recognized by the antiendomysium antibodies and, furthermore, catalyzes the deamidation of strategic glutamine to glutamic acid within the sequence of immunodominant gliadin epitopes. recently, another unexpected role for surface tg , in the innate immune response in the celiac disease, has been suggested. it follows that tg inhibitors might represent a potential attractive pharmacological alternative to the gluten-free diet that, nowadays, is the only possible therapy for celiac patients. starting from the sequence of the heptapeptide pqpqlpy, known to be a high affinity substrate of human tg , we have synthesized new analogs replacing pro with different constrained amino acids (d-pro, pip, chg, ind, deg, inp, hyp, thz)) with the aim to develop specific inhibitors of tg . actually, proline residues present in the gluten epitopes are important in determining the immunogenicity of the epitopes and the specificity of tg . herein, we describe the preliminary conformational studies of the synthesized analogs by cd and nmr spectroscopy and molecular modeling methods. the structural features of the peptides have analyzed in different environment. given the role of the domain pqpqlpy in the gliadin proteins, structural analysis on its analogs are of considerable interest. the results of our studies might be useful to clarify the role of the proline residues in the interaction of the gluten epitopes with tg and, consequently, to gain new insight in the molecular mechanism of celiac disease. carnosine and related histidine-containing dipeptides are known to react with high efficiency with the products of lipid peroxidation, namely -hydroxy-trans- , nonenal (hne) and other alpha, beta-unsaturated aldehydes, preventing their reaction with nucleophilic residues in proteins and nucleic acids. histidyl hydrazide alone or conjugated with aminoacids, long chain fatty acids, cholesterol and alpha-tocopherol synthesized in our laboratories demonstrated higher aldehydesequestering efficiency than carnosine, and were also efficient in protecting sh-sy y neuroblastoma cells and rat hippocampal neurons from hne-mediated death. the cytoprotective efficacy of these compounds suggests their potential as therapeutic agents for disorders that involve excessive membrane lipid peroxidation. lantibiotics are antibiotic natural products that are synthesised ribosomally and undergo extensive post-translational modification, resulting in multiple thioether bridges and dehydro amino acids in the mature peptide. one such lantibiotic is mersacidin, which is produced by bacillus sp. and exhibits extremely promising in vitro and in vivo efficacy versus a number of clinically relevant pathogens, most notably methicillin-resistant staphylococcus aureus (mrsa). mersacidin is believed to function by binding to the bacterial cell wall precursor lipid ii, thereby preventing its incorporation into the growing peptidoglycan network. in an attempt to better understand this mode of action and develop more active analogues, we have embarked upon its chemical derivatisation. some of these modifications resulted in an altered antibacterial spectrum, permitting some insight into tentative structure-activity relationships. the characterisation of these structurally complex compounds via a combination of multidimensional nmr and tandem mass spectrometry will also be presented. conjugation of peptides with different moieties, such as peg, lipids, carrier proteins and toll-like receptor ligands is an established approach to improve their pharmacokinetic profile for drug use and/or to enhance their immunogenicity as subunit vaccines. the development of suitable conjugation strategies for peptides of any complexity is therefore fundamental for their effective use in human therapeutic applications. here we describe our strategy to engineer a trimeric coiled coil to obtain a covalently linked, structurally stable construct endowed of extra functionality for further derivatization. we previously showed that covalent stabilization of the designed trimeric coiled coil izn , by interchain disulfide bonds, yielded an extremely potent and broad inhibitor of viral infection, (ccizn ) , [ ] . this potent inhibitory activity makes (ccizn ) not only attractive as an antiviral compound, but also as an immunogen to elicit a neutralizing antibody response [ ] . we have now developed an alternative synthetic strategy to obtain the covalently-linked izn trimer, which allows the presence in the molecule of a free thiol for subsequent chemoselective reactions. first, we showed that stable interchain thioether bonds can be effectively substituted for the disulfides. second, we devised an orthogonal cysteine protection scheme which allows formation of the thioether bonds, while leaving an extra free cysteine on demand. this thiol group can be used for conjugation of the trimeric coiled coil to adjuvant/carrier proteins or, as reported more specifically here, to a peg kda moiety. human igg is a most abundant type of immunoglobulin in serum and most of antibody drugs applied for therapy of cancer and autoimmune diseases also belong to this group of human immunoglobulin. specifically binding peptide to human igg is very useful for detection and purification as an affinity ligand of human igg. although some previous reports described such peptides, we tried here to isolate high-specific and high affinity ligands by employing random peptide-displayed t phage library. our t random peptide phage library possesses a total diversity of powered th consisting of different sequence length constrained by disulfide bond. by biopanning against human igg, we isolated several igg specific clones from our library. the peptides displayed on these phages shared some common sequences in the limited region surrounded by cys residues, which suggests they are essential for binding. these clones bound only to fc region of igg and did neither other types of human immunoglobulin nor igg of other animals. a synthetic peptide derived from a phage clone showed a sub-nanomolar of kd value in binding to human igg fc on spr analysis. these results indicate that the peptide motifs obtained here are a strong candidate of human igg-specific affinity ligand for detection and purification of igg. therefore, we are now going on constructing detection and purification system using modified and improved peptide motifs. synthesis of glp- analogues as potential agents for blood glucose control p. kanda, r.p. sharma, c.p. hodgkinson in this study, we synthesised a panel of glp- analogues stabilised against dpp-iv degradation through either selective amino acid substitutions for ala , or introduction of amide bond surrogates into the peptide backbone between ala and glu . each was made by standard fmoc or boc chemistry, purified by hplc, and characterized by electrospray mass spectroscopy. all derivatives except one bearing a hydrazine modification were stable to dpp-iv proteolysis for up to hours at ph . , o. each was tested for its ability to augment insulin release from a glucose-sensitive, murine insulinoma-derived tc- cell line in culture. it was found that each compound acted as a glp- agonist to varying degrees, with some exhibiting higher activity than native glp- toward promoting insulin release. the most active analogues have been chosen as candidates for stabilisation against renal clearance in efforts to develop new glp- analogues with therapeutic potential. the high propensity of the glucose regulatory hormone human islet amyloid polypeptide (iapp) to misfold and aggregate into cytotoxic beta-sheets and fibrils is strongly associated with beta-cell degeneration in type ii diabetes (t d) and precludes its pharmacological use for the treatment of diabetes. iapp analogs that combine solubility, lack of cytotoxicity, and bioactivity with the ability to block iapp aggregation and cytotoxicity could thus be of high biomedical interest. here we apply a minimalistic conformational restriction strategy to redesign the extremely insoluble and amyloidogenic -residue iapp sequence into a soluble, nonamyloidogenic, noncytotoxic, and bioactive iapp mimic (yan et al., pnas ( ) ). the designed mimic has nearly the same sequence as iapp but is highly soluble, nonamyloidogenic, noncytotoxic and a full iapp receptor agonist. in addition, the mimic binds with high affinity iapp and blocks and reverses with nanomolar activity its cytotoxic self-assembly which makes it to the most potent known iapp cytotoxic self-assembly inhibitor. due to its bifunctional nature, the mimic might find therapeutic application for the treatment of diabetes both as an inhibitor of amyloid cytotoxicity and as a soluble iapp receptor agonist. our findings offer a proof-of-principle of a chemical design approach for generating a novel class of highly potent inhibitors of polypeptide cytotoxic selfassembly which are nonamyloidogenic mimics of the native amyloidogenic sequence as well. such reengineered biomolecules -the design of novel mimics is in progress-are of high biomedical significance for understanding the mechanism of protein aggregation diseases and for the development of prospective therapeutic treatments. peptides that are capable of targeting abnormal changes of living tissue can be very useful in early detection or diagnosis of, e.g., cancer. conjugating a functional agent, an effector unit, to such a peptide may provide the agent with improved pharmacodynamic properties.the specificity of a thiol group for reactive groups offer a unique way to attach effector units to cysteine-free linear or cyclic peptides. tumor targeting peptides were synthesized by fmoc-type solid phase methods. peptides cyclic by cystine were modified by lactam bridges. fluorescein, metal (e.g. lanthanide) chelates and cytotoxins were coupled to tumor targeting peptides via e.g. a peg-type spacer, or the conjugates were immobilized on plates for adhesion assays. the method was uncomplicated and gave stable conjugates with good solubility. the approach is useful in making stable peptide-effector conjugates and sets of them for e.g. detection assays such as delfia method and has prospective use in development of diagnosis and therapy. antibacterial proline-rich peptides -synthesis and antibacterial activity d. knappe, r. hoffmann the antibacterial proline-rich peptide family, originally isolated from insects, shows remarkable activity against diverse bacterial and fungal pathogens. while more and more bacterial pathogens become resistant to common drugs, this part of insect innate immunity provides a new promising approach to develop future peptide-drugs. proline-rich peptides possess a significant sequence homology and share a common mechanism of action. in addition oglycosylated threonine residues of drosocin and formaecin appear to be necessary for full antimicrobial activity, although the significance of the carbohydrate moiety in interaction with intracellular targets is still unknown. we synthesized analogues of different antibacterial peptides on solid phase by the fmoc-tbustrategy. the combination or insertion of sequence regions from different native antibacterial peptide sequences offers several advantageous effects, including further reduction of toxicity and broadening of the antimicrobial activity. furthermore, mimicking the o-glycosylation site and changing the carbohydrate moieties, may yield new synthetic approaches to increase both the activity and the selectivity of these oligopeptides. new immunotherapeutic approaches have been developed for treatment of neurodegenerative diseases of the alzheimer's dementia (ad) type. the identification of a ß-amyloid-plaque specific epitope, aß( - ) (frhdsgy) [ ] , recognised by therapeutically active antibodies from transgenic ad mice, provides the basis for development of new ad vaccines and for molecular ad diagnostics. in order to produce immunogenic conjugates, the aß - epitope was attached via thioether linkage to synthetic carriers of well-defined structures, such as tetratuftsin derivative (ac-[tkpk(clac)g] -nh ) and its elongated version by a helper t-cell epitope (ac-fflltriltipqsld-[tkpk(clac)g] -nh ); sequential oligopeptide carrier (ac-[lys(clac)-aib-gly] -oh) and multiple antigenic peptide (clac-lys(clac)-lys(clac-lys(clac))-arg-arg-ßala-nh ). the epitope conjugates containing a cysteine residue either at the c-or n-terminus, and the chloroacetylated carriers were prepared by spps according to boc/bzl strategy. conjugation reactions were performed in solution under slightly alkaline conditions, and monitored by hplc and high resolution-ms. structures and molecular homogeneities of all epitope peptides, carriers and conjugates were ascertained by hplc, maldi and esi-fticr-ms. conformational preferences of the synthesized compounds in water and in tfe were examined by cd spectroscopy. comparative binding studies of the conjugates with a mouse anti-amyloid protein beta-( - ) monoclonal antibody were performed by indirect elisa. experimental data showed that the chemical nature of the carrier, the epitope topology and the presence of a pentaglycine spacer between the epitope peptide and the carrier, had significant effects on the antibody recognition and on the secondary structures of the conjugates. the new cla analogue , containing ethylene bridge between phe nitrogen atoms, was found to exhibit unexpected stimulatory effect in the model of the in vitro humoral immune response in mice. to disclose the structure-activity relationship the nmr solution conformational analysis was carried out. the solid-state and solution conformational analysis of native cla indicated the existence of this cyclic system as a complex mixture of conformations [ ] . the nmr spectra recorded at mhz in chloroform at k showed the different conformational behaviour of both cyclopeptides: cla exists as one isomer [ ] , peptide is in an equilibrium among at least of three conformers. the picornaviruses are small nonenveloped rna viruses with a single positive strand rna. the virus replication cycle starts after the penetration of the virus in the cytoplasm of the host cell. there are several stages of the virus life cycle used for attack. one of the most useful strategies for attacking of the virus includes inhibition of important for the virus lifecycle enzymes. the key enzymes in the replication of the picornaviruses are c and a proteases. changes in the active center of these enzymes make them incapable to produce polyprotein in vitro, therefore the inhibition by low molecular weight molecules could stop the viral replication in vivo. c protease plays a major roll in the enzymatic proteolysis of the initial viral polyprotein. the target compounds were based on structural modifications in the known as crucial for the cp inhibition activity dipeptides phe-gln by incorporation of additional amino acid and pyrrole moiety. the synthesis was cared out as multiple-peptide synthesis in parallel using stepwise spps, fmoc-strategy. the obtained compounds were tested for antiviral activity by agar-diffusion plaque inhibition test against coxsackievirus b replication in fl cell and on this base some structure-activity interpretations were made. histone deacetylases (hdac) play important roles in various aspects of regulation such as proliferation, differentiation, and aging by counteracting with histone acetyl transferases. the hdacs categorized in class-i and ii have a metalloprotease-related mechanism in its catalytic activity. these enzymes could be inhibited by small molecules bearing various zinc ligands such as hydroxamic acid and mercaptan. based on the structure of chlamydocin, which has a cyclic tetrapeptide framework, cyclo(-l-aoe-aib-l-phe-d-pro-), where aoe is ( s, s)- -amino- -oxo- , -epoxydecanoyl, we have developed potent hdac inhibitors as shown in the figure. in the present study, we examined the effects of the chlamydocin hydroxamic acid ( ) and ss-hybrid ( ) on the diabetes model mice, kk-ay. the peptide ( ) exhibited satisfactory activity to reduce both the blood glucose and blood insulin levels comparable to or even superior to those by pioglitazone, a pparγ agonist. the ss-hybrid ( ) , which is expected to be reduced inside of cells to generate the corresponding thiol-containing cyclic tetrapeptide, also showed a significant effect but less than ( ). the effect was dose-dependent from mg/kg to mg/kg. the effects of hdac inhibitors were also confirmed by the observation of in vivo histone hyperacetylation induced in the lymphocyte cells. synthetic peptides have a number of advantages over current vaccines. however, exploitation of synthetic peptides as vaccines has been limited by the small size; copy number, inefficient delivery, poor peptide immunogenicity and mhc restriction. we have developed chemical methodologies that overcome these limitations by synthesising and polymerising vinyl-peptides. protective b-cell and t-cell peptide epitopes from the oral pathogen porphyromonas gingivalis were identified for three different mhc restricted mouse strains using pepscan techniques. fmoc chemistry for spps was used to synthesize these peptides and a vinyl moiety was incorporated at the n-terminus using acryloyl chloride. after rp-hplc purification free radical polymerisation using ammonium persulphate and temed was used to polymerise the vinyl-peptides in the presence of either acrylamide or other vinyl-monomers to produce single peptide or multi-peptide polymers. size exclusion chromatography indicated that the peptide polymers were > million da. the peptide polymers were used to immunise each mouse strain (balb/c, cba and c bl ) and the t-cell response induced was evaluated using proliferation and cytokine (elispot) assays. the peptide polymers were found to be highly immunogenic, the single peptide polymers were found to only induce a response in their respective mouse strain, however, the multi-peptide polymer containing all of the t-cell epitopes was found to induce a response in all three mouse strains. in conclusion, our data shows that the polymerisation method overcomes all of the limitations in developing a peptide vaccine and most importantly that of mhc restriction. cytotoxic substances are auspicious weapons in tumour therapy. this compounds inhibit cell division and proliferation, hence, they affect all cells that are able to divide. however, all these compounds act intracellularly, i.e. at first they have to enter the tumour cell efficiently. this is a serious obstacle when using highly effective cytostatica and the cause of severe adverse effects by using higher doses. our aim is to overcome this problem by using synthetic hybride molecules composed of the cytostatic agent, in our case derivatives of arglabin, covalently linked to shuttle peptides. in order to identify the most effective possibility we tested two different strategies. by using the peptide hormone npy, whose specific y receptors are often overexpressed by tumour tissues, we intended to address the chemotherapeutic selectively to y receptor expressing tumour cells via receptor mediated endocytosis. on the other hand, the cytostatic agent was covalently bound to a cell penetrating peptide derived from human calcitonin (hct). recently, a c-terminal fragment of human calcitonin was found to internalize into excised nasal epithelium while the receptor activating n-terminal part is lacking. for the class of hct derived carrier peptides previous studies suggested a receptor independent "lipid raft" mediated endocytotic mechanism of uptake. here we present comparative data investigating both strategies, the highly selective receptor mediated delivery and the highly efficient receptor independent delivery. we investigated the peptide uptake by various cell lines and examined the release of the cytostatic agents inside the cells and its toxic effects. background and aims: synthetic peptides provide a straight-forward access to rationally designed inhibitors of molecular interactions based on structural information of proteins. poor membrane permeability may be overcome via cell-penetrating peptides, but low stability remains a major drawback. an increase of stability and bioactivity of peptides by coupling to polymers is intended. methods: for the development of peptide-functionalized cell-penetrating polymer conjugates, peptides were coupled chemoselectivly to hpma (n-( hydroxypropyl)methacrylamide, average mw . kda), as an inert backbone polymer by native chemical ligation. hpma is water soluble and its capacity in drug delivery has been demonstrated. peptide-functionalized polymers and free peptides were incubated with proteases or cell lysates and proteolytic break-down was determined by fluorescence correlation spectroscopy (fcs) deriving information on the number and size of fluorescent particles based on temporal fluctuations of a fluorescence signal caused by diffusion of particles through a femtoliter-size confocal detection volume. the diffusion time depends on molecular size. therefore this technique is suited for the detection of proteolytic fragments. results: efficient chemoselective conjugation of unprotected fluorescein-labelled peptides was accomplished by means of native chemical ligation. our fcs investigations revealed that conjugation of peptides to hpma increased their biostability. this data also indicate that this effect is peptide-dependent. a proapoptotic peptide coupled to hpma and introduced into mammalian cells by electroporation retained its bioactivity. cofunctionalization of hpma with this peptide and nonaargine yielded an efficient cellular import. macrolides, a rather large group of natural or semi-synthetic antibiotics, are widely known translation inhibitors whose structure is based on - -member lactones with carbohydrate substitutes attached. macrolides bind to ribosomal tunnel (rt) in a way that their lactone ring is located orthogonally to the long axis of the rt, covering most of its cleft. carbohydrate residues of the macrolides are spread along the walls of the rt. hence, the mechanism of protein synthesis inhibition by macrolides relies on the mechanical obstruction they provide to the passage of nascent polypeptide chain through the rt. the goal of this study was to design and obtain peptide derivatives of macrolides interesting both as antibacterial agents and potential probes for investigation of nascent peptide chain topography in the rt. tylosin ( in order to reach target sites inside the cns, neurotherapeutic candidates must overcome the blood-brain barrier (bbb). while several transport mechanisms occur at the bbb, this work has focused on the passive diffusion mechanism. the prediction of a peptide's ability to cross the bbb is not a simple task; hence there exists the need for the rational study of the relevant factors that affect the movement across this physiological barrier. here we present two new approaches based on in vitro non cellular assays for this type of studies. firstly, the evaluation of compound mixtures on parallel artificial membrane permeability assays (pampa). this approach increases the throughput of the study and structure-activity relationships can be easily establish. secondly, the transport and biological activity evaluation in a single assay. this second approach has been applied to the search of inhibitors for cns proteases involved in different neurological diseases (such as prolyl oligopeptidase for schizophrenia) able to cross the bbb. these two new approaches allow assaying compound's permeability in the early stages of a drug development project, and then designing novel analogues with improved bbb transport properties or using blood-brain-barrier shuttles for their delivery. two newly synthesized compounds are derivatives of l-valin and are positional isomers of nicotinic (m- ) and isonicotinic acid (p- ). these functional groups, as well as established good lipid solubility suggest that the main target for their biological action probably will be central nervous system. the presence of aminoacid l -valin, supposes their low toxicity, confirmed by our earlier experiments. the aim of the present work is to study their pharmacological activity as putative drugs. methods: male albino mice ( - g, in groups) were used. next neuropharmacological parameters were studied: analgesic effect (acetic acid test), neuromuscular coordination (rota-rod test), orientation ("hole board" test) and learning and memory (passive avoidance-step down test). results: significant analgesic effect of both compounds was established (more pronounced by dose mg/kg i.p.). slight depressing effect on the orientation reactions in animals was registered, but the neuromuscular coordination and locomotor activity of treated animals were not changed. good dose-dependent effect on learning and memory was established and М- had stronger effect than Р- . the compounds modified the effects of some model substances with central nervous activity. hexobarbital sleeping time was significantly prolonged by p- , but was antagonized by m- . pentileneterazole threshold was increased significantly and suggests some anticonvulsant activity of both compounds. conclusion: as positional isomers m- and p- demonstrated some variations in their pharmacological activity probably due to the differences in their kinetics, metabolism and excretion. registered significant neuropharmacological activity, accompanied by low toxicity motivates the new synthesis and future experimental studies. the glyproline family of regulatory peptides includes pro-gly-pro, gly-pro, pro-gly and also the simplest peptides with hyp substituted for pro. a distinctive feature of these peptides is that they exhibit a broad spectrum of biological activities: the antiulcer activity, the inhibition of blood clotting and thrombosis, the reduction of degranulation activity of mast cells, and the normalization of stressogenic behavioral disorders. alzheimer's (ad) and parkinson's (pd) disease are progressive neurodegenerative disorders which are characterized by amyloid plaques. the main components of the plaques are β-amyloid peptides (aβ - and aβ - ) and α-synuclein. we have previously shown that small peptides structurally related to the sequence of aβ( - ) protect against the neurotoxicity of aβ peptides. recent studies by other groups have shown that β-synuclein can counteract the aggregation of α-synuclein in the neurodegenerative process of pd, hereby might protect the central nervous system from the neurotoxic effects of alpha-synuclein. it was found that a tetrapeptide (kegv) protected against the neurotoxicity of aβ peptides in vivo. based on the previous findings, the following sequences of β-synuclein have been synthesized: kegv-nh and kegv-oh. after comparing the sequences of α-and β-synuclein, we found common sequences which are keqv, regv, kqgv, keqa. we have synthesized these tetrapeptides in amide forms at their c-termini. the peptides have been synthesized on mbha resin using a manual solid phase peptide synthesis equipment and boc-chemistry. the neuroprotective effects of peptides have been investigated in vitro in mtt test in differentiated neuroblastoma cell culture (sh-sy y) and in electrophysiological test on rats using multibarrel electrodes in vivo. the neuroprotective peptides might stop neuronal death and can influence ad and pd progression. the present study was carried out to determine antinociceptive effect in vivo of plant peptide hormone phytosulfokine-alpha (h-tyr( -so h)-ile-tyr( -so h)-thr-gln-oh ) (i) and its selected analogues, such as h-phe( -no )-ile-tyr( -so h)-thr-gln-oh (ii), h-d-phe( -so h)-ile-tyr( -so h)-thr-gln-oh(iii), h-tyr-ile-tyr( -so h)-thr-gln-oh(iv), h-tyr( -so h)-ile-tyr-thr-gln-oh(v) and h-tyr-ile-tyr-thr-gln-oh (vi) in rats. peptides were injected into the lateral brain ventricle at the dose of nmol. in the preliminary investigation we found the psk-as well analogues ii and iii induced a significant antinociceptive effect determined in the test of hot plate. the probable mechanism of this effect was discussed. a.b. bozhilova-pastirova , b.a. landzhov , p.v. yotovski , e.b. dzambazova , a.i. bocheva the tyr-mif- family of peptides (tyr-mif- `s) includes mif- , tyr-mif- , tyr-w-mif- and tyr-k-mif- , which have been isolated from bovine hypothalamus and human brain cortex. all these peptides interact with opioid receptors and in addition bind to non-opiate sites specific for each of the peptides. data in the literature suggest that tyr-mif- `s have antiopioid and opioid -like effects. we used wistar rats to study distribution and density of the tyrozine hydroxylase (th) imunoreactive fibres and nadph-d reactive neurons in the rat ventral and dorsal striatum. our results showed that neuropeptides mif- and tyr-mif- may affect them. opioid peptides have been recognized as modulators of reactive oxygen species (ros) in mouse macrophages and human neutrophils. data in the literature suggest that peptides of the tyr-mif- family -mif- and tyr-mif- have antiopioid and opioid -like effects. these neuropeptides are isolated from bovine hypothalamus and human brain cortex. so far no data about direct scavenger properties of tyr-mifs peptides were available. in this study we tested the hypothesis that they may scavenge ros in vitro. the antioxidant activity of these two substances was studied in the concentration range of - - - mol/l. we investigated the luminol-dependent chemiluminescence to test their ability to scavenge the biologically relevant oxygen-derived species: hydroxyl radical, superoxide radical, hypochlorous acid in vitro. we found that tyr-mif- was a powerful scavenger in all tested systems. the effects were higher for hypochlorous anion and weaker for superoxide radical. mif- had no scavenge activity against the hydroxyl and superoxide radicals and showed a moderate scavenger effect on hypochlorous anion. we have compared different strategies to increase the immunogenicity of an antigenic hiv peptide as a vaccine candidate. our selected b-cell epitope comprises amino acids ( - ) of the v region of hiv- , jy isolate (subtype d), and is in tandem with a t-helper epitope corresponding to the - region of tetanus toxoid. several presentations, including oligomerization, map dendrimer, conjugation to dextran beads or to other macromolecular carriers, have been synthesized and evaluated. murine sera from the different presentations of the v epitope have been compared with regard to antibody titers and cross-reactivity with heterologous hiv subtypes. the map dendrimer version of the peptide, conjugated to recombinant hepatitis b surface antigen protein, was a better immunogen than the dendrimer alone, and showed higher immunogenicity than other multimeric presentations, or than the peptide alone conjugated to dextran beads. the map dendrimer version, either alone or conjugated to hbsag, enhanced cross-reactivity towards heterologous v sequences relative to monomeric peptide. group a streptococcus (gas) responsible for critical diseases (eg. acute rheumatic fever and rheumatic heart disease) are classified over serotypes according to their surface virulence m proteins. development of vaccine to prevent infection with gas is hampered by the widespread diversity of circulating gas strains and m protein serotypes, and multivalent vaccine strategy would contribute to prevention against various gas infections and provide better protective immunity. we have studied the efficacy of incorporating four different epitopes derived from gas m protein into a single synthetic lipid core peptide (lcp) construct, in inducing broadly protective immune responses against gas following parenteral delivery to mice. peptide vaccine was synthesized on mbha resin by manual spps in situ neutralization/hbtu activation in boc-chemistry. immunisation with the mono-or multi-epitopic lcp vaccine led to high titers of antigen-specific systemic igg responses, and the production of broad protective immune responses as demonstrated by the ability of immune sera to opsonise multiple gas strains. systemic challenge of mice after primary vaccination showed that mice were significantly protected against gas infection in comparison with control mice demonstrating that vaccination stimulated long-lasting protective immunity. these data support to the usefulness of lcp system in the development of synthetic multiepitope vaccine to prevent gas infection. glycoprotein d represents a major immunogenic component of the virion envelope of herpes simplex virus and able to induce high titres of neutralizing antibodies. one of its optimal epitopes is the - region ( lkmadpnrfrgkdl ). several cyclic peptides possessing thioether bond and different ring size have been already prepared and some of them were conjugated with tetratuftsin derivative (ac-[tkpkg] -nh ) by thioether bond formation using selectively removable cys protecting groups. antibody recognition results suggested that the size of the cycle has considerable influence on antibody recognition, however, the replacement of met in position by nle is permitted. conjugation of cyclic peptide might increase the antibody recognition, but the binding depends on the structure and/or conjugation site of the cyclic peptide. conjugate with the best binding capacity ( . pmol/ ul) as well as the conjugate containing the linear ( - c) epitope ( . pmol/ ul) were selected for immunization. in order to increase the production of antibodies a new group of conjugates was prepared. in these constructs promiscouos t cell epitope peptide derived from tetanus toxoid (ysyfpsv) was attached to both amino groups of lysine residue coupled to the n-terminus of the carrier (ac-ysyfpsv-k(ac-ysyfpsv)-[tkpk(clac)g] -nh ). the cys containing linear and cyclic epitope peptides were conjugated to the carrier in solution ( . m tris buffer, ph ). this work was supported by grants of the spanish-hungarian intergovernmental program and cost chemistry action. background. primary hyperparathyroidism (pht) is characterized with increased parathyroid hormone (pth) secretion and in % of pht patients with hypertension. it was previously shown that pro-analogue of pth with a reversed sequence (which include strong alkali sequence -arg-lys-lys-) induced significant hypertensive response at dose - m/kg b.w. one of the hypothesis attributed hypertension in pht patients to the presence of fragments of degraded pth possessing -arg-lys-lys-sequence. aim. to compare influence on mean arterial blood pressure (map) of analogue of - pth fragment (amide) and - fragment of pth, with -arg-lys-lys-sequence and also responsible for binding to pth receptor. methods. chosen peptides were synthesized manually by a solid phase peptide synthesis method. the purity of the products was tested by reversed-phase high performance liquid chromatography. the synthesized peptides showed the right molecular mass. the influence on map of synthesized peptides was tested in wistar rats. sequential increasing boluses of each peptide: - , - and - m/kg.b.w. in the same animal were given i.v. blood pressure was measured continuously in carotid artery. results. injection of synthesized analogue of - fragment of pth does not show influence on map vs. control group. synthesized - fragment of pth increased map in min. of experiment for mmhg ± mmhg vs. time of administration of first dose and for mmhg vs. control group. conclusion. it seems to be possible, that in case of alternate degradation of pth, accumulation of - fragment of pth may partially play role in the mechanism inducing hypertension in pht patients. biologically active domains of a high affinity receptor for ig Е (fcεri) were determined, the fragments - and - of the receptor. the program of research of biological properties of synthesized tetrapeptide rnwd and heptapeptide rnwdvyk included the study of their binding with ige, which was contained in standard solutions and in patients' blood serum. the binding of peptides with ige was explored by the ifa method using ige antibodies labeled with horse-radish peroxidase (hrpo). peptides in the concentration of mkg/ml were used for sorption on immunological plotting boards. higher correlation between the ige concentration and the optical density of the solution after introducing monoclonal antibodies labeled with hrpo and substrate chromogenic mixture (r= . ) was found in the diagnostic system with sorption rnwdvyk-peptide than in the diagnostic system with sorption rnwd-peptide (r= . ). similar investigations were conducted with the diagnostic systems with sorption rnwd and rnwdvyk peptides, but rwnd peptides conjugated with hrpo were used as antibodies against immunoglobulin e, instead of hrpo-labeled monoclonal antibodies. almost equal correlation was found between the concentration of ige in standard serum and serum of allergy patients with the known concentration of ige and the optical density of the solutions after introducing the rnwd peptide, conjugated with hrpo. after introducing allergy patients' blood serum in the holes on the plotting board, the heptapeptide bound . % more ige than the tetrapeptide. our experiments demonstrated a high ige binding activity of synthetic rnwd-and rnwdvyk-peptides. in this study, the information spectrum method (ism) of the ha subunit of the h hemagglutinin protein of the influenza virus, h n , of different reference isolates was performed in order to identify possible antigenic determinants resistant to virus mutations. results of this analysis demonstrated that the primary structures of ha subunit of h hemagglutinins encode a common information corresponding to one characteristic frequency in their iss, which is probably important for the biological function of these proteins, including their possible recognition by the immune proteins targeting this molecule. besides, comparison of the iss of ha proteins of h "spanish flu" and h n isolates demonstrated an informational similarity between them. based on these results, a segment of the n-terminus of ha h n was identified to play a crucial role in the inhibitory and immunological properties of all possible h n variants. the identified core segment, being highly conserved in all h strains, was selected as an antigenic determinant and coupled to the sequential oligopeptide carrier (socn), (lys-aib-gly)n, to the lys-nεh groups, in order to develop a diagnostic immunoassay and formulate a vaccine candidate for the highly pathogenic h n influenza virus. background and aims: thrombin plays a key role in various disorders such as arterial thrombosis, atherosclerosis, restenosis, inflammation and myocardial infarction. insights into the way in which thrombin interacts with its many substrates and cofactors have been clarified by crystal structure and site-directed mutagenesis analyses, but until recently there has been little consideration of how its non-proteolytic functions are performed. we investigated cardiovascular effects of seven modified proline-and rgd-containing peptides designed from three surface-exposed sites of prothrombin, corresponding to residues - , - , and - . methods: cardioprotective effects of synthetic peptides were tested on the two rat models of heart failure produced by coronary artery occlusion ( -or -min) and reperfusion ( -or -min). arterial blood pressures from left carotid artery, heart rate and ecg ii standard lead were measured throughout experiment. at the end of second experiment hearts were morphologically investigated by light microscopy and electron microscopy methods. results: on animal model with short-term ischemia investigated peptides did not protected from myocardium ischemia during occlusion, however, tp-l , bk-mc and tp-h protected rat hearts from ventricular fibrillation, contributed more significant functional recovery during reperfusion and raising survival rate. on the model with prolong ischemia, acceptable cardioprotective effect revealed tp-h and bk-mc. these peptides significantly diminished necrotic zone of left ventricle, protected hearts from ischemia-reperfusion induced functional and morphological changes. conclusions: investigated proline-containing peptides revealed activity on cardiovascular system -decreasing of blood pressure, cardioprotective properties and improved recovery from ischemia. r. mansi, d. tesauro, c. pedone, e. benedetti, g. morelli the widespread use of compounds containing the gamma-emitting radionuclide mtc in nuclear medicine for the scintigraphic imaging, as well as the recent introduction of the beta-emitting radionuclides re and re in radiotherapy, have led to a rapid development of their chemistries, in order to produce novel radiopharmaceuticals. we have developed new peptide based radiopharmaceuticals based on a scaffold in which the radioactive metal ion is complexed by two different peptides that are able to bind two target receptors (see figure) . the + mixed-ligand approach has been used for the preparation of neutral oxotechnetium(v) and oxorhenium(v) peptide complexes. the complex preparation requires the simultaneous action of a dianionic tridentate ligand and a monoanionic monodentate thiolato on a suitable metal precursor. the dianionic tridentate ligand is based on the snn donor set able to stabilize the metal complex. the chelating agent (hsc(ch ) conhch ch(co-r)nh ) was coupled step by step to a bioactive peptide synthesized on solid phase. the second ligand, based on monodentate thiolato moiety, was coupled on n-terminus of the second peptide. labelling procedures and biological tests on tumour cells overexpressing receptors are described for mtc(o) complexed by cck and octreotide peptide derivatives. background: endostatin inhibits the proliferation of endothelial cells and induces their apoptosis. the measurement of serum endostatin can predict tumor vascularity. tumor angiogenesis is a strong prognostic factor in patients with hepatocellular carcinoma(hcc). significantly high levels of endostatin were noted in patients with trabecular-type tumors , and with hepatitis infection. methods : patients with hcc, patients with git malignancies, patients with liver metastasis and without metastasis, and normal persons . all subjects were tested for alfa-feto protein (afp) , ca . , carcinoemberyonic antigen (cea), and endostatin by elisa results : endostatin in normal control persons was . ± . ng/ml with a significant elevation (p< . ) between the hcc group and all the other tested groups .afp was . ± . ng/ml in normal persons with a significant elevation between hcc and all the other tested groups ( p< . ). ca . was . ± . u/ml in normal persons with a significance elevation ( p< . ) relative to hcc., and a significance of ( p< . ) relative to git cancers with metastases. cea was tested to be . ± . µg/l in normal persons , and had a significance of ( p< . ) relative to git metastases. endostatin was elevated in of patients with git cancers not proved to have metastasis. conclusion : endostatin can be used to denote metaplasia and can also detect possibilities of metastasis or liver cell affection even before the frank development of metaplasia affibody® molecules are a novel class of affinity proteins which are generated by combinatorial engineering of the aa three-helix bundle scaffold, originating from the b domain of staphylococcal protein a. we have used fmoc/tbu chemistry for total chemical synthesis of the affibody zher : , binding with picomolar affinity to the cell surface receptor her . the synthetic protein was investigated for molecular imaging of her -overexpressing tumours. in vivo detection of her in malignant tumours provides important diagnostic information which may influence patient management. to enable gamma camera imaging of the tumours, a panel of potential mtc-chelating sequences was designed and introduced into the affibody. the well-studied tc-chelating sequence mercaptoacetyltriglycyl (mag ) was compared to serine-containing sequences with increased hydrophilicity, such as mercaptoacetyltriserinyl (mas ). the total synthetic yield was - % and the her -binding affinity of the affibody conjugates were all in the range - pm. binding specificity of tc-labelled affibody molecules was determined on her -expressing skov- ovarian carcinoma cells. all variants showed receptor-specific binding. the tumour-targeting properties were studied in skov- tumour-bearing nude mice. all conjugates demonstrated high tumour uptake, quick blood clearance and low uptake in most other organs. the biodistribution results further showed that the more hydrophilic, serine-containing chelators resulted in a reduced hepatobiliary excretion, which significantly decrease the background in the abdomen area and provide for more sensitive detection. gamma camera images of mice with grafted tumours showed clear visualization of her -expressing tumours using the mtclabelled mas -affibody conjugate, suggesting a potential future application of this agent for diagnostic imaging. antimicrobial peptides are molecules with a unique mechanism of action. they are widespread in nature and play the role of an effective weapon of innate immune system against bacteria, fungi and viruses. the purpose of this study was to investigate the in vitro activity of natural antimicrobial peptides: citropin, piscidin, protegrin, temporin, uperin and the analogues of antimicrobial peptides: iseganan, pexiganan and omiganan. the peptides were synthesized using the solid-phase method and purified by high-performance liquid chromatography. the peptides were subjected to microbiological tests [mic (minimal inhibitory concentration) and mbc (minimal bactericidal concentration)] on reference strains of bacteria, according to the procedures outlined by the national committee for clinical laboratory standards (nccls). for comparison, conventional antibiotics (vancomycin, rifampycin, piperacillin, chloramphenicol) were included in this research. both the natural antimicrobial peptides and the analogues inhibited the growth of bacteria, but at higher concentrations than did conventional antibiotics. nevertheless, both natural origin of antimicrobial peptides and their low toxicity constitute a considerable advantage and this is an argument for considering the antimicrobial peptides as good candidates for medicines. the linear hexapeptide cypate-grdspk (compound ; the cypate moiety is a near-infrared fluorescent label) whose rgd sequence was rearranged to grd showed high uptake in the αvβ integrin-positive tumor tissues in vitro and in vivo. despite low affinity of to the integrin in the binding assays, the uptake was inhibited by equimolar amounts of the cyclic peptide c(rgdfv), which possesses high affinity to αvβ integrin. these observations led to hypothesis that cell internalization of compound may be mediated mostly by only one of the integrin subunits, as the β one. indeed, blocking of αv integrin by the specific antibody did not inhibit the internalization of in tumor cells, which was in the contrast with successful blocking the cell internalization by the anti-β integrin antibody. similar results were obtained in immunocytochemical assays employing the anti-αv and anti-β integrin antibodies. also, studies utilizing the β -knockout and wild-type mouse cell lines demonstrated that deletion of the β subunit markedly decreased internalization of compound in the β knockout cells. the preferential interaction of compound with the β subunit of integrins relative to the αv subunit was supported also by molecular modeling studies. summarizing, the bulk of our experimental and modeling data emphasizes interaction with the β integrin as the primary mechanism of the uptake of cypate-grdspk by tumors. since this compound showed the superior biodistribution profile in vivo, our results may provide a strategy to image and monitor the functional status of the β integrin in cells and live animals. background and aim: a growing tumor is accompanied by tumor intoxication development. intoxication independs on tumor size and intensity of its break-up. tumor intoxication is one of variant of endogenous intoxication. concentration of tyrosine-contained peptides (tcp) in blood plasma have been proposed as biochemical marker of endogenous intoxication at different organs cancers. our aim was to determine the tcp concentration in blood plasma patients with ovary tumor and its association with the severity of tumor. materials and methods: patients with ovary tumor, mean age is years, were studied. the control group consisted of healthy women without tumor. patients were divided into groups: people with non-malignant and people with malignant ovary tumor. tcp content in blood plasma was estimated by our technique. results: tcp concentration in the control group were , ± , mmol. the tested marker was present in increased concentration in blood plasma of the patients with ovary tumor. the mean concentration tcp in patients with non-malignant tumor was , ± , mmol. the content of this marker in blood plasma of patients from second group was increased , ± , mmol compared with healthy control group. after treatment a significant decrease in tcp content was observed. conclusions: the result indicate that content tcp in blood plasma depends of the type of tumor. it could be suggested that determination of tcp concentration in blood plasma could be useful for improve the diagnostic of ovary tumor and monitoring of its progression. c. strongylis , ch. papadopoulos , k. naka , l. michalis , k. soteriadou , v. tsikaris troponin is a structural protein complex, which is responsible for the regulation of skeletal and cardiac muscle contraction. it consists of three components: troponin i ( kda), troponin c ( kda), and troponin t ( kda), each of which carries out different functions in the striated muscles. cardiac troponins are released into the bloodstream of patients after the onset of a cardiovascular damage. even minimal elevations over the normal values, of serum troponin t and i are being used to diagnose acute myocardial infarction and also to rule out the patients' condition. the development and commercialization of highly specific biological assays for the detection of cardiac troponins is based on the production of specific antibodies against the whole complex or individual subunits. however, the specificity and sensitivity of these assays vary due to problems mainly originated from the fact that cardiac troponins have a high homology with the skeletal isoforms. the aim of this work is to select and synthesize appropriate regions of the cardiac isoforms of troponin i, c and t, suitable for the production of more sensitive and specific cardiac troponin detecting reagents. in order to construct the immunogenic complexes, the selected sequences were conjugated to the tetrameric sequential oligopeptide carrier (soc ), either by the classic solid phase step-by-step methodology or by chemoselective ligation reactions. using the carrier conjugated troponin sequences, anti cardiac troponin complex specific antibodies in high titers were produced. the increasing problems with the reproductive systems of man and animals are recently linked to the presence of polluting chemicals with endocrine activity, the so called endocrine disrupting chemicals or edc's . the family of edc's is a heterogeneous one and consists of natural and synthetic hormones (like estradiol, ethynylestradiol and diethylstilbesterol), phyto-estrogens (like genistein and coumestrol) and industrial chemicals (like bisfenol-a, ftalates and various pesticides). because of the complexity of the environmental matrices and the low physiologically active concentrations of the edc's there is still a need for an efficient routine analysis protocol. we want to develop a solid-phase bound receptor that possesses a high selectivity for edc's and thus can be used in a simple solid-phase extraction protocol. this receptor must have the right functional groups that bind the edc's with a strong affinity and must be able to create a cavity in which the edc's can fit. by looking at nature's own estrogenic receptor for humans we have found the different amino acids responsible for the specific interactions . in order to create the cavity which mimics the behaviour of the hormone-binding domain of the human estrogenic receptor we have made a tripodal scaffold. this tripodal scaffold has three orthogonal protected amino groups that will allow the generation of three independent peptide chains. milk proteins are a source of opioid peptides. these peptides are liberated from milk proteins during enzymatic hydrolysis. some of these peptides are characterized with agonistic (β-casomorphins) and some with antagonistic (casoxins) properties. the aim of the investigations was to determine the presence of opioid peptides with antagonistic properties in milk products. the experimental material included cheeses, yogurts and kefirs. peptides were extracted with a methanol-chloroform mixture ( : v/v). the peptide extracts were purified by spe method on c or stratax columns and characterized by sds-page electrophoresis. the agonist opioid peptides (casoxins) were identified by hplc using standard agonist peptides. the opioid activity was measured by examining the effects of peptide extracts on the motor activity of isolated rabbit intestine. the results of sds-page electrophoresis indicated the presence of to fractions in peptide extract derived from cheeses and yogurts and to ones from kefirs. the presence of casoxin a ( . - . µg/mg of extract) was proved in all examined the milk products. lactoferroxin a ( . - . µg/mg of extract) was identified only in kefirs and yogurts. those products were also found to contain trace amounts of casoxin c. all peptide extracts showed the antagonistic activity in the relation to motor activity of isolated rabbit intestine. the highest antagonistic activity was reported of peptide extract from kefirs ( . - . %) and gouda cheese ( . - . %), as compared to morphine. the physiological and nutritional function of these antagonist peptides requires elucidation. a. péter , r. berkecz , f. fülöp the past decade has seen a growing interest in β-amino acids, which are important intermediates for the synthesis of compounds of pharmaceutical interest and can be used as building blocks for peptidomimetics. oligomers of β-amino acids (β-peptides) fold into compact helices in solution. recently, a novel class of β-peptide analogues adopting predictable and reproducible folding patterns (foldamers) was evaluated as a potential source of new drugs and catalysts. studies on synthetic β-amino acids can be facilitated by versatile and robust methods for determining the enantiomeric purity of starting materials and products. highperformance liquid chromatography (hplc) is one of the most useful techniques for the recognition and/or separation of stereoisomers including enantiomers. the aim of the present work was to evaluate hplc methods for the separation of enantiomers of eighteen -amino- -aryl-substituted propanoic acids (β-amino acids). direct separations were carried out on different macrocyclic glycopeptide based stationary phases, such as ristocetin a containing chirobiotic r, teicoplanin containing chirobiotic t, teicoplanin aglycon containing chirobiotic tag, vancomycin containing chirobiotic v columns and on a chiral crown ether based column. the effects of different parameters on selectivity, such as the nature of the organic modifier, the mobile phase composition, the flow rate and the structure of the analytes are examined and discussed. the separation of the stereoisomers was optimized by variation of the chromatographic parameters. the efficiency of the different methods and the role of molecular structure in the enantioseparation were noted. the elution sequence of the enantiomers was determined in most cases. a rational approach to evaluating peptide purity a. swietlow , r. lax amgen, inc., pharmaceutics, thousand oaks, ca polypeptide laboratories, inc., torrance, ca, usa recent years have seen an enormous increase in interest in peptide therapeutics. new peptide leads are often chosen by screening procedures using microgram to milligram quantities of peptides, frequently provided by specialized manufacturers utilizing automatic synthesizers to maximize output. the purity of the resulting compounds is often not very high. the use of spps synthetic procedures predetermines that most impurities are closely related and difficult to resolve by reversephase purification. these factors, combined with the use of generic analytical methods not specifically optimized for the peptide in question (e.g. the ubiquitous . % tfa/water/acetonitrile system), lead to erroneous results that frequently severely overestimate the purity of the peptide. the use of poorly characterized materials in pharmaceutical development leads to significant risks of obtaining false negative or false positive results that may cause potential leads to be overlooked or misinterpretation of their pharmacological profiles. we describe a rapid, systematic and reliable hplc procedure for evaluation of peptide purity. utilizing the increased separation efficiency by increasing the column temperature and adjusting the gradient in two steps in reverse-phase buffers containing tfa, naclo , or ion-exchange buffers containing kcl, we demonstrate that methods -suitable for preclinical research -can be developed rapidly. the proposed approach will be illustrated with examples of peptides ranging between and amino acids and a model peptide vypnga. it will be demonstrated that peptides showing an hplc purity close to % are often - % less pure. to approach a high-throughput cell assay format using peptides, we attempt to design and construct a peptide microarray for examination of cell activities of peptides including apoptotic cell death. peptides were immobilized onto solid surfaces via a novel multi-functional linker. the linker enable us to examine various types of peptide cell assays in an array format. we also designed and synthesized peptidyl capture agents on the basis of the cell-active sequences suitable for the peptide microarray. the utility of targeted nanoparticles as fluorescent probes for tissue imaging has recently been subject to widespread interest. one exciting prospect is the further development of nanoparticles conjugated to both targeting peptides and cytotoxic cargoes. these nanodevices could preferentially bind to specific cells and/or tissues to provide effective tools for drug delivery. hence, such multifunctional nanoparticles could provide both diagnostic and therapeutic functions by acting as fluorescent probes that offer targeted delivery of therapeutic agents. we have coated the surface of quantum dots (qdots) with cell-penetrating peptides (cpp) to target and label u m cells for fluorescence imaging. qdots were initially coupled to polyethylene glycol linkers via carboxyl functionalities on their surface. a heterogeneous mixture of poly-arginine peptides of varying lengths (arg( )-arg( )) were covalently coupled via amide bonds to the polyethylene glycol linkers, conferring a cell penetrating capacity to the modified qdots. fluorescence imaging of u m cells, after incubation with the conjugated qdots at concentrations of nm, gave clear signals indicating cell binding and internalisation of the modified qdots across the plasma membrane. we aim to further expand this work by employing racemic mixtures of cpp and cytotoxic agents to engineer conjugates that will facilitate both imaging and the therapeutic delivery of cytotoxic moieties. the ability of cell penetrating peptides (cpp) to deliver biologically active cargoes into different cell types has been successfully applied in several experimental systems. despite the progress and growing number of described cpps, reports about the internalization mechanisms and the intracellular routes of cpps still remain controversial. we have characterized the membrane interaction and cellular localization of proteins delivered into hela cells by cell penetrating peptide transportan (tp) and its shorter analogue tp on ultrastructural level. our previous results obtained by transmission electron microscopy showed that complexes of transportans with gold-labeled streptavidin translocated into cells inducing large invagination of plasmamembrane, suggesting the uptake by macropinocytosis. the complexes of transportan with gold-labeled neutravidin, in contrary, were taken into cells mostly via caveosomes and clathrin-coated vesicles. the cell-transduced transportanprotein complexes localized mainly in the vesicular structures of different size and morphology. most of the complexes-containing vesicles in the perinuclear area contained also lamp protein, marker of late endosomes and lysosomes. still, the transportan-protein complexes were not confined in the membrane-surrounded vesicles, but spread in the cytosol suggesting the escape of transportan-protein complexes from endosomes. our findings show the involvement of different endocytic pathways in the transportan-mediated uptake process of proteins. the concentration of a cpp and the properties of cargo protein seem to determine the pathway for the cellular uptake of a particular construct. rgd peptides (r = arginine; g = glycine; d = aspartic acid) have been found to promote cell adhesion upon interaction with alphav-beta receptors, which are strongly overexpressed during neoangiogenesis by solid tumor associated cells compared to healthy cells. in this study we designed new targeting motifs aimed to deliver various antitumoral drugs specifically to cells involved in tumor vascularization. we inserted this short rgd sequence in tetracyclopeptides closed with various means. we expect these new cyclotetrapeptides to be more specific for the targeted receptor. moreover, these new type of cyclic peptides were multimerized on different scaffold to further improve the receptor avidity. our purpose is first to scrutinize and to quantify the efficient cellular uptake of these molecules and second, to address the specific cell targeting of a fluorescent cargo by differents tools such as fluorescence activated cells sorting (facs) analysis or fluorescence microscopy. these new targeting units were evaluated on two different cell lines: human umbilical vein endothelial cells (huvec) with an over expression of the alphav-beta integrin receptor and a cells expressing a much lower level of this receptor. preliminary results about the selectivity and the efficacy of these new targeting units will be presented. we have recently developed new approaches for obtaining highly immunogenic peptide conjugates: synthetic polyelectrolytes (pe) were used for the conjugation with peptide molecules in which pe carry out the carrier and adjuvant roles simultaneously. in this study, epitopes of antigenic parts of surface antigen of hepatitis b virus ( - , - , - and - of the s gene.) had been synthesized.the synthesis of peptides was performed by explorer pls ® automated microwave synthesis workstation (cem). peptide conjugates of synthetic anionic polyelectrolytes (copolymers of acrylic asid and n-vinylpyrrolidone) were synthesized by carbodiimide condensation following the modification procedures described early. composition and structure of bioconjugates were characterized by hplc (shimadzu), nanospr- , zetasizer nano zs, steady state fluorescence spectrometer qm- and viscotek tda size exclusion chromatography. it was obtained that a single immunization of mice with pe-peptide conjugates without classical adjuvant increased the primary and secondary peptide-specific immune response to hbsag. moreover, these conjugates possess own selectivity for recognizing the antibody in blood sera of hepatitis virus injected people. tissue engineering requires delivery of transplanted cells to organ sites needing repair/regeneration. we have demonstrated that several active laminin peptide-conjugated chitosan membranes enhanced the biological activity and promoted cell adhesion in a cell-type specific manner. the most active laminin peptide (ag : rkrlqvqlsirt)-conjugated chitosan membrane could deliver keratinocytes to a wound bed. when human keratinocytes were seeded onto the ag -chitosan membranes under serum-free condition, more than % of the cells attached within hrs. the membranes carrying keratinocytes were stable enough for handling with forceps and were inverted onto the muscle fascia exposed on the trunk of nude mice. keratinocyte sheets were observed after days and colonies appeared after days on the fascia of host mice. these cells were multilayered on day- and expressed various keratinocyte markers, including cytokeratin- , involculin, and laminin ? -chain. these results suggest that the ag -conjugated chitosan membrane is useful as a therapeutic formulation and is applicable as a cell delivery system, such as delivering keratinocytes to the wound bed. the peptide-chitosan approach may be a powerful cell transplantation tool for various tissues and organs. the fluorescent semi-conductive (cdse/zns) nanocristals possess very attractive optical properties that could be used for tracking individually biological receptors in vivo. our aim is to design functionalized water-soluble semi-conductive nanocristals (or quantum dots) that interact selectively with lipidic or biological membranes. to valid our approach, the interaction between the decorated qd and giant vesicles were observed by optical fluorescent and dark field microscopies. in view to solubilize and selectively bind fluorescent nanocristals to a lipid membrane, heterobifunctionalized peptidic ligands (liipe) that presented an adhesion domain for the nanocristal surface, an hydrophilic spacer and a terminal recognition function, were synthetized. the colloïdal stability of the water-soluble nanocristals (nc-lipe) was checked by dynamic light scattering, optical and electron microscopies the interaction of grafted nanocristals (nc-lipe) with positive or neutral giant vesicles was observed by optical fluorescence and dark field microscopies. as shown in figure, negatively charged nanocristals (nc-lipe) selectively adsorbed onto the surface of positively charged giant vesicles without altering the morphology of the vesicle. the nanocristals appeared as fluorescent patches growing on the surface of the vesicle until completely recovering. therefore these ligands (lipe) permitted to chemically functionalize the nanocristals by keeping their colloïdal stability and their fluorescence in water. furthermore it was possible to selectively label vesicle membrane. creatine analogues for treatment of obesity: us patent bioactive peptides containing pairs of basic amino acids are rapidly metabolized as a result of cleavage by trypsin-like enzymes. to increase the metabolic stability of opioid peptides containing arg-arg and arg-lys pairs, the arg residues were replaced by homoarginine (har) kenes international leprince , f. cavelier , p. gandolfo , m. diallo , h. castel , l. desrues m new bradykinin analogues modified with -aminocyclopentane- -carboxylic acid effect on rat blood pressure and rat uterus o. labudda-dawidowska , d. sobolewski , m Śleszyńska , i derdowska synthesis and heparin binding sites identification f. baleux , f. arenzana-seisdedos chemokines are small proteins involved in numerous biological processes (inflammation, immunity, morphogenesis, tissue repair, and tumour development) the general goal of our project is to elucidate the role that hs plays in vivo in the physiologic and pathologic activities of the chemokine cxcl /stromal derived factor- , and to characterise the molecular and structural determinants accounting for the interaction. three sdf isoforms, alpha ( aa), beta ( aa) and gamma ( aa) have been identified. we previously identified the major heparin binding site on sdf alpha and demonstrated the importance of hs/sdf interaction in hiv entry cell inhibition ( , ). sdf gamma amino acids sequence corresponds to the sdf alpha sequence extended by a c-ter amino acids sequence containing putative heparin binding sites. in order to determine sdf gamma heparin binding sites, wild type and mutants proteins were synthesised by stepwise solid-phase peptide synthesis using fmoc chemistry prusiner proc. natl. acad. sci here we describe xenome's drug development process for the chi family, conopeptide -mria[ ] of the predatory marine snail conus marmoreus, leading to a suitable drug candidate (xen ). xen is highly selective for the norepinephrine transporter (net) compared to other transporters, such as dopamine and serotonin, and inhibits net via an allosteric mechanism. xen is currently in a phase i/iia clinical trial for the treatment of severe pain. an intensive synthetic analogue and screening program around -mria, incorporating early stage animal data xen isomers were synthesized via selective disulfide bond formation to identify the active connectivity. data from alanine-scans, single amino acid mutations and probing of backbone interactions combined with the full d nmr structure, led to the development of a pharmacophore for xen . this model is refined from further studies where structure-activity relationships were developed utilising binding and functional assay data for a range of peptides anti-cancer drug design anti-cancer drug design published structural data for hdac-like protein, a bacterial enzyme sharing high homology to the hdacs in its active site, confirmed that this protein contains a zinc in the active site. for the discovery of specific hdac inhibitors, a number of hydroxamic acis and related compounds have been designed based on the ligating function to the zinc atom. the mechanism also involves an appropriate nucleophile in the active site. chlamydocin is a cyclic tetrapeptide on the other hand, we have been focusing the cyclic tetrapeptide to develop the potent and specific inhibitors of hdacs. in the present report, we employed the chlamydocin scaffold and successfully introduced the series of thioether as the functional group to the cyclic tetrapeptide. it is well argued that the strong inhibition of hdac requires the best combination of zinc ligand, capping group, and appropriate spacer between them jerusalem department of biological regulation department of organic chemistry department of biological chemistry, weizmann institute of science, rehovot, israel estrogen has a key role in the regulation of skeletal growth and maintenance of bone mass. the use of estrogen and selective estrogen receptor (er) modulators in treatment of osteoporosis is limited due to substantial risks for breast cancer. recently, we developed peptides having estrogen-like activity peptide emp- had no effect on bone growth in normal mice, and did not influence the ovx-induced bone-loss. we then developed a new µct methodology to evaluate uncalcified and calcified growth-plate parameters. in the ovx mice, peptide emp- reduced volume and thickness of the uncalcified growth-plate, a possible cause for the inhibition of bone longitudinal growth. based on a reported enhancement of er-in female mice during protein biosynthesis, after the release of the nascent polypeptide chain, ribosome recycling factor (rrf) disassembles the post-translational complex. rrf has been shown to be essential for bacterial growth. thus, we are attempting to design suitable compounds to inhibit the rrf function as candidates for new-type antibiotics. we have determined the structure of rrf with amino acid residues by nmr and x-ray analyses and shown that rrf has two domains domain i with three stranded helical bundles and domain ii with &beta;/&alpha;/&beta furthermore, we recently determined the structures of the s ribosome-rrf complex by cryo-electron microscopy and the s ribosome-rrf domain i complex by x-ray analysis using the results of these experiments, we elucidated the interaction profiles between rrf and ribosome and found that the cationic center consisting of three arginine residues on the surface of the helical bundle, which we have shown to be essential for the activity of rrf, is bound to helix - of s ribosomal rna. we synthesized the rna and peptide fragments around this interacting site and characterized them by physico-chemical analyses. the results of cd and biacore experiments to investigate the details of the interactions between them showed that a mer of rna fragment is bound to rrf <i>biochemistry</i> <b> </b> causes an increase of pain by inhibiting the opioid response [ ]. recent research has shown further that melancortin receptors, mainly subtype mc r, produce an increase in response to pain stimuli [ ]. based on this previous work, we are developing chimeric ligands which will be of benefit to therapeutic pain treatment with enhanced opioid efficacy by acting as agonists at opioid receptors and antagonists at both cck and melanocortin receptors cck (i) and melanocortin (ii) pharmacophores were overlapped by trp, and different profiles of opioid pharmacophores (iii) were linked to the n-terminal of the melanocortin pharmacophore (figure). the designed ligands showed moderate to high biological activity at all three receptors depending on their respective structures. design considerations and structure-activity relationships will be discussed in detail along with in-vivo assay results china synthetic exendin- is a -amino acid peptide that exhibits potent anti-diabetic and dose-dependent glucose-regulatory activity. exendin- is susceptible to degradation in plasma, so its activity is limited. our aim is to find sites in exendin- that are susceptible to cleavage and provide information for designing new exendin- analogues. in this study the stability of exendin- in human plasma was evaluated in vitro. exendin- was incubated in plasma at ℃, extracted with sep-pak octadecyl columns and subsequently analyzed using high performance liquid chromatography (hplc). the results showed that exendin- was slowly broken down in plasma with a half-life of . h. the degradation products were identified by quadrupole time of flight mass spectrometry (q-tof-ms) with electrospray ionization pharmacology of exenatide(synthetic exendin- ): a potential therapeutic for improved glycemic control of type diabetes one of the proposed solutions for the pharmacotherapy of obesity, a major health problem in the western world, is to regulate the biochemical pathways that control the metabolic balance in the body. the melanocortin pathway regulates energy balance by binding of the catabolic endogenic neuropeptide αmsh to its mc receptor and thus causes a decrease in food intake. we have synthesized a backbone cyclic peptide library, based on the minimal αmsh sequence phe -d-phe-arg-trp [ ], that activates the mc receptor. all the members of the library shared the same sequence analysis using colorimetric liposomes confirmed that bbc- penetrate the intestinal cells by the transcellular mechanism. moreover, bbc- have high metabolic stability to intestinal enzymes ( % recovery after hr). ec analysis showed that bbc- selectively binds and activate the mc and mc receptors (ec . ± . and . ± . respectively). oral administration of bbc- in mice showed reduces food intake melanocortin tetrapeptides modified at the n-terminus, his, phe,arg and trp positions department of experimental and health sciences graduate school of pharmaceutical sciences graduate school of agriculture graduate school of medicine consisting of amino acids, is a product of the metastasis suppressor gene kiss- . this c-terminally amidated peptide was identified as the endogenous ligand of an orphan g-protein coupled receptor metastin-gpr signaling may regulate gonadotropin secretion and negatively regulate cancer metastasis. it is interesting that activation of gpr signaling negatively regulates the function of sdf- -cxcr axis in cho and hela cell transfectants we conducted the structure-activity relationship (sar) study on kisspeptin- using the neuropeptide-derived rw-amide scaffold to identify five-residue peptide amides as novel gpr agonists equipotent to kisspeptin pro-), where aoe is ( s, s)- -amino- -oxo- , -epoxydecanoyl. in continuation of our study to design and synthesize analogues bearing a zinc ligand to develop potent inhibitors of histone deacetylase (hdac) inhibitors, we shifted the aromatic ring of phenylalanine at the aminoisobutric acid (aib) position and also at the imino acid position. the aim is to explore the interaction of cyclic scaffold with the rim of hdac paralogs. we replaced the epoxyketone moiety of aoe with sulfhydryl group, which is protected as disulfide hybrid, as zinc ligand. benzene ring was introduced to aib structure to design amino- -indane carboxylic acid and amino- -indane carboxylic acid. aromatic ring-containing imino acids, such as d-tic were also employed to replace d-pro. the cyclic tetrapeptides were profiled by the inhibition of hdac , hdac , and hdac in adult goats, however, the infection remains unapparent and the virus may cause abortion, vulvovaginitis or balanoposthitis. the use of a vaccine could provide a powerful tool for the control of cphv- infection. synthetic peptide-based vaccines have advantages of being selective, chemically defined and safe. in order to further localize immunogenic epitopes, glycoproteins b, c and d of cphv- were analyzed with several prediction programs. peptide conjugates incorporating t and b cell determinants in multiple copies in branched architecture are better immunogens for the preparation of goat vaccines, we synthesized peptide conjugates bearing t cell epitope on the n-terminal of the core. b cell epitopes were conjugated via a thioether bond on the ne-amino group of four choroacetylated lysine residues of the core. elisas confirm that the b cell epitopes and the conjugates t celltetratuftsin induce epitope-specific and antibody responses two recorded electrodes implanted bilaterally. eeg recorded after the mirror focus was arises. trh applicated intranasal in ultra low doses ( - m and - m) or intravenously in high doses ( mg, mg, mg). for eeg registration and analysis the computer system conan was used and new modification of fractal analyze of quantized eeg. results: the synchronization of epileptic activity between primary and mirror focuses observed on the third day after operation. intranasal application of trh induced reduction of spontaneous focal epileptic activity as in primary cobalt damage focus as in the mirror focus more than h. the inhibition of mirror focus was more expressed. quite the contrary intravenous trh administration provocated the epileptic discharges in both local focus. the intense synchronized generalized activity was record during - min deraos , t.v. tselios , i. mylonas , g.n. deraos it is known that cyclic peptides are more stable in enzymatic degradation and conformationally restricted compared to linear. the cyclization was achieved using o-benzotriazol- -yl-n,n,n',n'-tetramethyluronium tetrafluoroborate (tbtu) and -hydroxy- -azabenzotriazole, , , collidine allowing fast reaction and high yield final product. the purification was achieved using reversed phase high performance liquid chromatography (rp-hplc) and the peptide purity was assessed by analytical hplc and mass spectrometry (esi-ms). the synthesized cyclic plp analogue was found to exhibit lower agonist eae activity compared to linear plp - epitope in sjl/j mice. this implies that the conformation of cyclic analogue does not trigger autoimmune reaction in the central nervous system and therefore encephalomyelitis the netherlands department of experimental and health sciences on the other hand, in both central and peripheral nervous system, cck acts as neurotransmitter. recently, cck is focused at modulation effects on feeding especially. in this study, we tried to establish a sensitive and specific enzyme immunoassay (eia) for detecting cck and to investigate the effect of some dopamine receptor antagonists using this eia, we measured plasma cck-like immunoreactive substance (is) levels in five healthy human subjects after single oral administration of some prokinetics. the minimum amount of cck detectable by our eia system was . pg/ml, and the ic of the calibration curve was pg/ml. we revealed that domperidone and itopride caused significant decreases in plasma cck-is levels but metoclopramide and sulpiride did not. we established a sensitive and specific eia for cck. furthermore pro-pro-phe-phe-), isolated from linseed oil [ ], possesses a strong immunosuppressive activity comparable at low doses with that of cyclosporin a [ ]. it has been postulated that the tetrapeptide sequence pro-pro-phe-phe is important for biological activity of cla on the basis of this information we have synthesized a series of cla analogues in which the alpha-proline residue was replaced by beta -iso-proline and beta -homo-proline residues, respectively (fig. ). the synthesis of titled beta-amino acids has been achieved according to the literature procedure italy synthetic peptides are largely used as antigens in solid-phase immunoenzymatic assays (elisa) for recognition of antibodies (abs) in biomedical research and, most importantly, in the set up of diagnostic methods. it is well known that the method of peptide immobilization on the solid support is very important for a correct ab recognition atherosclerosis in patients infected with helicobacter pylori (hp) we synthesized appropriate oligopeptides immobilized on cellulose via n-or c-termini, using standard -alanine linkers as well as a new linker, developed for this particular studies glc)ghsvflapygwmvk) we found the strongest recognition when the peptide was linked to the cellulose support via the c-terminus. however, in the case of ureb f hp urease smallest epitope (sikedvqf), and epitope ub- ( - hp fragment: chhldksikedvqfadsri) the strongest reactions with sera of atherosclerosis suffering patients were obtained for n-terminally anchored peptides the synthetic dipeptide gamma-d-glutamyl-l-tryptophan (scv- ) has been shown to stimulate t-lymphocyte differentiation and specific immune responses, and enhance il- and inf-gamma production. due to this preferential activation of th cytokine production, scv- may show utility in treatment of infectious diseases cba mice at a dose of , µg/kg. the same animals were used for all three methods of administration with a dosing interval of weeks. blood samples were taken from the right retro-orbital sinus. for determination of the scv- concentration in blood samples, an "eia-scv- " competitive solid-phase immuno-enzyme assay was developed (loq ng/ml) with a mean residence time of minutes. minutes postdose, indicating very rapid absorption. mean concentrations then declined and were measurable through . and hours postdose (mrt and minutes, for i/p and p/o, respectively). the estimated bioavailability of scv- after i/p and p/o administration was almost equivalent gastrin- (g ) is a peptide which promotes gastric acid secretion, cell proliferation, and occasionally gastrointestinal cancer in the gastric antrum. g also promotes the growth of cancerous colonic epithelial cells, but the cck /gastrin receptor, which mediates its activity, is largely not expressed on such cells. instead, our previous studies have shown that some other receptor mediates stimulation of proliferation of dld- and ht human colonic carcinoma cells by ncarboxymethyl gastrin (g gly) at namomolar concentrations and inhibition at micromolar concentrations, indication separate binding sites. we have shown previously that g ( - )-oh stimulates cell proliferation of ht- cells - )-nh , in order to determine their selectivity for and activation of the putative proliferation-stimulatory receptor. the results revealed that g ( - )-oh is not selective for a single receptor, but binds both sites as do g and g gly. g ( - )-nh promotes dose-dependent and non-biphasic proliferation of dld- cells and binds a single receptor with low affinity. m comparative study of ige-binding activity of synthetic peptides rnwd and rnwdvyk v th involvement of l-name in the antinociceptive effects of newly synthesized analogues of tyr-mif- peptide in rats tyr-mif- is able to interact with opioid receptors with a higher potency at m sites as well as to its specific non-opiate receptors in the brain. nitric oxide and tyr-mif- `s are potent modulators of opioid activities. involvement of no in nociceptive effects is well documented in various physiological and pathological processes in the cns. l-name when administrated i.c.v. or systemically exhibit antinociceptive activity in rats as evaluated by the pp test. in the present study, we investigated the involvement of l-name in the antinociceptive action of newly synthesized analogues of tyr-mif- peptide: nα-(me)tyr-mif- , d-tyr-(me)-mif- , tyr(cl )-mif- and tyr(br )-mif- . the experiments were carried out on male wistar rats ( - g). the changes in the mechanical nociceptive greece paclitaxel is one of the most important anticancer drugs used mainly in treatment of breast, lung, and ovarian cancer and is being investigated for use as a single agent for treatment of lung cancer, advanced head and neck cancers, and adenocarcinomas of the upper gastrointestinal tract. however, the development of resistance to paclitaxel, the side effects and low solubility of this drug remain major obstacles for its optimal use in the clinical practice. in this work, we present the synthesis of various analogues in which paclitaxel is covalently bound to peptides or as multiple copies to synthetic carriers. these peptide-paclitaxel derivatives possess greater solubility in water, could be suitable in producing anti-paclitaxel antibodies and inhibit the proliferation of human breast, prostate and cervical cancer cell lines. although, no major differences were found concerning the extent of the antiproliferative effect between various paclitaxel derivatives and paclitaxel, the analogue with four molecules of paclitaxel covalently bound to synthetic carrier [ac-(lys-aib-cys) -nh ] when used at low concentrations inhibited cell proliferation more potently than paclitaxel th involvement of the histaminergic system in the nociceptin-induced pain-related behaviors in the mouse spinal cord the purpose of the present study was to determine whether histamine-containing neurons in the spinal cord are involved in nociceptin-induced behaviors in mice. the i.t. injection procedure was adapted from the method of hylden and wilcox. immediately following the i.t. injection, the time spent for nociceptive behaviors including scratching, biting and licking were measured. the i.t. administration of nociceptin resulted in nociceptive behavioral responses, which were eliminated by the i.t. co-administration of opioid receptor like- (orl- ) receptor antagonists. the nociceptive behaviors were significantly attenuated by the i.t. co-administration of the h receptor antagonists, but not the h receptor antagonists. i.t. co-administration of the h receptor antagonist significantly increased the behavioral responses, whereas the behavioral responses were completely attenuated by the i.t. co-administration of the h receptor agonist. an antiserum against histamine injected i.t. reduced the nociceptin-induced behavioral responses. the same result was observed by i.p. pretreatment with histidine decarboxylase inhibitor. in conclusion, i.t.-administered nociceptin elicits the orl- receptor-mediated nociceptive behavioral responses. the activation of the orl- receptor by nociceptin may induce the release of histamine in previous studies, we demonstrated that highly constraint cyclic (s,s)-cxaac-containing peptides inhibit platelet aggregation and fg binding [ , ]. cyclization reduces the allowed conformations, of both the backbone and the side chains, and possibly induces a favourable for the biological activity orientation of the charged side chains. conformational studies revealed that orientation of the charged side chains toward the same side of the molecule increase the anti-aggregatory activity of the inhibitor. in this work we present the synthesis and the inhibitory activity of new cyclic compounds. for the design of the studied compounds we combined the available information from the -cdc-containing inhibitors and the gpiib - (ymesradr) sequence which has been shown to inhibit the adp induced human platelet activation however, its pharmacological effects and physiological functions are still unclear. the present study was designed to characterize the nociceptive behaviours induced by intrathecal (i.t.) administration of hk- in mice. the i.t. administration was made in conscious mice according to the method described by hylden and wilcox. immediately after the i.t. administration, the accumulated time for nociceptive behaviours was measured for min. the i.t. administration of hk- dose-dependently produced characteristic nociceptive behaviours consisting of scratching, biting and licking, which peaked at - min and almost disappeared by min after injection. the subcutaneous pretreatment with morphine dose-dependently attenuated the hk- -induced nociceptive behaviours. the nociceptive behaviours elicited by low-dose of hk- were significantly inhibited by i.t. co-administration with nk receptor antagonist, however, the nociceptive behaviours elicited by high-dose of hk- were not affected by i.t. coadministration with nk receptor antagonist. on the other hand, nmda receptor antagonists significantly suppressed both high-and low-doses of hk- -induced nociceptive behaviours in a dose-dependent manner. these results suggest that the nociceptive behaviours induced by low-dose of hk- may be mediated through both nk and nmda receptors, whereas high-dose of hk- may induce the nociceptive behaviours through nmda receptor the bacterial lp are strong modulators of the innate immune system. until recently, it was generally assumed that triacylated lp, like the synthetic pam cys-sk , are recognized by tlr /tlr heteromers, whereas diacylated lp, like fsl- , induce signalling through tlr /tlr heteromers. contrary to this model, we could show that depending on the peptide moiety, diacylated lp also signal in a tlr -independent and tlr -dependent manner. the aim of this study was to analyse more closely the structural basis of this heteromer usage. the synthesis of lp was carried out by fully automated solid phase peptide synthesis and fmoc/tbu chemistry on tcp or rink amide resin. information on the structural factors determining the tlr /tlr versus tlr /tlr heteromer usage was obtained by testing of ligands with cells obtained from tlr , tlr , and tlr -deficient mice. when stimulating b-lymphocytes of wild-type mice we found that ester-bound long-chain fatty acids are necessary to induce considerable responses. for triacylated lp with long chain length ester-bound acyl residues (like pam c-ssnask ) the response in tlr -deficient cells was only slightly decreased, whereas for lp with short length ester-bound fatty acids (like pamoct c-ssnask ) the response was completely abolished. in summary, a tri-acylation pattern is necessary but not sufficient to render an lp tlr -dependent and a di naples department of organic chemistry "ugo schiff sera from patients suffering from autoimmune disorders often contain multiple types of autoantibodies, some of which can be exclusive of a disease and thus used as biomarkers for diagnosis. identification of these autoantibodies, as disease biomarkers, should be achieved using native antigens in simple biological assays. however, post-translational modifications, such as glycosylation, may play a fundamental role for specific autoantibody recognition. in line with these observations, we previously described synthetic glycopeptides able to detect high autoantibody titers in sera of patients affected by multiple sclerosis, an inflammatory, demyelinating disease of the central nervous system. we also demonstrated that glycopeptides able to reveal high antibody titers in multiple sclerosis sera are characterized by a type i we describe here the result of a conformationally driven rational design exercise, which led to the preparation of new, optimized glycopeptides endowed with enhanced antigenic properties. most importantly, the same approach, based on structure alignment, was used to shed light on the native antigen(s), target of pathogenetic autoantibodies involved in demyelination processes vitro and in vivo evaluation for cholecystokinin-b receptor imaging istituto nazionale per lo studio e la cura dei tumori ome (f )<br> (aib, α-aminoisobutyric acid) to investigate its binding properties to tb(iii) ions. according to our published spectroscopic results f populate a set of ordered conformations involving /α-helical segments and compact structures generated by the formation of a turn around the flexible gly -gly central motif. cd experiments showed that the binding of tb(iii) to f gives rise to a structural transition of the peptide chain from a helical to a folded conformation. peptide binding is also responsible for the dramatic increase in the tb(iii) fluorescence intensity, suggesting that the tb(iii)/f complex may represent an interesting system for imaging applications or bioanalytical sensing the kda protein of mycobacterium tuberculosis provokes specific immune response, therefore related epitope peptides and peptide-conjugates can be considered as potential diagnostics. in our previous study we have determined the functional human t-cell epitope within the - region. based on this we synthesised two groups of peptides: a) nand c-terminal alanine and beta-alanine elongated variants of the - epitope and b) - peptides with alanine substitution at different position according to the hla dr and tcr binding sites. peptides were prepared by solid phase synthesis using boc/bzl or fmoc/tbu strategy. the homogeneity and the primary structure of peptides were checked by analytical rp-hplc, amino acid analysis and esi-ms. the t-cell stimulatory activity of the compounds was investigated using in vitro assays (proliferation and ifn-gamma production) on the - epitope specific human t-cell clones and pbmc (peripherial blood mononuclear cells) from patients and healthy (ppd+, ppd-) subjects. the effective peptides were conjugated to branched polypeptides with polylysine backbone (sak, eak), tetratuftsin derivative (h-[thr-lys-pro-lys-gly] -nh ) and lysine dendrimer (h-lys-lys(h-lys)-arg-arg-beta-ala-nh ) (map) carrier via thioether bond formation. the subtitution degree of the conjugates was determined by amino acid analysis. pbmc and human t-cell clones were stimulated with the free peptide alone or with peptide-conjugates containing an equimolar amount of peptide or with a mixture of free peptide and carrier italy we demonstrated, for the first time, that an aberrant post-translational modification (ptm, n-glucosylation) is possibly triggering autoantibody response in multiple sclerosis. this was possible because of a "reverse approach", which led to csf (glc), a structure based designed glycopeptide, as the first multiple sclerosis antigenic probe accurately measuring high affinity autoantibodies (biomarkers of disease activity) in sera of a statistically significant patients' population universal peptide scaffold" to be modified with a series of glycosyl amino acids (different in sugars and linkages), in the aim of developing personalized diagnostic/prognostic tests. the csf beta-turn structure, exposing at the best the aberrant ptm specific for antibody-mediated forms of other autoimmune diseases, will lead to a family of antigenic probes to be used in diagnostic this information is encoded by the distribution of the electron-ion potential (eiip) of amino acids along the sequence and is represented by the frequency components in is. proteins with the same biological functions or interacting proteins (e.g. antibody/antigen) share the information corresponding to the common frequency components in their iss. investigation of the hiv- envelope glycoprotein gp , as a model system for hypervariable proteins, revealed that this information is strongly conserved and is not significantly affected by natural mutations. the c-terminus of the second conserved region (c ) of gp , encompassing ntm peptide is important for infectivity and neutralization of hiv- , while human natural anti-vasoactive intestinal peptide (vip) antibodies reactive with gp play an important role in control of hiv disease progression. ntm/vip multiple copies were coupled to an artificial sequential oligopeptide carrier for developing an immunoassay (elisa) as a reproducible, reliable and sensitive tool for the detection of anti-ntm/vip derived antibodies these peptides have been utilized in an immunopeptidometric assay for specific measurement of active, noncomplexed psa. however, this assay has not been sensitive enough for the measurement of active psa in clinical samples. therefore, we aimed to develop an improved assay utilizing the same principle as previously, but using a more sensitive detection method based on proximity ligation assay. methods: in the assay, psa is first captured on a solid phase by a psa antibody czech republic rapidly increasing knowledge of new gonadotropin-releasing hormones (gnrh)of different species of the animal kingdom induces the need to prepare new synthetic derivatives and fragments of these peptides with higher potency and metabolic stability and suitable for the formulation of new immunogens. the species related differences in the sequence of the native mammalian gnrh pglu-his-trp-ser-tyr-gly-leu-arg-pro-glynh concern predominantly the positions , and , particularly tyr in position is replaced for his or leu, leu in position by val or trp, and arg in position is substituted by lys, ser, asn or gln. several gnrh derivatives with with the above substitution and gnrh fragments were prepared by solid phase peptide synthesis and purified by rp-hplc. purity of the synthetic peptides was checked by capillary zone electrophoresis (cze); peptides were analysed as cations in acidic backround electrolytes (ph . - .quantitative analyses for determination of their effective electrophoretic mobilities and the estimation of their effective charges.supported by grant of ministry of agriculture of cr-nazv qf by rants of ga cr nos we use peptaibiomics for the structural determination of peptaibiotics from fungi grown on single agar plates thus avoiding time-consuming isolation and purification procedures. the method comprises fast and effective solid-phase extraction followed by on-line rp-hplc coupled to tandem esi-ms. here we present a survey of the peptaibiome of hypocrea species. in extracts of hypocrea semiorbis, h. vinosa, h. dichromospora, h. gelatinosa, h. nigricans, h. muroiana and h. lactea a multitude of short and long-chain peptides containing aib could be characterized. the formation of new and known peptaibiotics could be established by comparison with sequences stored in data bases japan process scale rp hplc purification of peptides and proteins is increasingly important in bio-pharmaceutical production. besides selectivity, other crucial factors are loadability, recovery loadability is believed to depend on the surface area of the packing material. consequently, smaller pores providing larger surface area should lead to increased loadability. this principle is misleading in the case of large molecules, because they cannot penetrate smaller pores. therefore the chromatographically accessible surface area has to be taken into account. recovery problems like irreversible adsorption or aggregation are frequently caused by hydrophobic surface properties of ods phases. the less hydrophobic c is a substituent to avoid considerably these problems. however c is less durable than ods under extreme acidic conditions. our new proprietary c modification technology combined with a perfect end-capping minimizes the presence of residual silanol groups and protects the silica surface sp- -c -bio demonstrates high mechanical stability by no obvious alteration of back pressure and particle size after repeated packing cycles in dac columns. by overcoming the common weaknesses of the conventional c rp silica phases, daisogel sp- -c -bio opens new avenues for process scale separation of peptides and proteins. m determination of peptide: protein molecular ratio in conjugates by seldi-ms method synthetic peptides are widely used as antigens in various research and practical areas of biology and medicine. peptides with molecular masses < kda should be conjugated with carrier proteins in order to ensure their immunogenicity and protect from proteolysis. in these cases the comparison of peptide immunogenicities and immunotest system development should be performed having in mind exact peptide-to-protein ratios. conjugates of peptide fragments of hepatitis c virus envelope protein e with ovalbumin, bovine serum albumin, and myoglobin were prepared using glutaraldehyde (ga), m-maleimidobenzoyl n-hydroxysuccinimide ester, dimethyl suberimidate (dms), -ethyl- -( -dimethylaminopropyl)-carbodiimide as conjugating reagents. the rough evidence of the peptide-protein conjugate formation was obtained by page. the exact peptide:protein molar ratio was estimated in all conjugates by seldi-ms. almost all conjugates had oligomeric structures due to the formation of intermolecular linkages between proteins. the peptide : protein molar ratio in conjugates varied from : to , : . conjugates obtained with the ga were more diversified in the number of peptide molecules linked to carrier proteins (peptide:protein ratios ranged from : to : ) than other conjugation reagents mazur-marzec poland posttranslational modifications (ptms) like phosphorylation, acetylation, or methylation have been shown to play a significant role in directing the function of various proteins [ ]. in eukaryotes, most of proteins have been shown to be posttranslationally regulated by a variety of different modifications. many effects of ptms include a change of enzymatic activity capillary electrophoresis (ce) has been used to study electrophoretic behavior of ptm-peptides gal-nh , gal( - )-nh by capillary electrophoresis. using a phosphate buffer most of ptm-peptides were poorly separated at acidic or neutral ph. the best results were obtained using trifluoroethanol containing separation buffers. optimization of ce separation of maps of peptides containing ptms should allow to detect ptm-proteins and characterize their role in the living cell. comparison of modification events occurring in diseased and healthy cell may iran the purpose of this study was to use the application of multiplex reverse transcription polymerase chain reaction(rt-pcr) assay for detecting the two most common leukemia translocations t( ; ) and t( ; ) in childhood acute lymphoblastic leukemia in iranian children. cases of leukemia patients were screened with the rt-pcr assay. this assay will identify the all type bcr-abl transcripts encoded by the t( ; ) and all described variants of the e a-pbx transcript encoded by the t( ; ). rna was isolated from leukocyte cells of patient's samples. through the construction and optimization of specific primers for each translocation,we have been able to set up multiplex rt-pcr reactions.then pcr products was electrophoresed on agaros gel and were compared with size markers and expected fragtments key words: acute lymphoblastic leukemia -multiplex rt-pcr tu study on the syntheses and lc/esi-ms analyses of the glutathione conjugates of bile acids t. wakamiya , m. sogabe a carboxylic acid-containing drug, is metabolized to a glutathione (gsh) conjugate in vivo, and the conjugate is excreted in human urine [ ]. although bile acids, compounds with carboxylic acid in molecules, are also expected to form gsh conjugates in liver, no evidence is so far obtained to confirm such metabolism, since there are no suitable standard samples for the research. in the present paper we report the syntheses of the gsh conjugates of main bile acids in human, i.e., cholic acid (ca), chenodeoxycholic acid (cdca), deoxycholic acid (dca), ursodeoxycholic acid (udca) and lithocholic acid (lca) as shown below, and the detailed analyses of these synthetic conjugates by means of linear ion trap lc/esi-ms. furthermore, the evidence for conversion of cholyl adenylate [ ] and ca-coa thioester into ca-gsh conjugate will be presented these peptides do no exhibit such strong side effects as csa, but their practical application is hindered because of their poor solubility in water. the - fragment of tat protein and its analogs, including oligoarginine sequences, are known for their unusual ability to cross cell membranes, skin, and blood-brain barriers. moreover, these peptides are able to transport other substances into cells. this strategy was successfully applied in cases of csa, taxol, and other drugs to improve their bioavailability. now we have synthesized a series of analogues of cyclolinopeptide a, clx, and the immunosuppressive fragment of ubiquitin, covalently bound to the cell-penetrating fragment of tat and its analogues. the ability to cross the biological membranes and the immunosuppressive activities of these conjugates were tested. the conformation of the peptides was determined by circular dichroism methods: we used fluorescein-labelled cationic cell-penetrating peptides and analyzed the uptake efficiency (flow cytometry) as well as the intracellular distribution (confocal laser scanning microscopy). the bioactivity of a proapoptotic cargo-peptide, delivered into the cells either via electroporation or via cpps was quantified using a caspase- activity assay and cellular assays. to address the integrity of cpps during their trafficking, a fluorescent double-labelled antp peptide was designed and used as an intracellular fret-sensor. results: endocytosis-mediated uptake of the cpp-cargo conjugate led to a significant reduction of cargo bioactivity compared to its direct transfer via transient membrane permeation. this finding was related to the sequestration of peptides within endocytic vesicles but also, in the case of the tnf response, to the induction of receptor internalization during cell entry. moreover, during endolysosomal passage peptides undergo significant proteolytical degradation. conclusions: the endocytosis-dependent uptake mechanism of cholesterol pullulan (cp), in which maltose moieties are partially modified by cholesterol, is unique in forming self-assembled nanoparticles ( - nm) in water. combination of these characteristics is considered to be promising for development of effective non-viral vectors without toxicity. a conjugate of hiv-tat and cp was synthesized and its gene expression efficiency was evaluated. fully protected hiv-tat-( - )-cys(snm)-gly-nh-r was obtained by conversion of the corresponding cys(acm) peptide which was synthesized by the solid-phase method [snm: (n-methyl-n-phenylcarbamoyl)-sulfenyl] [ ]. the sulfhydryl function was introduced to the hydroxyl groups of cp by acylation with trt- -mercaptopropionic acid followed by acid treatment. resulting -mercaptopropionyl-cp was coupled with cys(snm) peptide to form disulfide bridge and the protecting groups of the peptide were removed to give the cp-tat conjugate. cp-tat and pcmv-luc complex was transfected into cos cells and luciferase activity was analyzed after h. cp-tat elicited remarkable cytoplasmic luciferase activity and low toxicity this finding provides a possibility to use gnrh-iii as a targeting moiety for intracellular drug delivery. therefore we have prepared methotrexate and doxorubicine conjugates of gnrh-iii. the drug molecules were attached to the lys side chain in position of gnrh-iii by thioether bond formation through the gflg spacer elongated with cys either at the c-or n-terminus. since we found earlier that the dimer derivative of gnrh-iii was more effective, new dimer derivatives with a combination of antitumour agents were also prepared. branched gnrh-iii derivative (pyrhwshdwk(clac-glfgc(acm))pg-nh ]) was synthesised by spps. the drug molecules were attached to this compound by thioether bond and finally disulfide bridge was formed between two peptide chains. the cytotoxicity of new derivatives was characterised by mtt test th oligopeptide antifungals are exceptionally active against multidrug-resistant yeast previous studies have demonstrated that longer sirnas that are processed by dicer can result in more potent knockdown than the corresponding standard -mer sirnas. dicer-substrate - -mer sirnas were conjugated with different structural classes of peptides and their cell uptake properties evaluated. peptides were conjugated to the ' end of the sirna sense or antisense strand via a thioether bond under denaturing conditions to prevent aggregation and precipitation. the ability of conjugates to translocate fluorescently-labeled sirna across the plasma membrane was evaluated by flow cytometry. the results indicate that some peptides can mediate higher efficiency uptake of sirna into cells compared with lipofectamine or cholesterol-conjugated sirna. the peptide-sirna formulation with -mer sirna conjugates showed higher knockdown of tnf-alpha mrna and protein levels in activated human monocytes in vitro compared to the conjugated -mer sirna species. the products resulting from in vitro digestion of peptide-conjugated rna duplexes with recombinant human dicer were identified using esi-ms and consisted predominantly of the desired -mer sirna several peptides containing the sequence arg-gly-asp (rgd) were studied and developed for their nanomolar affinity to the membrane receptor alfa v beta and alfa v beta integrins, which are over-expressed by endothelial cells during proliferation and by tumor cells. to improve the pharmacological profile of some camptothecin derivatives (cpts), five conjugates were designed, where the cytotoxic drugs were covalently attached to the rgd peptide analogues for preferential uptake into tumor cells. the peptides to be used have been selected among a series of new pentacyclic peptides bearing at -position a trifunctional pseudoamino acid with a carboxy-terminal side-chain. peptide analogues showing the highest affinity to alfa v beta and alfa v beta integrins were coupled with cpts at different positions. the conjugates have been optimized for binding to the receptors, proteolytic stability and an overall improvement in tumor selectivity. the nature of the linkage between rgds and cpts has a major impact on stability and biological activity of the conjugates. the conjugates with amide bond, but not those with ester bond, are sufficiently stable and show in vitro antitumor activity against a and a cell lines combination of amaranth protein with other plant proteins (cereals) enables to formulate the composite protein (near to milk or beef protein, but exclusively on vegetal basis). it is shown on graphs. the aim of the projects' proposals is a development and realization of the technology for fractionation of amaranths defatted flour product is a top protein obtained by removing starches and next polysaccharides decomposed on soluble monosaccharide by specific enzymes. there are shown the chromatograph measuring. we can see complete disintegration of starch and the unchanged proteins. the separate solution monosaccharide is usable for others fermentative processes or as a nutrient solution for yeasts there are description methods for isolation amaranth protein -extraction processes, enzymatic removal starch. the product is a isolate protein rich in essential amino acids. the waste monosaccharide solution was used to production yeasts biomass rich in proteins vitamins amaranth protein isolate have high nutritional value and can be used as food ingredients, for functional, probiotic formulation to begin with, we made epitope mapping with the highly sensitive spot array method ( ) in order to study antigenic regions of parvovirus b vp and vp capsid proteins. epitope mapping identified highly reactive, immunodominant early epitope on parvovirus surface that centered to kyvtgin residues of vp . in the subsequent phases we developed the kyvtgin epitope type specific (ets) igg serodiagnostics. a correlation between enhancing igg avidity to b capsid and a transient reactivity with the point-of-care kyvtgin peptide was clear. together the two assays enhanced the value of early diagnosis of b infections ( ) acute phase-specific heptapeptide epitope for parvovirus b diagnosis synthetic peptide arrays on membrane supports-principles and applications human parvovirus b infection during pregnancy--value of modern molecular and serological diagnostics laboratory of peptide & protein chemistry & biology department of organic chemistry "ugo schiff" and cnr-iccom department of chemistry department of pharmaceutical sciences glc) able to recognize specific autoantibodies in multiple sclerosis (ms) patients' sera has been developed by the laboratory of peptide & protein chemistry and biology [ and bio-plex suspension array system, biorad. the biosensor technology and bio-plex suspension array system will offer advantages such as rapid analysis, and high sensitivity for a high throughput screening. immobilisation will be based on different strategies that are anchoring the synthetic antigen on different solid supports such as polystyrene well plates (elisa) optimisation of the different techniques was performed with anti-csf (glc) autoantibodies isolated using affinity chromatography from ms patients' sera. the analytical parameters such as specificity, sensitivity, and matrix effect were evaluated. the different technologies have been used for a high throughput screening of ms sera which control specific cell adhesion. here we discuss the route for preparation of amphiphilic block copolymers composed of hydrophobic polylactide and hydrophilic polyethylene oxide (peo) blocks, carrying various cell-adhesion oligopeptide sequences at the end of peo block. fully protected peptide fragments were prepared by solid-phase peptide synthesis by using fmoc strategy and chlortrityl resin. the side-chain protected peptides were cleaved from resin by % hfip solution in dcm. the copolymers peptide-polytehyleneoxide were prepared by coupling of the activated peptide fragments with α-amino-ω-hydroxy-peo in dmf using pypop as an activation reagent. subsequently, the polylactide block was grafted to the ω-hydroxy end group of the peptide-peo copolymer via a controlled rop polymerisation of lactide r)- -aminocyclopentanecarboxylic acid (acpc) and beta-methylphenylalanine (beta-mephe) were designed and synthesized to obtain more potent and selective mu-opioid receptor ligands with higher stability against proteolytic enzymes. we have prepared the peptides by spps methods using racemic amino acids. the diastereomeric peptides were separated by hplc. the configuration of the unnatural amino acids in the peptides was determined by chiral tlc using enantiomeric standards. radioligand binding assays and in vitro gpi and mvd assays indicated that several analogues showed high, subnanomol affinity and high selectivity for mu-opioid receptors having agonist or antagonist properties. the incorporation of alicyclic amino acids into the endomorphins resulted in enzyme resistant peptides. the most promishing analogues (dmt-pro-phe-phe-nh and tyr-( s, r)acpc-phe-phe-nh ) were labeled with tritium using precursor peptides containing dehydroproline or dehydro-( s, r)acpc amino acids and tritium gas and pd/baso catalyst. the novel peptides and their radiolabelled analogues with high specific radioactivity ( . - . tbq/mmmol) have become useful pharmacological and biochemical tools for the opioid research iran background and aims: injectable drug delivery based on polymer solution platforms has gained in resent years, particulary for protein-based therapies. the influence of polymer molecular weight (rg h, rg h) on the morphology, erosion of matrices and also on their in-vitro drug release behavior over a period of days was assessed for leuprolide acetate in this study. methods: each formulation was composed of % (w/w) polymer and % (w/w) leuprolide acetate dissolved in nmp. release studies were performed in a home-made diffusion cell at °c. the polymer erosion was studied using two different methods as follows. (a): l-lactic acid detection (b): ph change study. the morphology of the matrices was then analyzed by scanning electron microscope as is shown, the lower molecular weight polymer formulation shows higher porosity and pore diameter due to a rapid phase inversion phase i) can be divided into three more phases with different release rates. results showed that burst effect for rg h, %, was significantly (p< . ) higher than rg h ( %) italy fabrication of photocurrent-generating systems based on bioinspired organic-inorganic hybrid materials is currently of great interest. more specifically, the photoelectronic properties of nanometric films formed by peptide self-assembled monolayers have been actively investigated. in this work interdigitated gold microelectrodes were modified by covalently linking a hexapeptide ester functionalized by a lipoic acid (lipo) at the n-terminus. the peptide chain [lipo-(aib) -trp-aib-otbu] comprises five α-aminoisobutyric acid (aib) residues and one trp, a fluorescent amino acid with strong absorptions in the uv region. due to the very high percentage of conformationally constrained aib residues in the chain, the peptide adopts a rigid - -helical structure. cyclic voltammetry measurements indicate that the peptide forms a homogeneously and densely packed monolayer on the gold surface, while current/voltage curves exhibit interesting rectifying properties of the peptide sam. photocurrent generation experiments, performed on the peptide-layered microelectrode, show peculiar modifications of the spectrum. at nm a notably higher photocurrent/voltage response was observed for the peptide-modified electrode, suggesting that a photoinduced electron transfer process from trp to gold does take place with high efficiency this may lead to randomly orientated enzymes and subsequently limited activity. the aim of this work is to selectively activate enzymes at their c-terminal position in order to allow specific immobilisation. we chose akr a , an enzyme of the aldo/keto reductase superfamily, for the synthesis of an artificially monolabeled redoxprotein. akr a is a monomeric enzyme and catalyzes the nadph dependent reduction of aliphatic/aromatic ketones and aldehydes. to produce monofunctionalized enzymes we applied the strategy of expressed protein ligation (epl). accordingly, we used the impact®-system and cloned the aldo/keto-reductase as fusion protein with an additional intein/chitin binding domain. through intein mediated splicing we could produce c-terminal thioester of the akr a . in the next step, the thioester was coupled to a biotin containing peptide by native chemical ligation. this specifically modified enzyme was immobilised on avidin coated surfaces. the attachment on the surface was tested by tryptic digestion, followed by maldi-tof-ms analysis since safe organic solvent waste disposal is an important environmental problem, we aimed to perform peptide synthesis in water. we have reported solid phase peptide synthesis in water using water-soluble n-protected amino acids, such as -[phenyl(methyl)sulfonio]ethoxycarbonyl and -( -sulfophenylsulfonyl)-ethoxycarbonyl amino acids. following to study on water-soluble n-protected amino acids, we developed a new technology based on nanochemistry for solid phase peptide synthesis in water. the new technology is based on coupling reaction of suspended nanoparticle reactants in water. fmoc-amino acids are used widely in peptide synthesis, but most of them show poor water-solubility. we prepared well-dispersible fmoc-amino acid nanoparticles in water by pulverization using a planetary ball mill in the presence of poly(ethylene glycol) (peg). the size of fmoc-amino acid particles was - nm. to evaluate the utility of this technique, leu-enkephalin was prepared using the nanoparticulate fmoc-amino acids on a peg-grafted rink amide resin in water supramolecular structures formed from n-lipidated oligopeptides immobilized in the regular pattern on the cellulose surface are able to bind ligand molecule, thus acting like artificial receptors. due to the conformational flexibility of lipidated oligopeptide chains, the supramolecular structure is highly flexible, forming the cavities with the shape and prosperities adjusted most effectively to requirements of the guest molecule. structural requirements for a peptide providing the most efficient fit the guest molecules are not known, therefore an array of the artificial receptors have been synthesized and used in the studies. thus, even in the case, when the single receptor in an array does not necessarily have selectivity for a particular analyte, the combined fingerprint response can be extracted as a diagnostic pattern visually, or using chemometric tools in order to improve the sensitivity of the competitive binding and to study the mechanism of molecular recognition, experiments involving fluoresceine and fluoresceine marked acp fragment were performed. we found that λmax and intensivity of fluorescence depends on the structure of the peptide motif and lipidic fragment of receptor this mts was linked with dhhp- by disulfide bond, and the new molecular was named mts~dhhp- . the peroxidase activity of mts~dhhp- ( . x u•µmol- ) was tested and similar to that of mp- ( . x u•µmol- ). mts~dhhp- coated with quantum dots (qds) [ ] were observed to accumulate into neonatal rat cardiomyocytes (nrcms) of wistar rats and co-localized with mitotracker red in mt. these results suggest that mts~dhhp- is an excellent apx mimics and may have potential proceedings of the twenty-eighth european peptide symposium, kenes international, israel, , . references: elastin-based polypeptide, poly(val-pro-gly-val-gly), undergoes self-assembly called coacervation, in which microcoacervate droplets with approximately nm diameters are formed [ ]. nanoparticles cross-linked by cobalt- γ-irradiation of these microcoacervate droplets are useful as drug release devices. to investigate the size optimization of nanoparticles, the stability of nanoparticles in the treatment of enzyme, and the drug release profiles from nanoparticles, the three copolymers; poly[ (val-pro-gly-val-gly), (val-ala-pro-gly-val-gly)], poly[ (val-pro-gly-val-gly) application of polyelectrolytes and theoretical models the synthetic heptapeptide rnwdvyk is a fragment of a high affinity receptor (fcεri) for immunoglobulin e (fragment - ). it is the active domain for binding with ige. the program of studies of biological properties of the heptapeptide included the investigation of its binding to ige contained in standard solutions and in patients' blood serum. the binding of rnwdvyk with ige was investigated by the ifa method using the ige antibodies labelled with horse-radish peroxidase (hrpo). we determined the optimum sorption concentration of the peptide in this experimental immunoenzyme system to be mkg/ml. the ability of synthetic rnwdvyk peptides to bind with ige was studied as a function of ige concentration in standard serum ( . to ng/ml ige). a high correlation was found between the ige concentration and the optical density of the solution after introducing monoclonal antibodies labeled with hrpo and the substrate chromogenic mixture (r= . ). similar investigations were conducted using the allergy patients' blood serum. the serum with a known concentration of ige was added to immunological plotting boards with sorbed synthetic rnwdvyk peptides. a high correlation was also found between the concentration of ige in the patients' blood serum and the optical density of the solution after introducing monoclonal antibodies labelled with hrpo and substrate chromogenic mixture (r= . ). our experiments showed the high ige binding activity of synthetic rnwdvyk peptides. we demonstrated the possibility of construction of diagnostic systems for the quantitative determination of ige and ige-antibodies. the fusion protein nucleocapsid-dutpase is present in virions of mason-pfizer monkey betaretrovirus and in virus-infected cells where it potentially contributes to rna/dna folding and reverse transcription (barabas, et al., ; bergman et al., ; berkowitz, et al., ) . in addition to trimeric dutpase core, the protein possesses flexible n-and c-termini consisting of the nucleocapsid segment, and a peptide motif conserved in dutpases. to analyze the function of the flexible cterminal peptide segment, reconstitution experiments were designed with truncated enzyme lacking the c-terminal mer oligopeptide and the synthetic oligopeptide prepared on rink-amid resin by solid-phase peptide synthesis, using fmoc strategy. the truncated enzyme proved to be practically inactive. addition of the synthetic mer (pyrgqgsfgssdiy) at fold molar excess resulted in partial complamentation of the catalytic activity (to % of original). a mixture of the truncated enzyme and the mer oligopeptide (this latter at fold excess) was put to crystallization trials. we conclude that the c-terminal mer is essential for catalytic activity. antifolate drugs are inhibitors directed to interfere with folate metabolic pathway. methotrexate (mtx) and pemetrexed (alimta®) are known folic acid analogues used in cancer treatment. different peptide conjugates of mtx have been prepared for intracellular delivery. ( ) in octaarginine conjugates one of the carboxylic groups of mtx was attached to the n-terminal of the peptides. ( ) however, as results showed, that both carboxylic groups are required to the biological effect of mtx. therefore we decided to synthesize peptide conjugates of folic acid analogues in which the carboxylic groups are untouched. octaarginine, penetratin and a cyclic peptide cgnkrtrgc, which can deliver a cargo molecule in the lymphoid system, were used as delivery peptides. we introduced squaric acid or aminoxy acetic acid as linker moiety between the peptides and cargo molecules. the conjugation was monitored by rp-hplc, the crude products were purified by rp-hplc and were identified by mass spectrometry. the biological activity of the conjugates was evaluated in vitro on sensitive and resistant human leukemia (hl- ) cell lines. besides its endocrine activity, trh (the tripeptide pglu-his-pro-nh<sub> </sub>) has also been long recognized as a modulatory neuropeptide with broad range of physiological and pharmacological activities in the central nervous system (cns). although numerous centrally active and metabolically stable analogues and peptidomimetics have been synthesized using trh as a template, selectivity of their cns action has remained an issue to be addressed. we aimed at discovering novel analogues with enhanced cns-selectivity by incorporating pyridinium building blocks. the design also allowed for enhancing transport across the blood-brain-barrier and increasing residence time in the cns through prodrug strategy. solid-phase chemistry was used to prepare the analogues and novel methods previously not used to incorporate pyridinium moieties into resin-bound peptides such as the zincke reaction were also introduced. comprehensive evaluation included measurement of affinity to trh-receptor, acetylcholine-releasing, analeptic and antidepressant-like activity in animal models, as well as prediction of membrane affinity, determination of in vitro metabolic stability, and pharmacokinetics and brain uptake/retention studies that employed in vivo microdialysis sampling. a strong connection between acetylcholine-releasing potency and analeptic effect in animals was obtained for close analogues of trh, while pyridinium compounds designed from the structurally related pglu-glu-pro-nh<sub> </sub> maintained the antidepressant-like effect of the parent peptide, while showing significant decrease in analeptic action.in conclusion, an increase in the selectivity of cns-activity profile was obtained by the incorporation of pyridinium moieties. we have also demonstrated the benefits of the prodrug approach on the pharmacokinetics, brain uptake and retention of the analogues upon systemic administration. the use of small radiolabelled compounds such as peptides is a very attractive tool for the diagnosis of several different pathologies, specially cancer, through the use of nuclear medicine techniques. among the various membrane receptors, the two cholecystokinin receptors ccka-r and cckb-r are very promising biological targets for radiolabelled compounds due to their overexpression in many tumours . in order to develop radiolabelled peptide derivatives able to target these receptors, the binding mode of the c-terminal cholecystokinin octapeptide (cck ), toward the two cholecystokinin receptors ccka-r and cckb-r has been, recently, structurally characterized. the structural data suggest that modifications on the n-terminal end of cck obtained by introducing chelating agents and their metal complexes should not affect the interaction of the derivatized cck peptides with both ccka-r and cckb-r. here we report the labelling procedures and the in vitro and in vivo characterization of new mtc cck derivatives. a stable mtc-nitrido complex is obtained by using the coordinative set formed by: ) the n-terminal amino group and the sh cystein of the cck derivative cys-gly-cck peptide; and ) a pnp aminodiphosphine used as coligand. several phosphines are used in order to define the best labelling procedures and to optimize the in vivo biodistribution properties of the mtc labelled peptides.references various combinations of pore size and chemistry of silica-based materials were investigated for high performance liquid chromatography (hplc) of peptide separation. incorrect pore size and ligands have been suggested to cause peak broadening, poor resolution and poor recovery. our study suggests that an appropriate combination of pore sizes and ligands is necessary to obtain the most efficient usage of reversed-phase hplc columns according to the molecular weights of peptides and proteins. we will also show the possibilities of an improved method development for the separation of complex peptide mixture by ph or additives.the development of new biopolymer materials as drug delivery systems is of enormous interest on biomedicine. dendrimers are polymers with particular properties; they are highly branched polymers with well-defined chemical composition, and show compact globular shape, monodisperse size and controllable surface functionalities. peptide dendrimers incorporates amino acids in their structures and have additional features such as biocompatibility and biodegradability.in previous studies we described the solid-phase synthesis of a new class of polyproline-based dendrimers. these biopolymers have the capacity to cross the mammalian cell membrane and moderate toxicity. these promising results open up the possibility to explore these dendrimers as delivery systems.to design more versatile polyproline dendrimers, we have developed a methodology that involves the combination of solid-phase and solution strategies. diverse multivalent peg-and proline-based cores were synthesized to attain dendrimers with distinct topologies. dendrimers were synthesized by iterative building block addition [(glypro ) imdoh], around an inner core, using a peptide solution convergent approach. a variety of coupling methodologies and protecting groups for the n-terminal function were explored. the novel high throughput protein detection system using designed peptide arrays has been successfully indicated on their capabilities as the "protein-chip" [ ] [ ] [ ] [ ] . our concept has many advantages especially for high-quality industrial production and practical applications compared to arrays with antibodies or recombinant proteins. the deposited peptide solution can be dried without covalent immobilization, although, when the resulted arrays are exposed in protein-solution they showed planned conformation [ ] . based on these basic results, several hundreds of α-helical, β-loop and β-sheet peptides, which involved a cysteinyl residue for covalent immobilization and tamra as a fluorescent label, were successfully synthesized, characterized and used as capture molecules. a novel material for chips made from amorphous carbon suitable for our concept has been developed, which has significant advantages over conventional glass or polymer plates, such as no selffluorescence, mechanically more stable, easy manufacturing in the aspects of precised and high throughput processing. thus, chip-plate with nano-l wells could also be easily manufactured. peptides were deposited on these chips surface covalently as well as non-covalently in picol/spot (diameter: ca µm). the resulted chips were used for protein detection. a part of this work was funded by the okinawa-bio-project and nedo-grant. key: cord- - x nx s authors: zhang, huaqun; keane, sarah c. title: advances that facilitate the study of large rna structure and dynamics by nuclear magnetic resonance spectroscopy date: - - journal: wiley interdiscip rev rna doi: . /wrna. sha: doc_id: cord_uid: x nx s the characterization of functional yet nonprotein coding (nc) rnas has expanded the role of rna in the cell from a passive player in the central dogma of molecular biology to an active regulator of gene expression. the misregulation of ncrna function has been linked with a variety of diseases and disorders ranging from cancers to neurodegeneration. however, a detailed molecular understanding of how ncrnas function has been limited; due, in part, to the difficulties associated with obtaining high‐resolution structures of large rnas. tertiary structure determination of rna as a whole is hampered by various technical challenges, all of which are exacerbated as the size of the rna increases. namely, rnas tend to be highly flexible and dynamic molecules, which are difficult to crystallize. biomolecular nuclear magnetic resonance (nmr) spectroscopy offers a viable alternative to determining the structure of large rna molecules that do not readily crystallize, but is itself hindered by some technical limitations. recently, a series of advancements have allowed the biomolecular nmr field to overcome, at least in part, some of these limitations. these advances include improvements in sample preparation strategies as well as methodological improvements. together, these innovations pave the way for the study of ever larger rna molecules that have important biological function. rna structure and dynamics > rna structure, dynamics, and chemistry. regulatory rnas/rnai/riboswitches > regulatory rnas. rna structure and dynamics > influence of rna structure in biological systems. functional long ncrnas has expanded our understanding of the roles that rna can play in the cell. though several classes of ncrnas are active players in translation-ribosomal rnas make up the bulk of the translation machinery while transfer rnas shuttle amino acids into the ribosome for incorporation into the growing polypeptide chain, there is a growing appreciation of the functional importance of other ncrna classes as well as noncoding regions of mrnas. these elements have been shown to be key players in the regulation of gene transcription and translation, epigenetic control, and rna turnover (bonasio & shiekhattar, ; guil & esteller, ; j. t. lee, ) . numerous studies have highlighted how complex structural folds in rna molecules confer specific functions including riboswitches (knappenberger, reiss, & strobel, ; liberman et al., ; peselis & serganov, ; vicens et al., ; b. zhao, guffy, williams, & zhang, ; also reviewed by jones & ferre-d'amare, ; roth & breaker, ), ribozymes (chan et al., ; costa, walbott, monachello, westhof, & michel, ; meyer et al., ; l. a. nguyen, wang, & steitz, ; qu et al., ; suslov et al., ; c. zhao, rajashankar, marcia, & pyle, ; also reviewed by pyle, ; ren, micura, & patel, ) , and viral elements (akiyama et al., ; au et al., ; imai, kumar, hellen, d'souza, & wagner, ; keane et al., ) . additionally, recent studies have identified highly structured ncrnas as important regulators in various cellular processes and linked misregulation of ncrnas to important human diseases (esteller, ) . for these reasons, ncrnas are attracting increased attention in both drug design and intermolecular recognition (warner, hajdin, & weeks, ) . structural study of these important rnas is becoming increasingly necessary to understand how highly structured rnas function and interact with other rnas, proteins, and small molecule ligands. elucidation of three-dimensional structures and conformational landscapes provides detailed information of their regulatory mechanisms. in this review, we will give a broad overview of different methods for structure determination and discuss some recent advancements in both sample preparation and data acquisition that have advanced the study of large rnas by nuclear magnetic resonance (nmr) spectroscopy. first developed more than years ago, chemical probing of rna can be a useful tool to rapidly gain nucleotide-level structural information (ehresmann et al., ; peattie & gilbert, ; stern, moazed, & noller, ) . base-pairing, hydrogen bonding, nucleotide accessibility and other secondary structure-related characteristics, can be obtained by treating rnas with specific chemical reagents (typically dimethyl sulfate [dms] and/or one or more shape reagents) and probing for the sites of modification by sequencing (cordero, kladwang, vanlang, & das, ; merino, wilkinson, coughlan, & weeks, ; tian & das, ; weeks, ) . reactivity data derived from chemical probing studies can be converted to pseudo free energy terms and then incorporated into a secondary structure prediction algorithms to bolster the quality of the resulting structure predictions (cordero et al., ; deigan, li, mathews, & weeks, ; reuter & mathews, ) . chemical probing remains the best method for high-throughput analysis of secondary structure within large (even genome-wide) rnas (bevilacqua, ritchey, su, & assmann, ; watts et al., ; zubradt et al., ) . however, chemical probing provides limited insight into rna tertiary structure (homan et al., ) and only approximations of fast time scale dynamics of the molecule (gherghe, shajani, wilkinson, varani, & weeks, ) . biomolecular structures at atomic resolution provide key insights into the molecular determinants of interactions, the mechanisms of catalysis, and molecular topology, among others. generally, x-ray crystallography is the most widely used method of structure determination for biological macromolecules. indeed, protein structure elucidation has been overwhelmingly conducted using x-ray crystallography, with approximately % of protein structures having been determined using x-ray crystallography (berman et al., ; http://www.rcsb.org) . crystallography, as the name implies, requires the formation of an ordered crystal capable of diffracting x-rays. this necessitates conformational homogeneity within the crystal lattice. therefore, x-ray crystallography has proven very useful for those rna molecules that adopt relatively rigid conformation, for instance, riboswitches and catalytic ribozymes (liberman & wedekind, ; marcia & pyle, ; pyle, ; roth & breaker, ; toor, keating, taylor, & pyle, ; toor, rajashankar, keating, & pyle, ) . however, many rna molecules, particularly larger rnas, are flexible and may sample multiple conformations and thus less likely to crystallize in the absence of stabilizing cofactors like protein binding partners. x-ray crystallography has therefore played an important, but significantly smaller (approximately % of rna structures were determined using x-ray crystallography) role in the threedimensional structure determination of rnas relative to proteins (berman et al., ; http://www.rcsb.org single particle cryo-electron microscopy (cryo-em) is a powerful technique for structure elucidation of large biomolecules and biomolecular machines, and recent technological advances have catapulted this technique to the forefront of structural biology (kuhlbrandt, a (kuhlbrandt, , b nogales & scheres, ; vinothkumar & henderson, ) . cryo-em importantly allows for the visualization of dynamic biological processes by direct visualization of biomolecules with multiple structural states (dong et al., ; nakanishi, kishikawa, tamakoshi, mitsuoka, & yokoyama, ; roh et al., ) . however, to date, cryo-em has largely been applied to protein only or protein-nucleic acid complexes (e.g., the ribosome and spliceosome). cryo-em has been used to study the overall size and shape of very large rnas, reporting at very low resolution on secondary structure and branching pattern of these rnas (garmann et al., ; gopal, zhou, knobler, & gelbart, ) . there are a few structures of relatively small rnas derived from cryo-em data, (baird et al., ; zhang et al., ) but these low resolution density maps harbor limited structural detail requiring higher resolution models determined via other methods to glean mechanistic insight. a recent software developed by the das lab facilitates the building of rna coordinates into low resolution cryo-em density (kappel et al., ) . while this technique is described for protein-rna complex datasets, it is likely applicable to rna-only density maps. nmr spectroscopy is another common method for determining rna structure at atomic resolution. nmr spectroscopy is particularly well-suited for the study of biomolecules that are not prone to crystallization or those which require specific sample conditions to maintain the structure. compared to proteins, nmr spectroscopy has played a large role in the structure determination of rna molecules, roughly % of rna structures have been solved using this method (berman et al., ; http:// www.rcsb.org). importantly, nmr spectroscopy provides far more than just structural information, as it allows the dynamics of the biomolecule to be studied as well. while nmr is a powerful tool for studying rna structure and dynamics at the atomic level, there are some very real challenges/limitations when using nmr spectroscopy to determine the structure and dynamics of large rnas. (a) labor intensive. structure elucidation by nmr spectroscopy requires that most resonances are assigned before beginning any structural analysis. this is a time-consuming process both as it relates to data acquisition and data analysis. (b) signal overlap. rnas are built from four nucleotides which have similar chemical structures. this results in a lack of dispersion in the chemical shifts (also known as signal degeneracy). (c) unfavorable relaxation properties. large rna molecules tumble very slowly in solution leading to severe broadening of signals and significant losses in signal intensity. these challenges have generally restricted the size of rna that can be studied by nmr spectroscopy. of the rna structures determined by nmr spectroscopy, the average size is nucleotides with only six structures of rna larger than nucleotides (nt) (figure ) (berman et al., ; http://www.rcsb.org) . while solution nmr spectroscopy has a number of technical challenges, it also has some distinct advantages. (a) tertiary structure information. while secondary structure is importantly represented in the three-dimensional structure of rnas, the tertiary structure provides key insights into the function of the rna. (b) study the native conformation of an rna. because nmr is not predicated on growing crystals of the sample, there are no concerns that the structure might be affected by crystal f i g u r e histogram of rna structures that were determined using nuclear magnetic resonance spectroscopy. the size of the rna molecule (nucleotides, nt) is correlated with the number of structures reported in the protein data bank (rcsb.org, june ) packing or the conditions under which crystals can be grown. (c) access to information about dynamics. while a number of biophysical techniques can be applied to study dynamic processes in rna molecules (hao et al., ; p. nguyen & qin, ; panja, hua, zegarra, ha, & woodson, ; ritchie & woodside, ) , nmr is uniquely poised to study the dynamics of biomolecules at atomic resolution and over a wide range of timescales (al-hashimi & walter, ; marion, ) . structure determination by nmr spectroscopy is first and foremost predicated on having assignments for all (or most) atoms within the molecule. chemical shift assignments can be made via analysis of a variety of data and are useful in the analysis of nuclear overhauser effect (noe) distance restraints, among others. the experimental restraints can then be combined with empirical restraints to generate initial geometry-based structural models. these initial structural models can then be refined using a simulated annealing strategy to generate an ensemble of lowest energy conformers. in order to simplify, or in some cases make possible, the analysis and collection of data for very large rnas, a number of both methodological and technical improvements have recently been developed. some of the key contributions are highlighted below. because of the limited chemical shift dispersion that rnas exhibit, nmr spectra of unlabeled large rnas are extremely crowded, prohibiting unambiguous resonance assignment ( figure a ). there are a number of ways to "edit" a spectrum, separating chemical shifts by a second nmr-active nucleus (typically n or c). however, the large c- h dipolar coupling has a significant effect on t relaxation mechanisms leading to broad line widths thereby limiting the sensitivity of heteronuclear correlation experiments, particularly for large rnas that have large rotational correlation times (a. l. hansen & al-hashimi, ; lu, miyazaki, & summers, ; tolbert et al., ) . one attractive alternative to the traditional heteronuclear experiments is to "edit" a spectrum by the nucleotide-specific incorporation of deuterium atoms throughout the rna. using this approach, protons are replaced with deuterium and because deuterium resonates at a different frequency than protons, the signals are not observed in a proton detected h- h noesy spectrum. the spectral complexity is therefore significantly reduced (figure b ). in addition to the reduced spectral overlap, incorporation of deuterium limits pathways for h- h spin diffusion resulting in narrower line widths. because this method relies on removing signals rather than separating the signals into a second or third dimension, no one sample contains f i g u r e deuterium labeling greatly improves spectral quality and simplicity. (a) h- h noesy spectrum of the full-protiated (h, unlabeled) nucleotides hiv- core encapsidation signal (keane et al., ) is characterized by severe signal overlap and broad line widths. (b) selective deuteration of the sample results in spectra of significantly higher quality. there is minimal signal overlap and resonances are now effectively identifiable by the respective labeling scheme. four different labeling schemes are shown: a h (adenosines protiated, cytosines, guanosines, and uracils deuterated), c h (cytosines protiated, adenosines, guanosines, and uracils deuterated), g h (guanosines protiated, adenosines, cytosines, and uracils deuterated), u r (uracils protiated on the ribose and at c , adenosines, cytosines, and guanosines deuterated) sufficient structural information ( figure b ) and many different samples, each with a unique labeling scheme, must be prepared to obtain full sequence coverage of the rna. this approach is labor intensive and requires considerable resources and spectrometer time. nevertheless, this method is universally applicable and has been used to study a variety of systems including viral rnas (d'souza, dey, habib, & summers, ; keane et al., ; lu et al., ; miller, yildiz, lo, wang, & d'souza, ) and tetraloop-receptor complexes (davis et al., ) . notably, this approach was recently used to probe the structure of the kda hiv leader and provide insight into its dimerization mechanism (keane et al., ) . partially or fully deuterated ribonucleotide triphosphates (rntps) can be purchased or produced in the laboratory using either chemical or enzymatic methods (huang, yu, leproust, & gao, ; lu et al., ; scott, tolbert, & williamson, ) . rntps may be deuterated at nonexchangeable positions within the base and/or the ribose and can subsequently be used in the enzymatic synthesis of rna using t rna polymerase in place of fully protiated rntps (batey, battiste, & williamson, ; batey, inada, kujawinski, puglisi, & williamson, ; scott et al., ) . in much the same way that partially or fully deuterated rntps can be incorporated into an rna using in vitro transcription, so too can other common nmr-active isotopes. isotopically enriched c and n rntps have widely been used in nmr experiments to study rna structure, dynamics, and ligand binding (batey et al., ; nikonowicz et al., ) . traditionally, these studies have been applied to relatively small rnas. the ability to site-specifically incorporate c labels within an rna ribose and/or base makes nmr spectroscopy a powerful tool for dynamics studies of large rnas, expanding the types of experiments that can be conducted and simplifying analysis (see data acquisition and analysis advancements below). compared to uniform c incorporation, site specific incorporation of c labels allows for reduced spectral crowding while eliminating the strong c- c scalar couplings thereby increasing signal-to-noise ratios and facilitating direct carbon detection experiments (alvarado et al., ; longhini et al., ; lukavsky & puglisi, ; marchant, bax, & summers, ) . drawing inspiration from earlier synthetic strategies (santalucia, shen, cai, lewis, & tinoco, ) , the dayie lab has developed a chemo-enzymatic method to produce isotopically enriched pyrimidines (alvarado et al., ) and purines (longhini et al., ) with different c and/or n patterns. the incorporation of site specifically labeled nucleotides into large rnas strongly reduced spectral crowding and removed c- c j-coupling, allowing study of large rnas by trosy (pervushin, riek, wider, & wuthrich, ) experiments and enabling new method for rna noesy assignment with improved signal intensity of c and c protons. in addition, dynamics studies of rna could also benefit from using those selectively labeled nucleotides (leblanc, longhini, tugarinov, & dayie, ). as discussed above, the uniform incorporation of specifically labeled nucleotides can greatly facilitate nmr studies of large rnas. however, the ability to incorporate a labeled nucleotide in a position-specific rather than nucleotide-specific manner could also be advantageous. some complementary biophysical techniques, like fluorescence resonance energy transfer and electron paramagnetic resonance spectroscopy, require labeling at specific positions rather than uniformly at a nucleotide type throughout the whole molecule. chemical synthesis is commonly used to make rnas with position-specific labeling; however, it is generally limited to relatively small rnas (< nt) (paredes, evans, & das, ) . it is possible to enzymatically ligate several smaller synthetic rnas together; however, the low efficiency of the ligation reactions limits the overall yield of full-length product. in order to circumvent these issues, the wang lab developed a hybrid solid-solution phase transcription method, position-selective labeling of rna (plor), that can theoretically synthesize any rna with labeling at any specific position using commercially available nucleotides (liu et al., (liu et al., , . the biggest advantage of this approach over other methods is that it can prepare large selectively labeled rna samples at a scale appropriate for nmr studies (liu et al., ) . the use of plor to position-specifically incorporate a labeled nucleotide into a large rna has many potential benefits including breaking ambiguity in resonance assignments and facilitating the identification of long-range interactions. a very common approach to tackling resonance assignments of large rnas is a "divide and conquer" strategy, where the large rna (figure a) is broken down into individual folded domains (figure b ) (imai et al., ; keane et al., ; ziegeler, cevec, richter, & schwalbe, ). these small, individual domains are amenable to rapid data collection and resonance assignment. the assignment data can then be "grafted" to the full-length data to help deconvolute the more complicated data. while this approach works in many situations, it is dependent on the fragments adopting the same structure in isolation as they do in the full-length construct. it is possible for the fragments to not fold properly, or to be poor representations of the region in the larger context. however, we generally find this approach exceptionally helpful in the early stages of fulllength resonance assignments. another approach that is widely used is an enzymatic ligation approach using either t dna or rna ligase to join either chemically synthesized or in vitro transcribed rnas with different labeling strategies (figure c) (duss, maris, von schroetter, & allain, ; i. kim, lukavsky, & puglisi, ; lu et al., ; nelissen et al., ; tzakos, easton, & lukavsky, ; xu, lapham, & crothers, ) . these methods require extensive optimization and troubleshooting and are notorious for low yield of product, although significant improvements have been made to improve the yield, eliminate specific sequence requirements, and streamline the process (duss et al., ) . the allain lab developed a robust and versatile segmental labeling method that uses ribozymes and rnaseh cleavage to generate fragments of rna for subsequent ligation. this approach was demonstrated on the nucleotide rsmz rna, which was fragmented into four components for reassembly in nmr spectral analysis (duss et al., ) . this method offers several distinct advantages over other methods for segmental labeling of rnas. first, this method is flexible-new ligation sites can be introduced by performing the rnaseh cleavage with a new splint-directed site. second, only two pools of full-length rna, isotopically labeled and unlabeled, are neededcloning of subfragments is not necessary. finally, the cleavage and ligation strategy are efficient even for highly structured rnas, making this approach virtually universal. one alternative to traditional enzymatic-based segmental labeling strategies is a fragmentation-based approach that relies on annealing rather than ligation to join the fragments together (figure d ). this approach was pioneered in the summers lab and was a requisite for the unambiguous identification of an unexpected long-range base pairing interaction within the hiv- f i g u r e methods for fragmentation or segmental labeling of large rnas. (a) secondary structure of a large rna with a complex multihelix junction. (b) "divide and conquer" approach. small oligo rnas are designed to mimic the structure of particular regions within the large rna. these oligo rnas are well-suited for rapid chemical shift assignment by traditional methods. (c) enzymatic ligation allows for segmental labeling of a large rnas. two rnas are independently synthesized and can therefore be independently labeled. the two rnas can then be ligated together to make a full-length construct with only one region of the rna containing nuclear magnetic resonance-active isotope labeling. (d) fragmentation-based segmental labeling. the rna of interest is fragmented at a hairpin loop. the loop sequence is replaced (in some cases) with a run of intermolecular c-g base pairs which serve as an annealing handle. this approach in theory can be applied at any hairpin loop structure within a large rna molecules packaging signal (keane et al., ) . in this work, the authors replaced a hairpin loop with a run of intermolecular c-g base pairs, fragmenting the rna into two discrete pieces that could be transcribed (and therefore isotopically labeled) independently and annealed together post purification (keane et al., ) . a similar approach was used to study a nucleotide transcriptional intermediate of the type i-a -deoxyguanosine-sensing transcriptional riboswitch (helmling et al., ) . here, a similar approach was used, a break in the sequence was introduced by removing a loop; however, a g-c handle was not included and may not be necessary for all sequences (helmling et al., ) . this approach is versatile as a fragmentation site can in principle be incorporated at the loop of any hairpin element, which are ubiquitous elements in rna structures. a caveat to this approach is that individual fragments might form a stable structure that inhibits intermolecular base pairing. this approach might not work for all rnas or at all hairpin loops and therefore must be rigorously tested to ensure proper formation of the native structure. one of the most telling characteristics of rna structure is the hydrogen bonding pattern, the base pairing within the molecule. an nmr spectrum that reports on the base pairing within an rna can serve as a "fingerprint" of the molecule. traditionally, hydrogen bonding within small rnas has been inferred through noe (through space) correlations of imino protons (varani, aboulela, & allain, ; wüthrich, ) . hydrogen bonding can also be identified directly using a scalar coupling hnn-cosy (through bond) experiment (dingley & grzesiek, ; pervushin et al., ) . additional improvements in sensitivity can be gained by implementation of longitudinal-relaxation-enhancement techniques such as best-hnn-cosy experiments which selectively excite only desired nuclei which shortens experimental acquisition time and improves signal:noise (farjon et al., ) . however, all of these experiments rely on the detection of the imino proton, a proton that is readily exchangeable with solvent and subject to conformational dynamics at elevated temperatures (gao & patel, ; j. h. lee, jucker, & pardi, ; wagner, rinnenthal, narberhaus, & schwalbe, ) . the imino protons of transient base pairs can be broadened beyond detection due to exchange with solvent therefore these experiments are typically conducted at temperatures below room temperature (typically around c). large rnas are generally not amenable to study at low temperature due to the slower molecular tumbling. nevertheless, imino assignments have been made for several large rnas including the nt u /u snrna complex (burke, sashital, zuo, wang, & butcher, ) , a nt region of the encephalomyocarditis virus ires (imai et al., ) , and the nt adenine riboswitch (reining et al., ) . the sattler lab made important improvements to the hydrogen bond correlation experiments, utilizing band-selective pulses coupled with best relaxation enhancement to enable detection of hydrogen bonds in dynamic regions of rna (dallmann et al., ) . these methodological improvements allow for work at high temperature without exchange concerns, because the detection occurs via nonexchangeable protons (figure ) (dallmann et al., ) . f i g u r e detection of hydrogen bonds in canonical a-u and g-c base pairs. traditionally, imino protons (blue) are used to infer base pairing within helical structural elements. improvements to pulse sequences, implemented by the sattler lab, allow for the detection of hydrogen-bonding on nonexchangeable c (adenosine) and c (cytosine and uracil) protons (dallmann et al., ) . magnetization transfer pathways are indicated with arrows . | global orientation of helical elements distance restraints (which are obtained via the analysis of noe spectroscopy experiments) provide high-resolution local structural information, however cannot generally define the relative orientation of secondary structure elements within a large rna. residual dipolar couplings (rdcs) provide important orientation restraints and inclusion of these restraints in structure refinement greatly improves the overall accuracy of the structure (bermejo, clore, & schwieters, ) . rdc measurements are conducted both in the absence and presence of an alignment media (pf phage is common for rna samples) (clore, starich, & gronenborn, ; m. r. hansen, hanson, & pardi, ) , and are measured on a variety of carbon-hydrogen and/or nitrogen-hydrogen correlations within the base and ribose (m. r. hansen, mueller, & pardi, ; yan, corpora, pradhan, & bushweller, ; zidek, wu, feigon, & sklenar, ) . rdcs provide important restraints in rna structure determination; however, slightly fewer than half of nmr-derived medium to large rna structures (rnas larger than nt) used rdc measurements in structure refinement (au et al., ; barnwal et al., ; burke et al., ; cornilescu et al., ; davis et al., ; houck-loomis et al., ; jain, morgan, rife, salemi, & tolbert, ; kang, eichhorn, & feigon, ; n. k. kim, zhang, & feigon, ; lukavsky, kim, otto, & puglisi, ; lukavsky & puglisi, ; miller et al., ; ulyanov et al., ) . the incorporation of c into large rnas results in severe line broadening, limiting the usefulness of this approach for the study of larger rnas. marchant et al. developed a new method for recording rdc measurements in large rnas that capitalizes on the large chemical shift dispersion and narrow line widths of adenosine c protons (marchant et al., ) . this method requires the preparation of rna with n-adenosine and uses a variable flip angle hmqc to obtain rdc measurements and was applied to the study of a nt element corresponding to the hiv- reve response element (marchant et al., ) . dynamic conformational exchange is important for the function of regulatory rnas in molecular processes (al-hashimi & walter, ; dethoff, chugh, mustoe, & al-hashimi, ; xue et al., ) . one major strength of nmr spectroscopy is the ability to capture dynamics information, reporting on the local conformational fluctuations at single nucleotide resolution (bothe et al., ; . conformational dynamics of macromolecules occurs over a broad range of time scale, from picosecond to second, and formation of new base pairs in rna conformational exchange lies in the scale of microsecond to second (bothe et al., ; rinnenthal et al., ) . numerous nmr methods have been developed to investigate biomolecular dynamics at different time scales (bailor et al., ; kloiber, spitzer, tollinger, konrat, & kreutz, ; vallurupalli, sekhar, yuwen, & kay, ; b. zhao, hansen, & zhang, ) . here, we will highlight three approaches for the quantification of conformational dynamic exchange in rna molecules. the chemical shift saturation transfer (cest) nmr experiments, which utilize magnetization "interaction" of one state with another state of molecules, has been developed to look into slow conformational exchange of biomolecules (reviewed in vallurupalli et al., ) . recently, the zhang lab optimized carbon cest experiments for nucleic acids and applied these methods as well as r ρ experiments (see below) to the study of a nt fluoride riboswitch (b. zhao et al., ) . in an effort to fully characterize riboswitch function, structural insight into the ligand free state is essential. their work revealed that the unliganded riboswitch formed a pseudoknot-like structure that is lowly populated, reminiscent of the ligand-bound structure, and was undetectable via conventional approaches (b. zhao et al., ) . cest experiments do not require specialized isotopic labeling strategies and can be conducted on rnas that are uniformly n/ c labeled. relaxation dispersion (rd) nmr experiments are frequently used to characterize excited states (ess) of biomolecules (reviewed in al-hashimi, ; bothe et al., ; xue et al., ) . one such rd experiment is the r ρ experiment (reviewed in bothe et al., ; palmer & massi, ) . r ρ experiments can provide structural and functional insight for low-populated ess that have short lifetimes. this approach was used to characterize the dynamics of a nt portion of ribosomal rna, the a-site, which must dynamically sample multiple conformations to decode a messenger rna (dethoff, petzold, chugh, casiano-negroni, & al-hashimi, ; a. l. hansen, nikolova, casiano-negroni, & al-hashimi, ). r ρ experiments have also provided insight into the roles of structured rna elements within the hiv- genome, including the transactivation response (tar) element apical loop and the primary dimer initiation site (sl ) (dethoff, petzold, et al., ) . the al-hashimi lab characterized the es of the nt tar rna apical loop, in which many of the loop nucleotides are sequestered in base pairing (dethoff, petzold, et al., ) . the sequestration of loop nucleotides in the tar es inhibits binding by the viral transactivator protein and human cyclin t , activating hiv- genome transcription (dethoff, petzold, et al., ) . the nt sl exhibited significant conformational exchange, consistent with not one, but two excited state structures and the authors propose that these ess play important roles in intermolecular dimerization (dethoff, petzold, et al., ) . similar to cest experiments, r ρ experiments can be carried out using uniformly n/ c-labeled or unlabeled (a. l. hansen et al., ) rnas but have thus far been applied to the study of relatively small rnas. another common rd-based experiment used to characterize protein and nucleic acid dynamics in the microsecond-tomillisecond timescale is the carr-purcell-meiboom-gill (cpmg) experiment (yamazaki, muhandiram, & kay, ) . cpmg can help extract parameters like population, life time and chemical shifts of es in slow and intermediate exchange. extensive studies have been conducted in protein dynamics by cpmg, but in nucleic acids cpmg study is highly limited due to the intrinsic characteristics of dna and rna molecules, such as c- c j coupling and signal degeneracy (johnson & hoogstraten, ; lundstrom, hansen, & kay, ) . recently, using nucleotides that are selectively labeled with c and/or n at specific atom positions (as described above), cpmg was used to study the dynamics of relatively large rnas including the nt frame-shifting element from a human coronavirus (longhini et al., ) . the combination of these newly optimized nmr experiments as well as efficient synthesis of position-selective labeled nucleotides, conformational exchange dynamics of larger rnas will become less challenging and novel mechanisms of rna regulation may be unveiled. one of the main hindrances of nmr analysis of large rnas is the arduous task of assigning chemical shifts. chemical shift assignment is a prerequisite for any quantitative analysis of nmr data for either structure determination or dynamics studies. in an effort to establish the sequence and structural dependence on chemical shift, johnson and colleagues analyzed the nonexchangeable h , h , h , h , h , h , and h chemical shifts for rnas that have been deposited in the publicly available biological magnetic resonance data bank and have associated structure coordinates (barton, heng, johnson, & summers, ; brown, summers, & johnson, ) . here, they sought to identify structural features of a given base pair triplet and examined how the chemical shift of a given nuclei is affected by changes in the local structural environment. these database prediction tools can facilitate the validation of experimentally derived assignments and aid in the manual assignment of nmr data collected on large rna molecules. while advancements have been made to facilitate the study of large rna structure and dynamics by nmr spectroscopy, complimentary structural techniques have proven incredibly useful for providing additional global structural restraints. small-angle x-ray scattering (saxs) has been widely used in combination with nmr spectroscopy to refine nmr-derived structural ensembles (burke et al., ; cornilescu et al., ; grishaev, ying, canny, pardi, & bax, ; imai et al., ; jain et al., ; zuo et al., ) . in fact, studies show that saxs can be combined with sparse rdc restraints to accurately refine idealized/modeled rna structures (grishaev et al., ; wang et al., ) . another approach is to refine nmr structures with a cryo-electron microscopy density map. this approach has been applied to both relatively large and small rnas (gong, schwieters, & tang, ; miyazaki et al., ; zhang et al., ) and will likely be a necessary complement to structural studies as the size of the rna being investigated increases. zika virus produces noncoding rnas using a multi-pseudoknot structure that confounds a cellular exonuclease nmr studies of nucleic acid dynamics rna dynamics: it is about time regio-selective chemical-enzymatic synthesis of pyrimidine nucleotides facilitates rna structure and dynamics studies global shape mimicry of trna within a viral internal ribosome entry site mediates translational reading frame selection characterizing the relative orientation and dynamics of rna a-form helices using nmr residual dipolar couplings discrete structure of an rna folding intermediate revealed by cryo-electron microscopy structure and mechanism of a molecular rheostat, an rna thermometer that modulates immune evasion by neisseria meningitidis database proton nmr chemical shifts for rna signal assignment and validation preparation of isotopically enriched rnas for heteronuclear nmr preparation of isotopically labeled ribonucleotides for multidimensional nmr spectroscopy of rna the protein data bank improving nmr structures of rna genome-wide analysis of rna secondary structure regulation of transcription by long noncoding rnas characterizing rna dynamics at atomic resolution using solution-state nmr spectroscopy prediction of hydrogen and carbon chemical shifts from rna using database mining and support vector regression structure of the yeast u /u snrna complex structural basis for the second step of group ii intron splicing measurement of residual dipolar couplings of macromolecules aligned in the nematic phase of a colloidal suspension of rod-shaped viruses quantitative dimethyl sulfate mapping for automated rna secondary structure inference structural analysis of multi-helical rnas by nmr-saxs/waxs: application to the u /u di-snrna crystal structures of a group ii intron lariat primed for reverse splicing efficient detection of hydrogen bonds in dynamic regions of rna by sensitivity-optimized nmr pulse sequences rna helical packing in solution: nmr structure of a kda gaaa tetraloop-receptor complex accurate shape-directed rna structure determination functional complexity and regulation through rna dynamics visualizing transient low-populated structures of rna direct observation of hydrogen bonds in nucleic acid base pairs by internucleotide ( )j(nn) couplings. journal of the cryo-em structures and dynamics of substrate-engaged human s proteasome nmr structure of the -nucleotide core encapsidation signal of the moloney murine leukemia virus structural basis for packaging the dimeric genome of moloney murine leukaemia virus a fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of rna using ribozyme and rnase h cleavage probing the structure of rnas in solution non-coding rnas in human disease longitudinal-relaxation-enhanced nmr experiments for the study of nucleic acids in solution nmr studies of a.c mismatches in dna dodecanucleotides at acidic ph. wobble a(anti).c(anti) pair formation visualizing the global secondary structure of a viral rna genome with cryo-electron microscopy strong correlation between shape chemistry and the generalized nmr order parameter (s ) in rna conjoined use of em and nmr in rna structure refinement visualizing large rna molecules in solution solution structure of trnaval from refinement of homology model against residual dipolar coupling and saxs data rna-rna interactions in gene regulation: the coding and noncoding players insight into the csa tensors of nucleobase carbons in rna polynucleotides from solution measurements of residual csa: towards new long-range orientational constraints extending the range of microsecond-to-millisecond chemical exchange detected in labeled and unlabeled nucleic acids by selective carbon r- rho nmr spectroscopy pf filamentous phage as an alignment tool for generating local and global structural information in nucleic acids tunable alignment of macromolecules by filamentous phage yields dipolar coupling interactions time-resolved hydroxyl radical footprinting of rna with x-rays nmr structural profiling of transcriptional intermediates reveals riboswitch regulation by metastable rna conformations single-molecule correlated chemical probing of rna an equilibrium-dependent retroviral mrna switch regulates translational recoding an efficient and economic site-specific deuteration strategy for nmr studies of homologous oligonucleotide repeat sequences an accurately preorganized ires rna structure enables eif g capture for initiation of viral translation solution structure of the hiv- intron splicing silencer and its interactions with the up domain of heterogeneous nuclear ribonucleoprotein (hnrnp) a extensive backbone dynamics in the gcaa rna tetraloop analyzed using c nmr spin relaxation and specific isotope labeling long-range interactions in riboswitch control of gene expression structural determinants for ligand capture by a class ii preq riboswitch de novo computational rna modeling into cryo-em maps of large ribonucleoprotein complexes rna structure. structure of the hiv- rna packaging signal nmr detection of intermolecular interaction sites in the dimeric -leader of the hiv- genome nmr study of kda hcv ires rna using segmental isotope labeling structure and sequence elements of the cr / domain of medaka telomerase rna important for telomerase function probing rna dynamics via longitudinal exchange and cpmg relaxation dispersion nmr spectroscopy using a sensitive c-methyl label structures of two aptamers with differing ligand specificity reveal ruggedness in the functional landscape of rna. elife biochemistry. the resolution revolution cryo-em enters a new era. elife, , e nmr probing of invisible excited states using selectively labeled rnas imino proton exchange rates imply an induced-fit binding mechanism for the vegf -targeting aptamer epigenetic regulation by long noncoding rnas structural analysis of a class iii preq riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics riboswitch structure in the ligand-free state incorporation of isotopic, fluorescent, and heavy-atommodified nucleotides into rnas by position-selective labeling of rna synthesis and applications of rnas with positionselective labelling and mosaic composition applications of plor in labeling large rnas at specific sites chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in nmr resonance assignment, dynamics and structural characterizations nmr detection of structures in the hiv- -leader rna that regulate genome packaging isotope labeling strategies for nmr studies of rna structure of hcv ires domain ii determined by nmr structure determination of large biological rnas measurement of carbonyl chemical shifts of excited protein states by relaxation dispersion nmr spectroscopy: comparison between uniformly and selectively c labeled samples accurate measurement of residual dipolar couplings in large rnas by variable flip angle nmr visualizing group ii intron catalysis through the stages of splicing an introduction to biological nmr spectroscopy rna structure analysis at single nucleotide resolution by selective -hydroxyl acylation and primer extension (shape) speciation of a group i intron into a lariat capping ribozyme a structure-based mechanism for trna and retroviral rna remodelling during primer annealing structure of a conserved retroviral rna packaging element by nmr spectroscopy and cryo-electron tomography cryo em structure of intact rotary h(+)-atpase/synthase from thermus thermophilus multiple segmental and selective isotope labeling of large rna for nmr structural studies crystal structure of pistol, a class of self-cleaving ribozyme rna dynamics: perspectives from spin labels preparation of c and n labelled rnas for heteronuclear multi-dimensional nmr studies cryo-em: a unique tool for the visualization of macromolecular complexity characterization of the dynamics of biomacromolecules using rotating-frame spin relaxation nmr spectroscopy metals induce transient folding and activation of the twister ribozyme rna labeling, conjugation and ligation chemical probes for higher-order structure in rna nmr scaler couplings across watson-crick base pair hydrogen bonds in dna observed by transverse relaxation optimized spectroscopy attenuated t relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to nmr structures of very large biological macromolecules in solution ykkc riboswitches employ an add-on helix to adjust specificity for polyanionic ligands group ii intron self-splicing structure of a group ii intron in complex with its reverse transcriptase three-state mechanism couples ligand and temperature sensing in riboswitches structure-based mechanistic insights into catalysis by small self-cleaving ribozymes rna structure: software for rna secondary structure prediction and analysis mapping the landscape of rna dynamics with nmr spectroscopy probing the structural dynamics of proteins and nucleic acids with optical tweezers subunit conformational variation within individual groel oligomers resolved by cryo-em the structural and functional diversity of metabolite-binding riboswitches synthesis and nmr of rna with selective isotopic enrichment in the bases preparation of specifically h-and c-labeled ribonucleotides structural analysis of rna using chemical and enzymatic probing monitored by primer extension crystal structure of the varkud satellite ribozyme rna structure through multidimensional chemical mapping major groove width variations in rna structures determined by nmr and impact of c residual chemical shift anisotropy and h- c residual dipolar coupling on refinement crystal structure of a self-spliced group ii intron structural basis for exon recognition by a group ii intron preparation of large rna oligonucleotides with complementary isotope-labeled segments for nmr structural studies nmr structure of the full-length linear dimer of stem-loop- rna in the hiv- dimer initiation site probing conformational dynamics in biomolecules via chemical exchange saturation transfer: a primer nmr investigation of rna structure structure-activity relationship of flavin analogues that target the flavin mononucleotide riboswitch single particle electron cryomicroscopy: trends, issues and future perspective mechanistic insights into temperature-dependent regulation of the simple cyanobacterial hsp rna thermometer at base-pair resolution a method for helical rna global structure determination in solution using small-angle x-ray scattering and nmr measurements principles for targeting rna with drug-like small molecules architecture and secondary structure of an entire hiv- rna genome advances in rna structure analysis by chemical probing nmr of proteins and nucleic acids determining rna solution structure by segmental isotopic labeling and nmr: application to caenorhabditis elegans spliced leader rna characterizing rna excited states using nmr relaxation dispersion nmr experiments for the measurement of carbon relaxation properties in highly enriched, uniformly c, n-labeled proteins-application to c(alpha) carbons mq-hcn-based pulse sequences for the measurement of c '- h structure of the kda hiv- rna dimerization signal by a hybrid cryo-em, nmr, and molecular dynamics approach an excited state underlies gene regulation of a transcriptional riboswitch characterizing slow chemical exchange in nucleic acids by carbon cest and low spin-lock field r ( rho) nmr spectroscopy crystal structure of group ii intron domain reveals a template for rna assembly measurement of small scalar and dipolar couplings in purine and pyrimidine bases nmr studies of har rna secondary structures reveal conformational dynamics in the human rna dms-mapseq for genome-wide or targeted rna structure probing in vivo solution structure of the cap-independent translational enhancer and ribosome-binding element in the ' utr of turnip crinkle virus advances that facilitate the study of large rna structure and dynamics by nuclear magnetic resonance spectroscopy key: cord- -a hnuxy authors: uversky, vladimir n title: a decade and a half of protein intrinsic disorder: biology still waits for physics date: - - journal: protein science doi: . /pro. sha: doc_id: cord_uid: a hnuxy the abundant existence of proteins and regions that possess specific functions without being uniquely folded into unique d structures has become accepted by a significant number of protein scientists. sequences of these intrinsically disordered proteins (idps) and idp regions (idprs) are characterized by a number of specific features, such as low overall hydrophobicity and high net charge which makes these proteins predictable. idps/idprs possess large hydrodynamic volumes, low contents of ordered secondary structure, and are characterized by high structural heterogeneity. they are very flexible, but some may undergo disorder to order transitions in the presence of natural ligands. the degree of these structural rearrangements varies over a very wide range. idps/idprs are tightly controlled under the normal conditions and have numerous specific functions that complement functions of ordered proteins and domains. when lacking proper control, they have multiple roles in pathogenesis of various human diseases. gaining structural and functional information about these proteins is a challenge, since they do not typically “freeze” while their “pictures are taken.” however, despite or perhaps because of the experimental challenges, these fuzzy objects with fuzzy structures and fuzzy functions are among the most interesting targets for modern protein research. this review briefly summarizes some of the recent advances in this exciting field and considers some of the basic lessons learned from the analysis of physics, chemistry, and biology of idps. a bit more than ten years ago, protein science published a review entitled "natively unfolded proteins: a point where biology waits for physics" (protein sci ( ): - ). the major goal of that article was to bring an intriguing protein family of natively unfolded proteins (which are recognized now to constitute a subset of a very broad class of intrinsically disordered proteins, idps) out of shadow, to emphasize their lack of ordered structure under physiological conditions (at least ordered structure that could be detected by traditional low resolution techniques), to systemize their major structural properties, and to highlight their biological significance. the introduction of such biologically active but essentially unstructured proteins was used to challenge the hitherto dominant structure-centric viewpoint (structure-function paradigm), according to which a specific function of a protein is determined by its unique and rigid three-dimensional ( d) structure. the title of the review ("a point where biology waits for physics") was inspired by the observations that many of such "structure-less" proteins analyzed by that time acted as "binders" that did undergo at least partial folding after interaction with their binding partners. these observations provoked an idea that these biologically important proteins with little or no ordered structure have to wait to become more folded (and functional) as a result of binding to their specific partners. in other words, for these proteins, "biology," that is, the ability to have biological functions, seemed to wait for "physics" which is manifested in their ability to undergo binding-induced folding (at least partial), which is necessary to bring the functional state of these proteins to life. at the beginning, the idea that structure-less proteins can be biologically active was taken as a complete heresy by many researchers, since it was absolutely alien to then dominated structure-function paradigm which represented a foundation of the long-standing belief that the specific functionality of a given protein is determined by its unique -d structure. this structure-function paradigm that describes reasonably well the catalytic behavior of enzymes was based on the "lock-and-key" hypothesis formulated in by emil fischer. this viewpoint was solidified by the successful solution of x-ray crystallographic structures of many proteins (as of february , there were , protein structures in the protein data bank, with , of these structures being determined by xray crystallography). these many crystal structures reinforced a static view of functional protein, where a rigid active site of an enzyme can be viewed as a sturdy lock that provides an exact fit to only one key, a specific and unique substrate. despite numerous limitations, this lock-and-key model was an extremely fruitful concept that was responsible for the creation of modern protein science. figure (a) shows some of the most obvious scientific consequences of the application of structure-function paradigm which is deservedly placed at the center of the "big bang" model that gives rise to the protein science universe. obviously, the consideration of a protein as a rigid crystal-like entity is an oversimplification, since even the most stable and well-folded proteins are dynamic systems that possess different degrees of conformational flexibility. this is because of the simple fact that so-called conformational forces, that is, forces stabilizing the secondary structure of a protein and its tertiary fold, are weak and can be broken even at ambient temperatures due to thermal fluctuations. the breaking of these weak interactions releases the groups that were involved in these interactions and gives them the possibility to be involved in the formation of new weak interactions of comparable energy. since these structural rearrangements are of relatively small scale and since they occur typically in a time scale that is faster than the time required for structure determination by x-ray crystallography and many other physical techniques, the -d structures of proteins determined by these techniques represent averaged pictures. furthermore, one should keep in mind that not all proteins structures which are deposited to pdb are structured throughout their entire lengths. instead, many pdb proteins have portions of their sequences missing from the determined structures (so-called regions of missing electron density) , due to the failure of the unobserved atom, side chain, residue, or region to scatter x-rays coherently caused by their flexible or disordered nature. such flexible/disordered regions are rather common in the pdb, since only about % of protein structures deposited in the pdb do not have such regions of missing electron density. in addition to ordered proteins possessing disordered regions of varying length, the literature contains numerous examples of biologically active proteins with flexible structures. therefore, there is another class of functional proteins and protein regions that contain smaller or larger highly dynamic fragments, and some proteins are even characterized by a complete or almost complete lack of ordered structure under physiological conditions (at least in vitro) which appears to be a critical aspect of these proteins' function in vivo. , [ ] [ ] [ ] [ ] [ ] [ ] these proteins and protein regions (which are known now as idps and idp regions (idprs)) have no single, well-defined equilibrium structure and exist as heterogeneous ensembles of conformers such that no single set of coordinates or backbone ramachandran angles is sufficient to describe their conformational properties. these proteins were independently discovered one-by-one over a long period of time and therefore they were considered as rare exceptions to the general rule. although the phenomenon of biological functionality without stable structure was repeatedly observed, for a long time it was unnoticed by a wide audience because the authors frequently invented new terms to describe their protein of interest. in fact, an incomplete list of terms coined in the literature to describe these proteins includes floppy, pliable, rheomorphic, flexible, mobile, partially folded, natively denatured, natively unfolded, , natively disordered, intrinsically unstructured, , intrinsically denatured, intrinsically unfolded, intrinsically disordered, vulnerable, chameleon, malleable, d, protein clouds, dancing proteins, proteins waiting for partners, and several other names often representing different combinations of "natively/naturally/inherently/intrinsically" with "unfolded/unstructured/disordered/denatured" among several others. therefore, the majority of the names used in the early literature express that the "unfolded, unstructured, disordered, and denatured" state is a "native, natural, inherent, and intrinsic" property of these proteins. although protein intrinsic disorder is considered now as an established concept and pubmed contains hundreds and hundreds of papers talking about different aspects of idps/idprs, the route to recognizing these proteins as a novel functional entity was complex and lengthy. as it is often the case for new scientific concepts, the idea of structure-less functionality went through the stages of passive ignorance and active denial to scrupulous examination and enthusiastic acceptance. for example, it took me more than a year to publish my first paper dedicated to the systematic analysis of such proteins, and the manuscript was successively rejected by journals before it was finally accepted by proteins. however, time showed that the concept of protein intrinsic disorder was a useful invention and could be considered as a universal lock-pick that helps in solving many of the seemingly unsolvable figure . a: protein structure-function paradigm is the "big bang" created universe of the modern protein science. some major directions based on the consideration of protein function as lock-and-key mechanism are shown. modified from ref. . b: paradigm shift caused by the introduction of the protein intrinsic disorder concept opened a wide array of new directions in protein science. in essence, introduction of this concept can be considered as a scientific revolution that, according to kuhn, "occurs when scientists encounter anomalies that cannot be explained by the universally accepted paradigm within which scientific progress has thereto been made" (http://en.wikipedia.org/wiki/paradigm_shift). uversky problems in protein science. one could say that this idea gave a new boost to the development of the protein science, generating a wide array of principally novel research directions [see fig. (b) ]. the goals of this review are: (i) to outline some recent advances in the field of idps/idprs; (ii) to illustrate the usefulness of intrinsic disorder for protein function; (iii) to show that intrinsic disorder can affect different levels of protein structural organization; (iv) to indicate intimate involvement of intrinsic disorder in pathogenesis of various maladies; (v) to emphasize the exceptional structural heterogeneity of idps/idprs and to show that idps are definitely much more structurally complex than random coillike polypeptides; (vi) to accentuate that although this structural heterogeneity is very important for protein functionality, it represents a crucial hurdle for structural characterization of idps; (vii) to stress that new experimental and computational approaches and new theories and models are crucially needed for future progression of this field and protein science in general. these and other points highlight the current state of the field, where further advances in understanding of the "biology" of idps still waits for "physics," with "physics" now being new theories, instrumentation, and analytical approaches. identification of idps as unique entities belonging to a new protein tribe is directly related to the recognition that their amino acid sequences are dramatically different from those of ordered proteins. , , , [ ] [ ] [ ] for example, it has been pointed out that the low content of hydrophobic residues combined with the high load of charged residues that often gives rise to high net charge of a polypeptide chain represents a characteristic feature of some idps (so called extended idps or natively unfolded proteins with coil-like or close to coil-like structures, see below). therefore, compact proteins and extended idps can be distinguished based only on their net charges and hydropathies using a simple charge-hydropathy (ch) plot, where the idps are specifically localized within a specific region of ch phase space and are reliably separated from compact ordered proteins. more detailed comparison of amino acid sequences revealed that in comparison with ordered proteins and domains, the idps/idprs are significantly depleted in order-promoting amino acids (trp, tyr, phe, ile, leu, val, cys, and asn), , being instead enriched in disorder-promoting residues, such as ala, arg, gly, gln, ser, glu, lys, and pro. , , , , difference between ordered and disordered proteins goes far beyond these differences in their amino acid compositions. in fact, based on the comparison of the amino acid physico-chemical property-based scales (such as hydropathy, net charge, flexibility index, helix propensities, strand propensities, aromaticity, etc.) and more than composition-based attributes (e.g., all possible combinations having one to four amino acids in the group) it has been concluded that ordered and disordered proteins and regions can be discriminated using many of these attributes. based on the analysis of amino acid scales, a novel amino acid scale, top-idp (trp, phe, tyr, ile met, leu, val, asn, cys, thr, ala, gly, arg, asp, his, gln, lys, ser, glu, and pro), was built to provide ranking for the tendencies of the amino acid residue to promote order or disorder. the fact that the sequences of ordered and disordered proteins and regions are noticeably different suggested that idps clearly constitute a separate entity inside the protein kingdom, that these proteins can be reliably predicted using various computational tools, [ ] [ ] [ ] [ ] [ ] [ ] and structurally, that idps should be very different from ordered globular proteins since peculiarities of amino acid sequence determine protein structure. natural abundance of idps: touching the tip of the iceberg initial systematic analyses revealed that intrinsic disorder in proteins is a rather common phenomenon. in fact, as of , the list of experimentally validated natively unfolded proteins with chain length greater than amino acid residues contained more than entries. it was also pointed out that this list would probably be doubled if shorter polypeptides - residues long were included, and that these experimentally validated natively unfolded have at least homologues, which are also expected to be natively unfolded. , it happened that these "large" numbers (which actually were large enough to make a crucial point that biologically active structure-less proteins represent the new rule and not mere rare exceptions) constitute just a small tip of an iceberg. in fact, using computational tools developed for sequence-based intrinsic disorder prediction the wide spread of idps and hybrid proteins containing idprs was convincingly shown. [ ] [ ] [ ] [ ] for example, more than , out of , proteins in the thencurrent swiss protein database were identified as having long idprs. the published in analysis of whole genomes that span the kingdoms of life revealed that many proteins contained segments predicted to have consecutive disordered residues and that the eukaryotes exhibited more disorder by these measures than either the prokaryotes or the archaea. other studies on the abundance of intrinsic disorder in various evolutionary distant species supported these findings and consistently showed that the eukaryotic proteomes had higher fraction of intrinsic disorder than prokaryotic proteomes. , [ ] [ ] [ ] [ ] [ ] this conclusion is in line with the results of a comprehensive bioinformatics investigation of the disorder distribution in almost proteomes from viruses and three kingdoms of life, results of which are shown in figure as the correlation between the intrinsic disorder content and proteome size for species from viruses, archaea, bacteria, and eukaryotes. surprisingly, figure shows that there is a well-defined gap between the prokaryotes and eukaryotes in the plot of fraction of disordered residues on proteome size, where almost all eukaryotes have % or more disordered residues, whereas the majority of the prokaryotic species have % or fewer disordered residues. therefore, it looks like the fraction of % disordered residues serves as a boundary between the prokaryotes and eukaryotes and reflects the existence of a complex step-wise correlation between the increase in the organism complexity and the increase in the amount of intrinsic disorder. a gap in the plot of fraction of disordered residues on proteome size parallels a morphological gap between prokaryotic and eukaryotic cells which contain many complex innovations that seemingly arose all at once. in other words, this sharp jump in the disorder content in proteomes associated with the transition from prokaryotic to eukaryotic cells suggests that the increase in the morphological complexity of the cell paralleled the increased usage of intrinsic disorder. the variability of disorder content in unicellular eukaryotes and rather weak correlation between disorder status and organism complexity (measured as the number of different cell types) is likely related to the wide variability of their habitats, with especially high levels of disorder being found in parasitic host-changing protozoa, the environment of which changes dramatically during their life-span. the further support for this hypothesis came from the fact that the intrinsic disorder content in multicellular eukaryotes (which are characterized by more stable and less variable environment of individual cells) was noticeably less variable than that in the unicellular eukaryotes. it was pointed out that idps possess noticeable amino acid biases, and many idps/idprs are characterized by sequence redundancy and low sequence complexity, containing long stretches of various repeats and being completely devoid of some (often many) types of amino acid residues. these observations seem to indicate that the sequence space of idps/idprs should be simpler than that of ordered proteins. however, the reality is more complex than conventional wisdom might suggest, and the sequence space attainable by simple idps/idprs is more diversified than that of the structurally more sophisticated ordered proteins. in fact, a residue-long protein in which any of the normally occurring amino acids can be found has a sequence space of ( ) sequences. obviously, not all random amino acid sequences can fold into unique structures. in other words, a sequence space of a foldable protein (or "foldable" sequence space) is noticeably smaller than the entire sequence space available for a random polypeptide chain. for decades, the actual size of "foldable" sequence space continues to be unsolved mystery despite a large body of theoretical, biochemical, and computational work that aims to unravel the relationship between a protein's primary sequence and its resulting d structure. however, the actual number of different amino acid residues in a given foldable sequence can be dramatically reduced, since all twenty residues are not necessary for protein folding and the actual physicochemical identity of most of the amino acids in a protein is irrelevant. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in other words, folding alphabet can be noticeably reduced, , and amino acids can be clustered based on some shared features such as homolog substitution frequency, local structural environments, or peculiarities of the tertiary structural environments. this simplified folding code further reduces the available "foldable" sequence space. figure . correlation between the intrinsic disorder content and proteome size for species from viruses, archaea, bacteria, and eukaryotes. each symbol indicates a species. there are totally six groups of species: viruses expressing one polyprotein precursor (small red circles filled with blue), other viruses (small red circles), bacteria (small green circles), archaea (blue circles), unicellular eukaryotes (brown squares), and multicellular eukaryotes (pink triangles). each viral polyprotein was analyzed as a single polypeptide chain, without parsing it into the individual proteins before predictions. the proteome size is the number of proteins in the proteome of that species and is shown in log base. the average fraction of disordered residues is calculated by averaging the fraction of disordered residues of each sequence over the all sequences of that species. disorder prediction is evaluated by pondr-vsl b. modified from ref. . simply by virtue of their existence, idps/idprs add a new level of complexity to the sequence-structure relationship, dividing the population of protein sequences into two categories, sequences that yield natively ordered, and sequences that code natively disordered proteins. idps/idprs cannot fold spontaneously and some of them require specific partners to gain more ordered structure. therefore, they do not possess an entire folding code that defines the ability of foldable proteins to fold spontaneously into a unique biologically active structure. the missing portion of the idp folding code (or at least part of it) is supplemented by binding partner(s). this defines a principal difference between structured proteins and idps/idprs: foldable proteins fold first and then bind to their partners whereas idps/idprs remain disordered until they interact with their partners. , furthermore, many idps/idprs do not require folding to be functional, , , , , [ ] [ ] [ ] [ ] and some of them form fuzzy complexes, in which they preserve significant amount of disorder. , all this suggests that the sequence space of idps (at least those which either do not fold at all or do not completely fold at binding) is noticeably greater than the "foldable" sequence space due to the removal of restrictions posed by the need to gain ordered structure spontaneously. this represents one of the conundrums of intrinsic disorder, where the apparent sequence redundancy and simplicity are combined with the lack of structural restrains leading to the increase in the dimensions and complexity of the available sequence space. also, the existence of a noticeable sequencestructure heterogeneity of idps should be emphasized. since the unique d-structure of an ordered single-domain protein is defined by the interplay between all (or almost all) of its residues, one could expect that the structure-coding potential is homogeneously distributed within its amino acid sequence. on the other hand, a sequence of an idp/idpr contains multiple, relatively short functional elements and therefore represents a very complex structural and functional mosaic. this important feature defines the known ability of an idp/idpr to interact, regulate, and be controlled by multiple structurally unrelated partners. such functional "anatomy" of idps/idprs is determined by the extremely high level of their sequence heterogeneity, which is further increased due to the ability of a single idpr to bind to multiple partners gaining very different structures in the bound state. one of the crucial consequences of an extended sequence space and non-homogeneous distribution of foldability (or the structure-coding potential) within amino acid sequences of idps and idprs is their astonishing structural heterogeneity. in fact, a typical idp/idpr contains a multitude of elements coding for potentially foldable, partially foldable, differently foldable, or not foldable at all protein segments. as a result, different parts of a molecule are ordered (or disordered) to a different degree. this distribution is constantly changing in time where a given segment of a protein molecule has different structures at different time points. as a result, at any given moment, an idp has a structure which is different from a structure viewed at another moment. another level of structural heterogeneity is determined by the fact that many proteins are hybrids of ordered and disordered domains and regions, and this mosaic structural organization is crucial for their functions. also, even when they do not possess ordered domains, idps are known to have various levels and depth of disorder. over a few past years, an understanding of the available conformational space of idps/idprs underwent significant evolution. in fact, for a long time, idps were considered mostly "unstructured" or "natively unfolded" polypeptide chains. this was mostly due to the fact that the majority of idps analyzed at early stages of the field contained very little ordered structure, that is, they were really mostly unstructured or unfolded. finding and characterization of such "structure-less" proteins was important to build up a strong case to counter-point the dominant view represented by the classical sequence-to-structureto-function paradigm, especially since such fully unstructured, yet functional proteins clearly represented the other extreme of the protein structurefunction spectrum. the top half of the figure illustrates this situation by opposing rock-like ordered proteins and cooked spaghetti-like idps. however, already in some early studies, it was indicated that idps/idrs could be crudely grouped into two major structural classes, proteins with compact and extended disorder. , , , , based on these observations, the protein functionality was ascribed to at least three major protein conformational states, ordered, molten globular, and coil-like, , indicating that functional idps can be less or more compact and possess smaller or larger amount of flexible secondary/tertiary structure. , , , , , roughly at the same time, it was emphasized that the extended idps (known as natively unfolded proteins) do not represent a uniform entity but contain two broad structural classes, native coils and native pre-molten globules. currently available data suggest that intrinsic disorder possesses multiple flavors, can have multiple faces, and can affect different levels of protein structural organization, where whole proteins, or various protein regions can be disordered to a different degree. this new view of structural space of functional proteins can be visualized to form a continuous spectrum of differently disordered conformations extending from fully ordered to completely structure-less proteins, with everything in between (fig. , bottom half) . here, functional proteins can be well-folded and be completely devoid of disordered regions (rock-like scenario). other functional proteins may contain limited number of disordered regions (a grass-on-the rock scenario), or have significant amount of disordered regions (a llama/camel hair scenario), or be molten globule-like (a greasy ball scenario), or behave as pre-molten globules (a spaghetti-and-meatballs/sausage scenario), or be mostly unstructured (a hairball scenario). notably, in this representation, there is no boundary between ordered proteins and idps, and, the structure-disorder space of a protein is considered as a continuum. it is important to remember that even the most ordered proteins do not resemble "solid rocks" and have some degree of flexibility. in fact, a protein molecule is an inherently flexible entity and the presence of this flexibility (even for the most ordered proteins) is crucial for its biological activity. also, another important point to remember is that due to their heteropolymeric nature, proteins are never random coils and always have some residual structure. protein biophysicists/biochemists working on different aspects of ordered proteins (e.g., analyzing their structural properties, functions, folding, etc.) would find biophysical properties of functional idps/idprs to be rather unusual since these highly dynamic proteins do not follow the well-accepted wisdom that a protein has to be well-folded to be biologically functional. however, the unusualness is a subjective feature, and from the viewpoint of polymer physics the extended idps/idprs possess the expected behavior . structural heterogeneity of idps/idprs. top half: bi-colored view of functional proteins which are considered to be either ordered (folded, blue) or completely structure-less (disordered, red). ordered proteins are taken as rigid rocks, whereas idps are considered as completely structure-less entities, kind of cooked noodles. bottom half: a continuous emission spectrum representing the fact that functional proteins can extend from fully ordered to completely structure-less proteins, with everything in between. intrinsic disorder can have multiple faces, can affect different levels of protein structural organization, and whole proteins, or various protein regions can be disordered to a different degree. some illustrative examples includes ordered proteins that are completely devoid of disordered regions (rock-like type), ordered proteins with limited number of disordered regions (grass-on-the rock type), ordered proteins with significant amount of disordered regions (lhama/camel hair type), molten globule-like collapsed idps (greasy ball type), pre-molten globule-like extended idps (spaghetti-and-sausage type), and unstructured extended idps (hairball type). of flexible and charged polymers, whereas the behavior of an ordered protein is rather unexpected (i.e., due to the existence of the native ensemble that for well-folded, ordered proteins can be approximated as a harmonic well around a unique, welldefined equilibrium structure). therefore, one definitely should keep in mind that the "unusual" biophysics of extended idps/idprs has its roots in the usual polymer physics of highly charged and flexible polypeptides. each protein is believed to be a unique entity that has quite unique primary sequence which governs its d structure (or lack thereof) and ensures specific biological function(s). therefore, understanding the effect of sequence variance on the biological performance presents a challenging task. however, natural polypeptides have originated as random copolymers of amino acids, which were adjusted or "selected" over evolution based on their functional capacities. , despite their differences in primary amino acid sequences, protein molecules in a number of conformational states behave as polymer homologues, suggesting that the volume interactions can be considered as a major driving force responsible for the formation of equilibrium structures or structural ensembles. for example, ordered globular proteins and molten globules (both as folding intermediates of globular proteins or as examples of collapsed idps) exhibit key properties of polymer globules, where the fluctuations of the molecular density are expected to be much less than the molecular density itself. extended idps (both intrinsic coils and intrinsic pre-molten globules) and ordered proteins in the pre-molten globule intermediate state possess properties of squeezed coils, since water is a poor solvent for a polypeptide. in fact, even high concentrations of strong denaturants (e.g., urea and gdmcl) are very likely to be bad solvents for protein chains, resulting in the preservation of extensive residual structure even under these harsh denaturing conditions. based on these and related observations, and taking into account the fact that many idps/idprs are characterized by significant amino acid composition biases, the overall polymeric behavior of these proteins and regions can be mimicked reasonably well by the behavior of low-complexity polypeptides (e.g., homopolypeptide and block copolypeptides). following these ideas, it was shown that water is a poor solvent for polypeptide backbone alone and for the idps containing long tracts of polar amino acid residues since polar homo-polypeptides without hydrophobic groups (e.g., polyglutamine or glycineserine block copolypeptides) were shown to prefer collapsed ensembles in aqueous media. [ ] [ ] [ ] [ ] [ ] [ ] furthermore, even polyglycine was shown to have a tendency to form heterogeneous ensembles of collapsed structures in water. a systematic analysis of the conformational behavior of protamines, arginine-rich idps involved in the condensation of chromatin during spermatogenesis, and protamine-like peptides revealed that there is a charge-driven coil-to-globule transition in these highly charged polypeptides, where the net charge per residue serves as the discriminating order parameter. overall, the increase in the hydrodynamic dimensions of a polypeptide chain with increase in its net charge per residue can be attributed to the increase in the intramolecular electrostatic repulsions between similarly charged sidechains and the favorable solvation of these moieties. based on these premises, at least three different classes of globule-forming polar/charged idps were proposed. the first class is comprised by polar tracts which collapse due to water being a poor solvent for a backbone and non-charged side chains. the second class is represented by weak polyelectrolytes and weak polyampholytes, which have low per residue net charge and low fractions of positively and/or negatively charged residues. these idps/ idprs form collapsed structures since the driving force responsible for the collapse is not overcome by the intramolecular electrostatic repulsion between the charged side-chains and by their favorable free energies of solvation. furthermore, if such idps/ idprs possess polyampholytic nature, their globular state could be additionally stabilized by electrostatic interactions between the oppositely charged sidechains. finally, idps/idprs from the third class are strong polyampholytes characterized by high fractions of positively and/or negatively charged residues but have low per residue net charge. such intrinsically disordered protein can form collapsed structures stabilized mostly by multiple electrostatic interactions between solvated side-chains of opposite sign. the extended idps/idprs were used as a model system for the analysis of the effect of electrostatic interactions on conformational properties of unfolded proteins, and for testing the quantitative descriptions and predictions of polymer theory related to the influence of charged amino acids on chain dimensions. for example, based on the analysis of the conformational equilibrium of coarse-grained polypeptides as a function of sequence hydrophobicity, charge, and length it has been concluded that the variations in sequence hydrophobicity and charge define a coil-to-globule transition comparable to that seeing in the empirical ch-plot, , suggesting that a minimal, polymer physics-based model can capture the elements of global protein conformation. idps/idprs with very high net charges are expected to be more extended and behave more similar to random coils (i.e., similar to conformations adopted by proteins in the denaturant gdmcl). the analysis of the gdmcl-induced expansion of the unfolded states suggested that protein charge density plays a crucial role in defining the hydrodynamic behavior of the unfolded polypeptide chain. here, highly charged proteins can exhibit a prominent expansion at low ionic strength that correlates with their net charges. it has been also hypothesized that the pronounced effect of charges on the dimensions of unfolded proteins might have important implications for their cellular functions. similarly, a comprehensive analysis of the hydrodynamic dimensions of fg-nucleoporins containing large idprs with multiple phenylalanineglycine repeats (fg-domains) revealed that under the physiologic conditions in vitro these domains adopt distinct categories of disordered structures, such as molten globule, pre-molten globule, relaxedcoil, extended-coil (as in urea), or very extended-coil (as in gdmcl). the category of intrinsically disordered structure in a given fg-domain was related to its amino acid composition, namely to the content of charged residues, where more charged fg-domains possessed larger hydrodynamic dimensions. furthermore, fg-nucleporins with higher charge density were shown to be more dynamic than the collapsed-coil fg-domains, being also prone to repel other fg-domains. on the other hand, the collapsedcoil fg-domains were prone to oligomerize. these observations suggested that different types of fgdomains with different aggregation propensities provide molecular basis for two different gating mechanisms operating at the nuclear pore complex at distinct locations; one acting as a hydrogel, and the other as an entropic brush. therefore, the abundance and peculiarities of the charged residues distribution within the protein sequences might determine physical and biological properties of extended idps and idprs. also, simple polymer physics-based reasoning can give reasonably well-justified explanation of the conformational behavior of extended idps. in general, the conformational behavior of idps is characterized by the low cooperativity (or the complete lack thereof) of the denaturant-induced unfolding, lack of the measurable excess heat absorption peak(s) characteristic for the melting of ordered proteins, "turned out" response to heat and changes in ph, and the ability to gain structure in the presence of various binding partners. the analysis of the temperature effects on structural properties of several extended idps revealed that native coils and native pre-molten globules partially fold as the temperature is increased. , , [ ] [ ] [ ] [ ] these heating-induced structural changes in extended idps were attributed to the increased strength of the hydrophobic interaction at higher temperatures, leading to a stronger hydrophobic attraction, which is the major driving force for folding. similarly, extended idps/idprs are characterized by the "turned out" response to changes in ph, , - where a decrease (or increase) in ph induces their partial folding due to the minimization of their high net charges viewed at neutral ph, thereby decreasing charge/charge intramolecular repulsion and permitting hydrophobicdriven collapse to the partially folded conformation. every disordered protein is disordered in its own way data accumulated so far indicate that intrinsic disorder exists at multiple structural levels and might differently affect different regions/domains of idps. this defines noted structural complexity and heterogeneity of idps/idprs which are further enhanced by the way different proteins/protein regions respond to their environments. furthermore, since intrinsic disorder is crucial for many biological functions and therefore must prevail in different environments, the amino acid sequences and compositions of idps and idprs are specifically shaped by the peculiarities of their global and local environments. all this makes the protein intrinsic disorder phenomenon to be so broad that one can even assume that every disordered protein (or at least every family of disordered proteins) is disordered in its own way. this hypothesis has far-reaching consequences since it implies that a general disorder predictor has limited accuracy and cannot predict with equally high accuracy disorder status of all protein sequences due to their heterogeneity. it also implies that some environmental factors definitely should be taken into account when assessing intrinsic disorder in proteins. several examples are presented below to support the overall validity of these statements. the first example is given by transmembrane (tm) proteins, in which disorder is widely observed (e.g., % of human integral plasma proteins were predicted to contain long idprs). [ ] [ ] [ ] [ ] [ ] furthermore, disorder is unevenly distributed between the cytoplasmic and the external surfaces of these proteins, with cytoplasmic domains being up to threefold more disordered than extracellular domains. although these analyses gave interesting hints on the abundance of disorder in tm proteins, the obvious weakness of such evaluations is in the fact that they were performed using the disorder predictors developed from structured and disordered regions found in water-soluble proteins. however, the major physico-chemical properties of water-soluble and integral membrane proteins are very different due to the differences in their environments. for example, similar to typical water soluble proteins, the tm regions of membrane proteins are often highly structured, containing a-helices or b-structure, which are especially likely to occur due to the low dielectric constant values within the membrane bilayers. , on the other hand, the exterior regions of tm proteins are much more apolar than the exteriors of water-soluble proteins. [ ] [ ] [ ] therefore, the peculiarities of the membrane environment, with its highly nonpolar nature originating either from lipids or from protein interiors, are especially unfavorable for intrinsic disorder, since propensity for intrinsic disorder is typically encoded in a high content of polar and charged residues. therefore, the idprs found in integral membrane proteins would be expected to be generally localized within the regions external to the membrane bilayer. also, the distinctive environment of the membrane bilayer imposes constraints on the amino acid composition of integral membrane proteins, even on the regions external to the membrane bilayer. , comprehensive bioinformatics analysis revealed that integral membrane proteins commonly possess idprs defined as regions of missing electron density in their crystal structures. comparison of the idprs found in the a-helical and the b-barrel bundle integral membrane proteins with the idprs viewed in typical water-soluble proteins revealed the existence of statistically distinct amino acid compositional biases characteristic for these three protein classes. therefore, the use of specific amino acid signatures of idprs found in tm helical bundles and b-barrels can potentially lead to significantly more accurate disorder predictions for these two classes of integral membrane proteins. another illustrative example of the specific disorderrelated and environment-dependent sequence features is given by archaeal proteins. , based on the levels of predicted disordered residues, archaeal proteins can be grouped into three classes, with ranges of the disordered residue content of - %, %- %, and %- % (see fig. ). the archaeal proteomes with the highest disorder contents are halophiles and methanophiles. , similar to tm proteins, the estimation of intrinsic disorder in the extremophilic proteins of the microorganisms surviving under hypersaline conditions using predictors developed for the "normal" non-halophilic proteins existing under the normal physiological conditions of - mm nacl may not be accurate. in fact, one of the strategies used by the halophilic archaea, which are salt-loving extremophilic organisms that grow optimally at high salt concentrations, to maintain proper osmotic pressure in their cytoplasm is a so-called "salt-in" strategy that involves accumulation of molar concentrations of potassium and chloride in their cytosoles. this strategy requires extensive adaptation of the intracellular proteins to the presence of near-saturating salt concentrations. the proteomes of such "salt-in" organisms are highly acidic, , and their proteins are characterized by remarkable instability at conditions of low salt concentrations and by maintaining soluble and active conformations in hypersaline conditions that are generally detrimental to the non-halophilic proteins. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] finally, peculiarities of disorder distributions in viral proteins can be used to further support the importance of considering environmental factors. , here, the comprehensive analysis of intrinsic disorder in various completed proteomes revealed that the viral proteomes have the largest variation of disorder content, which ranges from . % disordered residues in the human coronavirus nl to . % disordered residues in the avian carcinoma virus proteome (see fig. ). the high predicted intrinsic disorder content in viruses has multiple functional implications, where some idprs are used in the functioning of viral proteins and help viruses to highjack various pathways of the host cells, others likely have evolved to help viruses accommodate to their hostile habitats, and still others evolved to help viruses in managing their economic usage of genetic material via alternative splicing, overlapping genes, and anti-sense transcription. these findings are in agreement with another study revealing that in comparison with archaea and bacteria, viral and bacteriophagic proteins were significantly more enriched in polar residues and depleted in hydrophobic residues and were close to eukaryotic proteins in terms of their amino acid compositions and the reduced content of the order-promoting residues. functional protein clouds: major functional advantages of being intrinsically disordered the high natural abundance of idpds/idprs and their specific structural features indicate that these proteins and regions might carry out important biological functions. this hypothesis has been confirmed by several comprehensive studies, , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , [ ] [ ] [ ] [ ] [ ] which revealed that these structure-less members of the protein kingdom are abundantly involved in numerous biological processes, where they are frequently found to play different roles in regulation of the functions of their binding partners and in promotion of the assembly of supra-molecular complexes. , , [ ] [ ] [ ] [ ] [ ] , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the conformational plasticity of idps/idprs provides them with a wide spectrum of exceptional functional advantages over the functional modes of ordered proteins and domains. , , , , , , , , , , , , , , , some of these advantages are: increased speed of interaction due to greater capture radius and the ability to spatially search through interaction space; increased interaction (surface) area per residue; strengthened encounter complex allows for less stringent spatial orientation requirements; efficient regulation via rapid degradation; the ability to be involved in one-to-many binding, where a single disordered region binds to several structurally diverse partners; the ability to be involved in many-to-one binding, where many distinct (structured) proteins may bind a single disordered region; the ability to overcome steric restrictions, enabling larger interaction surfaces in protein-protein and protein-ligand complexes than those obtained with rigid partners; the ability to fold upon binding (completely or partially); the ability of some idps/idprs to form very stable intertwined complexes; the ability of some idps/idprs to stay substantially disordered in bound state; binding fuzziness, where different binding mechanisms (e.g., via stabilizing the binding-competent secondary structure elements within the contacting region, or by establishing the longrange electrostatic interactions, or being involved in transient physical contacts with the partner, or even without any apparent ordering) can be employed to accommodate peculiarities of interaction with various partners; binding plasticity, where an idpr folds to specific bound conformations (which can be very different) according to the template provided by binding partners; high accessibility of sites targeted for posttranslational modifications (ptms); efficient structural and functional regulation via ptms such as phosphorylation, acetylation, lipidation, ubiquitination, sumoylation, and so forth, allowing for a simple means of modulation of their biological functions; efficient functional control via regulatory proteolytic attack sites of which are frequently associated with idprs; ease of regulation/redirection and production of otherwise diverse forms by alternative splicing (given the existence of multiple functions in a single disordered protein, and given that each functional element is typically relatively short, alternative splicing could readily generate a set of protein isoforms with a highly diverse set of regulatory elements ); the possibility of overlapping binding sites due to extended linear conformation; decoupled binding affinity and specificity, where, due to the induced folding, idp/idpr can be involved in the formation of specific but weak complexes. in other words, idp/idpr might possess high specificity for given partners combined with high k on and k off rates that enable rapid association with the partner without an excessive binding strength. this combination of high specificity with low affinity defines the broad utilization of intrinsic disorder in regulatory interactions where turning a signal off is as important as turning it on; diverse evolutionary rates with some id proteins being highly conserved and other id proteins possessing high evolutionary rates. the latter ones can evolve into sophisticated and complex interaction centers (scaffolds) that can be easily tailored to the needs of divergent organisms; flexibility that allows masking (or not) of interaction sites or that allows interaction between bound partners; the ability to be involved in the cascade interactions, where idp binding to the first partner induces partial folding generating a new binding site suitable for interaction with the second partner, and so forth. many disorderrelated functions (e.g., signaling, control, regulation, and recognition) are incompatible with well-defined, stable -d structures. , [ ] [ ] [ ] [ ] , , , , , , [ ] [ ] [ ] , , functions of many idps/idprs rely on interactions with specific binding partners, and many idps/idprs tend to undergo disorder-to-order transitions as a result of binding to their specific targets. functionally, idps/idprs were grouped in at least six broad classes based on the mode of action. , these broad classes included protein and rna chaperones, entropic chains, effectors, scavengers, assemblers and display sites, , and separate functions, including molecular recognition via binding to other proteins, or to nucleic acids, were assigned for idprs in early studies. , later, a rich spectrum of biological functions associated with idps/idprs was found based on a comprehensive computational study of a correlation between the functional annotations in the swiss-prot database and predicted intrinsic disorder. [ ] [ ] [ ] the approach was based on the hypothesis that if a function described by a given keyword relies on intrinsic disorder, then the keyword-associated protein would be expected to have a greater level of predicted disorder compared to the protein randomly chosen from the swiss-prot. this analysis revealed that % and % of swiss-prot functional keywords were associated with ordered and disordered proteins, respectively, whereas % functional keywords yielded ambiguity in the likely function-structure associations. [ ] [ ] [ ] interestingly, most of the structured protein-associated key words were shown to be related to enzymatic activities, whereas the majority of the disordered protein-associated keywords were related to signaling and regulation. these results agree well with the notion that enzymatic catalysis requires ordered structure and that effectiveness of signaling is dependent on binding reversibility, a property directly associated with the thermodynamics of disorder-to-order transition induced by binding. [ ] [ ] [ ] many idps and idprs undergo a disorderto-order transition upon functioning. , , , , , , , [ ] [ ] [ ] , [ ] [ ] [ ] [ ] when disordered regions bind to signaling partners, the free energy required to bring about the disorder to order transition takes away from the interfacial, contact free energy, with the net result that a highly specific interaction can be combined with a low net free energy of association. , high specificity coupled with low affinity is a useful pair of properties for a reversible signaling interaction. furthermore, a disordered protein can readily bind to multiple partners by changing shape to associate with different targets. , , all this clearly suggests that there is a new twopathway protein structure-function paradigm, with sequence-to-structure-to-function for enzymes and membrane transport proteins, and sequence-to-disordered ensemble-to-function for proteins and protein regions involved in signaling, regulation, and control. , , , , one of the first generalization of this concept was given by the protein trinity hypothesis, which suggested that native proteins can be in one of three states, the solid-like ordered state, the liquid-like collapsed-disordered state, or the gas-like extended-disordered state. function is then viewed to arise from any one of the three states or from transitions between them. this model was subsequently expanded to include a fourth state (pre-molten globule) and transitions between all four states. in reality, based on the outlined above idea of the continuous spectrum of protein structures, functional proteins contain various amounts of intrinsic disorder and this continuous structural spectrum of protein defines their limitless functional variability. among intriguing protein functions relying on intrinsic disorder are moonlighting activities, actions of hub proteins, , , , [ ] [ ] [ ] [ ] [ ] and scaffolding functions. , since all these functions illustrate the notions that the intrinsic disorder concept represents a universal skeleton key (or lock-pick) that helps unlocking seemingly unresolvable mysteries of protein science and therefore can be considered as a new ariadne's thread that helps navigate the unusual twists of the sophisticated relationships between protein sequence, structure, and function, they are considered in some detail below. moonlighting proteins. moonlighting is the ability of a protein to fulfill more than one function. often, these functions are unrelated or at least are not obviously related to each other. , [ ] [ ] [ ] the capability of a protein to be involved in moonlighting or multi-tasking activities represent one of the solutions used by the nature to increase the organism's complexity without the expansion of the genome size, where by acting differently at distinct points of metabolic networks proteins increase network complexity without increasing the actual size of the network. , [ ] [ ] [ ] among various molecular mechanisms used by the moonlighting proteins to switch between functions are changes in cellular localization, changes in ligand binding, expression in different cell types, and variations of the oligomerization state. in addition to these mechanisms that can be explained within the frames of the traditional structure-function paradigm, consideration of the intrinsic disorder phenomenon opens new possibilities. in fact, one of the peculiar functional advantages of idps/idprs is their binding promiscuity and ability to be involved in one-to-many signaling, whereby an idp/idpr binds structurally different partners in a template-induced folding process. , , , therefore, idps/idprs can use the same region or overlapping interaction regions/surfaces to exert distinct effects and employ the disorderbased mechanisms to switch function that relies on their capability to form different conformations upon binding. such structural malleability of idps/ idprs defines their ability to participate in unprecedented moonlighting events, where these disordered moonlighting proteins or regions produce the opposing effects (inhibition and activation) on different partners or even the same partner molecule. hub proteins. signaling interactions inside the cell can be described as specific and complex networks that can be considered as "scale-free" or "small-world" networks, which have hubs, with many connections, and ends, that have the only connection to just one neighbor. , such scale-free networks combine the local clustering of connections characteristic of regular networks with occasional long-range connections between clusters, as can be expected to occur in random networks. in other words, the distance between nodes in these scalefree networks follows a power-law distribution. based on their spatiotemporal peculiarities protein hubs were grouped into two broad categories, "date hubs" that binds their numerous partners sequentially, and "party hubs" simultaneously interacting with their partners. since many idps are known to be involved in interaction with large number of distinct partners, they clearly can be considered as hubs in the scale-free protein-protein interaction networks. , based on the systematic analysis of several know hub proteins followed by a series of robust bioinformatics studies, , - it was concluded that hubs commonly use disordered regions to bind to multiple partners and that there are at least two primary mechanisms by which disorder is utilized in protein-protein interaction networks where one disordered region binds to many partners or many disordered region bind to one partner. scaffold proteins. scaffold proteins constitute an important subclass of hubs that typically have a modest number of interacting partners and that are commonly found at the central parts of functional complexes, where they interact with most of their partners at the same time and therefore act as party hubs. besides being responsible for bringing together specific proteins within a signaling pathway and providing selective spatial orientation and temporal coordination to facilitate and promote interactions among interacting proteins, some scaffolds can influence the specificity and kinetics of signaling interactions via simultaneous binding to multiple participants in a particular pathway and facilitation and/or modifying the specificity of pathway interactions, other scaffold can change conformations of individual proteins and thus modulate their activities, still other scaffold proteins may modulate the activation of alternative pathways by promoting interactions between various signaling proteins. analysis of several well-characterized signaling scaffold proteins reveled that their large idprs are crucial for the successful scaffold function. a more global bioinformatics analysis revealed that a typical design of a scaffold protein includes a set of short globular domains ( amino acids on average) connected by long linker regions ( residues on average) with crucial binding functions. this gave further support to the notion that signaling scaffold proteins utilize the various features of highly flexible id regions to obtain more functionality from less structure. disorder and transcription regulation. conformational plasticity and adaptability associated with intrinsic disorder are crucial for various protein functions. among the proteins whose functional life is strongly disorder-dependent are transcription factors (tfs) , and other proteins involved in transcriptional regulation, such as the mediator complex, , core and linker histones, and ribosomal proteins. for example, from to % of tfs might possess long idprs, with the degree of disorder in eukaryotic tfs being significantly higher than in prokaryotic tfs. , also, tfs were shown to be depleted in order-promoting residues and enriched in disorder-promoting residues, and were characterized by high levels of a-molecular recognition feature (morf). furthermore, disorder is unevenly distributed within the tfs, with the degree of disorder in their activation regions being much higher than that in dna-binding domains. however, the at-hooks (which are dna-binding motifs present in many proteins which binds to the (ataa) and (tatt) repeats of dna) and basic regions of tf dna-binding domains are highly disordered suggesting that eukaryotes with their well-developed gene transcription machinery require transcription factor flexibility to be more efficient. a number of interesting and important roles were also ascribed to intrinsic disorder in tfs related to the regulation of heat shock response (so called heat shock factors, hsfs) and in the reprogramming tfs (the yamanaka factors, namely sox , oct / (pou f ), klf , and c-myc, and the thomson factors, namely sox , oct , lin , and nanog) overexpression of which is known to generate induced pluripotent stem (ips) cells from terminally differentiated somatic cells. disorder in the regulation of cellular pathways. of special interests are the vital roles of intrinsic disorder in regulation and orchestration of various cellular pathways. one of the illustrative examples of this regulatory role of intrinsic disorder is the canonical wnt-pathway that involves five proteins, axin, cki-a, gsk- b, apc (adenomatous polyposis coli, also known as deleted in polyposis . protein), and b-catenin (all shown to contain long idprs). this pathway is known to play a number of crucial roles in the development of organism, and the malfunctions of which might lead to various diseases including cancer. the comprehensive analysis of published data revealed that idprs found in wntpathway proteins orchestrate protein-protein interactions, and facilitate ptms and signaling. furthermore, the scaffold protein axin and another large protein, apc, are heavily enriched in disorder and act as flexible concentrators in gathering together all other proteins involved in the wnt-pathway. intriguingly, the multifarious roles of highly disordered apc in regulation of b-catenin function were established by showing that disordered apc helps the collection of b-catenin from cytoplasm, facilitates the bcatenin delivery to the binding sites on axin, and controls the final detachment of b-catenin from axin. another important illustration of the involvement of intrinsic disorder in regulation of crucial pathway is given by the process of the programmed cell death (pcd), which is one of the most intricate cellular processes where the cell uses specialized uversky cellular machinery and intracellular programs to kill itself and which enables metazoans to control cell numbers and eliminate cells that threaten the animal's survival. pcd includes several specific modules, such as apoptosis, autophagy, and programmed necrosis (necroptosis). these modules are not only tightly regulated but also intimately interconnected and are jointly controlled via a complex set of protein-protein interactions. recently, several large sets of pcd-related proteins across species were analyzed using a wide array of modern bioinformatics tools to understand the role of the intrinsic disorder in controlling and regulating the pcd. this analysis revealed that proteins involved in regulation and execution of pcd possess substantial amount of intrinsic disorder and idprs were implemented in a number of crucial functions, such as protein-protein interactions, interactions with other partners including nucleic acids and other ligands, were shown to be enriched in post-translational modification sites, and were characterized by specific evolutionary patterns. unique catalytic function of a protein is believed to be dictated by its unique d structure. this axiom constitutes a cornerstone of the lock-and-key paradigm and it seemed to be able to sustain the furious attack on protein structure-function relationship initiated by the discovery of idps and hybrid proteins with ordered domains and idprs. in fact, from the vast majority of experimental and computational studies a general conclusion was drawn over and over again, where the functional repertoire of idps complemented the functional arsenal of ordered proteins, with ordered proteins being mostly responsible for catalysis and transport and with idps doing the majority of other jobs in the cell. on the other hand, all proteins (even the most ordered and tightly folded ones) are intrinsically flexible molecules that undergo conformational changes over a wide range of timescales and amplitudes. in fact, the combination of active site reactivity with the dynamic character of proteins allows enzymes to be promiscuous and remarkably efficient at the same time. furthermore, in general, dynamic fluctuations are crucial for enzyme catalysis, since they can influence substrate binding and product release, and may even adjust the effective barriers of the catalyzed reactions. [ ] [ ] [ ] [ ] [ ] often, dynamic changes in the enzyme during the catalytic reaction can be described using the induced-fit model, where a conversion of one tight conformational ensemble (free enzyme) to another distinct ensemble (bound enzyme) takes place through a series of local substrate-mediated structural rearrangements. despite this crucial role of local flexibility in the enzymatic catalysis, enzymes are still relatively stable molecules whose dynamic character is restricted to a small set of tightly folded conformations and whose unique (albeit locally flexible) structures are needed for efficient catalysis. from this viewpoint, the presence of intrinsic disorder is expected to be poorly compatible with enzymatic catalysis, which requires a well-organized environment in the active site of the enzyme in order to facilitate the formation of the transition state of the chemical reaction to be catalyzed. in a sharp contrast to this common wisdom supported by a wide array of specific examples, several enzymes were shown to be much more dynamic than the catalytic machines are expected to be, clearly possessing, in their precatalytic states, many characteristic properties of molten globules and retaining unusually high flexibility in structurally defined enzyme-ligand complexes. one of the best characterized examples of such molten globular enzymes is the engineered monomeric form of chorismate mutase from methanococcus jannaschii (mjcm). , [ ] [ ] [ ] here, a functional monomer (mmjcm) was created by inserting the hinge-loop sequence into the long, dimer-spanning n-terminal helix. in its unbound form, mmjcm was shown to exists as a native molten globule that was described as a dynamic ensemble of a-helical conformers rapidly interconverting on the millisecond timescale. interaction with natural ligand induced global conformational changes in the molten globular mmjcm promoting formation of a defined enzyme-ligand complex, which, however, preserved unusually high flexibility. catalytic mechanism of the molten globular mmjcm was described as follows: "though probably stochastic in nature, internal motions in the complex may generate a collective dynamic matrix that samples catalytically active conformation(s) often enough to achieve rapid turnover in the presence of the true transition state." therefore, some enzymes can represent a highly dynamic heterogeneous conformational ensemble which is still compatible with efficient catalysis. in agreement with this hypothesis, a molten globular character was described for circularly permuted dihydrofolate reductase (dhfr), , and urease g from bacillus pasteurii (bpureg). [ ] [ ] [ ] of these three enzymatic molten globules ureg is the only natural molten globular enzyme known to date, since both circularly permuted dhfr and monomeric mjcm were obtained as a result of some genetic manipulations. although the number of known native molten globules with enzymatic activity is small, their existence provides an interesting hint on early protein evolution. in fact, simple logics suggests that well-ordered enzymes appear as a result of long evolutionary process, whose very likely starting point was a partially folded polypeptide with some general properties of the molten globule. idps/idprs can form highly stable complexes, or be involved in signaling interactions where they undergo constant "bound-unbound" transitions, thus acting as dynamic and sensitive "on-off" switches. the ability of these proteins to return to the highly flexible conformations after the completion of a particular function, and their predisposition to gain different conformations depending on the environmental peculiarities, are unique physiological properties of idps which allow them to exert different functions in different cellular contests according to a specific conformational state. due to their lack of rigid structure, combined with the high level of intrinsic dynamics and almost unrestricted flexibility at various structure levels in the non-bound state, as well as due to the unique capability to adjust to structure of the binding partner, idps are characterized by a very diverse range of binding modes, creating a multitude of unusual complexes, many of which are not attainable by ordered proteins. some of these complexes are relatively static, resemble complexes of ordered proteins, and, therefore are suitable for the structure determination by x-ray crystallography. among these static complexes are: morfs, wrappers, chameleons, penetrators, huggers, intertwined strings, long cylindrical containers, connectors, armature, tweezers and forceps, grabbers, tentacles, pullers, and stackers or b-arcs. these binding modes are shown in supporting information figure s and briefly described in the supporting information materials. in addition to the static complexes, where bound partners have fixed structures, some idps/idprs do not fold even in their bound state, forming so-called disordered, dynamic, or fuzzy complexes with ordered proteins, , [ ] [ ] [ ] [ ] [ ] other disordered proteins, [ ] [ ] [ ] or biological membranes. , in complexes of some of these idps with their binding partners, the disordered regions flanking the interaction interface but not the interface itself remain disordered. such mode of interaction was recently described as "the flanking fuzziness" in contrast to "the random fuzziness" when the disordered protein remains entirely disordered in the bound state. , it is also expected that the similar binding mode can be utilized by disordered protein while interacting with nucleic acids and other biological macromolecules. physically, binding is considered as joining objects together and suggests spatial and temporal fixation of bound partners. the formation of protein complexes with specific binding partners is expected to bring some fixation (at least at the binding site). therefore, disordered complexes where interaction of a disordered protein with the binding partners is not accompanied by a disorder-to-order transition within the interaction interface clearly cannot be described by the classical binding paradigm. this contradiction can be resolved assuming that the ordered binding partner and/or disordered protein contain multiple low affinity binding sites. the existence of several similar binding sites combined with a highly flexible and dynamic structure of disordered protein creates a unique situation where any binding site of disordered protein can interact with any binding site of its partner with almost equal probability, in a staccato manner. the low affinity of each individual contact implies that each of them is not stable and can be readily broken. therefore, such disordered or fuzzy complex can be envisioned as a highly dynamic ensemble in which a disordered protein does not present a single binding site to its partner but resemble a "binding cloud," in which multiple identical binding sites are dynamically distributed in a diffuse manner. in other words, in this staccato-type interaction mode, an disordered protein rapidly changes multiple binding sites while probing binding site(s) of its partner. an additional factor which can help holding a dynamic complex together could be a weak longrange attraction between protein molecules. this long-range attraction is universal for all protein solutions and has a range several times that of the diameter of the protein molecule, much greater than the range of the screened electrostatic repulsion. the most common outcome of these function-related structural changes is the overall increase in the amount of ordered structure. however, functions of some ordered proteins require local or even global unfolding of a unique protein structure. among specific features of these structural alterations are their induced nature and transient character combined with a wide range of molecular mechanisms by which they can be promoted. these functional unfolding-activating factors include light; mechanical force; changes in ph, temperature, or redox potential; interaction with membrane, ligands, nucleic acids, and proteins; various ptms; release of autoinhibition due to the unfolding of autoinhibitory domains induced by their interaction with nucleic acids, proteins, membranes, ptms, and so forth. among rather unusual factors used by nature to activate proteins via functional unfolding are light and mechanical force. for example, exposure to blue light results in the activation of the photoactive yellow protein (pyp), which is an ordered, water-soluble kda protein that contains a thioester linked uversky p-coumaric acid cofactor and serves as a photosensor in ectothiorhodospira halophila. , pyp is a bacterial blue light sensor that undergoes conformational changes upon signal transduction. the absorption of a photon triggers substantial protein unfolding and leads to the formation of the transient signaling state that interacts with the partner molecules. this allows the swimming bacterium to operate the directional switch that protects it from harmful illumination. comprehensive analysis combining double electron electron resonance spectroscopy (deer), high resolution nmr, and timeresolved pump-probe x-ray solution scattering (tr-saxs/waxs) revealed that the transiently activated and short-lived signaling state of the pyp possessed a large degree of disorder and existed as an ensemble of multiple conformers that exchange on a millisecond time scale. , this unusual behavior is illustrated in figure that shows structures of inactive folded pyp and its light-activated functional form, which is highly disordered. some proteins undergo local unfolding induced by the mechanical force and therefore can serve as force sensors. among these natural force sensors are mechanosensitive ion channels that recognize and respond to the membrane tension, which is the mechanical forces applied along the plane of the cell membrane, rather than to the hydrostatic pressure perpendicular to the membrane plane. these ion channels are activated via partial unfolding of some of their functional parts induced by membrane tension. for a long time, the fact that idps/idprs undergo disorder-to-order transitions either during their functions or in order to be functional was used as one of the strongest arguments against the idea of protein intrinsic disorder. it was stated that most idps (those which are not the artifacts of current methods of protein production) are in fact proteins waiting for a partner (pwps) that serve as parts of a multi-component complex and that do not fold correctly in the absence of other components. therefore, when folded after binding to their partners, these proteins are not too different from typical ordered proteins. however, one need to keep in mind that a portion of "folding code" that defines the ability of ordered proteins to spontaneously gain a unique biologically active structure is missing for idps/idprs since they cannot fold spontaneously. this missing portion of the "folding code" (or a part of it) can be supplemented by binding partner(s). as a result, ordered and disordered proteins can be discriminated on a simple basis of temporal correlation between their folding and binding: ordered proteins fold first and then bind to their partners while the idps/idprs remain disordered until they bind their partners and often preserve substantial disorder in the bound state. furthermore, numerous cases of functional unfolding (or transient disorder, or upside-down functionality) represent further support to the concept of functional disorder by clearly showing that many proteins possess dormant disorder that needs to be awakened in order to make these proteins functional. it is clear now that the idps and idprs are real, abundant, diversified, and vital. the highly dynamic nature of idps and idprs is a visual illustration of the chaos. however, the evolutionary persistence of these highly dynamic proteins (see below), their unique functionality, and involvement in all the major cellular processes evidence that this chaos is tightly controlled. to answer the question as to . ground state structure was determined by multidimensional nmr spectroscopy. this structure is in agreement with an earlier published . Å crystal structure, and modeled structure based on combined deer, tr-saxs/waxs, and nmr data. it consists of an open, twisted, -stranded, antiparallel b-sheet, which is flanked by four ahelices on both sides. [ ] [ ] [ ] on the contrary, the light-activated form is highly disordered. this structure satisfies deer, saxs/ waxs, and nmr data simultaneously. how these proteins are governed and regulated inside the cell, gsponer et al. conducted a detailed study focused on the intricate mechanisms of idp regulation. to this end, all the saccharomyces cerevisiae proteins were grouped into three classes using one of the available disorder predictors, dis-opred : (i) highly ordered proteins containing - % of the predicted disorder; (ii) moderately disordered proteins with - % predicted disordered residues; and (iii) highly disordered proteins containing - % of the predicted disorder. then, the correlations between intrinsic disorder and the various regulation steps of protein synthesis and degradation were evaluated. this analysis revealed that the transcriptional rates of mrnas encoding idps and ordered proteins were comparable. however the idp-encoding transcripts were generally less abundant than transcripts encoding ordered proteins due to the increased decay rates of the transcripts of genes encoding idps. furthermore, idps were shown to be less abundant than ordered proteins due to the lower rate of protein synthesis and shorter protein half-lives. as the abundance and half-life in a cell of certain proteins can be further modulated via their ptms such as phosphorylation, the experimentally determined yeast kinase-substrate network was also analyzed. idps were shown to be substrates of twice as many kinases as were ordered proteins. furthermore, the vast majority of kinases whose substrates were idps were either regulated in a cell-cycle dependent manner, or activated upon exposure to particular stimuli or stress. therefore, ptms may not only serve as important mechanism for the fine-tuning of the idp functions but possibly they are necessary to tune the idp availability under the different cellular conditions. in addition to s. cerevisiae, similar regulation trends were also found in schizosaccharomyces pombe and homo sapiens. based on these observations it has been concluded that both unicellular and multicellular organisms appear to use similar mechanisms for regulation of the intrinsically disordered protein availability. overall, this study clearly demonstrated that in eukaryotes, there is an evolutionarily conserved tight control of synthesis and clearance of most idps. this tight control is directly related to the major roles of idps in signaling, where it is crucial to be available in appropriate amounts and not to be present longer than needed. it has been also pointed out that although the abundance of many idps is under strict control, some idps could be present in cells in large amounts or/and for long periods of time due to either specific ptms or via interactions with other factors, which could promote changes in cellular localization of idps or protect them from the degradation machinery. , , , , overall, this study clearly showed that the chaos seemingly introduced into the protein world by the discovery of idps is under the tight control. in an independent study, a global scale relationship between the predicted fraction of protein disorder and protein expression in e. coli was analyzed. this study showed that the fraction of protein disorder was positively correlated with both measured rna expression levels of e. coli genes in three different growth media and with predicted abundance levels of e. coli proteins. when a subset of e. coli proteins that are known to be essential for the survival and growth of this bacterium were analyzed, the correlation between protein disorder and expression level became even more evident. in fact, essential proteins had on average a much higher fraction of disorder ( . ), had a higher number of proteins classified as completely disordered ( % vs. % for e. coli proteome), and were expressed at a higher level in all three media than an average e. coli gene. the manual literature mining for a group of e. coli proteins that had high levels of predicted intrinsic disorder revealed that the disorder predictions matched well with the experimentally elucidated regions of protein flexibility and disorder. a direct link between protein disorder and protein level in e. coli cells could be because the idps may carry out the essential control and regulation functions that are needed to respond to the various environmental conditions. another possibility is that idps might undergo more rapid degradation compared to structured proteins, which cells can counter by increasing mrna levels of the corresponding genes. in this case, higher synthesis and degradation rates could make the levels of these proteins very sensitive to the environment, with slight changes in either production or degradation leading to significant shifts in protein levels. even more support for the tight control of idps inside the cell came from the analysis of cellular regulation of so-called "vulnerable" proteins. the integrity of the soluble protein functional structures is maintained in part by a precise network of hydrogen bonds linking the backbone amide and carbonyl groups. in a well-ordered protein, hydrogen bonds are shielded from water attack, preventing backbone hydration and the total or partial unfolding of the soluble structure under physiological conditions. , since soluble protein structures may be more or less vulnerable to water attack depending on their packing quality, a structural attribute, protein vulnerability, was introduced as the ratio of solvent-exposed backbone hydrogen bonds (which represent local weaknesses of the structure) to the overall number of hydrogen bonds. it has been also pointed out that structural vulnerability can be related to protein intrinsic disorder as the inability of a particular protein fold to protect intramolecular uversky hydrogen bonds from water attack may result in backbone hydration leading to local or global unfolding. since binding of a partner can help to exclude water molecules from the microenvironment of the preformed bonds, a vulnerable soluble structure gains extra protection of its backbone hydrogen bonds through the complex formation. to understand the role of structure vulnerability in transcriptome organization, the relationship between the structural vulnerability of a protein and the extent of co-expression of genes encoding its binding partners was analyzed. this study revealed that structural vulnerability can be considered as a determinant of transcriptome organization across tissues and temporal phases. finally, by interrelating vulnerability, disorder propensity and co-expression patterns, the role of protein intrinsic disorder in transcriptome organization was confirmed, since the correlation between the extent of intrinsic disorder of the most disordered domain in an interacting pair and the expression correlation of the two genes encoding the respective interacting domains was evident. because of the fact that idps are highly abundant and play crucial roles in numerous biological processes, it was not too surprising to find that some of them are involved in human diseases. for example, a number of human diseases originate from the deposition of stable, ordered, filamentous protein aggregates, commonly referred to as amyloid fibrils. in each of these pathological states, a specific protein or protein fragment changes from its natural soluble form into insoluble fibrils, which accumulate in a variety of organs and tissues. [ ] [ ] [ ] [ ] [ ] [ ] [ ] several unrelated proteins including many idps are known to be involved in these protein deposition diseases. , an illustrative examples of human neurodegenerative diseases associated with idps includes alzheimer's disease (deposition of amyloid-b, tau-protein, a-synuclein fragment nac) [ ] [ ] [ ] [ ] ; various taupathies (accumulation of tau-protein in the form of neurofibrillary tangles) ; down's syndrome (nonfilamentous amyloid-b deposits) ; parkinson's disease and other synucleinopathies (deposition of asynuclein) ; prion diseases (deposition of prp sc ) ; and a family of polyq diseases, a group of neurodegenerative disorders caused by expansion of gac trinucleotide repeats coding for polyq in the gene products. furthermore, most mutations in rigid globular proteins associated with accelerated fibrillation and protein deposition diseases have been shown to destabilize the native structure, increasing the steady-state concentration of partially folded (disordered) conformers. [ ] [ ] [ ] [ ] [ ] [ ] [ ] the maladies given above have been called conformational diseases, as they are characterized by the conformational changes, misfolding, and aggregation of an underlying protein. however, there is another side to this coin: protein functionality. in fact, many of the proteins associated with the conformational disorders are also involved in recognition, regulation, and cell signaling. for example, functions ascribed to a-synuclein, a protein involved in several neurodegenerative disorders, include binding fatty acids and metal ions; regulation of certain enzymes, transporters, and neurotransmitter vesicles; and regulation of neuronal survival (reviewed in ref. ) . overall, there are about proteins and ligands that interact and/or co-localize with this protein. furthermore, a-synuclein has amazing structural plasticity and adopts a series of different monomeric, oligomeric, and insoluble conformations (reviewed in ref. ) . the choice between these conformations is determined by the peculiarities of the protein environment, suggesting that asynuclein has an exceptional ability to fold in a template-dependent manner. therefore, the development of the conformational diseases may originate not only from misfolding but also from the misidentification, misregulation, and missignaling of the related proteins. analysis of so-called polyglutamine diseases gives support to this hypothesis. polyglutamine diseases are a specific group of hereditary neurodegeneration caused by expansion of cag triplet repeats in an exon of disease genes which leads to the production of a disease protein containing an expanded polyglutamine, polyq, stretch. nine neurodegenerative disorders, including kennedy's disease, huntington's diseases, spinocerebellar atrophy- , , , , , , and dentatorubral pallidoluysian atrophy are known to belong to this class of diseases. [ ] [ ] [ ] [ ] in most polyq diseases, expansion to over repeats leads to the onset. it has been emphasized that such molecular processes as unfolded protein response, protein transport, synaptic transmission, and transcription are implicated in the pathology of polyq diseases. importantly, more than transcription-related factors have been reported to interact with pathological polyq proteins. furthermore, these interactions were shown to repress the transcription, leading finally to the neuronal dysfunction and death (reviewed in ref. ) . these results suggest that polyq diseases represent kind of transcriptional disorder, supporting our misidentification hypothesis for at least some of the conformational disorders. disorder is very common in cancer-associated proteins too. in a study, it was found that % of cancer-associated and % of cell-signaling proteins contain predicted regions of disorder of residues or longer. in contrast, only % of a set of proteins with well-defined ordered structures contained such long regions of predicted disorder. in experimental studies, the presence of disorder has been directly observed in several cancer-associated proteins, including p , p kip , bcl-x l and bcl- , c-fos, a thyroid cancer associated protein tc- , ews-fli fusion protein that includes a potent transcriptional activator, the ews domain, alongside the highly conserved dna-binding domain fli , , among many other examples. the best characterized example of the important cancerrelated idp is the tumor suppressor protein p , which occupies the center of a large signaling network. p regulates expression of genes involved in numerous cellular processes, including cell cycle progression, apoptosis induction, dna repair, as well as others involved in responding to cellular stress. when p function is lost, either directly through mutation or indirectly through several other mechanisms, the cell often undergoes cancerous transformation. , cancers showing mutations in p are found in colon, lung, esophagus, breast, liver, brain, reticuloendothelial tissues, and hemopoietic tissues. p is regulated by several different mechanisms including inhibition of its activity by interaction with e ubiquitin ligase mdm , which binds to a short stretch of p located within the transactivation domain. mdm -bound p cannot activate or inhibit other genes. mdm ubiquitinates p and thus targets it for destruction. mdm also contains a nuclear export signal that causes p to be transported out of the nucleus. , the possibility of interrupting the action of diseaseassociated proteins (including through modulation of protein-protein interactions) presents an extremely attractive objective for the development of new drugs. since many proteins associated with various human diseases are either completely disordered or contain long disordered regions, , and since some of these disease-related idps/idprs are involved in recognition, regulation, and signaling, these proteins/regions clearly represent novel potential drug targets. due to failure to recognize the important role of disorder in protein function, current and evolving methods of drug discovery suffer from an overly rigid view of protein function. in fact, the rational design of enzyme inhibitors depends on the classical view where d-structure is an obligatory prerequisite for function. while generally applicable to many enzymatic domains, this view has persisted to influence thinking concerning all protein functions despite numerous examples to the contrary. this is most apparent in the observation that the vast majority of currently available drugs target the active site of enzymes, presumably since these are the only proteins for which the "unique structure-unique function" paradigm is generally applicable. idps often bind their partners with relatively short regions that become ordered upon binding. [ ] [ ] [ ] targeting disorder-based interactions should enable the development of more effective drug discovery techniques. there are at least two potential approaches for the inhibition of the disorder-based interactions, where small molecule either bind to the binding site of the ordered partner to outcompete the idps/idprs or interacts directly with the idp/ idpr. the principles of small molecule binding to idprs have not been well studied, but sequence specific, small molecule binding to short peptides has been observed. an interesting twist here is that small molecules can inhibit disorder-based proteinprotein interactions via induction of the dysfunctional ordered structures in targeted idpr, that is, via the drug-induced misfolding. in agreement with these concepts, small molecules "nutlins" have been discovered that inhibited the p -mdm interaction by mimicking the inducible a-helix in p (residues - ) that binds to mdm . , although x-ray crystallographic studies of the p -mdm complex revealed that the mdm binding region of p forms an a-helical structure that binds into a deep groove on the surface of mdm , nmr studies showed that the unbound n-terminal region of p lacks fixed structure, although it does possess an amphipathic helix part of the time. a close examination of the interface between the proteins reveals that phe , trp , and leu of p are the major contributors to the interaction, with the side chains of these three amino acids pointing down into a crevice on the mdm surface. , the structure of nutlin- was shown to mimic the crucial residues of p , with two bromophenyl groups fitting into mdm in the same pockets as trp and leu , and an ethyl-ether side chain filling the spot normally taken by phe . [ ] [ ] [ ] nutlins and related small molecules increased the level of p in cancer cell lines. this drastically decreased the viability of these cells, causing most of them to undergo apoptosis. when one of the nutlins was given orally to mice, a % inhibition of tumor growth compared to the control was induced. , [ ] [ ] [ ] this successful nutlin story marks the potential beginning of a new era, the signaling-modulation era, in targeting drugs to protein-protein interactions. importantly, this druggable p -mdm interaction involves a disorder-to-order transition. principles of such transitions are generally understood and therefore can use to find similar drug targets, which are inducible a-helices. in addition to nutlins inhibiting p -mdm interaction, several other small molecules also act by blocking proteinprotein interactions. , some of these interactions involve one structured partner and one disordered partner, with disordered segments becoming a-helix upon binding. therefore, the p -mdm complex is not a unique exception and many other disorderbased protein-protein interactions are blocked by a small molecule. all this suggest that there is a cornucopia of new drug targets that would operate by blocking disorder-based protein-protein interactions. for these p -mdm -type examples, the drug molecules mimic a critical region of the disordered partner (which folds upon binding) and compete with this region for its binding site on the structured partner. these druggable interaction sites operate by the coupled binding and folding mechanism. they are small enough and compact enough to be easily mimicked by small molecules. methods for predicting such binding sites in disordered regions have been developed and the bioinformatics tools to identify which disordered binding regions can be easily mimicked by small molecules have been elaborated. a complementary approach for small molecules to inhibit the disorder-based protein-protein interactions relies on the direct binding of drugs to the idps/idprs, which is illustrated by the c-myc-max story. in order to bind dna, regulate expression of target genes, and function in most biological contexts, c-myc transcription factor must dimerize with its obligate heterodimerization partner, max, which lacks a transactivation segment. both c-myc and max are intrinsically disordered in their monomeric forms. upon heterodimerization, they undergo coupled binding and folding of their basic-helix-loophelix-leucine zipper domains (bhlhzips). since the deregulation of c-myc is related to many types of cancer, the disruption of the c-myc-max dimeric complex is one of the approaches for c-myc inhibition. several small molecules were found to inhibit the c-myc-max dimer formation. these molecules were shown to bind to one of the three discrete sites within the -residue bhlhzip domain of c-myc, which are composed of short contiguous stretches of amino acids that can selectively and independently bind small molecules. inhibitor binding induces only local conformational changes, preserves the overall disorder of c-myc, and inhibits interaction with max. furthermore, binding of inhibitors to c-myc was shown to occur simultaneously and independently on the three independent sites. based on these observations it has been concluded that a rational and generic approach to the inhibition of protein-protein interactions involving idps may therefore be possible through the targeting of intrinsically disordered sequence. recently, a functional misfolding concept was introduced to describe a mechanism preventing idps from unwanted interactions with non-native partners. idps/idprs are characterized by high conformational dynamics and flexibility, the presence of sticky preformed binding elements, and the ability to morph into differently-shaped bound configurations. however, detailed analyses of the conformational behavior and fine structure of several idps revealed that the preformed binding elements might be involved in a set of non-native intramolecular interactions. based on these observations it was proposed that an intrinsically disordered polypeptide chain in its unbound state can be misfolded to sequester the preformed elements inside the noninteractive or less-interactive cage, therefore preventing these elements from the unnecessary and unwanted interactions with non-native binding partners. it is important to remember, however, that the mentioned functional misfolding is related to the ensemble behavior of transiently populated elements of structure. in other words, it describes the behavior of a globally disordered polypeptide chain containing highly dynamic elements of residual structure, so-called interaction-prone preformed fragments, some of which could potentially be related to protein function. this ability of idrps/idprs to functionally misfold can be used for finding small molecules which would potentially stabilize different members of the functionally misfolded ensemble, and therefore prevent the targeted protein from establishing biological interactions. this approach is very different from the discussed above direct targeting of short idprs since it is based on a small molecule binding to a highly dynamic surface created via the transient interaction of preformed interaction-prone fragments. in essence, this approach can be considered as an extension of the well-established structure-based rational drug design elaborated for ordered proteins. in fact, if the structure of a member(s) of the functionally misfolded ensemble can be guessed, then this structure can be used to find small molecules that are potentially able to interact with this structure, utilizing tools originally developed for the rational structure-based drug design for ordered proteins. ideally, a drug that targets a given protein-protein interaction should be tissue specific. although some proteins are unique for a given tissue, many more proteins have very wide distribution, being present in several tissues and organs. how can one develop tissue-specific drugs targeting such abundant proteins? often, tissue specificity for many of the abundant proteins is achieved via the alternative splicing of the corresponding pre-mrnas, which generates two or more protein isoforms from a single gene. estimates indicate that between and % of human genes yield protein isoforms by means of alternatively spliced mrna. the added protein diversity from alternative splicing is thought to be important for tissue-specific signaling and regulatory networks in the multicellular organisms. the regions of alternative splicing in proteins are enriched in intrinsic disorder, and it was proposed that associating alternative splicing with protein disorder enables the time-and tissue-specific modulation of protein function. since disorder is frequently utilized in protein binding regions, having alternative splicing of pre-mrna coupled to idprs can define tissue-specific signaling and regulatory diversity. these findings open a unique opportunity to develop tissue-specific drugs modulating the function of a given id protein/region (with a unique profile of disorder distribution) in a target tissue and not affecting the functionality of this same protein (with different disorder distribution profile) in other tissues. wavy pattern of global evolution of intrinsic disorder idps/idprs are more common in eukaryotes than in less complex organisms. , , [ ] [ ] [ ] [ ] [ ] this suggests that disorder, with its ability to be implemented in various signaling, recognition, and regulation pathways and networks, is important for the maintenance of life in eukaryotic and especially muticellular eukaryotic organisms. , , , also, the finding that alternatively spliced regions of mrna code for idprs much more often than for structured regions suggests that there is a linkage between alternative splicing and signaling by idprs that constitutes a plausible mechanism that could underlie and support cell differentiation, which ultimately gave rise to the multicellular eukaryotic organisms. therefore, one can assume that intrinsic disorder represents a relatively recent evolutionary invention. however, this hypothesis obviously would be wrong if earlier stages of evolution would be taken into account. in fact, the chances that the first polypeptides that appeared in the primordial soup of the primitive earth possessed well-developed and unique d structures are minimal. the earth formed about . billion years ago. scientists dated the first fossils to . billion years ago. there are still debates and different theories about what happened in those years between the time the earth was cool enough to spawn life and the time the first fossils were formed. at the beginning of the th century, oparin and haldane proposed that some organic molecules could have been spontaneously produced from the gases of the primitive earth atmosphere, assuming that this primitive atmosphere was reducing (as opposed to oxygen-rich), and there was an appropriate supply of energy, such as lightning or ultraviolet light. thirty year later, this hypothesis (that constitutes a cornerstone of the theory of molecular evolution) received strong support from the elegant experiments of stanley l. miller and harold c. urey who were able to synthesize various organic compounds including some amino acids from non-organic compounds which were believed to represent the major components of the early earth's atmosphere (water vapor, hydrogen, methane, and ammonia) by putting them into a closed system and running a continuous electric current through the system, to simulate lightning storms believed to be common on the early earth. , however, the miller-urey experiment yielded only about half of the modern amino acids , suggesting that the first proteins on earth may have contained only a few amino acids. these findings go in parallel with the biosynthetic theory of the genetic code evolution suggesting that the genetic code evolved from a simpler form that encoded fewer amino acids, probably paralleled by the invention of biosynthetic pathways for new and chemically more complex amino acids. furthermore, some additional support of the validity of this hypothesis can be found in the standard genetic code (that consists of triplets of nucleotides, codons), which is redundant ( codons encodes for amino acids). in fact, with only two exceptions, codons encoding one amino acid may differ in any of their three positions. however, only the third positions of some codons may be fourfold degenerate, that is, any nucleotide at this position specifies the same amino acid and all nucleotide substitutions at this site are synonymous. using these observations as a reflection of the evolutionary development, it was proposed that there was a period during code evolution where the third position was not needed at all and a doublet code preceded the triplet code, giving rise to codons encoding for or fewer amino acids, if a termination codon is taken into account. based on these and many other premises, one can discriminate evolutionary old and new amino acids. in , eduard n. trifonov combined different single-factor criteria into a consensus scale and proposed the following temporal order of addition for the amino acids: g/a, v/d, p, s, e/l, t, r, n, k, q, i, c, h, f, m, y, w. even superficial analysis of this sequence reveals that many of the early amino acids (such as g, d, e, p, and s) are disorder-promoting, as they are very abundant in modern idps. on the other hand, the major orderpromoting residues (c, w, y, and f) were added to the genetic code late. this observation is further illustrated by figure (a) which represents modern genetic code, contains information on the early and late codons (shown by light red and light blue colors, respectively), and on corresponding disorder-and order-promoting residues (shown by red and blue colors, respectively). codons with intermediate age and disorder-neutral residues are shown by light pink and pink colors, respectively. figure uversky illustrates that there is relatively good agreement between the "age" of the residue and its disorderpromoting capacity, with early residues being mostly disorder-promoting, and with the majority of late residues being mostly order-promoting. this conclusion follows from the abundance of the matching colors (light red-red, light blue-blue, and light pinkpink). there are only two noticeable exceptions from these rule, valine and leucine, which are early but order-promoting residues. this strongly suggests that the primordial polypeptides were intrinsically disordered. it is very unlikely that these disordered primordial polypeptides possessed catalytic activity. this hypothesis is in line with the rna world theory suggesting that during the evolution of enzymatic activity, catalysis was transferred from rna first to ribonucleoprotein (rnp) and only then to protein. therefore, the first proteins in the "breakthrough organism" (the first to have encoded protein synthesis) would be nonspecific chaperone-like proteins rather than catalysts. , such rna chaperone activities of early proteins conferred to their carriers a significant selective advantage in the rna world, where rna, which is especially prone to misfolding, , was used for both information storage and catalysis. since the variability of physicochemical properties of amino acids greatly exceeds that of figure . peculiarities of disorder evolution. a: modern genetic code with information on the early and late codons (shown by light red and light blue colors, respectively) and disorder-and order-promoting residues (shown by red and blue colors, respectively). codons with intermediate ages (i.e., those located between early and late codons) are shown by light pink color, whereas disorder-neutral residues are shown by pink color. b: wavy pattern of the global disorder evolution. x-axis represents evolutionary time and y-axis shows disorder content in proteins at given evolutionary time point. here, primordial proteins are expected to be mostly disordered (left-hand side of the plot), proteins in lua likely are mostly structured (center of the plot), whereas many protein in eukaryotes are either totally disordered or hybrids containing both ordered and disordered regions (right-hand side of the plot). nucleotides and since protein structures are noticeably more stable than rna structures, the transition from rnas (ribozymes) to proteins as carriers of enzymatic activity was a logical evolutionary step. however, efficient catalysis relies on the proper spatial arrangement of catalytic residues which requires a stable structure. therefore, grafting of the enzymatic activity to proteins generated strong evolutionary pressure toward the well-folded structures. in other words, the global evolution of intrinsic disorder is characterized by a wavy pattern [see fig. (b)], where highly disordered primordial proteins with primarily rna-chaperone activities were gradually substituted by the well-folded, highly ordered enzymes that evolved to catalyze the production of all the complex "goodies" crucial for the independent existence of the first cellular organisms. due to its specific features crucial for the regulation of complex processes, protein intrinsic disorder was reinvented at the subsequent evolutionary steps leading to the development of more complex organisms from the last universal ancestor (i.e., the most recent organism from which all organisms now living on earth descend , ) , and culminating in the appearance of the highly elaborated eukaryotic cells [see there is no simple answer to the question on the comparative evolutionary rates of ordered and idps and regions in modern organisms. in fact, it looks like everything is possible, and intrinsically disordered sequences may evolve faster, slower or similar to ordered sequences. for example, disordered and ordered domains of the same protein (e.g., papillomavirus e oncoprotein) were shown to possess similar degrees of conservation and co-evolution. many other idps/idprs were shown to be characterized by high evolutionary rates , , determined by the lack of specific structural restrictions. in fact, the analysis of calcineurins, topoisomerase, ribosomal protein s , b-subunits of the potassium channel kvb . , and many other proteins showed that disordered regions in these proteins contained more amino acid substitutions, insertions, and deletions than the ordered regions of the same proteins. , furthermore, based on the observation that a significantly higher degree of positive darwinian selection was observed in idprs of proteins compared to regions of a-helix, b-sheet or tertiary structures, it was hypothesized that idprs may be required for the genetic variation with adaptive potential and that these regions may be of "central significance for the evolvability of the organism or cell in which they occur." on the other hand, some idps and idprs are highly conserved. human a-synuclein (a canonical idp of residues , ) differs from its mouse counterpart by merely six residues ( %), and there are just residue differences ( %, which include residue differences at positions and insertions/ deletions) between the human and canary a-synucleins. in flagellin, the ordered central region has greater sequence diversity than its disordered termini. functionally important conserved regions of predicted disorder were shown to be rather common in proteins from all kingdoms of life, including viruses. , furthermore, many functional domains of a significant size were shown to be intrinsically disordered. overall, a systematic study of several families of proteins with at least one structurally characterized disordered region revealed that their idprs are characterized by highly heterogeneous evolutionary rates, with some disordered amino acid sequences evolving slowly, and others evolving more rapidly than ordered sequences. also, even different parts of the same disordered region can possess noticeable variability in their divergence during the evolutionary process. finally, in some disordered proteins, peculiarities of the amino acid composition, and not the amino acid sequence might be conserved. , some future directions the last years witnessed a real revolution in our understanding of the protein structure-function relationships. the fact that there is an entire class of polypeptides which do not have rigid structures but possess crucial biological function was heavily underappreciated and ignored for a very long time despite numerous examples scattered in literature. the work which started in my group as an attempt to understand what is so special about several natively unfolded proteins produced a real explosion of interest to structure-less proteins with biological functions. a new field was created and a lot of intriguing information was produced related to structures and functions of idps/idprs. there is no need to list once again all the discoveries and findings made in this field-they are subjects of many recent reviews and some of them are briefly covered in this article. although the amount of data generated during the past decade and a half on specific features related to the structural properties of idps and idprs, their abundance, distribution, functional repertoire, regulation, involvement into the disease pathogenesis, and so forth is vast, it seems that this mass of data produced so far is just a small tip of a humongous iceberg. idps/idprs continue to bring discoveries almost on a daily basis and even more breakthroughs are expected in future. modern protein science is at the turning point, but biology still waits for physics. new models explaining various functions of idps, their evolution, and involvement in diseases are in great demand, together with the general theory unifying current knowledge on 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i am grateful to all former and current colleagues for their priceless contributions, assistance and support. key: cord- -z kx h authors: métifiot, mathieu; amrane, samir; litvak, simon; andreola, marie-line title: g-quadruplexes in viruses: function and potential therapeutic applications date: - - journal: nucleic acids res doi: . /nar/gku sha: doc_id: cord_uid: z kx h g-rich nucleic acids can form non-canonical g-quadruplex structures (g s) in which four guanines fold in a planar arrangement through hoogsteen hydrogen bonds. although many biochemical and structural studies have focused on dna sequences containing successive, adjacent guanines that spontaneously fold into g s, evidence for their in vivo relevance has recently begun to accumulate. complete sequencing of the human genome highlighted the presence of ∼ sequences that can potentially form g s. likewise, the presence of putative g -sequences has been reported in various viruses genomes [e.g., human immunodeficiency virus (hiv- ), epstein–barr virus (ebv), papillomavirus (hpv)]. many studies have focused on telomeric g s and how their dynamics are regulated to enable telomere synthesis. moreover, a role for g s has been proposed in cellular and viral replication, recombination and gene expression control. in parallel, dna aptamers that form g s have been described as inhibitors and diagnostic tools to detect viruses [e.g., hepatitis a virus (hav), ebv, cauliflower mosaic virus (camv), severe acute respiratory syndrome virus (sars), simian virus (sv )]. here, special emphasis will be given to the possible role of these structures in a virus life cycle as well as the use of g -forming oligonucleotides as potential antiviral agents and innovative tools. almost a century ago, the ability of guanosine, but not guanine, to form viscous gels was described ( ) . fifty years later, x-ray diffraction data clearly showed that the guanosine moieties in these gels were arranged in a tetrameric organization linked by eight hoogsteen hydrogen bonds ( figure ) ( , ) . these hydrogen bonds differ from the bonds observed in canonical watson-crick pairing and involve the interaction of the n group from one guanine with the exocyclic amino group from a neighboring base (figure a ). therefore, a g-tetrad or a g-quartet results from planar association between four guanines that are held together by eight hydrogen bonds and coordinated with a central na+ or k+ cation ( ) ( ) ( ) ( ) ( ) . in addition, nucleoside derivatives were also used to confirm the structural properties of g-quartets ( ) ( ) ( ) ( ) ( ) ( ) . conversely, a g-quadruplex or g is formed by nucleic acid sequences (dna or rna) containing g-tracts or gblocks (adjacent runs of guanines) and composed of various numbers of guanines. depending on the nucleotide sequence, the way g s can be formed presents a high degree of diversity. the core of a g is based on stacking between two or more g-tetrads, wherein the guanines can adopt either a syn or an anti glycosidic bond angle conformation. consequently, each of the four g-tracts that form the core of the structure can run in the same or opposite direction with respect to its two neighbors, forming parallel, antiparallel or hybrid core conformations. depending on these orientations, the g-blocks delimit four negatively charged grooves of different sizes: narrow, medium or wide (figure b-e) . for intra-molecular structures (figure b and c) , the four g-tracts belong to the same oligonucleotide and are attached by linkers with variable nucleotide sequences and lengths. these loops can adopt three different conformations: lateral, diagonal or propeller (figure b-d) . the bi-or tetra-molecular g structures (figure d -e) are assembled from g-tracts belonging to two or four different strands. the g-blocks can also be interrupted by one to seven non-g nucleotides, which result in bulges that protrude from the g core ( figure e ). in contrast to the almost mono-morphic canonical duplex, these variable structural parameters are directly related to the nucleotide primary sequence. this unique family of globular-shaped nucleic acid structures (figure f -j) presents a high level of plasticity that enables various applications (see 'g s as antiviral agents' section). however, a potential physiological role for g s has remained controversial for many years. nonetheless, interest in g-quaduplexes has increased over time, with thousands of reports and reviews published on several aspects of g s, including the biophysical and chemical characteristics as well as biological function in prokaryotes and eukaryotes ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . with an increasing volume of sequencing data, databases and algorithms have also been developed to enable search and mapping of g in a few mammalians as well as hundreds of bacterial species ( ) ( ) ( ) ( ) . additionally, g s present an intrinsic resistance to 'regular' nucleases. however, g specific nucleases have been isolated, first in yeast with kem /sep ( ) and later in humans with gqn ( ) . later, a dna binding protein (g r /rhau) that can unfold rna/dna quadruplexes was isolated in human cells ( , ) , which further highlights the biological relevance of g s. finally, development of g specific probes such as monoclonal antibodies ( ) ( ) ( ) and small chemical ligands ( ) ( ) ( ) ( ) ( ) ( ) facilitated in vivo studies that support quadruplex formation in cells. taken together, g s are likely present in several significant genomic regions and may be a key component in important cellular processes ( , ) . the sequencing of the human genome highlighted the presence of many sequences enriched in guanines that can potentially form quadruplexes [for a review, see ( ) ]. in theory, more than putative g-tetrads may be formed with loops of - nucleotides, and over , with loops of up to nucleotides. however, the genomic dna is dynamic, and quadruplexes, duplexes or other structural forms at those sites will be influenced by chromatin and other dna-binding proteins. thus, g formation depends on the cell type and cell cycle, ultimately impacted by environmental conditions and stresses. without clear signaling and tight regulation, extremities present in linear chromosomes could be recognized as damage dna and it would be deleterious for the cell if processed as such by repair mechanisms ( ) . telomere length has also been linked to the lifespan of organisms. telomeres are nucleoprotein structures found at the end of chromosomes protecting the genome from instability. this terminal region of chromosomes contains long tracts (several kilobases) of double stranded ttaggg:ccctaa repeats ending with a protrusion of single stranded ttaggg repeats ( - repeats) . telomerase is a ribonucleoprotein enzyme that adds ttaggg repeats to the end of dna strands in the telomere regions. human telomerase is a heterodimeric complex composed of (i) telomerase reverse transcriptase (tert), a dna-polymerase rna-dependent, (ii) a singlestranded rna template referred to as telomerase rna component (terc) and (iii) dyskerin [dyskeratosis congenita (dkc )], a pseudouridine synthase binding terc through the h/aca motif and stabilizing the telomerase complex ( , ) . accumulated experimental data indicate the presence of g s in telomeric dna ( , ( ) ( ) ( ) ( ) ( ) . for example, dna , a helicase/nuclease that cleaves g s, is involved in maintaining telomere integrity ( , ) . altogether, these observations clearly establish that telomeric g s are crucial structures for regulating telomere maintenance, thus providing a mechanism for controlling cell proliferation ( , ) . at the ends of telomeres, if the g-rich overhang is longer than four ttaggg repeats (> nucleotides), it can fold over itself and form secondary structures, including g s (figure a) . those structures prevent telomere elongation by the telomerase complex ( ) . accordingly, small g -ligands (e.g. telomestatin) and g -binding proteins (e.g. trf ) show anti-proliferative and potential anti-tumor activities through telomere interference ( , ( ) ( ) ( ) ( ) . however, the exact mechanism is more complicated as telomestatin derivatives present telomerase independent activity, most likely through targeting g s involved in tumor growth elsewhere in the genome ( ) ( ) ( ) . dna replication is highly regulated, which ensures faithful reproduction of genetic information through each cell cycle. in eukaryotes, this process is initiated at thousands of dna replication origins (oris) distributed along each chromosome ( ) . initially, a pre-replicative complex (pre-rc) is assembled during late mitosis and the early g phase; it evolves during the s phase to a pre-initiation complex through initiator protein activity (e.g., cyclin-dependent kinases, cdc , cdc and minichromosome maintenance complex (mcm) proteins). this step involves unwinding the origin and dna polymerase recruitment. a tissuespecific temporal system exists, and only a few origins undergo 'firing' (replication fork elongation). the remaining origins are dormant backups used under stress (e.g., dna damage and collisions with transcription machinery). studies on organisms ranging from drosophila to humans have highlighted that repeated elements are enriched with g residues ( ) . in vitro, origin g-rich repeated element (ogre) sequences form g s, and point mutations that affect the stability of these g s also impair origin function. even if a -bp cis-regulatory element is necessary for efficient initiation, g structure formation at oris might be the key to selecting the firing origins ( , ) . additionally, g s formed in the lagging strand can lead to the stalling of the replication fork ( figure d ). on the other side, formation of g s in the leading strand can displaced it from the template and causes polymerase slippage (figure d ). such events could participate to the unusual expansions of g forming mini-satellite sequences ( ) . altered gene expression levels are implicated in many human diseases. g s have been found in the promoter region of many genes and implicated in transcription and protein translation regulation [for a review, see ( ) ]. particularly interesting, the p region, which is upstream of the c-myc promoter, is highly sensitive to dnase i and s nucleases (nuclease-hypersensitive element or nhe). this purine-rich sequence can form an intramolecular g ( ) ( ) ( ) ( ) , which structure has been determined by nuclear magnetic resonance (nmr) ( ) . this nheiii region controls up to % of gene transcription activation, and the g acts as a transcriptional repressor element ( , ) . because interactions between dna-binding proteins and local dna supercoiling can impact the equilibrium between duplex/singlestranded dna (active transcription) and quadruplex dna (silent), c-myc expression and, ultimately, cell proliferation may be modulated. nucleolin (a nucleolar phosphoprotein) and adar (a z-dna binding/rna editing protein) both bind the c-myc promoter in vivo and stabilize the g structure of the promoter ( , ) . due to the high therapeutic potential of genomic g s, studies have also been performed on promoters of other genes implicated in cancer biology. for example, kras is one of the most frequently mutated genes in human cancer. its promoter contains a g-rich nuclease hypersensitive element that is critical for transcription ( ) . the polypurine strand forms g structures that bind crucial dna repair proteins (e.g., parp- , ku ) ( ) . direct evidence for g formation was also reported for the proximal promoter region of the ret proto-oncogene, and targeting this region with a small molecule represses ret proto-oncogene transcription ( ) . although many g-rich sequences have been identified in the genome, they enable generation of corresponding grich rnas when the sequences are located in transcription units. ultimately, these rnas can fold into quadruplex structures and impact gene expression ( ) . in untranslated regions ( -utrs), g s can act as translation repressors ( ) ( ) ( ) . g structures have also been proposed to regulate translation driven by ires domains in the absence of an mrna cap [for a recent review, see ( ) and the references therein]. finally, it has recently been shown that g rnas in coding regions can stimulate ribosomal frameshifts in vitro and in cultured cells providing a role for g in translational regulation ( , ) . a major challenge for future studies is to better understand genome instability, cancer and severe neurological/neuromuscular defects origins. repeated sequences, such as polynucleotide repeats (three and above), mini-and mega-satellites, are source of genome instability through either expansion or contraction ( ) . however, dna secondary structures associated with those tandem repeats may be crucial elements in the expansion phenomenon. for progressive myoclonus epilepsy type- (epm ), expansion of the dodecamer sequence (cgcg cg ) within the cystatin b promoter is thought to depend on g formation ( ) . in addition to this potential deleterious consequence, g s have also been implicated in development of specific immune responses. in antigen-activated b cells, class switch recombination promotes deletion of a dna fragment (several kilobases) that joins a new constant region to the variable immunoglobulin chain ( ) . these switch regions (s regions) are intronic, g-rich and repetitive sequences that form g-loops upon transcription (figure e ). in vivo, class switch recombination depends on the activity of aid (a cytidine deaminase) and muts␣ (msh /msh , and con-served dna repair factors involved in mismatch repair). aid promotes creation of u:g mismatches, and muts␣ binds with high affinity the g dna formed upon transcription of the s regions. in parallel, pathogens have also developed countermeasures to evade the immune system. the role of gquartets in this evasion is related to antigenic variation. for plasmodium falciparum, g-rich sequences have been found in the upstream region of group b var genes that can form in vitro stable g s ( ) . similarly, a g -forming sequence ( -g tg ttg tg ) is located upstream of the pile expression locus in neisseria gonorrhoeae ( ) . formation of g s is necessary for initiating this pilin antigenic variation through recombination, and interactions with reca could facilitate this specialized reaction. overall, immune evasion and immunoglobulin gene class switch recombination in vertebrates are clearly analogous mechanistically ( ) . searches for g s and elucidation of their function in the viruses' genome have mainly focused on the human immunodeficiency virus (hiv), which causes acquired immunodeficiency syndrome (aids). currently, ∼ million people are infected with hiv worldwide. with over million new infections and ∼ . million deaths from aids per year, the pandemic continues to spread. even without a vaccine, development of highly active anti-retroviral therapies have allowed people to live with hiv as a chronic disease ( ) . however, viruses within t cells remain fully capable of replicating and infecting other cells if the drug pressure is removed or when resistance emerges. thus, new drugs must be developed to overcome the treatment's genetic barrier. hiv- is an rna virus in the lentivirus genus and is part of the retroviridae family. lentiviruses are singlestranded, positive-sense, enveloped rna viruses. hiv- particles contain two molecules of genomic rna that are converted into double-stranded dna by the viral reverse transcriptase (rt). the resulting viral dna is then imported into the nucleus and insertion into the cellular dna is catalyzed by the virally encoded integrase (in). once integrated, transcription from the viral promoter at the long terminal repeat generates mrnas that code various viral proteins and genomic rna. alternatively, the provirus may become latent, which allows the virus and its host cell to avoid detection by the immune system. the presence of g structures has been highlighted at both rna and dna levels with implications throughout the viral life cycle. retroviral rnas dimerize in the cytoplasm of an infected cell allowing two copies of the genome to be encapsidated in the newly produced virion ( ) . while a single copy of the genome is sufficient for viral replication, the second copy is also used during reverse transcription, and the viral rt switches multiple times between the two rna molecules ( , ) . the strand transfers are partially responsible for the viral variability through production of recombinant molecules. therefore, understanding the mechanisms that drive dimerization and recombination is essential. dimerization is a two-step process that involves sequences upstream of the splice donor site ( , ) . the sequences involved in initial dimerization and encapsidation partially overlap at the end of the viral genome. one of the sequences is a highly conserved dimer initiation site (dis) that forms a stem loop. a concentration-dependent kissing-loop interaction is initiated from contacts between consecutive guanines ( ); the interaction then spreads to the stems. however, this interaction does not seem sufficiently strong to keep the two copies together during reverse transcription. several studies have identified a g-rich sequence that form bi-molecular g structures in the gag region of the hiv- genome, near the dis ( - ). localization of these g -forming sequences correlates with recombination hot spots and exhibits an increased rate of template switching that highlights a potential role for these structures ( ) . supporting this hypothesis, recombination in the u domain is cation-dependent and is lower in the presence of li + , which is a metal ion that fails to stabilize g s ( ). short rna templates from the central region of the hiv- genome contain g-rich sequences near the central polypurine tract (cppt) at the end of the pol gene (in coding sequence); this is a region where one of the two primers used for synthesizing the (−) strand dna is produced during reverse transcription. these sequences can form both intramolecular and dimeric g structures. moreover, reconstituted systems have confirmed that g structures near the cppt facilitate strand transfer and promote template switching by the rt ( ) . interestingly, certain elements from the cppt region are involved in forming the cppt flap, which is a region that plays an important role in nuclear entry of the double-stranded dna ( , ) . taken together, the g-rich regions located at the end of the genome and in the central region are likely maintained in proximity through inter-rna g formation with a crucial role in hiv- replication. retroviral nucleocapsid proteins (ncp) are multifunctional elements encoded in the gag gene. notably, ncp participate in many retroviral cycle steps by remodeling nucleic acid structures to favor thermodynamically stable conformations. they are referred to as nucleic acid chaperones and interact with nucleic acid phosphodiester backbones through electrostatic interactions thanks to basic residues (especially residues in the n-terminus) [for reviews, see ( , ) ]. moreover, hiv- ncp (ncp ) exhibits sequence-specific binding to runs of gs, ugs or tgs through interactions involving its two zinc fingers (cchc motifs separated by a proline-rich linker). although there is no doubt that ncp tightly binds g sequences, data in the literature shows that a hydrophobic interaction engaged by the c-terminal zinc finger of the protein may lead to g stabilization ( ) , while high concentration of ncp promotes g unfolding ( ) . a recent biophysical study using high-speed atomic force microscopy (hs-afm) addressed direct and real-time investigations on the molecular chaperone activity at the single-molecule level ( ) . ncp can efficiently promote bimolecular g formation and is able to anneal the g structures. the g structure is induced by both unprocessed ncp and mature ncp , which indicates that both proteins may participate in genome recognition, recombination, dimerization and packaging. ncp could act through a potential mechanism that involves synaptic g intermediates, as illustrated in figure ( ) . transcription of the viral dna is performed by the cellular rna polymerase ii from the viral promoter located in the -long terminal repeat (ltr) of the proviral genome. the u region (figure a) (tss) and close to the tata box (figure b ). this sequence overlaps with the so-called minimum promoter, which is composed of three sp as well as two nf-kb binding sites and is crucial for transcription initiation. the presence of eight blocks of guanines suggests that this region is a good candidate for g formation. two independent biophysical studies based on dmsmediated foot-printing assays or nmr recently evaluated the ability of this -nt g-rich sequence to form g structures ( , ) . dna fragments spanning this region (corresponding to two or three sp or nf-kb binding sites with oligonucleotides ranging from to bases) were all able to form stable parallel and anti-parallel g topologies with melting temperatures ranging from • c to • c in -mm kcl solutions. the three models are formed with rather long sequences (up to nucleotides), allowing the formation of long loops of to nucleotides (figure d, e and f). interestingly, a g structure was also observed in the nmr study with an anti-parallel g core composed of only two tetrads and an additional watson-crick base pair that stacks on top of the upper tetrad (figure g) . notably, these four topologies are mutually exclusive, and forming one of these g s in the promoter will prevent formation of the three alternative conformations. thus, the equilibrium between these forms may play a role in regulating promoter activity. recently, the interaction between the sp protein and a fragment of the hiv- promoter sequence folded into a g was studied ( ) . piekna-przybylska et al. used an affinitybased selection approach using biotinylated g s immobilized on streptavidin-coated magnetic beads. the pull down experiments followed by western blotting revealed that the sp protein can bind the hiv- promoter sequence when it adopts a g conformation. perrone et al. analyzed the effect of point mutations that disrupt the g structures formed in the promoter ( ) . the wild-type (wt) and mutated ltr promoters were cloned upstream of a firefly gene in a promoter-free plasmid. in hek t cells, the promoter activity of the mutated sequences (unable to form g ) was twice as high as the wt ltr. these data suggest that g s act as repressor elements in the transcriptional activation of hiv- . therefore, g structures might be critical for hiv- fitness and represent novel targets for antiviral drug development (see below). a better understanding of the role for g s in regulating the hiv- promoter activity would also shed light on hiv- latency and reactivation mechanisms, from which a new landscape may emerge in clinical research to eradicate hiv from reservoirs ( ) . a g-rich sequence composed of three conserved clusters has been identified in the reading frame of the negative regulatory factor (nef) ( ) . this coding sequence is located at the -end of the genome and overlaps with the -ltr (figure a ). isolated nef g-stretches can form g s in vitro. moreover, their stabilization represses nef expression and decreases viral replication. nef has multiple roles during hiv infection and has been implicated in immune evasion by promoting cd and major histocompatibility complex (mhc) molecule down-regulation. importantly, the absence of nef seems to correlate with low viral load and inhibition of disease progression. thus, targeting the g s located in the nef coding sequence is a new attractive therapeutic opportunity. sv is a polyomavirus with a -kb, closed circular and double-stranded dna genome that codes for six proteins and includes a non-coding regulatory region (ncrr). this latter regulatory region contains the ori and the encapsidation sequence (ses) but also controls the transcription direction (early versus late transcription). notably, six gc boxes (gggcgg) are present in this region, which can form an unusual quadruplex structure containing a ctetrad stacked between two g-tetrads as determined by nmr ( ) . these repeated sequences are binding motifs for sp and, therefore, play an important role in early transcription. on the other hand, sv genome replication requires the large t-antigen (tag), which is a multifunctional protein that binds the ori, has adenosine triphosphatedependent helicase activity and interacts with cellular proteins such as p and rb ( ) . interestingly, tag can unwind g dna structures ( , ) ; thus, it might play a crucial role in regulating replication as well as early and late transcription. perylene di-imide derivatives (pdi) stabilize g structures and inhibit both the g and tag duplex dna helicase activities. hence, pdi provide tools for probing the role of the g helicase activity in sv replication ( , ) and introduces new insights into the link between helicases and tumorigenesis ( ) or other human genetic diseases ( ) . the human papillomaviruses (hpv) family consists of more than viruses; approximately half are sexually transmissible and are considered high risk due to their carcinogenic properties. these viruses are one of the most common sexually transmitted infections and induce cervical cancer. notably, hpv and hpv contribute to % of cervical cancer induced by hpv infections. the hpv genome consists of kb, circular, double-stranded dna and integration into the infected cell induces dramatic genome instability ( ) . the open reading frames encode six 'early' proteins (e , e , e , e , e and e ) and two 'late' structural proteins (l and l ). the late proteins are major and minor capsids, respectively, and association with l /l heterodimers forms star-shaped capsomeres. however, l can self-assemble into - nanometers, virus-like particles, which can be used in prophylactic hpv vaccines to protect against an initial hpv infection. g-rich regions have been found in hpv genomes, and their potential to fold into a g structure has been described ( ) . g-rich loci that fulfill the criteria for g formation have only been found in eight types of hpv. however, a strong argument for the relevance of g s in hpv biology is that the viral protein e is a helicase that resembles sv tag ( ) . consequently, e may also present a g unwinding activity. for hpv and hpv , potential g -forming sequences are located in the long control region (lcr), which is a regulatory sequence composed of nearly kb, suggesting a potential role in transcription and replication. the presence of g s in the sequence coding for the l protein (hpv ), e (hpv , hpv ) and e (hpv , hpv , hpv ) suggests that g formation may also alter alternative splicing necessary for producing viral proteins from the overlapping open reading frame (orfs). targeting these g s could potentially serve as a basis for novel antiviral therapies. hodgkin's lymphoma, burkitt's lymphoma and nasopharyngeal carcinoma ( ) . in the case of hiv- co-infection, ebv is also associated with hairy leukoplakia and central nervous system lymphoma. the virus is ∼ to nm in diameter and is composed of a kb dna double helix that circularizes upon entry into the nucleus and becomes a viral episome. human herpes viruses are mostly asymptomatic due to latency. the epstein-barr virus encodes a genome maintenance protein (nuclear antigen , ebna ), expressed in all ebvassociated malignancies. ebna binds g-rich sequences at the viral replication origin, recruits the replication complex and is involved in metaphase chromosome attachment, which insures maintenance throughout mitosis ( ) . thus, formation of secondary structures, such as g s, may play a role in regulating ebv replication. the level of ebna synthesized is tightly controlled; it is sufficiently high to maintain viral infection but sufficiently low to avoid immune recognition by the host's virus-specific t cells. regulation occurs during translation due to secondary structures present in the mrna ( ) . destabilization of the g -forming sequence through antisense oligonucleotide annealing increases the translation rate and, consequently, promotes antigen presentation. on the other hand, stabilization of the g with a g ligand (pyridostatin) decreases ebna synthesis and allows immune evasion. interestingly, g s are similarly observed in mrna for other maintenance genes in the gammaherpesvirus family. these findings suggest that this mode of translational regulation may be more general among proteins that self-regulate synthesis. in addition, one could imagine alternative therapeutic strategies focused on targeting rna structures within viral orfs to interfere with the virus cycle as well as to promote antigen presentation and to stimulate the host immune response. recently, a g ligand called quarfloxin (cx- ) entered phase ii clinical trials. it is capable to suppress myc transcription by inhibiting the interaction between nucleolin and the c-myc g motifs ( ) . even if cx- failed trials because of bioavailability issues, original anti-cancer applications based on such ligands seem underway ( ) . taken together, all these studies suggest that targeting these g s could potentially serve as a basis for novel antiviral therapies. hence, as described for cellular targets ( ) , small ligands that can stabilize the g structure may compose a potential new class of therapeutic agents to fight viral infections ( , ) [for reviews, see ( , ) ]. recent studies showed that g ligands, such as braco- and tmpyp , inhibited hiv- replication ( , ) . exhaustive viral assays demonstrated that braco- acts at both reversetranscription and post-integration steps by targeting of the viral promoter ( ) . several laboratories are already working to identify g ligands that preferentially interact with the pathogen's g over cellular g s. this new antiviral strategy presents many advantages: (i) these compounds target viral dnas and rnas, which are the source of the disease; (ii) high conservation of these targets across subspecies suggests that they are important for viruses and (iii) mutations will likely impact viral fitness, limiting the emergence of resistant strains. nucleic acids have already been validated as therapeutics ( ) . the first example is formivirsen (vitravene r ), a phosphorothioate anti-sense oligonucleotide approved from to to treat retinitis caused by cytomegalovirus (cmv) ( ) . the second is pegaptanib (macugen r ), used in the clinic from to . this -fluoropyrimidine rna-based aptamer targets vegf to treat neovascular age-related macular degeneration ( ) . however, those two drugs have been withdrawn due to the insufficient benefice associated with their use. regarding g s, certain g oligonucleotides present therapeutic potential, including the thrombin-binding aptamer (tba, g t g tgtg t g ) ( , ) . the most promising g to date is as (agro ), a bases oligonucleotide targeting nucleolin with anti-proliferative properties ( ) . as recently completed phase ii clinical trials as anticancer drug (nct ) with low toxicity, highlighting the high therapeutic potential of g ( ) . for most applications, g -forming molecules have been selected through combinatorial methods (e.g. pegaptanib, anti-tba). two key protocols have been developed. first, polynucleotide arrays facilitated rapid detection of molecules with an affinity to the target. then, the development of selex (systematic evolution of ligands by exponential enrichment) facilitates isolation of oligonucleotide sequences with the capacity to recognize virtually any class of target molecule with high affinity and specificity ( ) . these oligonucleotides, 'aptamers', are emerging as a class of molecules that rival antibodies in both therapeutic and diagnostic applications ( , ) . in this section, we describe a few examples of aptamers specifically selected to interact with viral components and interfere with viral replication. hepatitis a virus (hav) belongs to the picornaviridae family. even without a specific treatment for hav infection, a vaccine has been produced to protect against initial infection. however, only a limited number of countries recommend vaccination because hav is rarely lethal. the hav genome comprises a . -kb single-stranded (+) rna with a -utr and poly(a) tail. it encodes for a single polyprotein, which includes the c protease. this enzyme is crucial for the virus because it cleaves the polyprotein into several capsid proteins and non-structural proteins. additionally, the c protease binds regulatory structural elements at the -utr, which control viral genome replication. thus, the c protease is an attractive target for develop a specific antiviral treatment. recently, hexadeoxyribonucleotides that specifically bind the hav c protease were identified through a hexanucleotide array ( ) . in vitro experiments showed that the hexanucleotide gggggt (g t) forms a tetramolecular g , binds the c-terminal domain of hav protease and is a potent protease inhibitor ( ) . every year in winter, influenza viruses cause seasonal respiratory disease epidemics ( ) . three subtypes have been defined (a, b and c), which depend on the surface proteins (hemagglutinin and neuraminidase). sixteen hemagglutinin (h) and nine neuraminidase (n) variants have been discovered, but only h , and as well as n and are commonly found in humans. these viruses belong to the orthomyxoviridae family, which composes enveloped viruses with a single-stranded negative-sense rna. the genome is to kb but is segmented into multiple ( or ) molecules. each rna is . to . kb and encodes for or proteins. the non-structural protein (ns ) is a kda multifunctional protein and participates in protein-protein and protein-rna interactions ( ) . during infection, ns interferes with cellular mrna biology (splicing, maturation and translation). as a result, ns prevents interferon (ifn) production, which leads to inhibition of the host's innate immunity ( ) . after cycles of selex, which employs a -base variable sequence, a g -forming aptamer was selected that presents high affinity for the ns rna binding domain (kd ≈ nm) ( ) . in a cellular context, this g can block ns and restore inf production, which results in antiviral activity without cellular toxicity. severe acute respiratory syndrome coronavirus (sars-cov) is an enveloped virus with a single-stranded rna genome ∼ kb long. it encodes poly-cistronic orfs, which code for non-structural proteins (nsp). since the outbreak, the three-dimensional structures of several key proteins have been determined (e.g. rdrp, c-like protease). the non-structural protein (nsp ) is one component of the viral replicase complex and contains a domain referred to as sars unique domain (sud), which interacts with g s. thus, g s are also relevant for the coronavirus and might be involved either in viral replication or host immune evasion ( ) . on the other hand, using a se-lex approach with oligonucleotides that harbor a nucleotide variable sequence and following rounds of selection, dna aptamers against the sars-cov helicase were isolated ( ) . these aptamers compose two distinct classes, g and non-g forming sequences, which were determined through circular dichroism and gel electrophoresis. their inhibitory effect on viral replication is being studied with encouraging results. combinatorial approaches have been used to identify several aptamers that target hiv- . the first rna aptamer isolated using the selex approach was an rna pseudoknot inhibitor of hiv- rt ( ) . using another procedure, the surf (synthetic unrandomization of randomized fragments) ( ) , isis was selected and exhibited sub-micromolar inhibition of hiv- . isis is a phosphorothioate containing dna octamer (t g t ) that forms g with anti-hiv properties. more specifically, it inhibited cell-to-cell and virus-to-cell spread of the hiv- by interacting with the v loop located on the viral glycoprotein gp ( ) ( ) ( ) . its g structure and phosphorothioate backbone were reported as essential to this inhibition. later studies showed that phosphodiester oligonucleotides containing only g and t inhibit hiv- replication and the most potent molecule, gtg tg tg tg t (t , ec at ∼ nm), formed a g in vitro ( ) ( ) ( ) . this oligonucleotide and related molecules, such as t (g tg tg tg t, figure j ), are potent hiv- in inhibitors in vitro. t was the first in inhibitor tested in clinical trials (zintevir tm developed by aronex pharmaceuticals in ) ( ) . however, the action mechanism in cells is more complex because it also targets viral entry ( ) . in attempts to obtain natural-type oligonucleotide, hotoda et al. identified a hexamer (tg ag) also targeting hiv- entry through gp binding ( ) . this 'hotoda's sequence' adopts a tetramolecular g structure and submicromolar hiv- inhibition was described for derivatives with -end substitutions. once more, the antiviral activity of the molecule was directly linked to its capability to form g s. later, selex approaches were developed to isolate dna aptamers with high affinity for the rnase h domain of hiv- rt. thus, the target protein used was either the isolated rnase h domain (p ) or the functional heterodimer p /p with a counter-selection that used the p /p form (truncated for the rnase h domain) ( , ) . interestingly, these selections mainly led to aptamers with g-rich sequences capable of forming g s. some, but not all, of these g inhibited the rnase h activity of hiv- rt in vitro with an ic in the nm range ( ) . surprisingly, these g aptamers ( del and del) were also potent in inhibitors in vitro with ic values in the range - nm ( ) . this dual inhibition can be explained by the structural similarities between the in active site and rt rnase h domain ( ) . the aptamer del can form an original dimeric interlocked g (figure i) , which is stable even at temperatures over • c ( ) . through comparing the structures of the g -forming aptamers that inhibit in, in inhibition likely requires a stack of gtetrads ( ) . similar to t , del is a potent antiviral agent with multimodal inhibition and, in cells, targets the viral entry step, reverse transcription and integration ( ) . additional in vitro studies indicated that free del and t can enter human cells, including epithelial (hela), hepatic (huh ) and lymphocytes (h ) cells ( ) . however, striking differences were observed in the presence of viral particles; hiv- strongly stimulates cellular uptake of aptamer ( ) . this latter observation opens an opportunity for specific drug delivery to cells that are infected, which may prevent intracellular side effects from g off-targeting. nucleic acids research, , vol. , no. given the remarkable physical properties of g s, scientists have engineered innovative tools using g folding to control ribozyme activity and constructed probes for state-ofthe-art imagery as well as quantification techniques, among other approaches. as an example, the aforementioned g del was engineered into aptamer beacons to visualize endogenous protein hiv- reverse transcriptase in living cells ( ) . the following section describes two such innovations related to viruses. hdv is a small, enveloped virus that depends on hepatitis b virus (hbv) for propagation ( ) . its genome is a circular, single-stranded (−) rna molecule of nucleotides. however, due to self-complementarity over ∼ % of its length, it folds into a partially double-stranded molecule (rod-like rna structure). because hdv does not encode for a polymerase, replication of its genome relies on cellular enzymes. upon polymerization following the 'double rolling circle' mechanism, genomic and anti-genomic circular rna is produced with linear polyadenylated mrna that only codes for the hdv protein, delta antigen (hdag). however, hdv also encompasses an nucleotide ribozyme with self-cleavage activity. molecular engineering led to development of a 'g-quartzyme', which is a ribozyme controlled by a g structure ( , ) . stabilization of the g at the end by monovalent ions (k + ) activates the ribozyme. cauliflower mosaic virus (camv) is a pararetrovirus that infects plants ( ) . its genome is an -kb, circular, doublestranded dna molecule produced through reverse transcription of a pre-genomic mrna. therefore, transcription of the genome is a crucial step for two reasons: (i) it is necessary to code for several viral proteins and (ii) it generates s rna that is slightly longer than the genome (terminally redundant), which serves as a matrix to replicate the dna genome. because the unique camv promoter, the s promoter, is a strong and constitutive promoter, it has been widely used to develop genetically modified organisms (gmos). consequently, there is a growing need to detect gmos in the food industry, and many methods have been developed based on elisa, polymerase chain reaction and other techniques. notably, a fluorescent assay has been engineered based on g formation. it involves two specific primers that recognize the s promoter. each primer is linked to a double repeat of the human telomeric sequence ttaggg. as a consequence, binding the two primers facilitates inter-strand quadruplex formation. upon adding berberine (a selective g -binder), a strong fluorescent signal is produced ( ) , which facilitates promoter detection. the x-ray structure of g-tetrads was determined in the early s; in the s, g s remained an intriguing deviation from the watson-crick canonical structure. however, a remarkable quantity of information has been obtained over the past two decades, which has allowed this field to grow from basic science to clinical application ( , ) . thus, the presence of g s in telomeres and oncogenic promoters has opened broad opportunities for understanding and generating new treatments against cancer. the viral world is extensive and includes viruses with replication schemes based on rna (e.g., flaviviruses), dna (e.g., papillomavirus) or intermediates thereof (e.g., retroviruses) and g s are part of their life cycle. first, they must address the presence of g s in their hosts, and second, they must also contain g -forming sequences that regulate important replication steps. several challenges remain to better define the features that provide specific g motifs with the ability to function as structural elements. nevertheless, g sequences and g -binders have been identified in some of the most pathogenic viruses; thus, they are attractive targets for controlling viral infections. pdb ids: kf , lpw, m p, y d and le . untersuchungenüber die guanylsäure helix formation by guanylic acid the structure of helical -guanosine monophosphate poly(inosinic acid) 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activation of an atr-dependent atm signaling pathway small-molecule-induced dna damage identifies alternative dna structures in human genes telomestatin, a novel telomerase inhibitor from streptomyces anulatus telomerase inhibition with a novel g-quadruplex-interactive agent, telomestatin: in vitro and in vivo studies in acute leukemia prevalence of quadruplexes in the human genome structural insights into g-quadruplexes: towards new anticancer drugs existence and consequences of g-quadruplex structures in dna telomeres: protecting chromosomes against genome instability a telomerase component is defective in the human disease dyskeratosis congenita human dyskerin: beyond telomeres all gene-sized dna molecules in four species of hypotrichs have the same terminal sequence and an unusual terminus genomic reorganization in ciliated protozoans telomeric dna oligonucleotides form novel intramolecular structures containing guanine-guanine base pairs an overhanging terminus is a conserved feature of telomeres monovalent cation-induced structure of telomeric dna: the g-quartet model processing of g dna by dna helicase/nuclease and replication protein a (rpa) provides insights into the mechanism of dna /rpa substrate recognition mammalian dna helicase/nuclease cleaves g-quadruplex dna and is required for telomere integrity telomerase: dr jekyll or mr hyde? telomerase extends the lifespan of virus-transformed human cells without net telomere lengthening g-quadruplex formation at the end of telomere dna inhibits its extension by telomerase, polymerase and unwinding by helicase discovery of g-quadruplex stabilizing ligands through direct elisa of a one-bead-one-compound library recent developments in the chemistry and biology of g-quadruplexes with reference to the dna groove binders an intramolecular g-quadruplex structure is required for binding of telomeric repeat-containing rna to the telomeric protein trf novel g-quadruplex stabilizing agents: in-silico approach and dynamics g-quadruplex stabilizer induces m-phase cell cycle arrest reevaluation of telomerase inhibition by quadruplex ligands and their mechanisms of action telomestatin impairs glioma stem cell survival and growth through the disruption of telomeric g-quadruplex and inhibition of the proto-oncogene, c-myb replication in context: dynamic regulation of dna replication patterns in metazoans new insights into replication origin characteristics in metazoans unraveling cell type-specific and reprogrammable human replication origin signatures associated with g-quadruplex consensus motifs g motifs affect origin positioning and efficiency in two vertebrate replicators stimulation of gross chromosomal rearrangements by the human ceb and ceb minisatellites in saccharomyces cerevisiae depends on g-quadruplexes or cdc making sense of g-quadruplex and i-motif functions in oncogene promoters dna tetraplex formation in the control region of c-myc the cationic porphyrin tmpyp down-regulates c-myc and human telomerase reverse transcriptase expression and inhibits tumor growth in vivo direct evidence for a g-quadruplex in a promoter region and its targeting with a small molecule to repress c-myc transcription the dynamic character of the g-quadruplex element in the c-myc promoter and modification by tmpyp propeller-type parallel-stranded g-quadruplexes in the human c-myc promoter ribonucleoprotein and protein factors bind to an h-dna-forming c-myc dna element: possible regulators of the c-myc gene puf/nm -h /ndpk-b transactivates a human c-myc promoter-cat gene via a functional nuclease hypersensitive element identification and characterization of nucleolin as a c-myc g-quadruplex-binding protein novel interaction of the z-dna binding domain of human adar with the oncogenic c-myc promoter g-quadruplex chromatin structure of the promoter region of the human c-k-ras gene g-rich oligonucleotide inhibits the binding of a nuclear protein to the ki-ras promoter and strongly reduces cell growth in human carcinoma pancreatic cells formation of pseudosymmetrical g-quadruplex and i-motif structures in the proximal promoter region of the ret oncogene an rna g-quadruplex in the utr of the nras proto-oncogene modulates translation an unusually stable g-quadruplex within the -utr of the mt matrix metalloproteinase mrna represses translation in eukaryotic cells a g-quadruplex structure within the -utr of trf mrna represses translation in human cells -utr g-quadruplex structures acting as translational repressors -utr rna g-quadruplexes: translation regulation and targeting stimulation of ribosomal frameshifting by rna g-quadruplex structures translational recoding induced by g-rich mrna sequences that form unusual structures repeat instability as the basis for human diseases and as a potential target for therapy tetraplex formation by the progressive myoclonus epilepsy type- repeat: implications for instability in the repeat expansion diseases mutsalpha binds to and promotes synapsis of transcriptionally activated immunoglobulin switch regions putative dna g-quadruplex formation within the promoters of plasmodium falciparum var genes reca-binding pile g sequence essential for pilin antigenic variation forms monomeric and end-stacked dimeric parallel g-quadruplexes the g genome hiv integrase inhibitors: -year landmark and challenges cell biology of retroviral rna packaging retroviral recombination and reverse transcription extensive recombination among human immunodeficiency virus type quasispecies makes an important contribution to viral diversity in individual patients moloney murine sarcoma virus genomic rnas dimerize via a two-step process: a concentration-dependent kissing-loop interaction is driven by initial contact between consecutive guanines functional characterization of the dimer linkage structure rna of moloney murine sarcoma virus a loop-loop 'kissing' complex is the essential part of the dimer linkage of genomic hiv- rna evidence for 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for tetramolecular antiparallel g-quadruplex formation solution structures of all parallel-stranded monomeric and dimeric g-quadruplex scaffolds of the human c-kit promoter a dynamic g-quadruplex region regulates the hiv- long terminal repeat promoter topology of a dna g-quadruplex structure formed in the hiv- promoter: a potential target for anti-hiv drug development u region in the hiv- genome adopts a g-quadruplex structure in its rna and dna sequence formation of a unique cluster of g-quadruplex structures in the hiv- nef coding region: implications for antiviral activity nmr observation of a novel c-tetrad in the structure of the sv repeat sequence gggcgg the large tumor antigen: a 'swiss army knife' protein possessing the functions required for the polyomavirus life cycle the sv large t-antigen helicase can unwind four stranded dna structures linked by g-quartets real-time investigation of sv large t-antigen helicase activity using surface plasmon resonance simian virus large 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anti-integrase agents closely related antiretroviral agents as inhibitors of two hiv- enzymes, ribonuclease h and integrase: 'killing two birds with one stone an interlocked dimeric parallel-stranded dna quadruplex: a potent inhibitor of hiv- integrase the guanine-quadruplex aptamer del inhibits hiv- replication ex vivo by interfering with viral entry, reverse transcription and integration cellular uptake of odns in hiv- human-infected cells: a role for viral particles in dna delivery? aptamer beacons for visualization of endogenous protein hiv- reverse transcriptase in living cells an update on hdv: virology, pathogenesis and treatment potassium ions modulate a g-quadruplex-ribozyme's activity modulating rna structure and catalysis: lessons from small cleaving ribozymes structure and replication of caulimovirus genomes a novel fluorescent biosensor for detection of target dna fragment from the transgene cauliflower mosaic virus s promoter quadruplex nucleic acids therapeutic applications of quadruplex nucleic acids weblogo: a sequence logo generator key: cord- -klb oe q authors: chen, serena h.; young, m. todd; gounley, john; stanley, christopher; bhowmik, debsindhu title: distinct structural flexibility within sars-cov- spike protein reveals potential therapeutic targets date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: klb oe q the emergence and rapid worldwide spread of the novel coronavirus disease, covid- , has prompted concerted efforts to find successful treatments. the causative virus, severe acute respiratory syndrome coronavirus (sars-cov- ), uses its spike (s) protein to gain entry into host cells. therefore, the s protein presents a viable target to develop a directed therapy. here, we deployed an integrated artificial intelligence with molecular dynamics simulation approach to provide new details of the s protein structure. based on a comprehensive structural analysis of s proteins from sars-cov- and previous human coronaviruses, we found that the protomer state of s proteins is structurally flexible. without the presence of a stabilizing beta sheet from another protomer chain, two regions in the s domain and the hinge connecting the s and s subunits lose their secondary structures. interestingly, the region in the s domain was previously identified as an immunodominant site in the sars-cov- s protein. we anticipate that the molecular details elucidated here will assist in effective therapeutic development for covid- . a new coronavirus, severe acute respiratory syndrome coronavirus (sars-cov- ), causes respiratory illness and has now reached a pandemic scale. denoted as coronavirus disease (covid- ) , its global spread is currently ongoing with symptoms that can range from mild flu-like to severe pneumonia, leading to death in certain cases. early investigations in china showed that sars-cov- has a high genomic sequence similarity to the previous sars-cov- , along with a bat coronavirus [ ] , [ ] . similar to sars-cov- , sars-cov- is a positive-sense, single-stranded rna virus of the betacoronavirus genus. given the health crisis caused by the mounting number of covid- cases worldwide, there is an urgent need to develop effective therapeutics and eventual vaccines. a critical step in developing targeted treatments is obtaining a detailed notice: this manuscript has been authored by ut-battelle, llc under contract no. de-ac - or with the u.s. department of energy. the united states government retains and the publisher, by accepting the article for publication, acknowledges that the united states government retains a non-exclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for united states government purposes. the department of energy will provide public access to these results of federally sponsored research in accordance with the doe public access plan (http://energy.gov/downloads/doe-public-accessplan). understanding of the molecular pathways of sars-cov- and constituent structures. the structural biology community has made rapid progress towards this goal by experimentally determining several sars-cov- proteins, including the spike (s) [ ] - [ ] , nucleocapsid (n) [ ] , and main protease (m pro ) [ ] . the s protein is particularly important since it resides on the viral envelope and is responsible for host cell entry by engaging angiotensin-converting enzyme (ace ) receptors [ ] , [ ] , [ ] . recent experimental structures of the sars-cov- s protein receptor binding domain (rbd) in complex with ace provide detailed interface information [ ] , [ ] ; targeting this interface represents an active area of research for therapeutic development [ ] . however, there may exist potential targets on the s protein besides the rbd domain. further motivating our work is the need to understand sars-cov- structure in the context of structures from previous coronaviruses. prior to the emergence of sars-cov- , comparisons of the trimeric s protein from different viruses showed they possess overall similar structures but with some local differences [ ] , [ ] . with the appearance of sars-cov- , it is necessary to investigate the s protein structure [ ] , and compare it to previous human coronaviruses. here, to obtain deeper insights into s protein structure for biological understanding and therapeutic targeting, we employ a combined molecular dynamics (md) simulation and artificial intelligence (ai) methodology on a series of coronavirus s proteins. specifically, we investigate the s proteins from the current sars-cov- , sars-cov- , middle east respiratory syndrome coronavirus (mers-cov), and human coronavirus hku (hcov-hku ). from structural analysis of extensive md simulations, we find substantial flexibility between the subunits of the s proteins. we find that reduced distance matrix representations, interpreted by unsupervised deep learning (dl), reveal important regions for s protein trimerization. these regions present potential targets for therapeutic development. human coronavirus spike (s) proteins are molecular complexes each formed by three protein chains [ ] , [ ] - [ ] . to better understand s protein structure [ ] , we started by studying the protomer, the structural unit of the trimeric complex, and compared the protomer structure of the current sars-cov- with protomers of previously identified human coronaviruses, including sars-cov- [ ] , mers-cov [ ] , and hcov-hku [ ] . we modeled each protomer system from the corresponding cryo-em s protein structure (see methods for more details). there are three major structural domains which constitute the protomer: the amino-terminal domain (ntd), receptor binding domain (rbd), and the s domain. the ntd and rbd are within the s subunit and are responsible for binding to host receptors, while the s domain is in the s subunit which mediates fusion of the viral and host membranes [ ] , [ ] . fig. a-d shows the initial protomer structures of the four s proteins, each highlighted by the three domains. to determine the dynamic change of the domain organization in the protomer structures, we measured the distribution of the interdomain distances from , structures of each system taken from independent ns md simulations (fig. e-g) . given the limited timescale, we made no attempt to reproduce the thermodynamics but focused on computing the structural features of the systems [ ] . the overall distributions of the three interdomain distances are broad, ranging from Å to Å between s and ntd/rbd domains and Å to Å between ntd and rbd domains. these wide distributions indicate enhanced structural flexibility in the domain arrangement of the four protomer systems compared to the cryo-em structures. to gain further insight into the , protomer structures, we applied convolutional variational autoencoders (cvaes) to encode the high dimensional protein structures from the md simulations into -d latent spaces for visualization. compared to commonly applied structural analysis methods, which are mostly limited to specific regions of the protein of interest, our dl approach allows us to evaluate a protein structure as a whole yet still capture detailed local structural features. we found individual clusters corresponding to each of the four protomer systems, suggesting that the structural features of the four systems embedded in the latent spaces are distinct from each other ( fig. a-c). representative structures selected from the clusters also show high structural flexibility in their domain arrangement ( fig. d-f). the hinge region connecting the s and s subunits opens up, causing broad distributions of the interdomain distances observed in fig. e-g. the exposed surface in the protomer may provide new targets for therapeutic action. therefore, we further explored the structural flexibility of the sars-cov- s protein. we compared the structures between the protomer and trimer to investigate whether the large structural arrangement of the domains in the protomer is affected by the trimeric state. we found that domain organization is stabilized by trimerization. unlike the protomer, the three domains are closely arranged in the trimer (figs. a-b and s ). in addition to their interdomain distances, the differences in structural flexibility between the protomer and trimer is captured in solventaccessible surface area (sasa) values (fig. c) . the sasa values of the protomer and one chain of the trimer are ± nm and ± nm , respectively. the difference in these values largely comes from their s domains, which have the values of ± nm and ± nm , respectively. again, we applied a cvae to encode the , protomer and trimer structures into -d latent spaces. we found two individual clusters, one corresponding to the protomer and the other to the trimer, suggesting that the structural features of the two oligomeric states embedded in the latent spaces are different (fig. d-g) . to locate the different structural features between the protomer and trimer in their -d structures, we compared the difference between the distance matrices of the representative protomer and trimer structures selected from the clusters in the latent dimensions (fig. a) . most differences between the distance matrices result from interdomain arrangement. however, there are some disparities within the s domain (fig. b) . one region of difference corresponds to residue numbers to in the pdb. in the -d structure, this region forms a beta sheet in the trimer while the structure is lost in the protomer (fig. c) . further investigation into this region reveals that the beta sheet in the trimer is stabilized by another beta sheet constituted by residues to of another chain (fig. d) . without the presence of other chains, this stability is lost; not only do residues to in the s domain lose the beta sheet structure, but residues to in the hinge region connecting the two subunits become an extended loop (fig. e) . oligomeric proteins are often stabilized by oligomerization and hold highly flexible regions in the protomer state [ ] . the transition between the structured and unstructured form of these flexible regions is sometime reversible, but due to the size of the s protein protomer and the timescale applied in this study, we did not observe reversible behavior of the two loop regions in the protomer state. interestingly, a fragment between residues and was identified previously as an immunodominant site in sars-cov- s protein [ ] . complementary antibodies acting on this site provided the dominant immune response for patients who recovered from the sars-cov infection. our results provide structural comprehension on the previous experimental observation and posit that targeting these residues might not only interfere with s protein trimerization and the subsequent viral activity but also aid antibody immune response. our findings provide insights into therapeutic design targeting the s protein beyond the oft-targeted rbd domain. here, we used unsupervised deep learning routines based on cvaes to systematically compare s protein ensembles from md simulations across lower-dimensional, latent spaces. by first comparing the s protein protomer structure of sars-cov- to those from previous human coronaviruses, we identified distinct clusters for each virus in the -d latent space, where representative structures from these clusters highlight their differences in domain flexibility. next, we compared the sars-cov- s protein protomer and trimer structures, which also displayed a clear separation of clusters in the latent space. while the main distinctions between these two states arise from the general gain in structural stability as the protomer self-assembles into the trimer state, we pinpointed structural transitions in specific flexible regions of the protomer that warrant consideration as potential therapeutic targets. these regions are promising as natural targets of immune recognition, but more importantly, they are involved in s protein oligomerization, suggesting they are susceptible to therapeutic action for protein destabilization. overall, our study provides a more complete molecular view of the sars-cov- s protein that may assist in accelerating both vaccine and drug design efforts. to generate initial systems for the structural study of human coronavirus s proteins, we built protomer structures from chain a of the cryo-em s protein structures of sars-cov- (pdb vsb [ ] ), sars-cov- (pdb crz [ ] ), mers-cov (pdb q [ ] ), and hcov-hku (pdb i [ ] ). the trimeric state of the sars-cov- s protein consists of all three chains in pdb vsb. each structure was solvated in the center of a water box with a minimum distance of Å from the edge of the box to the nearest protein atom, neutralized with counter ions and ionized with mm nacl. table summarizes the details of the five molecular systems studied. following a similar protocol to our previous studies [ ] , [ ] , the resulting systems were each subjected to , steps of energy minimization, followed by ns equilibration with harmonic restraints placed on the heavy atoms of the protein. the force constant was kcal/mol/Å [ ] . after equilibration, the restraints were released, and a ns trajectory was generated in a production run. for each system, independent ns trajectories were performed. structures were taken every ns for analysis, yielding a total of , structures for each system. all md simulations were performed with namd [ ] in npt ensemble at atm and k with a time step of fs. the charmm m force field [ ] and tip p water model [ ] were used. the nonbonding interactions were calculated with a typical cutoff distance of Å, while the long-range electrostatic interactions were enumerated with the particle mesh ewald algorithm [ ] . to further understand the molecular structures of different human coronavirus s proteins and the oligomeric state of sars-cov- s protein, we deployed a custom-built deep learning architecture, a convolutional variational autoencoder (cvae), to encode the high dimensional protein structures from the md simulations into lower dimensional latent spaces. the goal of our ai method is to reduce the high dimensionality of the molecular system while preserving the inherent characteristics of the system and learning novel behavior in a latent space that is normally distributed. the direct comparison between the decoded and original input data ensures the accuracy of the latent space representation. this customized cvae approach has been successfully applied to study the folding pathways of small proteins and structural clustering of biomolecules [ ] - [ ] . ) data preparation: we used translation and rotation invariant input data for the dl networks. we represented each md structure by a distance matrix using the c ↵ atoms of the protein and generated two input datasets for the cvaes. input included the matrices of the protomer structures of sars-cov- , sars-cov- , mers-cov, and hcov-hku . input included the matrices of the protomer structure and the chain a of the trimer structure of sars-cov- . for input , as the proteins are different in length, we first performed a multiple sequence alignment of the protomer structures of sars-cov- , sars-cov- , mers-cov, and hcov-hku by clustal omega [ ] . based on the alignment, we inserted gaps into the corresponding aligned residue location in the distance matrix and set the distance between gaps to be Å. the size of each distance matrix after the alignment was , ⇥ , . to reduce the matrix size, we applied a convolution layer with padding of and a ⇥ filter with strides of size in both the x and y directions. the size of each resulting matrix became ⇥ . finally, after alignment and size reduction we merged a total of , distance matrices of the four s proteins. an example of a distance matrix following the alignment and size reduction is represented in fig. s . for input , as the proteins are of the same length, no alignment was required. the size of each matrix was ⇥ . we again applied a convolution layer with padding of and a ⇥ filter with strides of size in both the x and y directions to reduce the matrix size. the size of each resulting matrix was also ⇥ , and we merged a total of , distance matrices of the protomer and trimer of sars-cov- s protein. ) cvae implementation: for each of the two input datasets, we randomly split the aligned matrices into training and validation datasets using the / ratio and applied a cvae to capture the important structural features and projected them in the three-dimensional ( -d) latent space for visualization. the encoder network of each cvae consisted of three convolutional layers and a fully connected layer. we used a ⇥ convolution kernel and a stride of , , and at the three convolutional layers, respectively. we trained each cvae until the training and validation loss converged. fig. s shows the loss curves along the epochs. the difference between decoded and original images is minimal, suggesting the models were trained successfully (fig. s ) . we then selected representative structures from the clusters in the latent space and visualized them using vmd [ ] . all md simulations and dl analysis were performed on the summit supercomputer at the oak ridge leadership computing facility. the distances calculated from the cryo-em structure (pdb vsb) are marked by a pentagon. a new coronavirus associated with human respiratory disease in china a pneumonia outbreak associated with a new coronavirus of probable bat origin cryo-em structure of the -ncov spike in the prefusion conformation structural basis for the recognition of sars-cov- by full-length human ace structure, function, and antigenicity of the sars-cov- spike glycoprotein structure of the sars-cov- spike receptor-binding domain bound to the ace receptor crystal structure of nsp endoribonuclease nendou from sars-cov- crystal structure of sars-cov- main protease provides a basis for design of improved -ketoamide inhibitors functional assessment of cell entry and receptor usage for sars-cov- and other lineage b betacoronaviruses sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor angiotensin-converting enzyme (ace ) as a sars-cov- receptor: molecular mechanisms and potential therapeutic target structure, function, and evolution of coronavirus spike proteins cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains pre-fusion structure of a human coronavirus spike protein stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis structures of mers-cov spike glycoprotein in complex with sialoside attachment receptors the energy landscape of a protein switch in silico design and validation of high-affinity rna aptamers targeting epithelial cellular adhesion molecule dimers b-cell responses in patients who have recovered from severe acute respiratory syndrome target a dominant site in the s domain of the surface spike glycoprotein graphene-extracted membrane lipids facilitate the activation of integrin ↵ v stability of ligands on nanoparticles regulating the integrity of biological membranes at the nano-lipid interface scalable molecular dynamics with namd charmm m: an improved force field for folded and intrinsically disordered proteins comparison of simple potential functions for simulating liquid water particle mesh ewald: an n log (n) method for ewald sums in large systems deep clustering of protein folding simulations mechanism of glucocerebrosidase activation and dysfunction in gaucher disease unraveled by molecular dynamics and deep learning visual analytics for deep embeddings of large scale molecular dynamics simulations the embl-ebi search and sequence analysis tools apis in vmd: visual molecular dynamics key: cord- -s kzp authors: zemla, adam t; lang, dorothy m; kostova, tanya; andino, raul; ecale zhou, carol l title: stralsv: assessment of sequence variability within similar d structures and application to polio rna-dependent rna polymerase date: - - journal: bmc bioinformatics doi: . / - - - sha: doc_id: cord_uid: s kzp background: most of the currently used methods for protein function prediction rely on sequence-based comparisons between a query protein and those for which a functional annotation is provided. a serious limitation of sequence similarity-based approaches for identifying residue conservation among proteins is the low confidence in assigning residue-residue correspondences among proteins when the level of sequence identity between the compared proteins is poor. multiple sequence alignment methods are more satisfactory--still, they cannot provide reliable results at low levels of sequence identity. our goal in the current work was to develop an algorithm that could help overcome these difficulties by facilitating the identification of structurally (and possibly functionally) relevant residue-residue correspondences between compared protein structures. results: here we present stralsv (structure-alignment sequence variability), a new algorithm for detecting closely related structure fragments and quantifying residue frequency from tight local structure alignments. we apply stralsv in a study of the rna-dependent rna polymerase of poliovirus, and we demonstrate that the algorithm can be used to determine regions of the protein that are relatively unique, or that share structural similarity with proteins that would be considered distantly related. by quantifying residue frequencies among many residue-residue pairs extracted from local structural alignments, one can infer potential structural or functional importance of specific residues that are determined to be highly conserved or that deviate from a consensus. we further demonstrate that considerable detailed structural and phylogenetic information can be derived from stralsv analyses. conclusions: stralsv is a new structure-based algorithm for identifying and aligning structure fragments that have similarity to a reference protein. stralsv analysis can be used to quantify residue-residue correspondences and identify residues that may be of particular structural or functional importance, as well as unusual or unexpected residues at a given sequence position. stralsv is provided as a web service at http://proteinmodel.org/as ts/stralsv/. results: here we present stralsv (structure-alignment sequence variability), a new algorithm for detecting closely related structure fragments and quantifying residue frequency from tight local structure alignments. we apply stralsv in a study of the rna-dependent rna polymerase of poliovirus, and we demonstrate that the algorithm can be used to determine regions of the protein that are relatively unique, or that share structural similarity with proteins that would be considered distantly related. by quantifying residue frequencies among many residueresidue pairs extracted from local structural alignments, one can infer potential structural or functional importance of specific residues that are determined to be highly conserved or that deviate from a consensus. we further demonstrate that considerable detailed structural and phylogenetic information can be derived from stralsv analyses. conclusions: stralsv is a new structure-based algorithm for identifying and aligning structure fragments that have similarity to a reference protein. stralsv analysis can be used to quantify residue-residue correspondences and identify residues that may be of particular structural or functional importance, as well as unusual or unexpected residues at a given sequence position. stralsv is provided as a web service at http://proteinmodel.org/as ts/ stralsv/. accurate sequence alignments between related proteins are important for many bioinformatics applications that involve comparative analysis. derived from calculated alignments, residue-residue correspondences allow construction of sequence motifs and profiles important in building homology models or in predicting protein functions. most of the currently used methods for protein function prediction rely on sequence-based comparisons between a query protein and those for which a functional annotation is provided. a serious limitation of sequence similarity-based approaches for identifying residue conservation among proteins is the lack of, or very low, confidence in assigning residue-residue correspondences among proteins when the level of sequence identity between the compared proteins is poor. indeed, it was shown by rost [ ] that more than % of all pair-wise alignments occurring in the so-called twilight zone ( - % sequence identity) may be incorrect [ ] . multiple sequence alignment methods are more satisfactory-still, they cannot provide reliable results at low levels of sequence identity, especially if the number of available closely related proteins is small (i.e., when the protein family has rather few members, or the list of related proteins that has been identified is short). having d structural information for a given protein can be especially useful in deriving functional annotation [ ] . structure comparison algorithms provide much higher confidence in assignment of residue-residue correspondences than do sequence-based algorithms. nevertheless even calculated structural alignments may be inaccurate: for some compared proteins, or regions therein, more than one possible superposition can reasonably be reported, and it may be difficult to decide which alignment is most satisfactory [ , ] . rigid body structural superpositions on the chain level have limitations when comparing multi-domain proteins with different conformations between domains. comparisons on the domain level may yield better results, but splitting of structures into domains can be problematic, and there is no reliable method that can do this automatically. even within compared structural domains, significant deviations can be observed in some loop regions, or due to large insertions or different conformations of structural motifs, all of which can significantly affect detection of structural residue-residue correspondences when rigid body approaches are used for alignment calculations. several algorithms have been proposed to facilitate flexible protein structure alignment calculations [ ] [ ] [ ] , but the complexity of such calculations remains a challenging development goal. another difficulty in identifying similar regions in compared protein structures lies in the possibility that analogous regions in structurally related proteins may display differences in sequential ordering of the motifs due to circular permutations or convergent evolution [ , ] . the majority of the existing flexible protein structure alignment algorithms report only sequential alignments, and there are very few (with varying levels of success) that can detect and align structures between which there are differences in the ordering of their structure motifs [ ] [ ] [ ] . the accuracy of calculated structural alignments can also depend on the nature of compared structural models. the atomic coordinates obtained from experimentally solved structures (x-ray crystallography or nuclear magnetic resonance spectroscopy) are always associated with some degree of uncertainty resulting from experimental errors from the intrinsic flexibility of the proteins or from atom vibrations [ ] . such structural deviations may sometimes significantly affect the calculated alignments and lead to incorrect conclusions about sequence motifs, profiles, or possible residue substitutions within analyzed functional regions in proteins. the accuracy of calculated residue-residue correspondences can be improved by refinement methods that evaluate results produced by different structure-based alignment programs or explore sequence-based alignments using, for example, the conserved domain database (cdd) as a set of reference alignments [ ] . our goal in the current work was to develop an algorithm that could address these difficulties and facilitate the identification of structurally (and possibly functionally) relevant residue-residue correspondences between compared protein structures. our approach is to first detect similar structural motifs, and consequently derive structure-based alignments from the calculated local superpositions of corresponding similar regions. our stralsv algorithm detects structurally similar regions within a given pair of protein structures, and reports residue-residue correspondences only from those local regions that are contained within a larger, similar structural context. when for a given reference structure a structure-based search is performed on a set of proteins from the protein data bank (pdb), stralsv identifies all structurally similar fragments from that set, evaluates the calculated structure-based alignments between the query (reference) motif (designated "segment" in this work) and the detected structure fragments, and quantifies the observed sequence variability at each residue position on the query structure. here we describe how the stralsv algorithm works, and we apply stralsv in a study of the rna-dependent rna polymerase of poliovirus. stralsv is an algorithm that identifies protein structural fragments having a d structure similar to that of a query structure, performs structure-based alignments between the query and the fragments, and quantifies at each position along the query structure the sequence variability represented among the selected fragments relative to the query. stralsv takes as input a query structure of interest, a database of protein structures, and various parameters (discussed below) that control the selection of fragments from the database and the sequence variability calculations. figure illustrates the steps in the algorithm. the algorithm uses a sliding-window approach for breaking the query structure into overlapping segments, each of which is independently used to identify from the input database protein structure fragments with d similarity. a recommended (default) window_size parameter is set to amino acids in length, although an arbitrary length can be chosen. the query structure is thus split into overlapping segments of length window_size; overlaps are by default / the length of the window_size. a final segment is taken one window_size in length extending from the c-terminus to ensure that all portions of the query structure are represented within a segment of exactly the window_size. each so-calculated query segment is then compared to all protein structures in the database using the lga (local-global alignment) code [ ] to identify structure fragments with sufficient structure similarity to the query segment. the lga_s score is used to evaluate structure similarity between a query segment and detected similar fragments. calculated lga_s scores range from % to % and reflect a percentage of residues from the query segment that are identified as structurally aligned with a given similar fragment. in the stralsv algorithm, a value of lga_s of at least % is used as a cutoff to ensure that there is sufficient structural similarity between the segment and the fragment over at least half the length of the segment. lga's distance cutoff parameter determines the maximum allowable distance between alpha carbons (cα) of superimposed amino acids within a calculated alignment; typically this parameter is set to . Å, and this default is used for stralsv calculations with win-dow_size values of residues. thus, fragments with sufficient structure similarity to the query segment are identified. each fragment is then evaluated to determine the tightness of its alignment to the query segment. the criteria for tight structure similarities in local regions (spans), described by zemla et al. [ ] , are used to identify ranges within the alignment that have tight local superpositions. each residue-residue pair from the alignment (closest superimposed residues from the query segment and database fragment) is assigned a score by calculating the local rmsd (root mean square deviation) among the surrounding residue-residue pairs. a continuous set of at least three residue-residue pairs that fulfil the rmsd cutoff of . Å comprises a span. a desired size of a calculated span (span_size; shortest acceptable tight local alignment without gaps) is used as an input parameter to stralsv, and is typically specified as , , or , but can be of arbitrary length. our previous experience [ ] , suggests that is a reasonable minimum length over which to impose a tight alignment; is the minimum value for span_size that is meaningful (since any ca atoms can be perfectly aligned), and imposes stringency that tends to eliminate capture of some related (desired) structure fragments. for the work reported here we selected a minimum span length of (span_size = ). from each alignment is extracted a set of spans. all alignments that contain at least one span of length no less than the specified minimum span length are deemed "qualified hits". (for an illustration of a "span", see additional file : stralsv-rdrp_suppl_figure .docx.) all residue-residue pairs that are contained within a span's alignment are used to calculate the sequence variability data at the corresponding position in the query structure. note that not all residues from calculated structure alignments contribute to the variability statistics at a given position in the query segment; regions in which the local rmsd distances between corresponding residues exceed . Å induce breakpoints between spans. also, because the algorithm uses overlapping segments, duplications are appropriately factored out in calculating the sequence variability. a frequency matrix (table ) is constructed for each position in the query structure by tallying the frequency at which each amino-acid (the standard amino acids plus 'x', corresponding to unusual or modified residues) is observed within the spans. from this matrix are extracted statistics describing the number of positional hits (residue-residue correspondences contributing sequence variability data) per position and a list of residues observed at each position, ordered by frequency of occurrence. these statistics are used to construct a variability profile (table ) , which can be used to identify positions at which there is relatively high or low sequence variability in structure context. the sample profile given in table shows the observed residues for positions through for polio rna-dependent rna polymerase (rdrp) run as the query protein against the complete pdb database (released on november , ). to illustrate the stralsv algorithm, we selected a minimum span length of and conducted analyses on poliovirus rdrp because poliovirus has been extensively studied and its polymerase is a member of a widely distributed protein family. in analyzing poliovirus rdrp, part of our effort involved determining suitable parameters for window_size and distance_cutoff. as win-dow_size is increased, the stringency with which similar structure fragments are selected is increased, due to the greater structural context that is provided by the query segment. as distance cutoff is increased, that stringency is reduced, because more laxity in the alignment is allowed. thus, for larger window_size values, a larger distance cutoff should be applied in order for the algorithm to not eliminate related structure fragments that have local structural deviations with respect to the query segment. likewise, as the window size is decreased, the distance cutoff should be reduced as well, to prevent capture of many small, less related structure fragments. to determine what combinations of win-dow_size and distance_cutoff values would provide com-parable results, we performed several benchmark tests involving various structure libraries (e.g., complete pdb, pdb-select , astral ) and input parameters (data not shown). the results from one such test involving the capture of structure fragments from a benchmark data set containing polymerase structures plus more than other structures randomly selected from the pdb is presented in figure . suitable parameters input to stralsv were expected to capture only structurally related fragments (i.e., the polymerases), whereas overly stringent parameters were expected to yield result sets lacking at least some of the polymerase fragments, and low stringency parameters were expected to capture less related fragments. in this way, we examined the dependency between window_size and distance_cutoff values to determine optimal (for the current study) parameter settings, and help define default parameter settings for stralsv. we conducted a test whereby window_size and distan-ce_cutoff parameters were varied from to and . Å to . Å, respectively ( figure ). we ran stralsv using each window_size/distance_cutoff combination for all query (poliovirus rdrp) segments enclosing residue g . we selected a region in the n-terminal portion of the protein upon which to focus this exercise. the region defined by residues - tended to be structurally unique, but at the same time contained a variety of well defined secondary structure elements of different sizes ( figure c ). furthermore, the selection of fragments to inspect from within this region was somewhat arbitrary; we selected those segments containing residue g due to its significance as a functional residue [ ] . we determined how many qualified hits were obtained for each window_size/distance_cutoff combination ( figure b ). parameter settings comprising window_size values ranging from to at distance cutoffs . Å to . Å gave satisfactory results; fragments from the seeded polymerase structures were reliably captured within a set of window_size and distance_cutoff parameter value combinations within these ranges (dotted oval in figure b ). window_size values smaller than tended to yield qualified hit sets missing some of the polymerases (i.e., true positives) for lesser (more stringent) values of dis-tance_cutoff and tended to capture increasing numbers of unrelated (false positive) structure fragments as the distance cutoff was increased. also, window_size values smaller than were sensitive to the distance cutoff value, yielding acceptable result sets only for narrow ranges of distance cutoff. very large window_size values (e.g., ) resulted in selection of all true positives for all but the very tightest (distance_cutoff < . Å) alignments. the parameter settings highlighted in red type in figure b were selected as default values for stralsv. based on parameters applied in zemla et al. [ ] and the analysis described above, we used stralsv to analyze sequence variability in structure context for poliovirus rdrp (pdb: ra ; [ ] ) using minimum span length of , lga_s cutoff %, and window_size/distance_cutoff combinations / . Å, / . Å, / . Å, and / . Å. stralsv produced variability matrices and sequence profiles (not shown; for excerpts see tables and ) from which were extracted data for analyzing related structure table excerpt of sample stralsv output "matrix" file for positions - from poliovirus rdrp analysis fragments and quantifying positional variability. plots containing data extracted directly from the matrix and profiles files are shown in figures , , and . we performed additional analyses in order to annotate the primary stralsv results: ) secondary structure assignments were calculated for poliovirus rdrp using dssp [ ] and were plotted along the x-axis of figure . dssp output was simplified as follows: helix, comprising alpha helix (h), pi helix (i), and / helix (g); strand, comprising extended strand (e) or residue in isolated beta bridge (b). ) to determine whether we could discern patterns with respect to positional hit frequency and previously table identified polymerase sequence motifs, we overlaid a plot comprising qualified hits versus sequence position with the positions of the well known motifs, extracted from the literature ( figure , gray boxes with labels a-g). this was accomplished by examining sequence alignments and extracting coordinates defining the known sequence motifs a through g from papers that included poliovirus rdrp among the aligned sequences [ ] [ ] [ ] [ ] . because there was considerable inconsistency regarding the boundaries of the sequence motifs, we defined inclusive boundaries for each motif whereby residues were included if they were identified within a motif in any of the sequence alignments reported in the literature. ) in order to inspect the most abundant sequence variants (i. e., the most conserved positions) in the context of the positional hit frequency along the reference protein, we extracted from the matrix files the frequencies of the most frequent residues and plotted them versus sequence position along with the positional hits for window size ( figure ). ) the literature was searched for evidence of functional annotation [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] for the most frequently observed residue positions (see additional file : stralsv-rdrp_suppl_table ) and positions for which functional annotation was identified were marked in figure . the qualified hits (structure fragments from pdb with detected local similarities to the query structure) for six selected sequence positions (positional hits) of polio rdrp (positions identified in figure ) were categorized and quantified based on scop (structure classification of proteins database; version . , june release) identifiers [ ] . note that because scop is a manually curated database of structure domains from the pdb, there is some delay (currently more than one year) before a new pdb entry is classified in scop. therefore, we have included in table and additional files , , , , , and (stralsv-rdrp_suppl_table , stralsv-rdrp_suppl_table , stralsv-rdrp_suppl_table , stralsv-rdrp_suppl_table , stralsv-rdrp_suppl_table , stralsv-rdrp_suppl_table ) data pertaining to qualified hits and sequence variability based on stralsv analysis using a complete tally of pdb identifiers in addition to those hits and variabilities that could be categorized by scop classification. the hits were evaluated separately for each of windows , , and , at six sequence positions selected to be representative of the types of frequency variation (of qualified hits) over the length of the polymerase (see specified positions in figure ). for each of the six selected positions, the following information was extracted from the stralsv matrix output file: all pdb templates (including chain information) that contributed to the profile at that position, the corresponding lga_s score, the sequence identity (seq id), the number of (template) amino acids that matched the query, the name of the corresponding amino acid of the template, and, when available, the scop identifier for each classified pdb template. effects of parameter settings on capture of database structure fragments numbers of positional hits (residues within spans) corresponding to each position along the poliovirus query . the regions of high variability were "locked" between regions of high specificity, which exhibited little or no change with the variation of win-dow_size. as window_size was increased, the numbers of positional hits tended to decrease in those regions for which window_size-dependent variability was observed. comparison of positional hit variabilities to secondary structure ( figure , along x-axis) revealed that the window-related variability tended to occur in regions of helical secondary structure. a polymerase "baseline", amounting to fewer than positional hits (corresponding primarily to polymerase structures) was observed along the entirety of the poliovirus chain ( figure xaxis). all polymerase baseline regions, with the exception of region - , were identifiable independently of window_size. the latter c-terminal region showed high specificity at window size . in this region, lowering the window_size value to just resulted in the capture of many (> ) structure fragments that have apparent structural similarity to poliovirus rdrp ( figure , tall red peaks at right side of plot). the n-terminal region of the poliovirus protein yielded the least positional hits, indicating relative structural uniqueness in this region. we selected six positions (figure , residues numbered at top of plot) at which to examine the diversity of structure fragments captured at each of four window_size values (table , at positions d , d , and l , qualification of structure fragments (and resulting positional hits) was largely independent of window_size, and few unrelated (non-e. ) fragments were captured, indicating that the structure motifs in the surrounding regions were largely limited to the families detected (e.g., e. . . and e. . . ) ( table , positions d and d ). for positions n , h , and h , at which there was considerable diversity in the scop families detected at smaller win-dow_size values (< ), omission of these more distantly related positional hits was observed as window_size increased. overall it was observed that window_size resulted predominantly in capture of structure fragments from the structure family to which poliovirus rdrp belongs (e. . . ) . therefore, at all six positions, window_size effectively filtered from among the nearly , pdb chains only those members of the polymerase/transcriptase families. to determine how sequence variability was distributed among the e. . families, we further categorized the amino-acid variabilities by scop family for position n at window_size (table ). in general approximately / of the positional hits could be categorized using scop; as an example, of the total positional hits were derived from structures that had been classified in scop. for this position (at which the greatest sequence variability was observed among the selected), the distribution of amino-acid variability contributed by e. superfamily members (e. . . , , , and ) was considerably more narrow ( amino acids (a, f, g, h, n) , of which half ( ) were n) than that observed overall ( amino acids). all of the positional hits that coincided in sequence with poliovirus rdrp at n were members of the e. . . (rna-dependent rnapolymerase) family, although a minority ( ; all of the remaining) of the hits (all rdrp of lambda ) from this family had h at position . thus, sequence variability was limited to n ( %) and h ( %) within family e. . . , in which poliovirus rdrp is categorized. (this family also includes hcv, fm, lambda , bvdv, norwalk virus, rhinovirus, rabbit hemorrhagic fever virus, and ibvd.) summarizing the amino-acid occurrences among qualified hits within fold e. (comprising superfamily and families), we observed the following amino-acid distributions at position n : n = %, f = %, g = %, h = %, a = . %, compared to a much broader distribution of amino-acid variabilities observed within templates outside of fold e. : v = %, r = %, q = %, i = %, h = %, y = %, and c, e, f, k, l, m, and t each < = % (comprising the "tail" in a distribution of sequence variability). for completeness, amino-acid variabilities derived from positional hits at all six positions examined (see figure ) are summarized in additional files , , , , , and : stralsv-rdrp_suppl_table , stralsv-rdrp_suppl_table , stralsv-rdrp_suppl_table , stralsv-rdrp_suppl_table , stralsv-rdrp_suppl_table , stralsv-rdrp_suppl_table . distributions of observed amino-acids are shown for all templates and grouped as within or outside of fold e. . from this detailed analysis it was possible to categorize the specific amino-acid variabilities per position-thus, stralsv can be used to detect positional trends and anomalies among structures related to the protein of interest. to determine how the window size parameter might affect the detection of amino-acid variability "globally" along the poliovirus rdrp chain, we plotted for each position along the reference structure the detected absolute amino-acid variability versus the number of qualified hits for window_size values , , and ( figure ) . as window_size decreased from to , there was observed an increase in the number of database structure fragments that contributed positional hits, and a corresponding increase in the amino-acid variabilities: at win-dow_size there were far more ( ) positions at which all amino-acids were observed among the positional hits compared to window_size ( position). at window_size there was considerably less sequence variability detected among the positional hits, with the most variable positions accounting for no more than distinct amino acids observed. the inset in figure displays the data points for those positions at which the dominant (most frequently observed) amino-acid occurred at a frequency of at least %. circled data points are those corresponding to window_size . there was considerable overlap among window_size values , , and with respect to positions at which the dominant residue occurred at frequency > = %. window_size produced positions, produced , and produced ; positions occurred in all three of these data sets. this plot demonstrates that the most dominant amino-acid residues occurred within the same (narrow) positional hit frequency range ( to < ) regardless of window_size, suggesting that highly conserved positions display relatively little sequence variability regardless of the selected window_size. regions corresponding to the well-known sequence motifs characteristic of polymerases were mapped to the positional hit frequency plot for window size ( figure ). as mentioned above, regions of high positional hit frequency tended to correspond with helix secondary structure ( figure ), but also with defined palm-domain motifs ( figure ). also plotted in figure are the frequencies of the dominant residues per position along poliovirus rdrp (lavender plot in figure) . included are residue-position labels for those positions at which the dominant residue frequency exceeded % and for which we were able to identify a functional annotation in the literature (see additional file : stralsv-rdrp_suppl_table ). although there is no clear criterion for identifying functional residues and short sequence or structure motifs based on stralsv profiles, it was evident that functionally relevant residues tended to emerge when selecting those positions displaying high degrees of conservation in structure context. furthermore, lowering the window size to just resulted in the capture of many structure fragments with common structure motifs (see last maxima in figure graph and table "other" column for positions h and h ), implying that stralsv may enable identification of common structure motifs shared among distantly related proteins. stralsv differs in several respects from other sequenceand structure-based algorithms for comparing proteins. first, by applying an overlapping sliding window to define segments of a structure of interest, the algorithm avoids the pitfalls of algorithms that compare proteins at a global level: by dividing a protein into segments, stralsv enables comparison of structures by using fragments corresponding approximately in size to super-secondary elements or structure motifs. in this way, portions of a structure can be compared at a local level to like fragments in the pdb without losing the greater structural context. stralsv applies a two-step approach to filtering pdb fragments in order to select those which are most likely to be relevant for a meaningful comparison. for example, the results presented in tables and and additional files , , , , , and : stralsv-rdrp_suppl_- table , stralsv-rdrp_suppl_table , stralsv-rdrp_suppl_table , stralsv-rdrp_suppl_table , stralsv-rdrp_suppl_table , stralsv-rdrp_suppl_table , illustrate how stralsv can be used to quantify and annotate sequence variability among structure fragments that form tight local alignments (determined by span and distance_cutoff parameters), yet have sufficient structure context (determined by window_size) to filter out unrelated fragments. larger window_size values effectively increase the stringency with which structure fragments are selected, whereas smaller distance_cutoff values also increase this stringency, but more locally, by enforcing tighter local alignments. as seen in table , considerably fewer fragments are selected at window_size than with smaller window_size values for those positions that are sensitive to window_size, but the result set is greatly enriched for fragments that are closely related to the reference structure (i.e., polio rdrp). how much stringency the user wishes to apply in running stralsv may depend on one's research interest and the protein being studied. smaller window_size (as well as greater distan-ce_cutoff) parameter values will result in capture of more structure fragments, many of which will be from structures that are more distantly related in terms of their scop classification and the taxonomy of the organisms that they are from (table ). it must be noted that as the window_size (or distance_cutoff) parameter is relaxed, more "noise" arises in the result set; however, at the same time there is potential for discovering structural relationships and sequence laxity that may not otherwise be discernable when comparing only closely related structures. effects of parameter setting on capture of database structure fragments not unexpectedly, the frequencies of positional hits tend toward maxima in regions of helical secondary structure, and as window_size is decreased the numbers of positional hits tend to increase (figures , ) . reduction in window_size relaxes constraints on the alignment, and therefore smaller fragments may align more tightly, thereby meeting the distance cutoff. in some regions, the numbers of positional hits do not change significantly with changes in window_size (e.g., peaks up to position and - ), indicating that in certain regions there exist conserved structure motifs that are shared only among a specific set of structures. for example, there is observed in the n-terminal regions up to approximately position (figures , ) a "polymerase baseline", which appears to be structurally unique. this region is external to the catalytic tunnel of the polymerase, and may define structural and functional specificity for polio and related viruses. window_size produced a polymerase baseline in the c-terminal region as well, although here the positional hit frequency was highly sensitive to window_size. it may be that only window_size (or greater) effectively filtered out non-polymerase structure fragments due to the stringency enforced by context, as it appeared that smaller segments of polio rdrp in this region resembled short segments in distantly related proteins. we observed considerable diversity among the scop families represented by fragments detected at different positions at window_size values less than (n , h , h ). this demonstrated a clear filtering of these positional hits as the window_size increased. one must be aware, however, that the two parameters, window_size and distance_cutoff, have an opposing relationship with respect to qualifying a hit: increase in alignment stringency is achieved by increasing window_size, but also by decreasing distance_cutoff. as window_size is decreased, it is necessary to also reduce the distance_cutoff in order to avoid an unacceptable increase in the number of "false positive" (considerably less structurally relevant) fragments captured. we achieved a reasonable selection of window-size/distance-cutoff parameter pairings by examining the relationship between these parameters ( figure ). the apparent (perhaps unexpected) decrease in the numbers of non-e. structure fragments captured by window compared to window at positions n and h (table "other" column) are explained by the high-stringency filtering achieved by the distance cutoff . Å applied at window . thus, it is desirable to strike a balance between window size and distance cutoff. small window_size values are useful for capturing shorter fragments, but to ensure that the result set is not populated by spurious hits corresponding to ubiquitous secondary structure elements (e.g., alpha helix), one must apply added stringency at the level of distance_cutoff. smaller window_size values are appropriate when the user is interested in focusing the analysis on a relatively small structure motif, which may occur with or without the surrounding structural context in the reference structure. in capturing these smaller structure fragments, one is cautioned to enforce tighter alignments in order to assure that the resulting qualified hits are relevant to the study. regions corresponding to the well-known sequence motifs a-g, characteristic of polymerases, were mapped to the positional hit frequency plot for window size ( figure ). all categories of polymerase (rdrp, rddp/rt, ddrp, dddp) are recognized to contain sequence motifs a-d. identification of motifs e, f, and g, however, has been somewhat obscured by the greater diversity among sequences assigned to these structure motifs. o'reilley and kao [ ] reported motif e as being exclusive to rddps and rdrps, and motifs f [ , ] and g [ ] were identified in poliovirus rdrp. motif f was identified in phi [ ] , bvdv and hcv [ ] , reovirus, phi , bvdv, hcv, rhinovirus, norwalk virus, and hiv [ ] , and fmdv, rhdv, and hiv [ ] . a detailed structurebased comparison of these polymerases using stralsv may clarify the assignment of motifs a-g among the polymerase classes. in examining the amino-acid variability versus qualified hit frequency (figure inset, circled data points) for highly conserved positions ( figure dots) that had been functionally annotated (see additional file : stralsv-rdrp_suppl_table ), it appeared that the numbers of positional hits and the degree of amino-acid variability observed for high-frequency positions was largely independent of window_size. this implies (at least for the positions that were examined in this study) that the functionally relevant positions are consistently detected as high-frequency-residue positions regardless of win-dow_size. therefore, the stralsv algorithm is sufficiently sensitive to detect structurally or functionally conserved residues even when the parameters may not be perfectly tuned. stralsv is especially useful for identifying highly conserved residues at positions that occur in regions in which there are large numbers of positional hits; dominant residue frequency cannot be considered significant in regions of structural conservation (e.g., the n-terminal region up to about position in poliovirus rdrp), whereas identification of dominant residues occurring with high frequency at positions with large numbers of positional hits may help identify residues at positions that are structurally and/or functionally significant. for example, more than positional hits contributed to residue counts at positions d and d (window_size , figure ), known to be critical for rdrp function. aspartic acid occurred at close to % frequency at these positions. the structure fragments contributing to these data points comprised rdrps and dsrna phage polymerases (scop family e. . . ). the combination of many positional hits and low amino acid variability may provide a means of identifying key functional residues. because the ability to detect possibly functional residues is of particular interest in protein functional annotation, we compared the results obtained using stralsv to those of another bioinformatics tool [ ] . firestar uses alignments to identify functional residues based on close atomic contacts in pdb structures and annotated residues in the catalytic site atlas (csa). we ran poliovirus rdrp (pdb: ra ) through the online firestar server (data not shown), and found considerable overlap between the functional residue list generated by firestar and the list of % dominance residues generated by stralsv (see additional file : stralsv-rdrp_suppl_table ): k , k , r , d , d , g , and k . stralsv identified the more broadly conserved residues within the functional set identified by firestar. a main difference between the two approaches is that firestar identifies functional residues involved in ligand bindingsome being highly conserved and others displaying considerable sequence variability. in particular we note n , which is determined by firestar to be associated with binding of cytidine-s-triphosphate and uridine-smonophosphate (sites and ). stralsv identified n as a residue that displays some degree of conservation, but also quantified the degree of conservation at this position across a wide range of structures (see additional file : stralsv-rdrp_suppl_table ). furthermore, stralsv is not limited to identification of residues that have been associated with a binding site, but can be used to infer structure and/or functional significance based on sequence conservation in structure context regardless of pre-existing annotation information. results from stralsv analysis can be used to characterize residue positions in a reference protein by detection of similar locations in other proteins (sometimes from quite distant organisms and different assigned structure classifications), in which corresponding residue positions within similar structural motifs are observed. such analyses may potentiate rapid identification of invariant (as well as unusual or unexpected) residues, which in many cases are essential to a protein's function. it also may enlighten studies of newly discovered natural or engineered mutations that have not yet been observed in the sequence databases. results from stralsv structure similarity searches performed against large sets of structurally related proteins can facilitate refinement of constructed homology models by suggesting corrections to the query-template alignments (using an approach similar to that of [ ] ) or by providing a list of possible conformational variants of corresponding structural fragments for loop-building procedures. calculated residue-residue correspondences can be used to evaluate pure sequence alignment methods and also to derive structural environment-specific substitution matrices, which have been shown to be useful for detection of remote homologs [ ] . when applied to experimentally solved structures, stralsv also could facilitate identification of structural motifs (local conformations) that have not yet been observed in pdb. such findings could aid in the discovery of previously unidentified structural motifs or suggest refinement of constructed structural models in particular regions. twilight zone of protein sequence alignments local alignment refinement using structural assessment structural proteomics: a tool for genome annotation the structural alignment between two proteins: is there a unique answer comparative analysis of protein structure alignments flexible protein alignment and hinge detection flexible structure alignment by chaining aligned fragment pairs allowing twists matt: local flexibility aids protein multiple structure alignment circular permutations of natural protein sequences: structural evidence common structural cliques: a tool for protein structure and function analysis connectivity independent protein-structure alignment: a hierarchical approach topology independent protein structural alignment flexsnap: flexible non-sequential protein structure alignment automated clustering of ensembles of alternative models in protein structure databases iterative refinement of structure-based sequence alignments by seed extension lga: a method for finding d similarities in protein structures stralcp-structure alignment-based clustering of proteins a single mutation in poliovirus rna-dependent rna polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity structural basis for proteolysis-dependent activation of the poliovirus rna-dependent rna polymerase dictionary of protein secondary structure: pattern recognition of hydrogen bonded and geometrical features structure of the rna-dependent rna polymerase of poliovirus a structural and primary sequence comparison of the viral rna-dependent rna polymerases analysis of rna-dependent rna polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure molecular model of sars coronavirus polymerase: implications for biochemical functions and drug design oligomeric structures of poliovirus polymerase are important for function clustered charged-to-alanine mutagenesis of poliovirus rna-dependent rna polymerase yields multiple temperature sensitive mutants defective in rna synthesis crystal structure of poliovirus cd protein: virally encoded protease and precursor to the rna-dependent rna polymerase mutation of lysine residues in the nucleotide binding segments of the poliovirus rna-dependent rna polymerase poliovirus rna-dependent rna polymerase ( dpol): structural, biochemical, and biological analysis of conserved structural motifs a and b crystal structure of the rna-dependent rna polymerase form hepatitis c virus reveals a fully encircled active site effects of mutations in poliovirus dpol on rna polymerase activity and on polyprotein cleavage scop: a structural classification of proteins database for the investigation of sequences and structures a mechanism for initiating rna-dependent rna polymerization the structure of the rna-dependent rna polymerase from bovine viral diarrhea virus establishes the role of gtp in de novo initiation the structure of a birnavirus polymerase reveals a distinct active site topology structure of foot-and-mouth disease virus rna-dependent rna polymerase and its complex with a template-primer rna firestar-prediction of functionally important residues using structural templates and alignment reliability evdtree: structure-dependent substitution profiles based on decision tree classification of d environments submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this work was conducted at lawrence livermore national laboratory under doe contract de-ac - na . the work was supported by an llnl internally funded grant to tv and cz through the laboratory directed research and development program, and by a uc-llns fees grant awarded to tv and cz. tv was also partially supported by the national science foundation, through the independent research and development program. any opinion, finding, and conclusions or recommendations expressed in this material are those of the author (tv) and do not necessarily reflect the views of the national science foundation. stralsv is a new algorithm for detecting closely related structure fragments from a structure database (pdb or user-defined) and quantifying residue frequency from tight local structure alignments. input parameters to stralsv (window_size, distance_cutoff, or span_size) can be varied in order to adjust the stringency with which structure fragments are selected or with which local alignments are made, thereby providing the user with flexibility in detecting similar structure fragments. highstringency parameter settings will effectively filter out all but highly structurally similar fragments and will impose very tight local alignments, whereas low-stringency parameters will enable detection of more distantly related structures, which may be of interest, for example, when the user wishes to detect distant evolutionary relationships among proteins or to test the range of possible sequence variability that might be expected to be tolerated within a given structure motif. it should be emphasized, however, that stralsv safeguards against degradation of sequence variability data quality by enforcing structure context upon local alignments in a twostep process of identifying "qualified" hits.it has long been recognized that peptides with very different sequences may have similar tertiary structures. in this work we applied stralsv in a study of the rnadependent rna polymerase of poliovirus and demonstrated that the algorithm could be used to determine regions of the protein that were relatively unique (e.g., the n-terminal region) or that shared structural similarity (e.g., c-terminal motifs) with structures that were distantly related (non-e. scop classifications), and that by quantifying residue frequencies among many (hundreds or even thousands) of residue-residue pairs extracted from local alignments, one can infer potential structural or functional importance of specific residues that are determined to be highly conserved or that deviate from a consensus. we further demonstrated that considerable detailed structural and phylogenetic information can be derived from stralsv profiles.stralsv is available as a web service at http://proteinmodel.org/as ts/stralsv/. authors' contributions az designed and developed the stralsv algorithm, performed calculations for poliovirus rdrp, and contributed the background material on structurebased methods. cz and dl wrote codes and developed methods for postprocessing of stralsv results, and performed literature searches for interpretation of biological significance of various residue positions of poliovirus rdrp. cz and az wrote the manuscript, with contributions from dl and tk. all authors participated in the discussions and shaped the ideas that led to the experimental design and results of this work. all authors read and approved the manuscript. key: cord- -i ecxgus authors: nan title: abstracts of publications related to qasr date: - - journal: nan doi: . /qsar. sha: doc_id: cord_uid: i ecxgus nan tive mechanisms p. - . edited by magee, p.s., henry, d.r., block, j.h., american chemical society, washington, . results: an overview is given on the approaches for the discovery and design concepts of bioactive molecules: a) natural products derived from plant extracts and their chemically modified derivatives (cardiac glycosides, atropine, cocaine, penicillins, cephalosporins, tetracyclines and actinomycins, pyrethrins and cyclosporin; b) biochemically active molecules and their synthetic derivatives: acetylcholine, histamine, cortisonelhydrocortisone, indole- -acetic acid (phenoxyacetic acid herbicides); c) principles of selective toxicity is discussed exemplified by trimethoprimlmethotrexate, tetracyclines, acylovir, azidothymidine, antifungal agents; d) metabolism of xenobiotics; e) exploitation of secondary effects (serendipity); f) receptor mapping; g) quantitative structure-activity relationship studies; h) empirical screening (shotgun approach). results: past and present of qsar is overviewed: a) historical roots; b) the role of qsar models in rational drug design, together with a simplified diagram of the steps involved in drug development, including the place of qsar investigations; c) classification of qsar models: structure-cryptic (property-activity) models, structure-implicit (quantum chemical) models, structure-explicit (structure-activity) and structure-graphics (computer graphics) models; d) a non-empirical qsar model based on quantities introduced for identification of chemical structures, using szymansk's and randic's identification (id) numbers, including applications for alkyltriazines. bioessays, , ( ) , - . results: a review is given on recent observations about receptor structure and the dynamic nature of drug receptors and the significance of receptor dynamics for drug design: a) receptors are classified according to structure and function (i) ion channels (nicotinic acetylcholine, gaba, glycine); (ii) g protein linked [adrenergic ( c x ,~) , muscarinic acetylcholine, angiotensin, substance k, rhodopsin]; (iii) tyrosine kinase (insulin, igf, egf, pdgf); (iv) guanylate cyclase (atrial natriuretic peptide, speractin); b) protein conformational changes can be best studied on allosteric proteins whose crystal structure is available (e.g. hemoglobin, aspartate transcarbamylase, tryptophan repressor) (no high resolution of a receptor structure is known); c) receptor conformational changes can be studied by several indirect approaches (i) spectral properties of covalent or reversibly bound fluorescent reporter groups; (ii) the sensitivity of the receptor to various enzymes; (iii) the sedimentation of chromatographic properties of the receptor; the affinity of binding of radioligands; (iv) the functional state of the receptor; d) there are many unanswered questions: e.g. (i) are there relatively few conformational states for receptors with fluctuations around them or many stable conformational states; (ii) how can static structural information be used in drug design when multiple receptor conformations exist. title: designing molecules and crystals by computer. (review) author: koide, a. ibm japan limited, tokyo scientific center, tokyo research laboratory - sanban-cho, chiyoda-ku, tokyo , japan. source: ibm systems journal , ( ), - . results: an overview is given on three computer aided design (cad) systems developed by ibm tokyo scientific center: a) molecular design support system providing a strategic combination of simulation programs for industrial research and development optimizing computational time involved and the depth of the resulting information; b) molworld on ibm personal systems intended to create an intelligent visual environment for rapidly building energetically stable d molecular geometries for further simulation study; c) molecular orbital graphics system designed to run on ibm mainframe computers offering highly interactive visualization environment for molecular electronic structures; d) the systems allow interactive data communication among the simulation programs for their strategically combined use; e) the structure and functions of molworld is illustrated on modeling the alanine molecule: (i) data model of molecular structures; (ii) chemical formula input; (iii) generation of d molecular structure; (iv) formulation of bonding model; (v) interactive molecular orbital graphics; (vi) methods of visualizing electronic structures; (vii) use of molecular orbital graphics for chemical reactions. title: interfacing statistics, quantum chemistry, and molecular modeling. (review) author: magee, p.s. biosar research project vallejo ca , usa. source: acs symposium series , no. . in: probing bioactive mechanisms p. - . edited by magee, p.s., henry, d.r., block, j.h., american chemical society, washington, . results: a review is given on the application and overlap of quantum chemical, classical modeling and statistical approaches for the quant. struct.-act. relat. , - ( ) abstr. - understanding of binding events at the molecular level. a new com-a) qsar of cns drugs has been systematically discussed according plementary method called statistical docking experiment is also to the following classes: (i) general (nonspecific) cns depressants: presented: general anesthetics; hypnotics and sedatives; (ii) general insights obtained using energy-minimized structures; activation in the bound state, types and energies of interactions at the receptor site and in crystal; four successful examples (significant regression equations) are given for the modeling of binding events using physico-chemical descriptors and correlation analysis: (i) binding of a diverse set of pyridines to silica gel during thin-layer chromatography; (ii) binding of meta-substituted n-methyl-arylcarbamates to bovine erythrocyte ache; (iii) binding of meta-substituted n-methyl-arylcarbamates to ache obtained from susceptible and resistant green rice leafhoppers; (iv) activity of phenols inhibiting oxidative phosphorylation of adp to atp in yeast; a new statistical method for mapping of binding sites has been developed based on the hypermolecule approach, identifying key positions of binding and nature of the energy exchange between the hypermolecule atoms and the receptor site; two examples are given on the successful application of statistical modeling (statistical docking experiment) based on the hypermolecule approach: (i) inhibition of housefly head ache by metasubstituted n-methyl-arylcarbamates (n = , r = . , s = . , f = . ); (ii) inhibition of housefly head ache by orthosubstituted n-methyl-arylcarbamates (n = , r = . , s = . , f = . ). (nonspecific) cns stimulants; (iii) selective modifiers of cns functions: anticonvulsants, antiparkinsonism drugs, analgetics and psychopharmacological agents; (iv) miscellaneous: drugs interacting with central a-adrenoreceptors, drugs interacting with histamine receptors, cholinergic and anticholinergic drugs; b) the review indicates that the fundamental property of the molecules which mostly influence the activity of cns drugs is hydrophobicity (they have to pass the cell membrane and the bloodbrain barrier); c) electronic parameters, indicative of dipole-dipole or charge-dipole interactions, charge-transfer phenomena, hydrogen-bond formation, are another important factor governing the activity of most cns agents; d) topographical, lipophylic and electronic structures of cns pharmacophores are reviewed; e) qsar equations, tables and figures from references are shown and discussed. the relevant template for each atom in the molecule is mapped into a bit array and the appropriate atomic position is marked; volume comparisons (e.g. common volume or excluded volume) are made by bit-wise boolean operations; the algorithm for the visualization of the molecular surface comprising the calculated van der walls volume is given; comparisons of cpu times required for the calculation of the van der waals molecular volumes of various compounds using the methods of stouch and jurs, pearlman, gavazotti and the new method showed that similar or better results can be achieved using the new algorithm with vax-class computers on molecules containing up to several hundred atoms. abtstr. - quant. struct.-act. relat. , - ( ) one of the important goal of protein engineering is the design of isosteric analogues of proteins; major software packages are available for molecular modeling are among others developed by (i) biodesign, inc., pasadena, california; (ii) biosym technologies, san diego, california; (iii) tripos, st. louis, missouri; (iv) polygen, waltham, massachusetts; (v) chemical design ltd. oxford; the molecular modelling packages use three basic parameters: (i) descriptive energy field; (ii)algorithm for performing molecular mechanics calculations; (iii) algorithm for performing molecular dynamics calculations; modelling study of the binding events occurring between the envelop protein (gp ) of the aids (hiv) virus and its cellular receptor (cd ) protein supported the hypothesis that this domain was directly involved in binding the gp envelop protein leading to the design of conformationally restricted synthetic peptides binding to cd . title: finding washington, . results: a new technique called "homology graphing" has been developed for the analysis of sequence-function relationships in proteins which can be used for sequence based drug design and the search for lead structures: a) as target protein is inhibited by the ligands of other proteins having sequence similarity, computer programs have been developed for the search of the similarity of proteins; b) proteins are organized into hierarchical groups of families and superfamilies based on their global sequence similarities; c) global sequence similarities were used to find inhibitors of acetolactate synthase (als) and resulted in a quinone derivative as a lead structure of new als inhibitors; d) local sequence similarities of bacterial and mammal glutathione synthase (gsh) were used to find inhibitors of gsh; e) it was shown that the sequence segment of gsh was similar to dihydrofolate reductase (dhfr) is part of the atp-binding site; f) biological bases of local similarity between sequences of different proteins were indicated: molecular evolution of proteins and functionally important local regions; g) homology graph, as a measure of sequence similarity was defined; h) sequence-chemical structure relationship based on homology graph and the procedure to find lead structures was illustrated by an example resulting in a list of potential inhibitors selected by the procedure based on the sequence segment from residue to of the sequence of tobacco als. source: acs symposium series , no. . in: probing bioactive mechanisms p. - . edited by magee, p.s.. henry, d.r., block, j.h., american chemical society, washington. . results: a review is given on the molecular design of the following major types of antifungal compound in relation to biochemistry, molecular modeling and target site fit: a) squalene epoxidase inhibitors (allilamines and thiocarbanilates) blocking conversion of squalene , -oxidosqualene; b) inhibitors of sterol c- demethylation by cytochrome p- (piperazines pyridines, pyrimidines, imidazoles and triazoles); c) inhibitors of sterol a' -t a' isornerization andlor sterol reductase inhibitors (morpholines); d) benzimidazoles specifically interfering with the formation of microtubules and the activity phenylcarbamates on benzimidazole resistant strains; e) carboxamides specifically blocking the membrane bound succinate ubiquinone oxidoreductase activity in the mitochondria electron transport chain in basidiomycetes; f) melanin biosynthesis inhibitors selectively interfering with the polyketide pathway to melanin in pyricularia oryzae by blocking nadph dependent reductase reactions of the pathway (fthalide, pcba, chlobentiazone, tricyclazole, pyroquilon, pp ). title: quantitative modeling of soil sorption for xenobiotic chemicals. (review) author: sabljic, a. theoretical chemistry group, department of physical chemistry, institute rudjer boskovic hpob , yu- zagreb, croatia, yugoslavia. source: environ. health perspect. , ( ), - . results: the environmental fate of organic pollutants depends strongly on their distribution between different environmental compartments. a review is given on modeling the soil sorption behavior of xenobiotic chemicals: a) distribution of xenobiotic chemicals in the environment and principles of its statistical modeling; b) quantitative structure-activity relationship (qsar) models relating chemical, biological or environmental activity of the pollutants to their structural descriptors or physico-chemical properties such as logp values and water solubilities; c) analysis of the qsar existing models showed (i) low precision of water solubility and logp data; (ii) violations of some basic statistical laws; d) molecular connectivity model has proved to be the most successful structural parameter modeling soil sorption; e) highly significant linear regression equations are cited between k , values and the first order molecular connectivity index ( ' x ) of a wide range of organic pollutants such as polycyclic aromatic hydrocarbons (pahs) and pesticides (organic phosphates, triazines, acetanilides, uracils, carbamates, etc.) with r values ranging from . to . and s values ranging from . to . ; f) the molecular connectivity model was extended by the addition of a single semiempirical variable (polarity correction factor) resulting in a highly significant linear regression equations between the calculated and measured ko, values of the total set of compounds (n = , r = . , s = . , f = ); g) molecular surface areas and the polarity of the compounds were found to be responsible for the majority of the variance in the soil sorption data of a set of structurally diverse compounds. title: strategies for the use of computational sar methods in assessing genotoxicity. (review) results: a review is given on the overall strategy and computational sar methods for the evaluation of the potential health effects of chemicals. the main features of this strategy are discussed as follows: a) generalized sar model outlining the strategy of developing information for the structure-activity assessment of the potential biological effects of a chemical or a class of chemicals; b) models for predicting health effects taking into account a multitude of possible mechanisms: c) theoretical models for the mechanism of the key steps of differential activity at the molecular level; d) sar strategies using linear-free energy methods such as the hansch approach; e) correlative sar methods using multivariate techniques for descriptor generation and an empirical analysis of data sets with large number of variables (simca, adapt, topkat, case, etc.); f) data base considerations describing three major peer-reviewed genetic toxicology data bases (i) national toxicology program (ntp) containing short term in vitro and in vivo genetic tests; (ii) data base developed by the epa gene-tox program containing different short term bioassays for more than compounds, used in conjunction with adapt, case and topkat; (iii) genetic activity profile (gap) in form of bar graphs displaying information on various tests using a given chemical. title: quantitative structure-activity relationships. principles, and authors: benigni,, r.; andreoli, c.; giuliani, a. applications to mutagenicity and carcinogenicity. (review) laboratory of toxicology and ecotoxicology, istituto superiore di sanita rome, italy. source: mutat. res. , ( ), - . results: methods developed for the investigation for the relationships between structure and toxic effects of compounds are summarized: a) the extra-thermodynamic approach: the hansch paradigm, physical chemical properties that influence biological activity and their parametrization, originality of the hansch approach, receptors and pharmacophores: the natural content of the hansch approach, predictive value of qsars, a statistifa tool: multiple linear regression analysis, the problem of correlations among molecular descriptors, other mathematical utilizations of extrathermodynamic parameters; b) the substructural approach: when topological (substructural) descriptors are needed, how to use topological decriptors; c) qsar in mutagenicity and carcinogenicity: general problems, specific versions of the substructural approach used for mutagenicity and carcinogenicity, applications to mutagenicity and carcinogenicity. title: linking structure and data. (review) author: bawden, d. source: chem. britain , (nov) , i - . address not given. results: the integration of information from different sources, particularly linking structural with non-structural information is an important consideration in chemical information technology. a review is given on integrated systems: a) socrates chemicallbiological data system for chemical structure and substructure searching combined with the retrieval of biological and physicochemical data, compound availability, testing history, etc.; b) psidom suite of pc based structure handling routines combining chemical structure with the retrieval of text and data; c) cambridge crystal structure databank on x-ray data of organic compounds integrating information on chemical structure, crystal conformation, numerical information on structure determination, bibliographic reference and keywording; d) computer aided organic synthesis for structure and substructure search, reaction retrieval, synthetic analysis and planning, stereochemical analysis, product prediction and thermal hazard analysis. title: determination of three-dimensional structures of proteins and nucleic acids in solution by nuclear magnetic resonance spectroscopy. source: critical rev. biochem. mol. biol. , ( ) , - . results: a comprehensive review is given on the use of nmr spectroscopy for the determination of d structures of proteins and nucleic acids in solution discussing the following subjects: a) theoretical basis of two-dimensional ( d) nmr and the nuclear overhauser effect (noe) measurements for the determination of d structures is given; b) sequential resonance assignment for identifying spin systems of protein nmr spectra and nucleic acid spectra, selective isotope labeling for extension to larger systems and the use of site specific mutagenesis; c) measurement and calculation of structural restraints of the molecules (i) interproton distances; (ii) torsion angle restrains; (iii) backbone torsion angle restraints; (iv) side chain torsion angle restraints; (v) stereospecific assignments; (vi) dihedral angle restraints in nucleic acids; d) determination of secondary structure in proteins; e) determination of tertiary structure in proteins using (i) metric matrix distance geometry (ii) minimization in torsion angle space; (iii) restrained molecular dynamics; (iv) dynamical simulated annealing; (v) folding an extended strand by dynamical simulated annealing; (vi) hybrid metric matrix distance geometry-dynamical simulated annealing method; (vii) dynamical simulated annealing starting from a random array of atoms; f) evaluation of the quality of structures generated from nmr data illustrated by studies for the structure determination of proteins and oligonucleotides using various algorithms and computer programs; g) comparisons of solution and x-ray structures of (i) globular proteins; (ii) related proteins; (iii) nonglobular proteins and polypeptides; h) evaluation of attainable precision of the determination of solution structures of proteins for which no x-ray structures exist (i) bds-i (small -residue protein from the sea anemone sulcata; (ii) hirudin (small -residue protein from leech which is a potent natural inhibitor of coagulation); i) structure determination by nmr is the starting point for the investigation of the dynamics of conformational changes upon ligand abtstr. - quant. struct.-act. relat. , - ( ) binding, unfolding kinetics, conformational equilibria between different conformational states, fast and slow internal dynamics and other phenomena. title: aladdin. an integrated tool for computer-assisted molecular design and pharmacophore recognition from geometric, steric, and substructure searching of three-dimensional molecular structures. ( aladdin has the ability to (i) objectively describe receptor map hypothesis; (ii) scan a database to retrieve untested compounds which is predicted to be active by a receptor map hypothesis; (iii) quantitatively compare receptor map hypotheses for the same biological activity; (iv) design compounds that probe the bioactive conformation of a flexible ligand; (v) design new compounds that a receptor map hypothesis predicts to be active; (vi) design compounds based on structures from protein x-ray crystallography; a search made by aladdin in a database for molecules that should have d dopaminergic activity recognized unexpected d dopamine agonist activity of existing molecules; a comparison of two superposition rules for d agonists was performed by aladdin resulted in a clear discrimination between active and inactive compounds; a compound set was designed that match each of the three lowenergy conformations of dopamine resulting in novel active analogues of known compounds; mimics of some sidc ~ . . p.piide beta turns were designed, in order to demonstrate that aladdin can find small molecules that match a portion of a peptide chain and/or backbone; results: lately a number of chemical information systems based on three-dimensional ( -d) molecular structures have been developed and used in many laboratories: a) concord uses empirical rules and simplified energy minimization to rapidly generate approximate but usually highly accurate -d molecular structures from chemical notation or molecular connection table input; b) chemical abstracts service (cas) has added -d coordinates for some million organic substances to the cas registry file; c) cambridge structural database system contains x-ray and neutron diffraction crystal structures for tens of thousands of compounds; d) maccs d developed by molecular design ltd., contains the standard maccs-i structures to which additional -d data, such as cartesian coordinates, partial atomic charges and molecular mechanics energy are added; maccs d allows exact match, geometric, submodel and substructure searching of -d models with geometric constrains specified to certain degree of tolerance; two -d databases are also available from molecular design that can be searched using maccs d [drug data report ( , models) and fine chemicals directory ( , models)]; e) aladdin (daylight chemical information systems) is also searches databases of -d structures to find compounds that meet biological, substructural and geometric criteria such as ranges of distances, angles defined by three points (dihedral angles) and plane angles that the geometric object must match. aladdin is one of a number of menus working within the framework provided by daylight's chemical information system. title: improved access to supercomputers boosts chemical applica-author: borman, s. c&en sixteenth st., n.w., washington dc , usa. source: c&en , ( ) , - . results: supercomputers have been much more accessible by scientists and engineers in the past few years in part as a result of the establishment of national science foundation (nsf) supercomputer centers. the most powerful class of supercomputers have program execution rates of million to billion floating-point operations per second, memory storage capacities of some ten million to miltion computer words and a standard digital word size of bits, the equivalent of about decimal digits. the following examples are given for the use of supercomputer resources for chemical calculations and modeling: a) modeling of key chromophores in the photosynthetic reaction center of rhodopseudomonas viridis showing the heme group, the iron atom and the chlorophyll which absorbs light and causes rapid transfer of electron to pheophtin and then to the quinone; modeling includes a significant part of the protein having about atoms out of a total of some , ; quant. struct.-act. relat. , - ( ) abstr. - b) modeling of transition state of reaction between chloride and methyl chloride including electron clouds and water molecules surrounding the reaction site; c) analysis of nucleic acid and protein sequences to evaluate the secondary structure of these biopolymers; d) construction of a graphical image of hexafluoropropylene oxide dimer, a model for dupont krytox high performance lubricant; e) calculation of the heats of formation of diaminobenzene isomers indicated that the target para isomer was kcal/mol less stable then the meta isomer byproduct therefore the development for its large scale catalytic synthesis was not undertaken (saving was estimated to be $ to $ million). , b) comparison of the newly defined eo, parameter with the taft-kutter-hansch e, (tkh e,) parameter showed characteristic steric effects of ortho-alkoxy and n-bonded planar type substituents (e.g. no,, ph); c) in various correlation analyses using retrospective data eo, satisfactorily represented the steric effects of ortho-substituents on reactivity and biological activity of various organic compounds; d) semi-empirical am calculations using a hydrocarbon model to study the steric effects of a number of ortho-substituents resulted in the calculation of the es value (difference in the heat of formation between ortho-substituted toluene and t-butylbenzene) which linearly correlated with the eo, and the tkh e, parameters; e) effects of di-ortho substitution on lipophilicity could be mostly expressed by the summed effect of the -and -position substituents; t) highly significant regression equations were calculated for the pk, values of di-ortho-substituted benzoic acids using various substituent parameters; g) quantitative analysis of the effect of ortho-substitution is difficult because it is a result of overlapping steric and electronic effects. title: calculation of partition coefficient of n-bridgehead com- ( i i ) is more lipophilic than propanolol- -sulphate (iv)]. fig. shows the relationship between lipophilicity and ph for the compounds (circle represents (i), triangle (ii), rhomboid (m) and square gv - ( ) abstr. - f (rekker's constant, characterizing hydrophobicity). results: a good agreement was found between the observed and calculated logp values of i ( . and . , respectively) and for iii. the hydrophobicity of i was found to be significantly lower than that of i ( . and . , respectively) . the large deviation was attributed to the surface reduction as a result of condensed ring formation in i. since interesting pharmacological activities have been reported for several derivatives of this type of compounds, the hydrophobicity of the unsubstituted lh-indolo[ , -c]quinoline has been calculated to be . : ( ) [interaction energy between a molecule and the binding site model was assumed to be the sum of its atomic contributions according to the expres-e , . , , ,~~(~) was the interaction energy parameter between the site region rand the atom-type of atom a and ag(b) was the total interaction energy for the binding mode b (binding mode was regarded as feasible when the molecule was in its energetically most favorable conformation)]. sion ag(b) = erelion reatomi a in r er,typc(a). where results: for development of the binding site model, first a simple geometry was proposed and agm- agm,calc i agm+) was calculated for the whole set of compounds. if the calculated binding energy of any of the compounds was outside of the above boundary, the proposed site geometry was rejected and a more complex one was considered. this procedure had been repeated until all molecules in the set could be fitted within the experimental data range. as a result a d, five-region voronoi binding site model has been developed for the pahs containing a trigonal pyramid (rl) in the center and portions r rs having infinite volumes and indicated by boundary planes. region r, represented access to the solvent and regions r rs were blocked for binding ( fig. ) : pyrene is shown in its optimal binding mode with its atom barely touching the boundary surfaces and edges: calculations showed that benzene and other monoaromatic ring compounds should be very weak competitors for the b[a]p site. the model correctly predicted the binding energy of nine competitors outside of the training set. '% (wiener index calculated as the sum of all unique shortest distances between atoms in the hydrogen suppressed graph of the compound); (wiener index calculated as the sum of all geometric distances between atoms in the hydrogen suppressed molecule of the compound). results: the traditional d wiener number is defined as the sum of the lengths of all possible routes in the molecular graph. here the length is proposed to be calculated as the real three-dimensional length between atoms: this is the d wiener number. this number has many of the advantageous features of the related and very much studied d wiener number. additionally, it is highly discriminative and its use in quantitative structure-property relation studies (qspr) appears to be encouraging, according to the preliminary calculations. of these the most convincing is the set of statistical parameters for the linear correlation between the experimental and calculated enthalpy functions of dw the lower alkanes not shown here. three different models have been tried and in all cases the d wiener number seemed to be superior to the d one as it is reflected in (eqs. - ). a) gaba receptors in human mouse, rat and bovine brain tissues, membrane preparations and cellular uptake systems; b) gaba receptors in cat and rat spinal cord preparations; c) cultured astrocytes. as in the equations nearly all indicator variables had negative regression coefficients it was concluded that instead of searching for better analogs, the research should be directed toward degradable pro-gaba or pro-muscimol derivatives that are efficiently taken up into the central nervous system (cns). . (+ . ) irng + . ( ) title: synthesis and qsar of -aryl- -(~- -quinolyi/l-isoqui-noly ethyl)piperazines and some related compounds as hypotensive agents. authors ( ) based on eq. , an optimal logp is predicted (logpo = . ). the highest activity was produced by the -( -methylphenyl)- -(~- -qui-data determined: chemical descriptors: abtstr. - quant. struct.-act. relat. , - ( ) nolylethyl) piperazine, its logp value being near to the optimal value ( . ). l.og(bph) values calculated by eq. agree well with the observed ones. source: toxicology , ( ), - . compounds: , -dimethoxyphenol, -chlorophenol, . -dichlorophenol, -methyl- -nitropheno , , dichlorophenol, , , -trichlorophenol, , , , -tetrachlorophenol, , , -triiodophenol, pentachlorophenol. biological material: chinese hamster ovary (cho) cells. data taken from the literature: ezoc; ecsoc; eczoa; ec~oa [concentration (mmol l) of the compound leading to a or % inhibition of the cell growth or adenosine uptake, respectively]. data determined: ego; ecso [concentration (mmol l) of the compound leading to a or % inhibition of the na+/k+-atpase activity, respectively]. chemical descriptors: logp (logarithm of the partition coefficient in i-octanollwater); u (hammett's constant, characterizing the electron-withdrawing power of the substituent); e, (taft's constant, characterizing steric effects of the substituent); x (molecular connectivity index, calculated by koch's method). results: highly significant linear relationships were calculated between log (eczo) and logp (r = - . ). the relationship between log(ec,,) and u being less good (r = - . ). combining the two parameters the relationship has improved (eq. i): ( ) (logarithm of the partition coefficient in i-octanollwater); (hansch-fujita's substituent constant characterizing hydrophobicity); (hammett's constant, characterizing the electron-withdrawing power of the substituent); (sterimol steric parameter, characterizing the steric effect of the meta substituents); (rplc derived hydrophobic substituent constant, defined by chen and horv th, and extrapolated to x methanol); (indicator variable for the present for the absence of hydrogen bonding substituents). results: logk' values were determined for the benzenesulfonamides and correlated with chemical descriptors. a highly significant linear relationship between logk' and logp was calculated (eq. ): ( pk, (negative logarithm of the acidic dissociation constant); logp (logarithm of the partition coefficient in i-octanol/water). results: relationships between ki values and the chemical descriptors were investigated for cpz and its listed metabolites. relationship between log(l/ki) and logp was calculated (eq. ) no numerical intercept (c) is given: ( in spite of the complexity of the full mechanism of inhibition involving at least six transition states and five distinct intermediates, a significant linear regression equation was calculated for ki (eq. ): since the crystal structure of the acyl-enzyme complex, the acylation and deacylation rate were available, it was concluded that the inhibition begins with the histidine catalyzed attack of serine , at the benzoxazinone c , while the carbonyl oxygen occupies the oxyanion hole formed by glycine and serine . title: antifolate and antibacterial activities of -substituted authors: harris, n.v.; smith, c.; bowden, k. rhone results: it was shown earlier that binding of diaminoquinazolines to dhfr correlated with the torsional angle of the -amino group of the quinazoline nucleus. it was postulated that the interaction between the adjacent -substituent and the -amino group was very important in determining dhfr binding of the compounds possibly, because of the influence on the hydrogen-bond formed between the -amino group and a residue at the active site. the existence of such interaction in -substituted , -diaminoquinazolines were shown by measuring a , , and ~~ values. the ui and uor electronic parameters correlated well with chemical shifts of the -nh, groups (eq. ) but showed poor correlation for the -nh, group (eq. ), respectively: ( ) the equations suggest that the through-ring resonance interactions between the -substituent and the adjacent -amino group are disrupted by some other effects which might have significance for binding. a) an extensive set of compounds based on the nalidixic acid structure of type i. where r', r , r and r are various substituents; x and x* = c, n (for nalidixic acid: x = c, x = n, r' = et, r' = cooh. r = h, r = me); b) subset of (i) (set a) containing fifty two , -disubstituted -alky l- , -dihydro- -oxoquinoline- -carboxylic acids; compounds: abtstr. - quant. struct.-act. relat. , - ( ) c) subset of (i) (set b) containing one hundred and sixty two xylic acids; d) subset of (i) (set c) containing eighty five , -dihydr - -oxo- , -naphthyridine- -carboxylic acids with substituted azetidinyl, pyrrolidinyl and piperidinyl rings at position , fluorine at position and ethyl, vinyl or -fluoroethyl substituent at position . biological material: ps. aeruginosa v- , e. coli nihj jc- , s. the study showed that the most active compounds have fluorine in position , r can be a wide variety of nitrogen containing substituent and the best predictor for r is its lipophilicity. compounds: phytoalexins: pisatin, , a-dihydroxy- , -(methylenedioxy)pterocarpan, a, la-dehydropisatin, -hydroxy- , -(methylenedioxy)- a, a-dehydropterocarpan, (*)- -hydroxy- -methoxypterocarpan, (+)- -hydroxy- -zmethoxypterocarpan, (-)- -zhydroxy- -methoxypterocarpan, vestitol, sativan, formonenetin, coumestrol, '-o-methylcoumestro , phaseoilin, phaseollinisoflavan, '-methoxyphaseollin-isoflavan, glyceollin, a- a-dehydroglyceollin, tuberosin, a, ladehydrotuberosin. (capacity factor determined rp-hplc). calculated for logp of six reference compounds using their k' values (eq. ): ( ) n = r = . s not given f not given the lipophilicity of the phytoalexins were within the range of log p = . - . . it was found that the antifungal activity of similar compounds positively correlated with antifungal activity but no equation could be calculated for the whole set of compounds. it was suggested, however, that compounds with logp values higher than . were retained in the membranes, therefore phytoalexins with slightly lower lipophilicity, as well as greater fungitoxicity and systemic activity should be searched. certain structural features seemed to correlate with antifungal activity such as the presence of phenolic oh and benzylic hydrogen. it was suggested that the ability of the ortho oh group to form fairly stable intramolecular hydrogen bond may contribute to the greater stability of the shiff base hnctional group and the higher biological activity of the substances (various subsets required different equations). results showed that compounds with increasing lipophilicity and electron donating substituents at the -and -positions have high inhibitory activity. i-[( '-allyl- '-hydroxybenzilidene)amino]- -hydroxyguanidine was found to be the most active compound. the use of parameter focusing of the substituent hydrophobic constant and electronic constants was suggested for the selection of further substituents to design effective compounds. biological material: a) rabbits; b) rats; c) guinea pig. data taken from the literature: analogue results: prp, ecjoh, ecsob, ecsot values were measured and presented for the c,, paf analogue and compared with that of other analogues. c,, paf analogue was less potent than the c or cis paf analogues and equivalent to c,, paf analogue, showing that the activity decreased with lipophilicity. a highly significant parabolic relationship was calculated between log(rps) and cf (eq. ): the maximum activity was calculated cf = . , this corresponds to the cl paf. (energy minimizatipn of the compounds were calculated using the free valence geometry energy minimization method); (molecular shape analysis according to hopfinger was used to quantitatively compare the shape similarity of analogs in their minimum energy conformer states (within kcal/mol of their global minimum energy ( fig. shows the superposition of the reference conformations of the phenylalanine and tryptophane analogues). quant. struct.-act. relat. , - ( ) chemical descriptors: logp (logarithm of the partition coefficient in -octanol/water); (hansch-fujita's substituent constant characterizing hydrophobicity of a substituent on the aromatic ring and the hydrophobicity of the aromatic ring itself, respectively) ; [common overlap steric volumes (a ) between pairs of superimposed molecules in a common low energy conformation]; [dipole moment (debeyes) of the whole molecule and of the aromatic ring, respectively, calculated using the cndoi method] ; quantum chemical indices (partial atomic charges calculated by the cndoi method); - [torsion angles (deg) (fig. ) rotated during the conformational analysis of the compounds]. results: significant parabolic regression equations were calculated for the antigelling activity of the phenylalanine and tryptophan analogues (eq. and eq. , respectively): the different qsar for the phenylalanine and tryptophan analogues indicated that they interact with hemoglobin in different ways or at different sites. for the phenylalanine analogues the hydrophobicity of the side chain, the aromatic dipole moment and the steric overlap volume explained about %, % and % of the variance in antigelling activity, respectively. for the tryptophan analogues the square of the dipole moment or the steric overlap volume explained % or % of the variance in ra, respectively, being the two descriptors highly correlated. the results show that the tryptophan analogs have a relatively tight fit with the receptor site. title: s-aryl (tetramethyl) isothiouronium salts as possible antimicrobial agents, iv. in both eq. and eq. , log(l/c) depended primarily on electronic factors (eu' ) and only secondarily on hydrophobicity (ctobsd). a threshold logp value for the active isothiuronium salts was indicated, as the compounds with logp values between - . and - . were found to be totally inactive with the exception of the nitro-derivatives. title: comparative qsar study of the chitin synthesis inhibitory activity of benzoyl-ureas versus benzoyl-biurets. source: tagungsbericht , no. \ r* ponents explaining . %, . % and . % of the variance. fig. shows the minimum energy conformation of a highly active representative of the urea analogs (dimilin) with . a distance between the and carbon atoms. fig. shows the low energy conformation of the corresponding biuret analog with the two benzene rings in appro:imately the same plane and with the same c -c distance ( . a) allowing to fit a hypothetical benzoylurea phamacophore. the similarity of the regression equations and the modelling study supported the hypothesis that the benzoylbiurets act by the same mechanism as the benzoylureas. biological material: insect species: aedes aegypti, musca domestica, chilo suppressalis, hylemya platura, oncopeltus suppressalis, oncopeltus fasciatus, pieris brassicae, leptinotarsa decemlineata. [concentration of the benzoylurea derivative (various dimensions) required to kill % of insect larvae (a. aegypti, m. domestica, c. suppressalis, h. platura, . suppressalis, . fasciatus, p. brassicae or l. decemlineata]. data determined: lcso [concentration of the biuret analogue (ppm) required to kill % of insect larvae (a. aegypti or m. domestica]; molecular modeling (models of the compounds were built using molidea); conformational analysis (minimum energy conformations of the compounds were calculated using molecular mechanics method). chemical descriptors: the thesis is devoted to the quantitative analysis of the uncoupling activity of substituted phenols using chemical descriptors in order to obtain further information on the mode of action of phenol uncouplers: the study of the partition coefficient of substituted phenols in liposomelwater system [p(l/w)] showed that (i) p(l/w) depended primarily on the logp value; (ii) influence of steric and electronic parameters depended on the type of the lipid involved; qsar analysis of uncoupling phenols in rat-liver mitochondria identified the relevant physicochemical parameters required for phenols being protonophore in inner mitochondrial membrane and quantitatively separated the potency as the protonophore in the inner mitochondrial membrane and the incorporation factor (iogp); protonophoric potency of substituted phenols was linearly related to uncoupling activity when certain critical physicochemical parameters of the experiment were taken into account; linear relationship was calculated between uncoupling activities of substituted phenols and related uncouplers in the mitochondria from the flight muscles of house flies and in spinach chloroplasts; the results indicated a shuttle type mechanism for the uncoupling action of substituted phenols. title: uncoupling properties of a chlorophenol series on acer cell - ( ) compounds: chlorinated phenols substituted with -c , -c , , , -cl, , , -ci, pentachlorophenol, -ci- -me. -c - -me, -c - , -me, -c - , -me, -ci- -ally , -c - -pr- -me, z , , -c , , , , -c , , -cl, , -c , , -ci, , , -c , -cl- -no , , -c - -no,, -ci- , -no,. biological material: acer pseudoplatanus l. cell suspensions. data determined: dso [concentration of the compound (pmolll) required for % uncoupling effect registered by measuring the oxygen consumption rate by polarography]; [minimal concentration of the compound (pnol/l) required for giving a full uncoupling effect]. chemical descriptors: logp (logarithm of the partition coefficient in -octanol/water); mr (molar refractivity); ed (steric parameter representing the perimeter of coplanary molecules projected onto the aromatic plane); a (angular parameter expressing the hindrance in the neighborhood of the hydroxyl group in positions and , respectively) ; ui, (hammett's constants, characterizing the electron-withdrawing power of the para-substituent and the ortho-or -nitro substituents, respectively). results: highly significant linear regression equations were calculated for the uncoupling effects of chlorophenols in acer cell suspensions: the equations for the uncoupling effects in the whole cells and those calculated previously for isolated mitochondria or chloroplasts possess similar structures. . (* . ) a, - . ( ) title: effects of ' substituents on diphenyl ether compounds. results: sar suggested that the space for the n' and nz substituents in the psi binding site is relatively large. the variation of the number of the carbon atoms of r on the photosynthetic inhibitory activity is shown in fig. (hansch-fujita's substituent constant characterizing hydrophobicity); chemical descriptors: . (* . ) ior + . (& . ) hb + . the biological activity of three out of the (dpe- , and ) substituted diphenyl esters were measured and listed. igr values were measured for the three compounds and compared with that of a- and methoprene. it was found that the position of acetamido group in the phenol moiety when it is in the ortho position abtstr. - - ( ) increases the lipophilicity of the compound with a logp value of . . if the same group is in mr para position, the logp values are . and . , respectively and they are comparatively ineffective. when both the ortho positions are substituted with tertiary butyl groups (dpe- ) the logp value is relatively higher ( . ) which increases the lipophilicity of the compound and explains the pronounced idr activity at relatively low concentrations. abstr. results: a highly significant linear regression equation was calculated for the descriptors of r' (r' = i-pro was eliminated as an outlier) (eq. ): the compound with r' = eto, r = me and z = was found to be an effective, broad spectrum insecticide. the replacement of the quaternary carbon with a silicon atom cansimplify the synthesis of test compounds and thus can be advantageously utilized for the preparation of large compound sets for qsar studies. the data suggest that the initial electron loss from the given compounds is the preeminent factor effecting the reaction rate. a single mechanism is suggested over the entire range of reactivities, where a transition state with a considerable positive charge is involved. title: connection models of structure and activity: ii. estimation of electronoacceptor and electronodonor functions of active centers in the molecules of physiologically active materials. research institute of physiology active materials chernogolovka, moskow district, ussr. engl. summary). authors chemical descriptors: logp (logarithm of hydrophobicity). results: calculations for electronoacceptor and electronodonor entharpic and free energy factors on the base of functional groups were made according to the principle of independence of active centers: data determined: linear correlation was found between the calculated and measured characteristics: the accuracy of the fitting was the same as the measurement error of ah,,, and agm.the entropy might be calculated from enthalpy, gibbs energy and temperature: the good linear correlations between the measured and calculated data show that the functional group approaches might be used for these compound types. the substituent effects for the a-acceptorlr-donor substituents (f, c , br, i) were found to be very much larger for the c fsr relative to the nitrobenzenes. these results indicate that the extra electron enters a o*-orbital, which is localized on the c-r atoms. for the structure-solubility relationship of aliphatic alcohols. the study indicated that solubility of aliphatic alcohols depends primarily on molecular connectivity ('x), the number of carbon atoms in the alkyl chain (n'), the number of hydrogens on the a-carbon atom (normal, iso, secondary, ternary) and the degree of branching (vg): ( ) n not given r not given s not given f not given eq. was found to be a highly significant predictor of s (eq. ): the result support kier's, furthermore kier and hall's earlier models on the structural dependence of water solubility of alcohols. -log(s) = 'x + ( )* sg - . title: linear free energy relationships for peroxy radical-phenol reactions. influence of the para-substituent, the orthodi-tert-butyl groups and the peroxy radical. k (reaction rate constant (m -'s -i ) of the reaction between the reaction of cumyl-, -phenylethyl-and t-butyl-peroxy radicals and ortho-para-substituted phenol inhibitors). data taken from the literature: chemical descriptors: u+ r. ui, ur (charton's electronic substituent constant and its decomposition to inductive and resonance components, respectively for the characterization of the para substituent); (indicator variable for the presence or absence of the t-bu groups in , -position of the phenols). results: highly significant linear regression equations were calculated by stepwise regression analysis for logk in spite of the diverse data set originating from different laboratories using different peroxy radicals (eq. , eq. ): quant. struct.-act. relat. , - ( ) ( ) n = r = . s = . f = . i c~" was not selected by stepwise regression indicating that the orthodi-t-bu substitution had no significant effect on the rate of hydrogen abstraction from phenols by the radicals. the form of the equations for different subsets of the phenols and radicals indicated that the reaction mechanism was the same for the different peroxy radicals. title: a fractal study of aliphatic compounds. a quantitative structure-property correlation through topological indices and bulk parameters. the following descriptors are considered as 'bulk parameters': vw (van der waals volume, calculated from the van der waals radii of the atoms); mw (molecular weight); sd (steric density of the functional group). results: highly significant equations are presented for calculating vw, sd and mw r values ranging from . to . , other statistics and the number of investigations are not given. q and values calculated by these equations were introduced to the equation given above and the physicochemical properties were calculated. the observed and calculated iogv,, d and p values are presented and compared for the alkanes, alcohols, acids and nitriles. the observed and calculated physicochemical parameters agreed well. fractal nature of the alkyl chain length was discussed and a relationship was presented between the fractal-dimensioned alkyl chain length and a generalized topological index. title: application of micellar liquid chromatography to modeling of organic compounds by quantitative structure-activity relationships. chemical descriptors: logp (logarithm of the partition coefficient in -octanol/water). results: in a series of experiment with the listed compounds micellar liquid chromatography has been applied to model hydrophobicity of organic compounds in a biological system. the measured logk' values of the substituted benzenes were found to be superior predictors of logp. fig. shows the plot of logp versus logk' of the substituted benzenes. highly significant correlation was calculated for the logk' values of phenols (open squares) (n = , r = . ), for the rest of the compounds (full squares) (n = , r = . ) and for the entire set (n = , r = . ). further experiments using various surfactant types in the mobil phase suggested that logk' values generated on a lamellar phase may be better predictors of hydrophilicity than logp obtained from binary solvent systems. title: isoxazolinyldioxepins. . the partitioning characteristics and the complexing ability of some oxazolinyldioxepin diastereoisomers. authors quant. struct.-act. relat. , - ( ) source: j. chem. soc. perkin trans. i . no. , - compounds: oxazolinyldioxepin derivatives of type i and , where x = h, f, ci, cf or ch . data determined: logk' [logarithm of the capacity factor, measured by reversed-phase liquid chromatography (rplc)]; mep (molecular electrostatic computed by geesner-prettre and pullman's vsspot procedure). chemical descriptor: logp (logarithm of the partition coefficient in -octanollwater). results: the logk' and logp values were measured for the two type of diastereomers and a highly significant linear relationship between logk' and logp was presented (r = . ): the meps of i and 's f-derivatives were determined and presented, "a" for type i, "b" for type ii: the complex forming ability of the diastereoisomers with mono-cations was investigated and explained in terms of the structures and electronic properties of the compounds. results: linear relationships are presented plotting y versus n for the hydrophobic sorbents (fig. ) and the slopes of these straight lines are suggested for experimental determination of . q, values. ~ values determined by the suggested method are listed. while no linear relationships were found between kd and n, y depend linearly on n for the test compounds [alkanols (i). alkane diols ( ) results: three linear models were fittedwith independent variabies of log(p), mr and o x . the best fitting parameters (independent of composition) were obtained from the following models (no statistical characteristics is presented): ( ) ( ) the two types of correlations (with structural and with moving phase parameters) together might be used for the optimization of chromatographic separation of complex mixtures of sulphur-containing substances. (zero order molecular bonding type connectivity index); ig(k) = a + a p' + a logp + a p' logp ig(k) = a + a tg(cm) + a, logp + a tg(cm) logp - - the kd values derived by the suggested method were compared by kd values calculated by martin's rule and a good agreement was found. title: mathematical description of the chromatographic behaviour of isosorbide esters separated by thin layer chromatography. compounds: isosorbide esters: isosorbide (l), - -monoacetate, - -monoacetate, - -mononitrate, - -mononitrate, l-diacetate, - -nitro- -acetate, - -nitro- -acetate, l-dinitrate. rn, r~i [retention factors obtained by thin-layer chromatography in benzene/ethylacetate/isopropanol/ ( : : . ) and in dichloromethane/diisopropylether/isopropanol ( : : : ) eluent systems, respectively]. data determined: chemical descriptors: (information index, based on the distribution of the elements in the topological distance matrix); (the geometrical analogue); (randic connectivity index); (maximum geometric distance in the molecule); compounds: highly diverse chemicals. grouped according to the following properties: contains (ester or amide or anhydride) or (heterocyclic n) or ( bound to c) or (unbranched alkyl group with greater than carbons). data determined: aerud chemical descriptors: (aerobic ultimate degradation in receiving waters). v x x, nci m, (molecular weight). results: the paper has aimed at developing a model for predicting aerud. the data sets were collected from biodegradation experts. the experts estimated the biodegradationtime that might be required for aerud on the time scales of days, weeks, months and longer. (valence second order molecular connectivity index); (fourth order path/cluster connectivity index); (number of covalently bound chlorine atoms); highly diverse chemicals but typical in wastewater treatment systems were examined. zero to six order molecular and cluster connectivity indexes were calculated using computer programs wrinen in for-tran for ibm pc/xt. the best fitted linear regression model is: [first order rate constant: transport or transformation parameter (mol/pa. h)]. results: the qwasi fugacity model describes the fate of a (contaminating) chemical, such as organo-chlorine compounds, pesticides or metals. the lake model consists of water, bottom and suspended sediments, and air. the model includes the following processes: advective flow, volatilization, sediment deposition, resuspension and burial, sediment-water diffusion, wet and dry atmospheric deposition, and degrading reactions. the steady state solution of the model is illustrated by application to pcbs in lake ontario using the equilibrium criterion of fugacity as the variable controlling environmental fate of the chemical. the applications are based upon inaccurate data. use of fugacity is inappropriate for involatile chemicals, such as metals, or ionic species, because fugacities are calculated from a basis of vapor phase concentrations. for these materials the use of the equilibrium concentration activity is more appropriate since activities are calculated from a water phase base. thus, a new equilibrium criterion, termed the "aquivalent" concentration (or equivalent aqueous concentration) is suggested as being preferable. this concentration has the advantage of being applicable in all phases, such as water, air and sediments. the formalism developed in the qwasi approach can also be applied, making possible a ready comparison of the relative rates (and thus, the importance) of diverse environmental fate processes. all these are illustrated by applying the model on a steady state basis to quant. struct.-act. relat. , - ( ) abstr. - the pcb example and to the fate of lead in lake ontario. the estimated and observed concentrations of pcbs and lead in lake ontario agree well: the largest difference in the case of pcbs in rain amounts to a factor of three. in other phases, and especially in the case of lead, the difference is usually less than per cent. although in order to judge the biological effects of a contaminant it is of fundamental importance to know its transport and transformations, and the present model has been proven to useful to describe this; direct biological implications are not deduced at the present stage. the similar slopes of the equations show that these compounds exert their cytotoxicity primarily by alkylation. while the majority of the tested compounds showed no hypoxia-selective cytotoxicity (ratio awa .o), the -n and -no substituted compounds were more toxic to uv cells under hypoxic conditions (ratio = . for the compound with r = -n ), indicating cellular reduction of the nitro-group. the measured hypoxic selectivity of the -no and -n , substituted compounds was a fraction of the calculated ratio (measured fold and calculated fold by eq. between the -n and -nh, substituted compounds). the main reason for the difference between the calculated and measured hypoxic selectivity is suggested to be the low reduction potential of the -n and -no, groups (e = - mv and e = - mv, respectively). title: quantitative structure-activity relationships for the cytotoxici- (hammett's constant, characterizing the electron-withdrawing power of the substituent); (hammett's polar electronic constant characterizing the electron withdrawing power of the substituent for anilines). results: significant linear regression equations were calculated for the halflife (t / ), growth inhibition ( ) and clonogenicity data (ctlo) using hammett constants (eq. , eq. , eq. ): ( ) n = r = . s = . f not given - ( ) type, test animals, the mean level of toxicity and the form of the equation. e.g. analysis of the toxicity of phenols showed a transition between simple dependence from logp to exclusive dependence to reactivity factors indicating two separate classes of phenol toxicity (eq. for mouse i.p. toxicity, and eq. for rat oral toxicity): results: an additivity model, plc = cni a ti -to, where ni is the number of ith substituents in a benzene derivative, at; is the toxicity contribution of the ilh substituent and to is the toxicity of the parent compound (benzene), was used for predicting toxicity of lo similar correlation was found between mutagenicity and u (fig. ) indicating that both biochemical and chemical processes involve a ph dependent nucleophilic ring opening (including the protonation of the aziridin nitrogen as rate controlling step) and in.,uenced by electronic and steric factors (equation not given). resonance effect). (electron density on n in the homo calculated by the mndo). results: highly significant linear relationships between log( ic) and logp, &homo (eq. ); iogp, qhomo (eq. ) are presented indicating that the more hydrophobic and more electron-rich triazines are more active according to the ames test: substructures [a total of fragments were generated from the compounds using the program case (computer-automated structure evaluation) system]. results: a comparative classification of the compounds were performed using case for identifying molecular fragments associated with cancerogenic activity (biophores) as well as deactivating fragments (biophobes). case identified biophores and biophobes from the fragments of the compounds with a less than . % probability of being associated with carcinogenicity as a chance. the sensitivity and specificity of the analysis was unexpectedly high: . and . , respectively. the predictive power of case biological material: chemical descriptors: was tested using the identified biophores and biophobes on a group of chemicals not present in the data base. the ability of case to correctly predict carcinogens and presumed non-carcinogens was found to be very good. it was suggested that non-genotoxic carcinogens may act by a broader mechanism rather than being chemical specific. compounds: compounds of type i where r = h, ch , c,h , czh , c h , czh , c h , c h , c h , sc h ; compounds of t y p i where r = h, c~heoh, sc h h. data determined: p t pi" (a priori probability of appearance of the i-th active compound); (a priori probability of appearance of the i-th nonactive compound). (the first order molecular connectivity index); (the second order molecular connectivity index); (information-theoretic index on graph distances calculated by the wiener index according to gutmann and platt); chemical descriptors: (rank of smell, where the rank is defined to equal with one for the most active compound). results: the authors' previously proposed structure-activity relationship approach was applied for structure-odor relationship. different compounds of groups i and i were examined using the topological indices w, r, i, x as independent variables and v as the dependent variable. the best correlation was obtained between r and v ( fig. i) results: logp and iogp, values were determined for the nitroimidazole derivatives. significant linear equations were calculated, the best one related for logp and logp,r (eq. ): ( ) logp = . logp,i + . n = r = . s not given f not given chemical descriptors: logp descriptors (logarithm of the partition coefficient in i-octanoll water); ( indicator variables taking the value of for the presence of cr/p-hydroxy , a-fluoro, a-methy , afluoro, -hydroxy, i a-fluoro. , -acetonide, -deoxy, -acetate, -propionate, i-butyrate or -isobutyrate, respectively). results: a data set of steroids were compiled after removing those ones containing unique substituents. the set was divided into two categories of approximately equal membership by defining a threshold logp value of . . a descriptor set was created and the non-significant ones were eliminated using the weight-sign change feature selection technique. linear leaning machine was applied to calculate the weight vectors and complete convergence was achieved in the training procedure. the predictive ability of the linear pattern classifier thus obtained was tested using the leave one out procedure. the predictive ability was found to be i . %. the predictive ability of the approach was found to be good and improvement was expected with larger data set. steroids, however, containing new substituents would have to be subjected to a repeated pattern-recognition calculation. lengthlbreadth descriptors ( descriptors)]. results: for modeling the shape of the compounds, simca was used: the approach was to generate disjoint principal models of clustered points in a multidimensional space. the number of clusters for each structure was determined by using hierarchical cluster analysis. fig. shows the orthogonal views of a schematic representation of the sim-ca models for the atom clusters in senecionine: each compound in turn was used as a reference structure. every other structure was superimposed on the reference using the ends of the corresponding binding moment vector plus the ring nitrogen atom. canonical correlation analysis was used for calculating the correlation between the five biological activity data and shape descriptors of structures. the best correlation was observed for jurs' shadow descriptors. the msa and simca descriptors were comparable. the model was able to express both the amount and direction of shape the differences, and also for encoding relevant information for correlation with the biological activity. compounds: n-substituted -methyl- -nitropyrazole- -carboxamides ( ), n-substituted -amino- -methylpyrazole- -carboxamides (iii), n-substituted -methyl- -diazopyrazole- -carboxamides and n-piperidiny -n-( , -dimethyl- -nitrosopyrazol- -yl)-urea (vii) . title: structure-activity correlations for psychotomimetics. . phenylalkylamines: electronic, volume, and hydrophobicity parameters. abtstr. quant. struct.-act. relat. , - ( ) data determined: edso conformational analysis g [dose of the compound (mg/kg) which causes % of the rats which were trained on rngfkg reference compound to respond as they would to the training drug]; (geometries of the compounds were calculated using mmf from starting geometries determined by the program euclid). discriminant analysis resulted in a function containing six variables which misclassified only one compound in the training set. when the data was repeatedly split randomty into a training and a test sb, the misclassification rate was % ( out of classifications). fig. shows the plot of the two canonical varieties from discriminant analysis visualizing the separation of hallucinogenic and nonhallucinogenic derivatives (meaning of symbols are the same as in fig. ). multiple regression analysis (mra) was found to be the most useful for identifying relevant and discarding redundant variables. highly significant parabolic regression equations were calculated for the human activity data (a) (n = , r ranging from . to . , f not given) and for animal data (edso) (n = , r = . and r = . , f not given). eight descriptors were found to be highly significant. among these the importance of directional hydrophobicity and volume effects indicated that steric and hydrophobic interactions participate in the interaction with the receptor.mra indicated a strong interaction between the meta-and para-substituents and the presence of the formation of charge transfer complex by accepting charge. data did not support the hypothesis that the human activity data and animal ( ) data taken from the literature: sweet(n) (sweet taste of the compound, where n represent the number of times a sample has to be diluted to match the taste of % sucrose solution). [class fit distances of a compound to sweet and nonsweet class (dimension not given) calculated by principal component analysis]. chemical descriptors: mr (molar refractivity); bi, l (sterimol steric parameters, characterizing the steric effect of the substituent); r (hansch-fujita's substituent constant characterizing hydrophobicity); urn, up (hammett's constants, characterizing the electron-withdrawing power of the substituent in meta-and para-position, respectively). results: no statistically significant regression equation was obtained by the hansch-fujita approach using the chemical descriptors listed. d', d quant. stact.-act. relat. , - ( ) abstr. - principal component analysis of the data set extracted principal components, explaining % of the variance of the sweet compounds. the sweet compounds clustered in a relatively confined region of the d space whereas the tasteless and bitter compounds were scattered around the sweet compounds. a coomans plot, however., indicated, when plotting d' versus d , that sweet and nonsweet compounds could be well separated along the d' axis ( fig. , title: conformation of cyclopeptides. factor analysis. a convenient tool for simplifying conformational studies of condensed poly-ring systems. prolyl-type cyclopeptides. authors conformations of the six-membered dop-ring family may be reproduced by means of a superposition of the canonical twist (t), boat (b) and chair (c) forms. physically, the coefficients have the meaning of relative contributions (amplitudes) of the t, b and c forms into the total conformation of the ring. here factor analysis (fa) and principal component analysis was used in conformational studies of various x-ray conformers of dop/pyr. a correspondence was found between factors identified and rpt, when the rings are considered separately. this fact allows a physical interpretation of the fa results: two or three puckering variables were found for the dop and pyr rings expressing the absolute amplitudes of the basic pucker modes. subse-quent fa treatment of the condensed system revealed five conformational variables necessary and sufficicnt to describe the twolring puckering completely. each of the basic pucker modes defines a unique pattern of conformational variation of the whole two-ring system. the results demonstrate that fa is a powerful technique in analysing condensed poly-ring systems, not amenable to the rpt treatment. title: preprocessing, variable selection, and classification rules in the application of simca pattern recognition to mass spectral data. authors: dunn m*, w.j.; emery, s.l.; glen, g.w; scott, d.r. college of pharmacy, the university of illinois at chicago south wood, chicago il , usa. source: environ. sci. technol. , ( ) , - . compounds: a diverse set of compounds observed in ambient air classified as ( ) nonhalogenated benzenes; ( ) chlorine containing compounds; ( ) bromo-and bromochloro compounds; ( ) aliphatic hydrocarbons; ( ) miscellaneous oxygen-containing hyhocarbon-like compounds (aliphatic alcohols, aldehydes and ketones). pattern recognition was applied to autocorrelation-transformed mass spectra of the compounds using providing chemical class assignment for an unknown]; m/z;, mlz,, m/zl (first three principal components scores of simca). results: simca pattern recognition method was applied on a training set of toxic compounds targeted for routine monitoring in ambient air. the analysis resulted in very good classification and identification of the compounds ( % and %, respectively). however, the training procedure proved to be inadequate as a number hydrocarbons from field samples (gcims analysis) were incorrectly classified as chlorocarbons. a new approaches for the preprocessing (scaling the ms data by taking the square root of the intensitiesfollowed by autocorrelation transform), variable selection (only the most intense ions in the ms spectrum were taken), and for the classification rules of simca has been introduced to improve results on real data. fig, and as a result of the revised rules the classification performance has been greatly improved for field data ( - %). title: a qsar model for the estimation of carcinogenicity. - ( ) it was suggested that the mechanism of mutagenicity of the cimeb[a]ps measured in the ames test is probably more complex than the simple reactivity of carbocation intermediates. [dipole interaction potential (dimension not given)]; [molecular electrostatic potential (kcallmol) in a plane]; [molecular electrostatic field map (kcall mol), mapping the e(r)j values of a molecule surface in a plane, predicting the directions and energies of the interactions with small polar molecules at distances greater than the van der waals sphere]; (construction of surfaces corresponding to a given value of potential); d mep and mef maps ( d maps weregenerated by superimposing the equipotential curves corresponding to a value of kcallmol in the case of mep. and . kcallmol in the case of mef, computed in several planes perpendicular to the mean plane of the analogues in low energy conformations, stacking over each other in a distance). results: the three vasopressin analogues differ significantly in their biological activities. both mep and mef maps of the of the biologically active (mpa')-avp and (cpp')-avp are similar, but they are different from that of the inactive (ths')-avp. fig. , fig. abtstr. - quant. struct.-act. relat. , - ( ) a new method for calculating the points of the equipotential curves was also presented. crystal structure (crystal coordinates of the molecules were determined by x-ray diffraction methods). data taken from the literature: [electrostatic molecular potential (ev) were calculated using am- type semiempirical mo calculations]; conformational analysis [minimum energy conformations were calculated using x-ray structures as input geometries followed by am -method (fletcher-powell algorithm)]. chemical descriptors: ui, u [rotational angles of the n-ally group (deg)]. results: four similar energy minima were located by am-i calculations for both namh+ and nlph+. the energy minima for the protonated nam + and nlph + were the most populated ones with conformational enantiomers relative to the involved n-allyl-piperidine moiety ( % and %, respectively). it was shown that the isopotential curve localization of emp contour maps were very similar for the corresponding conformations of both nlph * and namh + indicating that both molecules should interact a the same anionic sites of the opioid receptor, ( p morphine receptor). fig. and fig. shows the emp contour maps of namh' and nlph + , respectively, in their preferred conformations: compounds: esfenvalerate (ss and sr isomers) of type i, -phenoxybenzyl -( -ethoxyphenyl)- , , -trifluoropropyl ether (r quant. struct.-act. relat. , - ( ) abstr. isomer) ( ). a-cyano- -phenoxybenzyl -( -chlorophenyl)- -methylpropionate (s isomer) (iii) and deltamethrin (iv). " cn data determined: conformational analysis (minimum energy conformations of the compounds in vacuum were calculated using am molecular orbital method and broyden-fletcher-goldfarb-shanno method integrated into mopac); (root mean square, indicating the goodness of fit between two conformers in d); (logarithm of the partition coefficient in i-octanol/water estimated using clogp program); [heat of formation of the most stable conformer (kcallmol)]. rms logp e chemical descriptors: - results: it was assumed that the d positions of the benzene rings of the pyrethroids are decisive for good insecticidal activity. the lower energy conformers of (i) (ss and sr isomers), ( ) (r isomer), ( ) (s isomer) and deltamethrin (iv) were compared by superimposition. inspite of their opposite configuration, esfenvalerate (i) (ss isomer) and the new type pyrethroid i (r isomer) were reasonably superimposed, indicating that the positions of the benzene rings in space are important and the bonds between them are not directly determinant (fig. ) crystal structure (x-ray crystal coordinates of penicillopepsin was obtained from the protein data bank). data determined: electrostatic potential [electrostatic potential of the protein atoms (kcallmol) is calculated using the partial charges in the amber united atom force field); docking (the dock program was used to find molecules that have a good geometric fit to the receptor). results: a second generation computer-assisted drug design method has been developed utilizing a rapid and automatic algorithm of locating sterically reasonable orientations of small molecules in a receptor site of known d structure. it includes also a scoring scheme ranking the orientations by how well the compounds fit the receptor site. in the first step a large database (cambridge crystallographic database) is searched for small molecules with shapes complementary to the receptor structure. the second step is a docking procedure investigating the electrostatic and hydrogen bonding properties of the receptor displayed by the midas graphics package. the steps of the design procedure is given. the algorithm includes a simple scoring function approximating a soft van der waals potential summing up the interaction between the receptor and ligand atoms. directional hydrogen bonding is localized using electrostatic potential of the receptor at contact points with the substrate. the shape search of (i) was described in detail. a new method has been developed for the construction of a hypothetical active site (hasl), and the estimation of the binding of potential inhibitors to this site. the molecules were quantitatively compared to one another through the use of their hasl representations. after repeated fitting one molecule lattice to another, they were merged to form a composite lattice reflecting spatial and atomic requirements of all the molecules simultaneously. the total pki value of an inhibitor was divided to additive values among its lattice points presumed to account for the binding of every part of the molecule. using an iterative method, a self consistent mathematical model was produced distributing the partial pki values of the training set in a predicting manner in the lattice. the hasl model could be used quantitatively and predictively model enzyme-inhibitor interaction. a lattice resolution of - a was found to be optimal. a learning set of e. coli dhfr inhibitors were chosen to test the predictive power of the hasl model at various resolutions. binding predictions (pki values) were calculated for the entire inhibitor set at each resolution and plotted separately for the learning and test set members at . a resolution (fig. i) : . ala-). data determined: molecular models ( d structure of the molecules have been constructed and displayed using the program geom communicating with cambridge x-ray data bank, brookhaven protein data bank, sandoz x-ray data bank sybyl and disman; quant. struct.-act. relat. , - ( ) abstr. - distance geometry [nuclear overhauser enhancements (noe) and spin-spin coupling constants were measured by d nmr methods, semiempirically calibrated as proton-proton distance (a) and dihedral angle (deg) constrains and used in distance geometry calculations (disman) andlor in restrained molecular dynamics calculations to determine d structure of molecules in solution]; crystal structure (atomic coordinates of the compounds were determined by x-ray crystallography); rms [root mean square deviation (a) of the corresponding atoms of two superimposed molecular structures]. chemical descriptors: results: distance geometry calculations were carried out using geom and disman, to identify all conformations of the compounds in solution which were consistent with experimental data obtained by noe measurements. the application of geom was demonstrated by modelling cycbsporin a with and without a limited set of h-bond constrains and with a full nmr data set. in case of cyclosporin a, randomly generated linear analogues of the cyclic structure were formed from the monomers. geometric cyclization was achieved using disman, resulting in many different but stereochemically correct conformations of cyclosporin a. superposition of the backbones of the best cyclic conformers showed rms deviations between . a and . a. fig. shows the superposition of a disman generated ring conformation (thick line) with its x-ray structure of cyclosporin a (thin line) with h-bond constraints (rms = . a): fig. distance and dihedrl-angle constraints have been extracted from noe and vicinal coupling data and used to generate the conformation and the cyclization conditions of the hexapeptide (fig. ) (position of residual distance violations and their direction is shown by arrows): although the described method is not exhaustive, it explores a much greater variety of initial structures than had been previously possible. title: a new model parameter set for @-lactams. authors: durkin, k.a.; sherrod, m.j.; liotta*, d. department of chemistry, emory university atlanta gl , usa. source: j. org. chem. , ( ) , - . compounds: @lactam antibiotics of diverse structure. data taken from the literature: crystal structures (crystal coordinates of the p-lactams were determined using x-ray diffraction method). results: superposition of the x-ray structures and the calculated geometries of -lactams using the original parameter set in the mm force field in model gave satisfactory rms values. a lack of planarity of the -lactam ring and significant differences in the calculated bond lengths and anglesaround the &lactam nitrogen were detected, however. = , s, so, so,]. in order to improve fit, a new atom type with new parameters has been developed for the p-lactam nitrogen (wild atom type coded with symbol in model). the new parameters were evaluated by comparison of the calculated and x-ray geometries of the -lactams. using the new parameter set, the x-ray data were satisfactorily reproduced except for the sulfone -lactams. it was indicated that the ampac data were not suitable for the sulfones as the hypervalent sulfur compounds are not well described in the am hamiltonian. an additional parameters was, however, derived giving good structural data unrelated to the ampac information. it is not known which the new parameter sets is the best for the sulfone / -lactams. title: a molecular modelling study of the interaction of compounds noradrenalin. biological material: a) cdna of the hamster lung ,-adrenergic receptor and &-adrenergic receptor kinase; b) bacterio-ovine-and bovine-rhodopsin and rhodopsin kinase. protein primary sequence (amino acid sequence of the hamster lung p,-adrenergic receptor has been deduced by cloning the gene and the cdna of the hamster lung &adrenergic receptor);, (the cosmic molecular modeling program was used for modeling a-helices in a hydrophobic environment using p and w torsion angles of - " and - ", respectively, according to blundell et al.); (the two highest lying occupied and the two lowest lying unoccupied orbitals, respectively, calculated using indo molecular orbital calculation); crystal structure (crystal coordinates of noradrenaline has been determined by x-ray diffractometry); conformation analysis (minimum energy conformation of the &-adrenergic receptor model has been calculated using molecular mechanics method). results: strong experimental evidences suggested that rhodopsin and ,-adrenergic receptor had similar secondary structure. thus, it was assumed, that similarly to bacterio-ovine-and bovine-rhodopsins, d -adr ener gic receptor -. . b -adrenergic receptor possesses a structure consisting of seven ahelices traversing the cell membrane. fig. shows the postulated arrangements of the a-helices of rhodopsin and the &-receptor. using the experimental data, a model of the &-adrenergic receptor has been generated for the study of its interaction with noradrenaline. a possible binding site was created. successful docking indicated that homo and lumo orbitals contributed to the binding in a chargetransfer interaction between trp- and noradrenaline. a hydrogen bond was detected between the threonine residue of the model receptor and the noradrenaline side chain hydroxyl explaining why chirality was found to be important for the activity of adrenergic substances. title: three-dimensional steric molecular modeling of the [binding affinity (nm) of the compounds to the -ht receptor]. data determined: molecular modeling ( d molecular models of each compound were made using camseqlm molecular modeling system); [distance (a) from the center of the aromatic ring to the ring-embedded nitrogen, when the nitrogen is placed in the same plane as the aromatic ring]. results: in order to derive rules for the -ht pharmacophore, a molecular graphics-based analysis was made using six core structures. the structures were aligned so as to overlay the aromatic rings and to place the ring embedded nitrogen atom in the same plane as the aromatic ring. nine steric rules were derived from the analysis common to all potent -ht agents. fig. shows the d representation of the six overlaid -ht core structures using camseqim: the -ht inactivity of atropine could be explained because its steric properties differed from those the active ics - only by a single atom and failed to meet two of the nine hypothetical criteria. uv-visible spectra [spectrophotometric studies of mixtures of the dyes and nicotine in % (vlv) aqueous ethanol mixture at "ci. results: cyanine dyes demonstrate a multitude of biological activities which may be due to the interference of the adsorbed dye molecule on active sites of the living cell. it was shown by uv-and visible spectrophotometry that the hydroxy styryl cyanine dyes and nicotine formed : charge-transfer complexes. the absorption band of the complex formed between nicotine and dye was detected at wavelengths longer than those of the individual pure substances having identical concentrations to those in mixture. fig. shows that the two partially positive centres of the dye ( -( -hydroxystyryl)-pyridinium-i-ethyliodide) were located at a similar distance than the two nitrogen atoms of pyridine or pyrrolidinyl moieties of nicotine allowing the suggested : i parallel stucking interaction between the two molecule: molecular modeling ( conformations were calculated using a distance geometry algorithm and energy minimized by a modified mm force field in moledit). results: all conformers within kcallmol of the lowest energy conformer were superposed on the x-ray structure of mk- . crystal structure (crystal coordinates of the proteins were determined using x-ray diffraction method). data taken from the literature: was less than a); (probability that a given tetrapeptide sequence is superimposable on the ribonuclease a structure); [probability that the ith residue (amino acid) will occur in the jth conformational state of the tetrapeptide which is superimposable to ribonuclease a]. results: it was suggested that the five tetrapeptides were essential components of larger peptides and might be responsible for their biological activity (binding to the cd receptor). earlier it was hypothesized that the critical tetrapeptide located in a segment of ribonuclease a, would assume low energy conformations (residues - , a @-bend, having a segment homologous to the sequence of peptide t). low energy conformers of the tetrapeptides could be superimposed to the native structure of segment - of ribonuclease a. fig. shows the superimposition of peptide t (full square): many low energy conformers could be calculated for the tetrapeptides but for the polio sequence. the p, value for most tetrapeptides were - times higher that the value of the less active polio sequence. the results supported the hypothesis that the active peptide t adopts the native ribonuclease @-bend. title: potential cardiotonics. . synthesis, cardiovascular activity, molecule-and crystal structure of -phenyl-and -(pyrid- -yl)- data determined: [dose of the compound (mollkg) required for % increase of the heart beat frequency of guinea pig or dog heart]; [dose of the compound (mollkg) required for % decrease of systolic or diastolic blood pressure of dog]; crystal structure (atomic coordinates of the compounds were determined by x-ray diffraction); molecular modeling (molecule models were built using molpac); mep [molecular electrostatic potential (mep) (dimension not given) was calculated using cndoiz]. results: milrinon and its oxygen containing bioisoster possess highly similar crystal structure and mep isopotential maps ( fig. and fig. ) both compounds show strong positive inotropic and vasodilatoric activity. it was suggested that the negative potential region around the thiocarbonyl group such as the carbonyl group in milrinon imitates the negative potential field around the phosphate group of camp. title: molecular mechanics calculations of cyclosporin a analogues. effect of chirality and degree of substitution on the side chain conformations of ( s, r, r, e)- -hydroxy- -methyl- -(meth~lamino)- octenoic acid and related derivatives. [solution conformation of csa in cdch has been elucidated via molecular dynamics simulation incorporating distance constrains obtained from ir spectroscopy and nuclear overhauser effect (noe) data]; (conformational analysis was performed using the search subroutine within sybyl); energy minimization (low energy conformers were calculated using molecular mechanics withinmacromodel ver. . applying an all-atom version of the amber force field). results a total of conformations of csa have been identified within kcalfmol of the minimum energy conformer. population analysis showed that one conformer dominates in solution. fig. shows the superposition of the peptide backbone of the crystal and solution structures of csa (crystal structure is drawn with thick line and the solution structure with thin line). it was shown that the boltzmann distribution between active and inactive conformers correlated with the order of the immunosuppressive activity. a common bioactive conformer serving as a standard for further design has been proposed for csa and its analogs. abtstr. quant. struct.-act. relat. , - ( ) data determined: molecular modeling (models of (i), ( ) and ( ) were built using sybyl based on x-ray coordinates of the compounds); conformational analysis (minimum energy conformations of the compounds were calculated using the search option of sybyl and mndo method; [interaction energy of the molecules (kcall mol) with a hypothetical receptor probe (negatively charged oxygen atom) calculated by grid]. results: the specific receptor area of the sodium channel was modeled with a negatively charged oxygen probe (carboxyl group), interacting with the positively charged (protonated) ligand. fig. shows areas for energetically favorable interaction (areas i, oh ho c *. . quant. struct.-act. relat. , - ( ) abstr. - biological material: a) aspergillus ochraceus; b) carbopeptidase a. data determined kobr [first order rate coefficient (io- /sec) of the hydrochloric acid hydrolysis of ochratoxin a and b]; x-ray crystallography (coordinates of the crystal structure of ochratoxin a and b was obtained using x-ray diffraction); (models of ochratoxin a and b was built using alchemy); ["c nmr chemical shifts (ppm) of the amide and ester carbonyls of the ochratoxins]. chemical descriptors: pka (negative logarithm of the acidic dissociation constant). results: a reversal of the hydrolysis rate between ochratoxin a and b was observed comparing the hydrolysis rates obtained in vitro (carbopeptidase a) and in vivo (hydrochloric acid). the difference in hydrolysis rates cannot be due to conformation since the two toxins have the same conformation in both crystal and in solution. fig. shows the fit of ochratoxin a and b based on superimposing the phenolic carbon atoms. it is suggested that the relative large steric bulk of the chloro atom hinders the fit between ochratoxin a and the receptor site of carbopeptidase a. thus, probably the slower metabolism is the reason, why ochratoxin a is more toxic than ochratoxin b. title: inhibitors of cholesterol biosynthesis. . trans- -( -pyrrol- charge distribution studies showed that compactin had two distinct regions of relatively large partial charges corresponding to the pyrrol ring and the isobutyric acid side chain. experiments for more closely mimicking the polar regions associated with the high activity of compactin indicated that potency of the new compounds was relatively insensitive to the polarity of the r' group. it was also suggested that an electron deficient pyrrole ring was required for high potency. title: synthesis and biological activity of new hmg-coa reductase inhibitors. . lactones of pyridine-and pyrimidine-substituted . dihydroxy- -heptenoicf-heptanoic) acids. chemical descriptors: results: an attempt was made to correlate electrophysiological activity with the effect of the position of the aryl group on the conformation of the side chain using molecular modeling. the study suggested that the compounds with class activity prefer a gauche (a in fig. ) and compounds in which class i activity prefer trans relationship of the nitrogens (b in fig. ) : the study indicated that the point of attachment of the aryl moiety had an effect on the side chain conformation which appeared to be a controlling factor of the electrophysiological profile of these compounds. title: a molecular mechanics analysis of molecular recognition by cyclodextrin mimics of a-chymotrypsin. authors ( ) quant. struct.-act. relat. . - ( ) biological material: chymotrypsin. data taken from the literature: crystal structure (crystal coordinates of the macrocycles determined using x-ray diffraction analysis). data determined: molecular modeling structure superposition (models of b-cd and in chains by nmethylformamide and n-dimethyl-formamide substituted (capped) b-cd were built using the amber program and the coordinates for building the n-methylformamide substituent were calculated using mndo in the mopac program); (energy minimization of the molecules were calculated in vacuo using molecular mechanics program with the amber force field); (energy minimized structures of b-cd and capped b-cd were separately fit to the xray structure of the b-cd complex); [molecular electrostatic potential (kcallmol) of b-cd and capped b-cd were approximated by the coulombic interaction between a positive point charge and the static charge distribution of the molecule, modeled by the potential derived atomic point charges at the nuclei and visualized as d mep map]. results: b-cd and capped b-cd were analyzed as biomimetic models of the active site of chymotrypsin. capped b-cd was shown to be the more effective biomimetic catalyst. capping also altered certain structural features of molecular recognition. the orientation of the secondary hydroxyls were altereddue to twisting of some of the glucose units. secondary hydroxyl oxygen mimics the ser- of chymotrypsin in initiating the acyl transfer event through nucleophilic attack on the substrate. fig. shows the energy minimized structures of b-cd (a) and capped b-cd (b) (fragment number is given in parenthesis). the mep maps of b-cd and capped b-cd showed that the qualitative features of the electrostatic recognition were practically the same in the two mimics. biologicai material: four monocotyledonous (johnson grass, yellow foxtail, barnyard grass, yellow millet) and four dicotyledonous weed species (velvetleaf, morning glory, prickly sida, sicklepod). data determined: [pre-emergence and postemergence herbicidal activities of the compounds were measured and rated using a scale ranging from (no activity) to (complete kill]; [measure of the compound's ability (dimension not given) to translocate upwards in plants through xylem vessels); [soil sorption coefficient calculated by the formula k, = c,/c,, where c, is the concentration of the compound (pg compoundlg soil) and c. is the concentration of the compound (pg compoundlml) in water solution in equilibrium with the soil]; (models of the compounds were built using maccs and prxbld programs); tscf kd molecular modeling quant. struct.-act. relat. , - ( ) abstr. - conformational analysis (minimum energy conformations of the compounds were calculated using mm molecular mechanics method); (molecules were visualized using program mogli on an evans and sutherland picture system ); [total energies, orbital eigenvalues, atomic charges and dipole moments of simple model analogs of type i were calculated using prddo (partial retention of diatomic overlap) level of approximation]. electronic structure chemical descriptors: logp (logarithm of the partition coefficient in -octanollwater). results: conformational analyses and high level quantum mechanical calculations of the conformational preferences showed that the compounds with r = -c and -ci substituents adopt a coplanar structure stabilized by intramolecular hydrogen bond, whereas the -c analogue does not (fig. ): higher logp values ( . - . logarithmic unit difference), higher kd and tscf values of the -ci and -ci substituted compounds relative to the -ci analog were interpreted as the result of the intramolecular hydrogen bond and were consistent with the observation that the -ci and -ci analogs were active as post-emergence but not pre-emergence herbicides while the -ci derivative was active in both modes. title: application of molecular modeling techniques to pheromones of the marine brown algae cutleria multifida and ectocarpus siliculosus (phaeophyceae). metalloproteins as chemoreceptors? (geometrical models of the compounds were constructed using information from the cambridge structural data base (csd) and calculated using molecular mechanics methods in sybyl); (minimum energy conformations of the compounds were calculated using molecular mechanics method within sybyl). chemical descriptors: kfcq [partition coefficient in fc /water (fc = fluorocarbon results: as both ectocarpene (i) and multifidene ( ) trigger mutual cross reactions between male gametes of ectocarpus siliculosus and cutleria multifida males it was supposed that a common mode of binding should exist for the two structurally different pheromones. the active analogue approach was applied to model the pheromone receptor by superposing the minimum energy conformations of active structural analogues (hi, iv, v, vi) on ectocarpene and multifidene. the common active conformation of (i) and ( ) was extracted by systematic superimposition of the analogues. to explain the function of the double bonds in the pheromones, the presence of a receptor bound metal cation was assumed. simultaneous optimization,of both structures without and with a receptor bound metal cation resulted in virtually the same conformations. fig. shows the mapping of multifidene onto ectocarpene in their biologically relevant conformations. solvent)]. title: critical differences in the binding of aryl phosphate and carbamate inhibitors of acetylcholinesterases. conformational analysis [minimum energy conformations of (asn-ala-asn-pro) was calculated using charmm (chemistry at harvard macromolecular mechanics), amber (assisted model building with energy refinement) and ecepp (empirical conformational energy program for peptides) potential energy functions]; [root mean square deviation (a) of the position of the corresponding atoms of two superimposed molecular sructures], results: low energy conformations of (asn-ala-asn-pro) has been determined using charmm, ecepp and amber in order to determine their final conformations and relative energies. the final conformations were compared calculating the rms values of their c" atoms and matching the parameters of the energy minimized (asn-ala-asn-pro), peptide to that of the ideal helix or coiled coil. the similarity of the final conformations obtained by using any two different potentials starting from the same conformation varied from the satisfactory to highly unacceptable. the extent of difference between any pairs of the final conformations generated by two different potential energy functions were not significantly different. the lowestenergy conformation calculated by each of the energy potentials for any starting conformation was a left handed helix and pair-wise superposition of the c" atoms in the final conformations showed small rms values ( .o - . a) . it was suggested that the native conformation of (asn-ala-asn-pro), in the cs protein may be a left-handed helix, since all three potential energy functions generated such conformation. - ( ) source: proteins proteins , ( ), - , . biological material: crambin. data determined phi-psi probability plot (probabilities of the occurrences of phi-psi dihedral angle pairs for each amino acid were determined and plotted using the data of approximately proteins from the brookhaven protein data bank); (optimization technique for the reproduction the folding process converging to the native minimum energy structure by dynamically sampling many different conformations of the simplified protein backbone). chemical descriptors: phi-psi values [dihedral angles (deg) defined by the bonds on either side of the a-carbon atom of the amino acid residue in a protein]. results: a simplified model has been developed for the representation of protein structures. protein folding was simulated assuming a freely rotating rigid chain where the effect of each side chain approximated by a single atom. phi-psi probabilities were used to determine the potentials representing the attraction or repulsion between the different amino acid residues. many characteristics of native proteins have been successfully reproduced by the model: (i) the optimization was started from protein models with random conformations and led to protein models with secondary structural features (a-helices and strands) similar by nature and site to that of the native protein; (ii) the formation of secondary structure was found to be sequence specific influenced by long-range interactions; (iii) the association of certain pairs of cysteine residues were preferred compared to other cysteine pairs depending on folding; (iv) the empirical potentials obtained from phi-ps probabilities led to the formation of a hydrophobic core of the model peptide. x [dihedral angle (deg) ]. results: four kinds of monte carlo simulations of about , steps of the conformations of crambin were carried out by using the second derivative matrix of energy functions (starting from native and unfolded conformations both in two kinds of systems, in vacuo and in solution). fig. shows the native (a) and theunfolded (b) conformation of crambin. starting from native conformation, the differences between the mean properties of the simulated crambin conformations obtained from in vacuo and solution calculations were not very large. the fluctuations around the mean conformation during simulation were smaller in solution than in vacuo, however. simulation starting from the unfolded conformation resulted in a more intensive fluctuation of the structure in solution than in vacuo indicating the importance of the hydration energy term in the model. the conformations generated in the simulations starting from the native conformation deviate slightly from the xray conformation (rms = . a and i . a for in vacuo and solution simulations, respectively). the results indicate that the simulations of the protein with hydration energyare more realistic that the simulations without hydration energy. fig. results: earlier studies overestimated the catalytic rate decrease of the hypothetical d a point mutant of thrombin ( orders of magnitude decrease calculated instead of the order of magnitude measured). the source of error was due to an overestimation of v and neglecting the effects of the surrounding water molecules and induced dipoles. to compensate for these errors, a scale factor of . was introduced into the calculations. as aresult of the rescaling, one magnitude increase of tat for the d mutant and two magnitudes decrease of k,, of the k mutant of ribonuclease a was predicted. it was shown that the effect of the mutations on the catalytic rate depended almost entirely on steric factors. it was suggested that in mutants of serine proteases where the buried asp is replaced by ala or asp, the kcat value will decrease between - orders of magnitude. title: high-resolution structure of an hiv zinc fingerlike domain via a new nmr-based distance geometry approach. authors: summers*, m.f.; south, t.l.; kim [root mean square deviation (a) of the corresponding atoms of two superimposed molecular sructures]. results: the atomic resolution structure of an hiv zinc fingerlike domain has been generated by a new nmr-based dg method using d noesy backcalculation. the quality of the structures thus obtained were evaluated on the basis of the consistence with the experimental data (comparison of measured and back-calculated nmr spectra) rather than comparing it tostructural informations from other sources (e.g. x-ray data). the method provided a quantitative measure of consistence between experimental and calculated data which allowed for the use of tighter interproton distance constraints. the folding of the c( l)-f( )-n( )-c( )-g(s)-k( ) residues were found to be virtually identical with the folding of the related residues in the x-ray structure of the iron domain of rubredoxin (rms values . and . a). the backbone folding of the peptide was found to be. significantly different from that of the "classical" dna-binding zn-finger. fig. shows the wire frame model of all the back%ne atoms and certain side chain atoms of the peptide (dg struciure) (dashed lines indicate hydrogen atoms): active site of the protease dimer in an extended conformation with extensive van der waals and hydrogen bonding and was more than % excluded from contact with the surrounding water (fig. i , where the inhibitor is shown in thicker lines and the hydrogen bonds in dashed lines): data determined: ago,, ago, [standard free energy (callmol) of transfer of a molecule from an apolar phase to an aqueous phase, observed or calculated by eq. : ago, = c aui ai, where aoi is the atomic solvation parameter of atomic group i, ai is the accessible surface area of atom i, respectively]. results: atomic solvation parameters (asps) characterizing the free energy change per unit area for transfer of a chemical group from the protein interior to aqueous surroundings were determined. ago, and ago, were determined and compared, and a highly significant linear relationship is presented (fig. ) . one letter symbols indicate amino acid side chains: fig . the binding of the inhibitor induced substantial movement in the en-' zyme around the residues to in both subunits at places exceeding i - the structure of glutamine synthetase is discussed. it was established that hydrophobic interactions are important for the intersubunit interactions, and the hydrophobic interactions between the two rings of subunits are stronger than between the subunits within a ring. the cterminal helix contribute strongly to the inter-ring hydrophobic interaction. asps are suggested to estimate the contribution of the hydrophobic energy to protein folding and subunit assembly and the binding of small molecules to proteins. title: determination of the complete three-dimensional structure of the trypsin inhibitor from squash seeds in aqueous solution by nuclear magnetic resonance and a combination of distance geometry and dynamical simulated annealing. authors: holak*, t.a.; gondol, d.; otlewski, j.; wilusz, t. max-planck-hstitut fiir biocbemie d- martinsried bei miinchen, federal republic of germany. title: interpretation of protein folding and binding with atomic crystal structure (atomic coordinates of cmti-i was determined by solvation parameters. x-ray diffraction method). results: in order to obtain information of the d structure of the free cmti-i in solution, a total of inhibitor structures were calculated by a combination of distance-geometry and dynamical simulated annealing methods, resulting in well defined d positions for the backbone and side-chain atoms. fig. shows the superposition of the backbone (n, c", c, ) atoms of the structures best fitted to residues to (binding loop): the average rms difference between the individual structures and the minimized mean stfucture was . (* . ) a for the backbone atoms and . (+ . ) a for all heavy atoms. title: electron transport in sulfate reducing bacteria. molecular modeling and nmr studies of the rubredoxin-tetraheme-cytochrome-c complex. biological material: a) sulfate reducing bacterium (desulfovibrio vulgaris); b) rubredoxin (iron-sulfur protein); c) tetraheme cytochrome c from d. vulgaris; e) flavodoxin. detected in the segments from the residues to and - . fig. shows the best superposition (residues to ) of the nmr and crystal structure of cmti-i indicating the backbone c, c", n, , as well as the disulfide c and s atoms: fig. it was demonstrated that uncertainty in nmr structure determination can be eliminated by including stereospecific assignments and precise distance constraints in the definition of the structure. crystal structure (coordinates of the crystal structure of the compounds were determined by x-ray crystallography). results: the speed of the homolysis of the organometallic bond is " times higher in the apoenzyme bound coenzyme biz than in a homogenous solution. structural changes occurring during the co-c bond homolysis of the coenzyme biz leading from cobalt(ii ) corrin to cobalt(i ) corrin were investigated. fig. shows the superposition of structures of the cobalt corrin part of the biz (dotted line) and of cob(ii)alamin (solid line): biological material: apoenzyme, binding the coenzyme biz and the data determined: . shows that the crystal structure of biz and cob(i )alamin are strikingly similar and offers no explanation for the mechanism of the protein-induced activation of homolysis. it was suggested that the co-c bond may be labiiized by the apoenzyme itself and in addition to a substrate-induced separation of the homolysis fragments (which mights be supported by a strong binding of the separated fragments to the protein). 'h nmr (complete stereospecific assignments were carried out and proton-proton distance constrains were determined by the analyses of dqf-cosy, hohaha and noesy spectra); nh, ah, oh [chemical shifts (ppm) of proton resonances of human eti ; d structure ( d structure of et was calculated using the distance geometry program dadas based upon the noesy proton-proton distance constrains determined by nmr spectroscopy); [root mean square distance (a) between et conformers calculated by distance geometry (dadas)]. results: the solution conformation of et has been determined by the combined use of d 'h nmr spectroscopy and distance geometry calculations. five structures of et have been calculated from different initial conformations. the superposition of the backbone atoms the calculated structures is shown in fig. . the average rms value in the core region for the main-cahin atoms was . a. quant. struct.-act. relat. , - ( ) the lack of specific interactions between the core and tail portions of et and a characteristic helix-like conformation in the region from lys' to cys" was shown. literature data indicated that neither the eti - nor the etl - truncated derivatives of et showed constricting or receptor binding activity suggesting that the et receptor recognizes an active conformation consisting of both the tail and core portion. the present study, however, suggested that the receptor bound conformation of et is probably different from that in solution because the lack of interaction between tail and core. the hydrophobic nature of the tail suggested the importance of a hydrophobic interaction with the receptor. compounds: triphenyl-methyl-phosphit cation (tpmp+). biological material: nicotinic acetylcholine receptor (achr), a prototype of the type i of membrane receptor protein from the electric tissue of torpedo and electrophorus. results: a computer model of the achr ion channel has been proposed. fig. shows the side view of the ion channel model with five pore-forming mz-helices and the channel blocking photoaffinity label (tpmp +) represented by the dotted sphere: fig. it was supported by electronmicroscopy, electrophysiological-and biochemical experiments that the mz-helices were formed by homologous amino acid sequences containing negatively charged amino acid side chains which act as the selectivity filter. the amino acid side chains may undergo conformational changes during the permeation of the cation. the predicted transmembrane folding of four transmembrane a-helices of type i receptors is shown in fig. : fig. energy profile calculations indicate that other transmembrane sequences of the receptor protein besides m may affect the ion channel. source: cabios , ( ), - . results: an interactive computer program tefoojj has been developed for drug design on ibm/pc and compatible computers. the program contains the following modules and performs the following calculations: a) series design for selecting an optimal starting set of compounds using a modified version of austel's method; b) regression analysis calculating the hansch's equation; c) hansch searching method using the equation calculated by the regression analysis routine or the use of an input equation for the identification of the most active compounds; d) geometrical searching methods for finding the optimum substituents in the parameter space using the sphere, ellipse, quadratic or polyhedric cross algorithms with or without directionality factors; e) space contraction for reducing the dimension of the parameter space by eliminating non-significant parameters; f) an example is given for the lead optimization of an aliphatic lead compound correctly predicting the n-pentane to be the optimum substituent. results: a new expert system sparc is being developed at epa and at the university of georgia to develop quantitative structure-activity relationships for broad compound classes: a) classical qsar approaches predict therapeutic response, environmental fate or toxicity from structure/property descriptors quantifying hydrophobicity, topological descriptors, electronic descriptors and steric effects; b) sparc (sparc performs automated reasoning in chemistry), an expert system written in prolog, models chemistry at the level of physical organic chemistry in terms of mechanism of interaction that contribute to the phenomena of interest; c) sparc uses algorithms based on fundamental chemical structure theory to estimate parameters such as acid dissociation constants (pk,s), hydrolysis rate constants, uv, visible and ir absorption spectra, and other properties; d) the information required to predict input data for broad classes of compounds is dispersed throughout the entire ir spectrum and can be extracted using fourier transforms; e ) the accuracy of sparc algorithm was demonstrated on the close match of calculated and experimental pk, values of carboxylic acid derivatives near to the noise level of measurement. abtstr. - quant. struct.-act. relat. , - ( ) results: a new stand-alone molecular simulation program, nmrgraf integrating molecular modeling and nmr techniques has been introduced by biodesign inc. a) nmrgraf is a molecular modeling program utilizing force fields which incorporate empirical properties such as bond lengths and angles, dihedral, inversion and nonbonded interactions, electrostatic charges and van der waals interactions; b) the molecular structural properties are combined with nuclear overhouser effect (noe) and j-coupling nmr data (experimental interproton distance constrains and bond angle data); c) the nmr proton-proton distance data are accurate only at relatively short distances ( to a) which restricts the use of nmr noe approaches only for the analysis of molecules with known x-ray structure; d) the combination of nmr and molecular modeling approaches, however, makes it possible to model virtually any molecule even if its structure does not exist in the databases. title: electronic structure calculations on workstation computers. results: the main features of the program system turbomole for large-scale calculation of scf molecular electronic structure on workstation computers is described: a) the program system allows for scf level treatments of energy, first-and second-order derivatives with respect to nuclear coordinates, and an evaluation of the me? correlation energy approximation; b) the most important modules of turbomole are (i) dscf performing closed and open shell rhf calculations; (ii) egrad used for analytical scf gradient evaluations; (iii) kora calculating direct two-electron integral transformation (iv) force for the computation and processing of integral derivatives and the solution of cphf equations; c) comparison and evaluation of timings of representative applications of turbomole on various workstations showed that apollo ds o.ooo and iris d were the fastest and comparable to the convex c in scalar mode. results: a new algorithm has been developed for the calculating and visualizing space filling models of molecules.the algorithm is about times faster than a conventional one and has an interesting transparency effect when using a stereo viewer. a) the algorithm is briefly described and and the result is visualized on modeling a ribonucleotide unit; b) in the order of increasing atomnumbers, the (x,y) sections of the hemispherical disks of the atoms are projected on the screen with decreasing value of the azimuthal angle (p) of the van der wads radius and as the value of p decreases the projection is increasingly whitened to obtain shading effect on the surfaces of the spheres; c) the transparency of the van der waals' surfaces of atoms of a molecule makes possible to perceive almost the whole space filling structure and not only the surface, hiding the underlying atoms. title: supercomputers and biological sequence comparison algo-authors: core*, n.g. ; edmiston, e.w. ; saltz, j.h. ; smith, r.m. rithms. yale university school of medicine new haven ct - , usa. source: computers biomed. res. , ( ) , - . compounds: dna and protein fragments. chemical descriptors: sequences of monomers. results: a dynamic programming algorithm to determine best matches betweenpairs of sequences or pairs of subsequences has been used on the intel ipsc/l hypercube and ,the connection machine (cm-i). parallel processing of the comparison on cm-i results in run times which are to times as fast as the vax , with this factor increasing as the problem size increases. the cm-i and the intel ipsc hypercube are comparable for smaller sequences, but the cm-i is several times quicker for larger sequences. a fast algorithm by karlin and his coworkers designed to determine all exact repeats greater than a given length among a set of strings has been tried out on the encore multimax/ o. the dynamic programming algorithms are normally used to compare two sequences, but are very expensive for multiple sequences. the karlin algorithm is well suited to comparing multiple sequences. calculating a multiple comparison of dna sequences each - nucleotides long results in a speedup roughly equal to the number of the processors used. source: cabios , ( ), . results: a program has been developed for the prediction and display of the secondary structure of proteins using the primary amino acid sequence as database. a) the program calculates and graphically demonstrates four predictive profiles of the proteins allowing interpretation and comparison with the results of other programs; b) as a demonstration the sliding averages of n sequential amino acids were calculated and plotted for four properties of human interleukin : (i) plot of the probabilities of a-helix, p-structure and p-turns according to chou and fasman; (ii) @-turn index of chou and fasman; (iii) plot of the hydrophobicity index of hopp and woods; (iv) flexibility index of karplus and schulz; c) the regions of primary structure having properties which usually go together agreed reasonably well with each other, i.e. loops and turns with bend probability and hydrophilicity with flexibility. title: dsearch. a system for three-dimensional substructure searching. quant. struct.-act. relat. , - ( ) sci. , ( ) , - . results: the search for threedimensional substructures is becoming widely used in d modeling and for the construction of pharmacophores for a variety of biological activities. a system ( dsearch) for the definition and search of three-dimensional substructures is described: a) representation of atom types consists of five fields (i) element (he-u); (ii) number of non hydrogen neighbors (bonded atoms); (iii) number of 'k electrons; (iv) expected number of attached hydrogens; (v) formal charge; (vi) four type of dummy atoms are also used to define geometric points in space (e.g. centroid of a b) definition of queries (i) definition of spatial relationship between atoms; (ii) matches in atom type (iii) preparation of keys (constituent descriptors); (iv) execution of key search; (v) geometric search including the handling of angleldihedral constrains and takes into account "excluded volume"; c) time tests showed that a search of d structures with to atoms in large databases with more than , entries took only a few minutes ( - s). results: a database containing about , compounds in connection tables and , experimentally determined structures from the cambridge structural database has been transformed into a database of low energy d molecular structures using the program concord. the strategy for building the d database consisted of the following four steps: a) generation of approximate d coordinates from connection tables (hydrogens were omitted); b) assignment of atom types from connection table information characterized by five descriptors: (i) element type (he-u); (ii) number of attached non-hydrogen neighbors ( - ); (iii) number of 'k electrons ( - ); (iv) calculated number of attached hydrogens ( - ); (v) formal charge ( - , , ); c) addition of three types of chemically meaningful dummy atoms for purposes of d substructure searching: (i) centroids of planar and -membered rings; (ii) dummy atoms representing the lone electron pairs; (iii) ring perpendiculars positioned orthogonal to and . a above and below each planar ring; d) efficient storage of the resultant coordinate database indexing the compounds with identification number; e) the database can be used among others to deduce pharmacophores essential for biological activity and to search for compounds containing a given pharmacophore. title source: c&en , ( ) , - . results: a complex carbohydrate structure database (ccsd) has been developed by the complex carbohydrate research center at the university of georgia, having more than structures and related text files, with about more records to be added over the next two years. the following are the most important features of ccsd: a) in ccsd, database records include full primary structures for each complex carbohydrate, citations to papers in which sequences were published, and suplementary information such as spectroscopic analysis, biological activity, information about binding studies, etc; b) structural display format visualize branching, points of attachment between glycosyl residues and substituents, anomeric configuration of glycosyl linkages, absolute configuration of glycosyl residues, ring size, identity of proteins or lipids to which carbohydrates are attached and other data; c) it is planned that ccsd will provide threedimensional coordinates, to visualize and rotate the structures in stereo and study their interaction with proteins or other biopolimers. probing bioactive mechanisms p commercial carbamate insecticides of type ii, where r' = h, s-bu biological material: acetylcholinesterase. data determined: d [distance (a) between the serine oxygen of acetylcholinesterase and the methyl substituents of the carbamate and phosphate inhibitors molecular modeling (models and minimum energy conformations of acetylcholine, aryl carbamate and phosphate ester inhibitors were created using the draw mode of a maccs database and the prxbld modeling program) results: transition state modeling of the reaction of the serine hydroxyl ion of acetylcholinesterase with the methylcarbamoyl and dimethyl phosphoryl derivatives of , -dimethyl-phenol showed that the active site binding for these two classes of acetylcholinesterase inhibitors should be different. the model shows that the distances between the serine oxygen and the ring substituents (meta-and para-me-thy groups) are different in both spacing and direction. fig. shows the transition state models of serine hydroxyl d values for the meta-and para-methyl substituents of n-methylcarbamate and dimethylphosphate were meta = . , para = . and meta = . , para = . a, respectively title: a comparison of the charmm, amber and ecepp potentials for peptides. . conformational predictions for the tandemly repeated peptide (asn-ala-asn-pro) biological material: tandemly repeated peptide (asn-ala-asn-pro) which is a major immunogenic epitope in the circumsporozoite (cs) protein of plasmodium falciparum conformational analysis dream or reality? a authors: nbray-szab *, g.; nagy, j.; bcrces, a. priori predictions for thrombin and ribonuclease mutants molecular modeling (geometric model of subtilisin and trypsin was built using protein data bank coordinates and model of thrombin was built using the theoretical coordinate set of graphic representations of the triad of the tetrahedral intermediate for the enzymes formed on the active side chain and residues (ser- , his- and asp in subtilisin; ser- , his- and asp-i in trypsin and thrombin; his- , lys- and his-i in ribonuclease a) were created using pcgeom electrostatic properties (electrostatic surfaces and fields of the molecules were calculated and displayed using amber and associated programs); [chemical shift (ppm) measured by nmr results: a hypothetical model between rubredoxin and cytochrome c was built as a model for the study of electron transfer between different redox centers, as observed in other systems. fig. i shows the main chains atoms of the proposed complex where the hemes of the cytochromes are shown along with the center of rubredoxin and stabilized by hydrogen bonds and charge-pair interactions (the nonheme iron of the rubredoxin is in close proximity to heme of cytochrome c ): spectroscopy].the model was consistent with the requirements of steric factors, complementary electrostatic interactions and nmr data of the complex. comparison of the new model and the nmr data of the previously proposed flavodoxin-cytochrome c complex showed that both proteins interacted with the same heme-group of cytochrome c . title: nuclear magnetic resonance solution and x-ray structures of squash trypsin inhibitor exhibit the same conformation of the proteinase binding loop.authors: holak*, t.a.; bode, w.; huber, r.; otlewski, j.; wilusz, t. max-planck-institut fiir biochemie d- martinsried bei munchen, federal republic of germany.source: j. mol. biol. , no. , - . biological material: a) trypsin inhibitor from the seeds of the squash cucurbita maxima; b) p-trypsin and trypsin inhibitor complex.title: retention prediction of analytes in reversed-phase high-performance liquid chromatography based on molecular structure. . quant. struct.-act. relat. , - ( ) results: an expert system (cripes) has been developed for the prediction of rp-hplc retention indices from molecular structure by combining a set of rules with retention coefficients stored in a database. the method underlying the system is based on the "alkyl aryl retention index scale" and aims to improve the reproducibility of prediction and compatibility between various instruments and column materials. the vp-expert system shell from microsoft was used for the development. the performance of cripes was demonstrated on several subtypes of substituted benzenes (phenacyl halides, substituted arylamines, arylamides and other types). in general the calculated and measured retention indices agreed well but relatively large deviations were observed between the ie and ic values for phenacyl bromides and chlorides, o-and p-bromo anilines, n-methylbenzamide and n,n-dimethylbenzamide and phthalate esters. the extension of the database with further interaction values was regarded as necessary for a consistently high accuracy at prediction. [out-of-plane bending energy (kcal/mol) given by the formula e, = kd', where d is the distance from the atom to the plane defined by its three attached atoms and k is a force constant]; eb e, [torsional energy (kcal/mol .deg ) associated with four consecutive bonded atoms i,j,k,l given by the formula e, = ki,j.k,l(l fs/lslcos(lslbi,j,k,~)), where b is the torsion angle between atoms i j , k and , s and k are constants]; e, [potential energy (kcallmol) (nonbonded energy term) associated with any pair of atoms which are neither directly bonded to a common atom or belong to substructures more than a specified cutoff distance away given by the formula e, = kij(l. la" - . /a ), where a is the distance between the two atoms divided by the sum of their radii, and k is the geometric mean of the k constants associated with each atom]. results: model geometries produced by the tripos . force field have been assessed by minimizing the crystall structures of three cyclic hexapeptides, crambin and diverse complex organic compounds. comparative force field studies of the tripos . , amber and amberlopls force fields were carried out by energy minimization of three cyclic hexapeptides starting from the crystal structures showed the tripos . force field superior to the others with the exception of amber, ecep and levb force fields as published by other workers. a direct comparison between the performance of tripos . and . amber using isolated crambin showed that the bond and torsion angles of tripos . averaged closer to the crystal structure than the angles calculated by amber (rms = . a, . deg and . deg for bond lengths, angles, and torsions, respectively, and rms = . a for heavy atoms). fig. shows the superimposed structures of crambin before and after energy minimization:fi . tripos . was assessed for general purpose applications by minimizing organic compounds starting from their crystal structures. the test showed that tripos . had a systematic error in overestimating the bond lengths of atoms in small rings. statistical analysis of the results showed that t r i p s . had an acceptable overall performance with both peptides and various organic molecules, however, its performance was not equal to the best specialized force fields. title: new software weds molecular modeling, nmr. author: krieger. j. c&en sixteenth st., n.w., washington dc , usa. source: c&en , ( ) , . key: cord- -d gcfepx authors: fan, samuel w; george, richard a; haworth, naomi l; feng, lina l; liu, jason y; wouters, merridee a title: conformational changes in redox pairs of protein structures date: - - journal: protein science doi: . /pro. sha: doc_id: cord_uid: d gcfepx disulfides are conventionally viewed as structurally stabilizing elements in proteins but emerging evidence suggests two disulfide subproteomes exist. one group mediates the well known role of structural stabilization. a second redox-active group are best known for their catalytic functions but are increasingly being recognized for their roles in regulation of protein function. redox-active disulfides are, by their very nature, more susceptible to reduction than structural disulfides; and conversely, the cys pairs that form them are more susceptible to oxidation. in this study, we searched for potentially redox-active cys pairs by scanning the protein data bank for structures of proteins in alternate redox states. the pdb contains over unique redox pairs of proteins, many of which exhibit conformational differences between alternate redox states. several classes of structural changes were observed, proteins that exhibit: disulfide oxidation following expulsion of metals such as zinc; major reorganisation of the polypeptide backbone in association with disulfide redox-activity; order/disorder transitions; and changes in quaternary structure. based on evidence gathered supporting disulfide redox activity, we propose disulfides present in alternate redox states are likely to have physiologically relevant redox activity. emerging evidence supports the concept of two distinct types of disulfides in protein structures which have different functional roles. the best known group act as structural stabilizers in proteins, significantly lowering the gibb's free energy of the protein chain by crosslinking it, resulting in proteins that are more resistant to denaturation. the second group are redoxactive and best known for their catalytic role in proteins such as thiol-disulfide oxidoreductases. an increasing role for the redox-active group in protein redox regulation is emerging. , these disulfides act as redox-sensitive switches and are best viewed as posttranslational modifications akin to phosphorylation. it has now been established that oxidative regulation: important in processes such as the cell cycle, apoptosis, and response to oxidative stress; is an important control mechanism in the cytosol. transient oxidation mediates control of enzymes, signaling pathways, as well as transcriptional and translational control. however, these characterized cytosolic proteins likely only represent the tip of the iceberg. increasingly, reduction of disulfides is also emerging as a key regulator of protein function in oxidizing environments. [ ] [ ] [ ] importantly, ageing and several major diseases including neurodegenerative diseases such as alzheimer's disease, type ii diabetes, cancer, and cardiovascular disease have been associated with abnormal redox conditions. structural and redox-active disulfides can be distinguished by their redox potentials. disulfide redox potentials measured in thiol-disulfide oxidoreductases range from À to À mv. [ ] [ ] [ ] [ ] for disulfides serving structural purposes, the redox potential can be as low as À mv. computational methods for distinguishing the two groups would be a boon. reactivity of a redox-active cys is modulated by its immediate protein environment. in addition to the effects of the electrostatic environment, physical stress applied to a disulfide bond both through stretching and twisting enhances its reactivity. these forces can be applied to a protein dynamically or may be captured during the protein folding process, rendering the disulfide poised to act upon exposure to variations in redox conditions. because of their enhanced reactivity, redoxactive disulfides are also more susceptible to cleavage/ oxidation under nonphysiologic conditions. for instance, redox-active disulfides are prone to cleavage by synchrotron radiation during the process of x-ray structure determination. [ ] [ ] [ ] other factors such as the ph of crystallization or the presence of mutations that change the electrostatic environment near a disulfide may also influence reactivity. here, we mined the protein data bank (pdb) for highly similar proteins that have been solved in multiple redox states, that is, disulfide-bonded in one structure and reduced in another. we present evidence that most of the disulfides present in our redox pairs dataset are likely to be physiologically redox-active. many of the proteins have known thiol-based redox activity, while others have a high likelihood of thiol-based redox activity based on their involvement with redox processes. we also observed and classified types of conformational changes which occur during redox transitions. the redox pairs dataset contains , pairs of protein structures consisting of protein chains, of which ( . %) are high-resolution structures (< . Å). many structures are of the same protein solved in different redox environments. a filtered dataset consists of unique protein groups clustered at % sequence identity. further evidence of disulfide redox activity was sought based on experimental data in the literature, association of protein function with known redox activity, and implication of the disulfide in redox activity by independent computational prediction methods. for proteins, direct experimental evidence of redox activity for the particular disulfide was available (des-ignated by ''k'' in table s , supporting information). the proteins belong to several major groups known to have redox activity. unique redox pairs are associated with aerobic metabolism in eukaryotes or carbon fixation in plants (in groups d and e of table s ); a further five are associated with maturation of proteins of these pathways (in groups e and g). four redox pairs are associated with ion channel activity (group a): two of these, the chloride channel clic and the detoxification enzyme arsenate reductase, have known redox activity , ; while a third, aquaporin, was recently implicated as a pore for the reactive oxygen species hydrogen peroxide, as part of the redox signaling process. seven redox pairs are associated with the cell cycle, a process known to be redox regulated (group b). formation of disulfides involving the catalytic cys in the phosphatases pr- and cdc b regulate their resident protein's function. , redox pairs (group d) are involved in transcriptional control, replication, and dna synthesis including the oxidative stress response transcription factor oxyr and ribonucleotide reductases from several species in multiple redox states. redox pairs generated or destroyed reactive oxygen species (groups f and y). the latter comprised three different peroxiredoxins as well as superoxide dismutases from several species. four different amine oxidases generate peroxide while inducible nitric oxide synthase generates no. other large groups consist of proteins devoted to cell entry figure . to further assess the likelihood that the presence of proteins in the pdb with disulfides in both redox states is an indicator of physiological disulfide redox activity, we looked for gene ontology (go) terms overrepresented in the redox pairs dataset compared to the entire pdb. go is a dynamic controlled vocabulary of over , terms used to describe molecular function, process and location of action of a protein in a generic cell. the analysis revealed many go terms consistent with redox function, including cell redox homeostasis and multiple pathways of cysteine biosynthesis (supporting information table ). interestingly, several of the enriched go terms are linked to proteins mediating cell entry, including bacterial toxins and viral entry proteins. reduction of disulfides, often accompanied by irreversible conformational changes, have previously been implicated as a general mechanism in cell entry proteins of diverse types. experimental evidence directly supporting such a general process exists for hiv gp , newcastle disease virus, sars coronavirus and chlamydia. [ ] [ ] [ ] [ ] thus the go terms support redox roles for proteins in the redox pair dataset. disulfides were tested computationally for likely redox activity by screening the dataset for oxidized structures where the disulfide had particular properties previously associated with redox activity. flagged disulfides were those (a) with high torsional energies; (b) found in known redox-active sequence motifs; or (c) which disobeyed known rules of protein stereochemistry. studies of disulfide-bonds in model proteins have shown that those bonds with high torsional energies are more easily cleaved than disulfide-bonds with lower stored energy. [ ] [ ] [ ] [ ] disulfides in proteins have torsional energies > kj mol À : a con-servative threshold associated with redox activity. these are designated by ''h'' in supporting information table . redox-pair disulfides were also found in sequence motifs previously shown to be associated with redox activity. proteins, designated by ''m'' in table s , contain known redox-active motifs such as the cxxc motif: a redox-active disulfide motif commonly found in the thioredoxin fold, but which has also arisen convergently in other folds. a further proteins, designated by ''f'' in table s , contain disulfides in protein structures that disobey known rules of protein stereochemistry. , these disulfides, which table . key to groups: a, ion channel, ion pump modifier; b, protein folding, isomerization; c, cell cycle; d, calvin cycle; e, photosynthesis, respiration, transport, maturation of proteins involved in these processes; f, destruction of oxygen radicals; g, redox metal homeostasis; h, amino acid metabolism; i, sulfur metabolism; j, nucleotide metabolism/homeostasis; k, isoprenoid biosynthesis; l, rna-interacting; m, proteases; n, dna repair; o, actin-related; p, carrier proteins; q, other metabolism; r, nadp þ -dependent oxidoreductase; s, other oxidoreductase; t, signaling; u, development; v, cell growth pathways; w, enzyme inhibitors; x, redox homeostasis; y, ros generating; z, apoptosis; a, protein fate; b, cell entry; c, cell adhesion; d, dna-interacting proteins; e, translation; f, immunity; g, carbohydrate metabolism; h, nad þ -dependent oxidoreductases; i, ca handling; j, methyltransferase; k, other. fan et al. we refer to as ''forbidden'' disulfides, conform to canonical structural motifs. deformation of the resident structure enhances disulfide reactivity by inducing stress in the protein chain. , forbidden disulfides are proposed to have a functional role through redox activity. in summary, a total of proteins in the redox pair dataset contain disulfides predicted to be redox-active by a second independent bioinformatic analysis method. nonetheless, it is possible that some proteins with alternate cys-pair redox states are non-native, that is, not encountered under normal, or abnormal, physiological conditions. we reasoned that proteins demonstrated selective reduction of disulfides were more likely to represent physiological states. accordingly, for proteins with more than one disulfide, the dataset was partitioned into pairs where all the disulfides were reduced and those where some disulfides were selectively reduced while others remained intact, to further test the likelihood of involvement in physiological redox processes. the enriched go terms for both the partially and fully reduced sets contained terms previously associated with thiol-based redox regulation indicating both the fully and partially reduced sets have likely redox activity (supporting information table ). having established the redox-pair dataset is enriched in proteins involved in redox-regulated processes, we studied conformational changes between redox pairs of proteins. for many proteins in the data set there were negligible conformational differences between the sg atoms of each cys residue of the reduced and oxidized states (see fig. ). for this group, other factors may be required for, or preclude, conformational changes in these proteins. crystal packing forces, for example, may prevent a subsequent conformational change after cleavage of the disulfide by synchrotron radiation. for another group, the small distance changes of - Å observed are most likely due to rotation of one or both cys sidechains toward each other to form the sas bond. some of the proteins exhibiting these small conformational changes are oxidoreductases with catalytic thiols such as thioredoxin, thioredoxin reductase, and protein disulfide isomerase. for proteins which exhibit major conformational changes, four classes were identified based on the nature of the change. these groups were: proteins that oxidize disulfides following expulsion of metals such as zn; proteins that exhibited major reorganization or ''morphing'' of portions of the polypeptide backbone in association with disulfide redox-activity; proteins that exhibited order/disorder transitions; and proteins that exhibited changes in quaternary structure. redox regulation of proteins that form disulfides following expulsion of zn is an emerging area of signaling. for example, reversible redox regulation of the zn-binding site has recently been proposed for small tim proteins of the mitochondrial intermembrane space (ims). small tim proteins have an essential role in the transport of proteins into the ims. these proteins can exist in both a reduced zn-bound state figure . change in disulfide separation in protein redox pairs. excluding interchain disulfides, around % of redox structure pairs have a change of over Å in the sulfur atom separation between the oxidized and reduced structures. maximal intrachain differences of up to . Å were apparent in -deoxy-d-xylulose- -phosphate reductoisomerase (dxp reductoisomerase) and the rna sulfuration enzyme trmu. and an oxidized disulfide-bridged state. both states likely represent physiological forms of the proteins which are adopted in response to changing redox conditions in the ims. in addition, expelled zn acts as a second messenger to effect further cellular responses. proteins that form disulfides following expulsion of metals such as zn were identified by searching for ligation of metals by cys in reduced structures. a total of redox pairs in proteins were found (table i , z in s ). proteins identified include transcriptional regulators such as smad and anti-trap; transcriptional silencers such as sirt ; and the enzymes of sulfur metabolism, bhmt, mete, and meth. also notable were a number of rna-interacting proteins including amino-acid-trna synthetases, rna/dna polymerases, ribonucleotide reductase and the ribosome. in addition to zn, a few other bound metal atoms were represented including fe, ni, cu. there were several examples of inorganic iron-sulfur (fe-s) clusters associated with redox-active disulfides including: xanthine dehydrogenase, a major source of ros in the failing heart; and components of respiratory complexes (table i) . other proteins containing fe-s clusters contained other redox-active disulfides that were not associated directly with the fe-s cluster. all proteins containing fe-s clusters (designated with ''c'' for cofactor) are listed in table i regardless of whether the fe-s cluster is directly associated with a labile dithiol pair or not. crystallographers typically differentiate two types of zn sites: catalytic zn sites, which in their simplest, mononuclear form have three amino-acid sidechains and one water molecule as ligands; and structural zn sites, which have four amino acid ligands. from a redox point of view, a second variable -lability, is important. some structural zn sites expel zn upon variation of physiological redox conditions, whereas others bind zn tightly, and would not be expected to release zn under physiological conditions. we refer to this first redox-regulated group as ''labile'' zn sites; and the second, purely structural group, as ''inert.' ' we wished to determine whether zn sites associated with redox pair disulfides detected in the study were labile or inert. the number of zn ligands and their identity are key indicators of zn site lability. for catalytic mononuclear zn sites, three amino acids, which are often his, ligate the zn. in binuclear zn sites there are usually six ligands, and the carboxylated residues asp and glu predominate over his. , cys is more commonly found as a ligand in structural zn sites which generally have four ligands. on consideration of ligand type alone, s sites -those with four cys ligands, are the most labile type of structural zn site, and are almost as reactive as a free thiol. most of the structural zn sites identified in this study were of the s type (table i) , suggesting they are labile. four catalytic zn sites were also found in the dataset. these catalytic zn sites are highly unusual because of their utilization of cys as a ligand instead of the more usual his, glu, or asp. two of the catalytic sites are within bhmt and meth: enzymes of sulfur metabolism, which is redox-regulated in plants. selection of sulfur ligands for zn in these enzymes may confer an additional level of redox control on sulfur metabolism. as an additional control, we checked whether metal atoms were expelled from oxidized structures. a metal atom is not present in over a third of the oxidized structures. for another structure, aspartate transcarbamylase ( tug), the metal site was only partially occupied in the oxidized chain. for the remaining structures where the metal atom is retained in the oxidized structure, the shortened sg-sg distance may be caused by a heterogeneous mix of oxidized and reduced, metal-bound structures in the ensemble. we reasoned that if the metal was expelled from some members of the ensemble, or partially expelled (still in the site but held, for example, by only two of four potential ligating cys), then the b-factor should be higher than for chains where no shortened sg-sg distances are observed. although b-factors cannot be compared between different structures, for some of the redox pairs, different chains of the same structure in different disulfide redox states could be compared. our analysis confirmed that the b-factor of the metal was higher in the oxidized chains for % of structures (see fig. ). a similar result was obtained for proteins with fe-s clusters. there were no instances of expulsion of the fe-s cluster from the oxidized structure but oxidation of fe-ligating cys residues to form disulfides was generally associated with a higher b-factor of the relevant fe atom than fe atoms where the ligating cys remained reduced. in contrast, in reduced structures all fe atoms of the fe-s cluster had similar b-factors. further inspection of proteins with likely redoxactive zn sites reveals that two forbidden disulfide motifs, the jump strand disulfide (jsd) and a type of b-diagonal disulfide (bdd), are repetitively associated with redox-active zn-binding sites in the dataset. oxidized structures that contain a bdd include isoleucyl trna synthetase ( ile - , - ), the apoptosis-related proteins birc and survivin ( xb , - ; f h, - ), aspartate transcarbamylase ( r b, - ) and the dna repair enzyme recr ( vdd, - ). oxidized structures that contain a jsd include the amino-acyl trna synthetases for leu and ile ( obh, - ; jzs, - ), the enzymes of sulfur metabolism meth and bhmt ( q m, - ; lt , - ) and gtp hydrolase gtphi ( gtp, . a large group of proteins in the dataset were observed to undergo plastic deformations involving large scale rearrangements of the polypeptide backbone. we refer to these conformational changes as morphing transitions. these transitions, which typically involve unraveling portions of secondary structure, are illustrated in figure and in animations in the supporting information. the well known redox-sensitive protein oxyr belongs to this group. in oxyr, a protein of residues, significant refolding of the c-terminus occurs involving residues - . in the reduced structure, residues - form a short helix flanked by regions of coil. upon oxidation, residues - adopt a different fold with residues - and - forming two parallel b-strands joined by a loop with a disulfide-linkage between cys and cys . in the reduced oxyr structure, the two cys that form the disulfide are separated by almost Å. cys in oxyr is known to undergo alternate modifications such as glutathionylation. because of the spectacular nature of the transition between the reduced and oxidized structures, and the existence of an alternate mode of redox regulation, the physiological relevance of the disulfide-linked structure has been called into question. to determine whether the oxidized structure of oxyr is indeed an oddity, or if other proteins exhibit similar conformational changes, we systematically searched for morphing proteins in the dataset by scanning for protein structure pairs that exhibited large differences of the backbone torsional angles over at least seven contiguous residues. changes in pseudo-dihedral angles calculated using four consecutive ca atoms exceeding a summed threshold of degrees over seven consecutive residues were used to detect changes in secondary structure. twenty-two parents were found in the fully reduced set and four in the partially reduced set (designated m in supporting information table ). proteins showing the largest conformational changes are in table ii . these proteins include the cl À channel clic : a protein that forms ion channels by inserting itself into membranes in response to oxidation; the detoxification enzyme arsenate reductase, which undergoes multiple conformational changes associated with a disulfide relay as part there is partial oxidation of the metal site, the b-factor of the metal should be higher because it has fewer ligands and hence more conformational freedom. this is true for most structures (bars above the axis). standard errors are depicted where they could be calculated. key to structures: clostridium acetobutylicum thymidine kinase - xx ; e. coli aspartate transcarbamylase - r b, - tug; thermotoga maritima sir - h ; homo sapiens sir - j f; equus caballus alcohol dehydrogenase - axg; bacillus subtilis tryptophan rna-binding attenuator protein-inhibitory protein (antitrap) - bx ; severe acute respiratory syndrome coronavirus nonstructural protein - g t, - ga ; homo sapiens estrogen receptor - hcq; deinococcus radiodurans recombinational repair protein - vdd; norovirus c-like protease - wqs; moorella thermoacetica bifunctional carbon monoxide dehydrogenase/acetyl-coa synthase , and cyclic phosphodiesterase, a trna-splicing enzyme where the disulfide modulates access to the active site. like oxyr, several of these proteins exhibit large changes in ca separations of the two cys between alternate redox states (table ii) . thus large physical separation of the reduced cys pair seems to be a common feature of many of these proteins and does not preclude disulfide formation. another recognizable group of conformational changes involves order/disorder transitions. in these transitions, alternate redox states of the protein correlated with differing amounts of disorder in the protein structure, as evidenced by missing electron density. a total of redox pair structures in parents exhibited order/disorder transitions correlated with the redox state of the disulfide (table iii, fig. ). proteins were classified into four groups based on the location of disulfide-forming cys residues with respect to the disordered region in the protein chain. proteins in group a had a single cys residue in a disordered region, with the other cys residing on a more stable region of structure. in group b, both cys residues reside on a single disordered region. in group c, both residues lie on disordered regions which are not contiguous in the sequence. in group d, disulfide reduc-tion is associated with multiple regions of disorder in the reduced protein chain. disorder-to-order transitions have previously been observed upon binding of ligands. acquisition of order upon binding of ligands concomitant with disulfide formation was apparent for the group b oxidoreductase gdhb, where the disulfide straddled part of the pqq binding site, and the group a rna sulfuration enzyme ectrmu, where the disulfide straddles the trna-binding site. however, the reverse was true for the thermotoga maritima trna-processing enzyme, psi s, where significant disorder of the protein chain concomitant with disulfide reduction occurred upon binding of the trna substrate fragment. the introduction of disorder in this case may facilitate further co-operative binding of the rna and protein after the initial docking step. analyses of the composition of disordered sequences have detected amino acid biases from global frequencies. in particular, aromatic amino acids and cys are depleted in disordered sequences, whereas lys is enriched. , the disordered sequences in the redox pair dataset are also depleted in aromatics, in agreement with more general studies of disordered sequences, but are not depleted in cys, or enriched in lys; and thus do not conform entirely to previously detected sequence patterns. redox pair disordered sequences are instead enriched in oxygen-bearing sidechains such as asp, glu, ser and thr. some changes in secondary structure are also apparent during the order/disorder transitions. in the oxidized state, both cys residues generally reside on coil in their proteins, but for a small number of pairs the oxidized form has a secondary structure that ''melts'' upon reduction of the disulfide. examples include vitamin d-binding protein , and the e. coli oxidoreductase ghdb, , where helices are lost upon reduction; and the bacteriophage cell entry protein prd where a small b-sheet disappears upon reduction. the amount of disorder in the chains is also of interest. for most reduced structures, the disorder introduced upon reduction of the disulfide affects less than % of the polypeptide chain. however, three of the four group d proteins exhibited large regions of disorder in excess of % of the involved protein chain. interestingly, all were associated with a second more-ordered monomer that was oxidized. for example, the thermotoga maritima trna-processing enzyme, psi s undergoes a spectacular loss of secondary structure in one monomer upon binding of rna. it is possible that the fully reduced state of these protein dimers is uncrystallisable and that only partially and fully oxidized oligomers are sufficiently ordered to enable structure solution. we also investigated whether formation of interchain disulfide bonds altered the quaternary state of proteins. different redox states of the interchain disulfides are associated with two general types of quaternary interaction: those where the number of oligomers differs between the reduced and oxidized state; and those where the number of subunits involved in the oligomer stays constant, but the oligomerization interface changes. some redox pairs exhibit a mixture of the two types of quaternary change. for example, disulfide-bonded dimers of the macrophage sialoadhesin in the more ordered oxidized structure: a disulfide is formed between cys and cys which straddles a key phosphorylation site at thr . formation of the disulfide is proposed to inhibit kinase activity by recruiting the cognate phosphatase. the t-loop was recently identified as a target for selective cancer drugs. b: a larger order/disorder transition associated with expulsion of zn from the oxidized structure of leucyl-trna synthetase. , in the more ordered reduced structure, several new secondary structures condense enabling rigid co-ordination of the zn. zn is represented by red spheres in the reduced structure. oxidized structures are shown on the left, reduced structures on the right. fan et al. form an interface distinct from that found in trimers formed upon binding of sialosides, and may negatively regulate ligand binding. twenty-nine redox pair protein clusters with intermolecular disulfide bonds exhibit changes in quaternary structure upon oxidation/reduction. redox regulation of the quaternary state has previously been associated with cooperative behavior in proteins. for example, the hoxb protein binds dna in vitro when either oxidized or reduced but formation of an intramolecular disulfide between hoxb monomers is necessary for co-operative binding. other well-known examples are oxyr and p . several other proteins in the dataset exhibit co-operative behavior. cytoglobin is a member of the vertebrate haemoglobin family residing in the nucleus and cytosol that is upregulated figure . quaternary changes between different redox states. a: changes in the number of subunits in the multimer between different redox states of chloroplast gapdh. the a subunit of spinach chloroplast gapdh in a reduced, monomeric form (pdb hki) is shown on the right and the oxidized homotetramer in complex with nad þ (pdb nbo) is shown on the left. in higher plants, photosynthetic gapdh exists in vivo mainly as heteromeric isoforms, a b and a b being the most abundant, interconvertible conformations. the b subunit is homologous to the a subunit but has an additional nonhomologous cterminal extension. stable homotetramers of a subunits (a -gapdh) are found at low levels in chloroplast preparations and may be the only form of photosynthetic gapdh present in unicellular green algae and cyanobacteria. calvin cycle enzymes are known, as a group, to be redox regulated. the activity of the ab isoform of chloroplast gapdh is redox regulated by formation of a disulfide between c-terminal thiols in the nonhomologous part of the b chain in light, and its reduction in the dark. the interchain disulfide between a subunits may be important for redox control of the a isoform, which is known to form complexes with the small chloroplast protein cp and phosphoribulosekinase in the dark. the disulfide-forming cys ( nbo numbering) is not present in the b subunit of chloroplast gapdh which is transcribed from a different gene, or in cytosolic gapdhs. b: changes in the oligomeric interface in cytoglobin, a member of the vertebrate haemoglobin family residing in the nucleus and cytosol that is upregulated under conditions of hypoxia and certain types of stress. intermolecular disulfide bonds in cytoglobin modulate the quaternary structure and may regulate the ligand-binding properties. both the reduced and oxidized states are dimers. under conditions of hypoxia and certain types of stress (see fig. ). it has recently been implicated as a tumor suppressor. intermolecular disulfide bonds in cyto-globin modulate the quaternary structure and may regulate the ligand-binding properties (table iv) . the homologous lamphrey haemoglobin is in an equilibrium italicized prefixes are species abbreviations. protein names in roman font. ao, aminooxidase; bcp, bacterial comigratory protein peroxiredoxin; bmp , bone morphogenetic protein ; dna pol iii k, dna polymerase iii k; ef-ts, elongation factor ts; gapdh, glyceraldehyde- -phosphate dehydrogenase; gef, guanine nucleotide exchange factor; gga , golgi-localized c adaptin; grp , guanine nucleotide exchange factor and integrin binding protein homolog grp ; grx, glutathione reductase; pcbp , poly(c)-binding protein- ; phsam domain, polyhomeotic-proximal chromatin protein sterile alpha motif domain; pnmt, phenylethanolamine n-methyltransferase; rubiscol, ribulose- , -bisphosphate carboxylase/oxygenase large subunit; sam-s-adenosyl-l-methionine; vegf, vascular endothelial growth factor; ae, aeropyrum pernix; af, archaeoglobus fulgidus; bpv, bovine papilloma virus; cr, crithidia fasciculata, dm, drosophila melangaster; fmdv, foot and mouth disease virus; hpv, human papilloma virus; hs, homo sapiens; me, methanobacterium thermoautotrophicum; mm, mus musculus; mp, mycoplasma pneumoniae; nt, nicotiana tabacum; ps, pisum sativum (pea); rn, rattus norvegus; sp, spinacia oleracea; td, themiste dyscritum; tt, thermus thermophilus. a nd cys in different monomer. state between a low o affinity oligomer and a high-affinity monomer which contributes to its co-operativity. there was also a small group of transmembrane receptors which form covalent dimers in a neck region close to the membrane. these include the low-density oxidized ldl receptor, and the asialoglycoprotein receptor. covalent dimer formation may promote further quaternary changes such as higher oligomer formation in response to bound ligands as has been recently demonstrated for the metabotropic glutamate receptors. relevance to disease importantly, ageing and several major diseases including neurodegenerative diseases such as alzheimer's disease, type ii diabetes, cancer and cardiovascular disease have been associated with abnormal redox conditions. inadvertent triggering of redox-active disulfide switches is a likely contributor to these phenotypes. this view is supported by the association of several redox pairs in the dataset with disease. sod (also known as cuzn-sod) is an enzyme mutated in familial lateral sclerosis (fals), an age-dependent degenerative disorder of motor neurons in the spinal cord, brain stem and brain. sod is largely localized to the cytosol but is also found in the intermembrane space of mitochondria. sod is activated by formation of the disulfide and insertion of cu by the copper carrier ccs. both of these proteins are represented in the redox pair dataset (tables i and s ). the conserved disulfide in sod is essential for activity and unusual because it remains oxidized in the cytosol. although the disulfide is intrachain, it is thought to play a critical role in stabilizing the sod homodimer. formation of the disulfide is part of the sod maturation process which is regulated by physiological oxidative stress. the three reduced proteins in the dataset are three distinct mutants associated with familial lateral sclerosis (fals): g r, h r, and i t. the exclusive association of the reduced proteins with the disease mutants suggests redox imbalance of this disulfide is implicated in the disease. this hypothesis is supported by the finding that fals mutants have the common property of being more susceptible to disulfide reduction than wild-type protein. toxicity associated with disulfide-linked multimerization of sod mutants has recently been implicated in the disease mechanism. several types of conformational change were observed in the redox pair dataset suggesting there may be common structural modes of redox regulation. proteins exhibited disulfide oxidation following expulsion of metals such as zn, morphing, order/disorder transitions, and changes in quaternary structure. in an individual protein more than one mode may be employed. for example in leu-trna synthetase, part of the protein chain becomes disordered upon zn expulsion but this is not the case for all proteins with labile zn. redox regulation of proteins that form disulfides following expulsion of zn is an emerging area of signaling. well known and characterized examples including hsp which is activated by zn expulsion as part of the oxidative stress response ; and the small tim proteins which are kept in a transport-competent state in the cytosol by conjugation with zn. upon delivery to mitochondria, zn is expelled from small tim proteins which are activated by disulfide formation. zn is not expelled from all protein zn-binding sites with equal facility, and is probably not expelled from some sites at all under physiologic conditions. for structural zn sites an analogous situation may exist to the bipartite distribution of redox-active and structural disulfides in proteins, with labile zn sites being redox-active and inert zn sites corresponding to the purely structural group. key to zn expulsion under oxidizing conditions is the presence of oxidation-prone cys as a zn ligand. sites with more cys are likely to be more facile. based on the number and nature of ligands, the zn sites detected in the redox pair dataset likely belong to the redox-active group. the repeated association of forbidden disulfides of the bdd type with labile zn binding sites is particularly interesting because of the recent characterization of the oxidative process that leads to activation of the bacterial chaperone hsp . the presence of the zn center keeps hsp monomeric and functionally inactive. upon exposure to elevated temperatures, the cterminal domain swings away from the protein's center of mass, exposing the zn centre. the n-terminal thiols in the zn site, cys and cys , are preferentially oxidatively modified. the remaining two cys, and , form a disulfide of the bdd type, releasing zn and unfolding the c-terminal domain. unfolding the c-terminus removes a steric barrier to dimerization, activating the protein's chaperone function. the function of disulfide formation between cys and in the zn binding site has not been fully delineated. potential benefits are protection from other oxidation modifications and restriction of the conformational freedom of the protein chain allowing easier refolding upon return of the protein's milieu to normal conditions. however, these benefits apply to any disulfide and the repeated appearance of the bdd in labile zn sites of proteins of the redox pair dataset suggests a more specific function, possibly recognition by a foldase or metallochaperone. we hypothesize the forbidden disulfide would become stressed upon condensation of the b-sheet in its vicinity, possibly allowing easier re-insertion of zn. without any detailed experiments as a guide, the significance of the other repeated forbidden disulfide motif, the jsd, is not clear, but the same arguments relating to its forbidden disulfide nature would apply. the jsd-containing proteins meth and bhmt are known to be reversibly inactivated by disulfide formation. in vitro, bhmt can be reactivated by dialysis against dtt and zn, but the specific method of reactivation in vivo has not been characterized. for most structures exhibiting order/disorder transitions in the dataset, the reduced structure is associated with more disorder than the oxidized structure. energetically, formation of a disulfide bond between cys residues would have a favorable entropic contribution to the stability of the chain. a notable exception is leu-trna synthetase where the more ordered reduced structure binds zn (fig. (b) ). this may be generally true for order/disorder transitions associated with labile zn sites: the zn-bound state is entropically favored over a single disulfide because the polypeptide chain is constrained at three or four points instead of two. a subset of redox pair proteins exhibiting order/ disorder transitions correlated with disulfide redox status contain regions of disorder in excess of % of the protein chain. all the proteins in this group exist as dimers with the other monomer being more ordered. the disordered monomers in these class d structures may correspond to the molten globule state. for these proteins, the molten globule state adopted may be physiologically relevant to their function. molten globules are collapsed forms of the protein chain which have some native-like secondary structure but a dynamic tertiary structure as seen by far and near circular dichroism spectroscopy, respectively. the radius of gyration of proteins in the molten globule state is $ % larger than the native state but precise tertiary structures at atomic resolution have not yet been obtained for commonly studied molten globule proteins such as lactoglobulin a. however, one of the proteins in group d: platelet factor , which like hepatocyte growth factor, binds heparin and regulates angiogenesis, has been shown to adopt the molten globule state upon reduction of its disulfides. it seems likely proteins in group d adopt a molten globule state in vivo but can also adopt more ordered states upon binding of appropriate ligands. several of the proteins bind large polymers such as rna and heparin and the order/disorder transitions may mediate co-operative behavior between monomers. general studies of disordered sequences suggest there may be several different types of disordered sequence, three of which have been previously detected using an automatic classifier. the oxygen-rich sequences of the disordered regions of proteins of the redox pair dataset seem to represent a novel type of disordered sequence not previously recognized. oxygen-rich sequences may confer bistable switch-like properties on the sequence: an oxygen-rich environment stabilizes the disulfide in the oxidized state by increasing its pka ; while the subset of negatively charged asp and glu residues in oxygen-rich sequences may contribute to disorder in the reduced state via likecharge repulsion. disordered regions are known to undergo post-translational modifications including acetylation, glycosylation, methylation, and phosphorylation which may regulate the order/disorder transition. redox-activity of resident cys residues should be added to this list of post-translational modifications. perhaps the most astonishing conformational changes associated with disulfide reduction are the morphing transitions. the current paradigm, due to anfinsen, is that the primary sequence of a protein encodes a unique three-dimensional structure. various discoveries that have challenged this notion include the spectacular ph-induced conformational change in influenza a haemagglutinin (ha), molecular chaperones, and the previously discussed disordered sequences. all of these discoveries have been difficult to reconcile with the concept that protein sequences adopt a unique minimally frustrated fold. ha is believed to be kinetically trapped in a higher energy fold by an n-terminal domain which is subsequently cleaved, allowing it to cascade into a minimally frustrated fold. the conformations of disordered sequences appear to be principally governed by entropic factors: weak sequence preferences for a variety of conformational substates or modes appear to be co-selected by ligands. morphing structures appear to be a new type of challenge to the concept of a unique minimally frustrated fold. for morphing sequences, the structure appears to be dependent on the environment. unlike influenza a ha, where kinetic trapping during the folding process clearly plays a part in the structure adopting two different folds, for morphing sequences the two folds are energetic minima for the sequence in a particular environment. as such structural changes are reversible and dependent on environment. we were previously aware of two instances where subdomain morphing of proteins has been associated with reversible disulfide reduction: a redox-controlled structural reorganization of the ion channel clic proposed to regulate its insertion into membranes, and sequential oxidation of the transcription factor oxyr in response to oxidative stress which modulates its quaternary structure and dna-binding properties. here we showed that morphing is a common type of major structural change associated with disulfide reduction. the most spectacular morphing transition is that of clic , where an entire subdomain of the protein rearranges in response to different redox conditions. several redox pairs were found exhibiting large morphing conformational transitions of similar magnitude to oxyr, supporting the notion that these types of transitions may be reasonably common. large separations of the cys residues in the reduced state are a typical feature of these proteins, and do not preclude disulfide linkages forming. possibly attack by a helper molecule, such as glutathione, may be required as an intermediate in disulfide formation to bridge the intervening distance. large physical separation of partner cys residues may represent an example of negative morphing transitions are not limited to changes in disulfide redox status. other morphing transitions not associated with disulfide redox state have been noted in mad and lymphotactin. [ ] [ ] [ ] ph and redox are probably only two of multiple environmental changes associated with structural morphing. the relative importance of these and other environmental changes in protein morphing remain to be determined. morphing transitions may also have some relevance to evolutionary bridging states. in , based on structures of arc repressor, sauer proposed the existence of evolutionary bridging states. a substructure in wild-type arc repressor adopts a b-strand conformation which forms a two-stranded b-sheet with the adjacent monomer. in the n l mutant of arc repressor, the substructure was shown to fluctuate between the native state and a helical state on a millisecond time scale. a double mutant known as ''switch arc'' (n l, l n) was shown to be stabilized in the alternate helical conformation. sauer proposed the fluctuating n l mutant represented an evolutionary bridge between two stable low energy folds. proteins that can adopt two different folds from the same sequence could evolve two different functions upon gene duplication and mutation hence acting as an evolutionary bridge between two different folds. it is clear morphing proteins could also act as bridge states: deselection of one state over the other by point mutation may lead to loss of the ability to morph in the daughters resulting in proteins with different but related folds. although we have concentrated solely on disulfide reduction in this study, several anecdotal examples of disulfide isomerization should be mentioned in the context of the aforementioned discussion. a second example of an evolutionary bridging state was proposed based on a study of cnidarian collagen. the nterminal domain of minicollagen has % sequence identity with the c-terminal domain, but the structures show a different disulfide bonding pattern and fold. at this stage it is not clear if the two forms are interconvertible or represent true one-state endpoints evolved from an evolutionary bridging state. a different example where major structural organization is regulated by disulfide isomerization is the activation of p lysozyme. this conformational change is irreversible and associated with release of a hydrophobic transmembrane sequence by membrane depolarization, suggesting the latent state may be a kinetically trapped state rather than a true morphing protein. our study mined pairs of protein structures which contain disulfides in both reduced and oxidized states and represents the first systematic compilation of such a dataset. quaternary changes, morphing and expulsion of transition metals, particularly zn, have previously been associated with redox-related changes in proteins and here we showed these conformational changes are more common than anecdotal examples suggest. to our knowledge order/disorder transitions have not previously been linked with redox changes but also seem to be a common type of conformational change. like morphing transitions, changing redox conditions appear to be only one of many mechanisms regulating order/disorder transitions. the oxygen-rich sequences of the disordered regions of proteins of the redox pair dataset seem to be a novel type of disordered sequence not previously recognized. finally, two forbidden disulfide motifs are associated with redoxregulated zn binding here for the first time. the physiological significance of redox-controlled structural changes is only beginning to be appreciated. previously, all oxidative changes were thought to be irreversible and damaging. it is now known that redox signaling pathways can modulate homeostatic and adaptive responses. however, continued stimulation of adaptive responses may lead to maladaptive responses. the redox pair dataset is a valuable resource for the investigation of physiological and pathological processes associated with disulfide redox activity. the pdb was mined for pairs of structures with cys in different redox states. first, all disulfide-containing structures were identified by querying their dssp records, as ssbond records in the pdb header were found to be unreliable. for each disulfide the atomic residue positions were mapped to the sequence position. the connectivity of the disulfides in the sequence was then used to assign a unique identifier, d mn score [eq. ( )], to each protein. the d mn score allows direct comparison of the disulfide connectivity between structures as structures with the same disulfide bonding will have the same d mn score. where i and j are the sequence positions of disulfidebonded cys. all protein sequences in the pdb were clustered at % sequence identity using blastclust. the % threshold was chose to capture the same protein with some natural and engineered mutations allowed. some highly conserved orthologs from closely related species were also captured in the same cluster, for example, the nf-jb orthologs in human and mouse. proteins within each group were compared to detect subgroups with different d mn scores-indicating alternative disulfide connectivity. for fully oxidized proteins, a set of ''pseudo-reduced'' d mn s were calculated by iteratively ignoring one or more disulfides. the set of pseudoreduced d mn s for the oxidized protein were matched against d mn s for the reduced proteins in the dataset to select redox pairs. as an additional control, proteins were split into two groups based on whether some or all disulfide bonds in the protein were reduced. proteins in which cys residues were engineered into proteins were filtered from the list. analysis of gene ontology term enrichment go terms that are over and under-represented in the oxidized proteins in the redox-pairs dataset compared to the pdb as a whole were identified using fisher's exact test, in the same way as applied in tools that analyze gene-expression data. go annotations for the entire pdb were taken from the goa resource, downloaded from the ebi. to ensure a fair comparison, the go annotations of a nonredundant set of the oxidized structures ( % sequence identity) were compared to go annotations of a nonredundant pdb data set ( % sequence identity). to improve overall annotation coverage, a representative pdb structure is assigned all the go terms belonging to the pdbs within its cluster group. because go is a hierarchical data structure, go terms are set to a single level in the hierarchy to enable a fair comparison. here we used go level three. at this level there is a compromise between information quality and the number of annotations available. where a go term is below this level we move up the hierarchy until we reach the third level go term(s). go terms above level three cannot be used. a p-value was calculated representing the probability that the observed number of each go term (n) could have resulted from randomly selecting this go term in the pdb as a whole (n). fisher's exact test was used in order to approximate this p-value. go terms that are most specific to the redox pairs set will have p-values near zero, and those that are under-represented will be near one. protein conformational changes were examined using a variety of complementary approaches. we used profit (www.bioinf.org.uk/software/profit) to search for large conformational changes such as hinge movements. profit superimposes two structures using ca atoms and was used to calculate rmsd and distances between residues. large movements were indicated by the change in distance between the sulfur groups of the cys residues in a disulfide. because rmsd is averaged over the entire molecule, large changes confined to a subdomain of protein may not be obvious using rmsd. large conformational changes between structure-pairs corresponding to rearrangements of the polypeptide backbone were also identified using an implementation of a method by flocco and mowbray. for pairs of structures, the method compares the change in a pseudo-torsional angles defined by four consecutive ca-atoms. conformational changes were identified by running a smoothing window (five residues long) over the torsional-difference plot. stretches of contiguous residues with seven or more pseudo-dihedral angles that exhibited individual movements in excess of were initially filtered from the dataset. these conformational changes were further classified by comparing dssp annotations, solvent accessibility and secondary structure, between the protein pairs. proteins with conformational changes where the summed changes in the pseudo-torsional angles exceeded a threshold of degrees with accompanying secondary structure changes were classified as morphing. the presence or absence of multiple chains in the pdb structure could cause errors when calculating changes in solvent accessibility; therefore, dssp was run on individual chains only. all solvent accessibility measures were normalized using values derived by chothia. each amino acid was assigned to one of two states; core or exposed. core residues are those with a solvent accessibility of less than % and exposed residues are those with a solvent accessibility equal to % or more. secondary structures were assigned to one of three states: helix, strand and coil. regulation of the protein disulfide proteome by mitochondria in mammalian cells redox regulation: a broadening horizon disulfide isomerization switches tissue factor from coagulation to cell signaling disulfide exchange in domain of cd is required for entry of hiv-i redox regulation facilitates optimal peptide selection by mhc class i during antigen processing a single dipeptide sequence modulates the redox properties of a whole enzyme family substitution of the conserved tryptophan- in escherichia coli thioredoxin by site-directed mutagenesis and structure-function analysis urea dependence of thiol disulfide equilibria in thioredoxin-confirmation of the linkage relationship and a sensitive assay for structure redox properties of protein disulfide isomerase (dsba) from escherichia coli molecular and cellular aspects of thiol disulfide exchange force-dependent chemical kinetics of disulfide bond reduction observed with single-molecule techniques forbidden'' disulfides: their role as redox switches tryparedoxins from crithidia fasciculata and trypanosoma brucei -photoreduction of the redox disulfide using synchrotron radiation and evidence for a conformational switch implicated in function oxidized and synchrotron cleaved structures of the disulfide redox center in the n-terminal domain of salmonella typhimurium ahpf specific chemical and structural damage to proteins produced by synchrotron radiation the protein data bank all intermediates of the arsenate reductase mechanism, including an intramolecular dynamic disulfide cascade the intracellular chloride ion channel protein clic undergoes a redox-controlled structural transition membrane transport of hydrogen peroxide a combined in vitro/bioinformatic investigation of redox regulatory mechanisms governing cell cycle progression structure and biochemical properties of prl- , a phosphatase implicated in cell growth, differentiation, and tumor invasion structural mechanism of oxidative regulation of the phosphatase cdc b via an intramolecular disulfide bond structural basis of the redox switch in the oxyr transcription factor a model for the role of multiple cysteine residues involved in ribonucleotide reduction-amazing and still confusing substrate specificity and redox potential of ahpc, a bacterial peroxiredoxin structure of superoxide reductase bound to ferrocyanide and active site expansion upon x-ray-induced photo-reduction cross-strand disulphides in cell entry proteins: poised to act the gene ontology annotation (goa) database: sharing knowledge in uniprot with gene ontology chlamydia attachment to mammalian cells requires protein disulfide isomerase thiol/disulfide exchange is required for membrane fusion directed by the newcastle disease virus fusion protein inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein and prevent hiv- entry protein-disulfide isomerase-mediated reduction of two disulfide bonds of hiv envelope glycoprotein occurs post-cxcr binding and is required for fusion the crystallographically determined structures of atypical strained disulfides engineered into subtilisin a new force-field for molecular mechanical simulation of nucleic-acids and proteins in vivo formation and stability of engineered disulfide bonds in subtilisin harnessing disulfide bonds using protein engineering the anatomy and taxonomy of protein structure disulfide bridges in globular proteins snapshots of trna sulphuration via an adenylated intermediate zinc coordination environments in proteins as redox sensors and signal transducers a disulfide relay system in the intermembrane space of mitochondria that mediates protein import betaine-homocysteine methyltransferase: zinc in a distorted barrel identification of plant glutaredoxin targets conformational changes in protein redox pairs protein thiol modifications visualized in vivo zinc plays a key role in human and bacterial gtp cyclohydrolase i nuclear and mitochondrial compartmentation of oxidative stress and redox signaling crystal structure of the atx metallochaperone protein at . 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Å resolution a structural basis for the unique binding features of the human vitamin dbinding protein the . a crystal structure of the apo form of the soluble quinoprotein glucose dehydrogenase from acinetobacter calcoaceticus reveals a novel internal conserved sequence repeat the receptor binding protein p of prd , a virus targeting antibiotic-resistant bacteria, has a novel fold suggesting multiple functions crystallographic and in silico analysis of the sialosidebinding characteristics of the siglec sialoadhesin cooperative dna binding of the human hoxb (hox- . ) protein is under redox regulation in vitro formation of disulfide bond in p correlates with inhibition of dna binding and tetramerization role of light in the regulation of chloroplast enzymes molecular mechanism of thioredoxin regulation in photosynthetic a b -glyceraldehyde- -phosphate dehydrogenase redox regulation of chloroplast enzymes in galdieria sulphuraria in view of eukaryotic evolution cytoglobin, the newest member of the globin family, functions as a tumor suppressor gene structural basis of human cytoglobin for ligand binding oxidative stress causes rapid membrane translocation and in vivo degradation of ribulose- , -bisphosphate carboxylase/oxygenase crystallization and structural analysis of gadph from spinacia oleracea in a new form the intermolecular disulfide bridge of human glial cell line-derived neurotrophic factor: its selective reduction and biological activity of the modified protein a closed conformation for the polk catalytic cycle crystal structure of the apoptosis-inducing human granzyme a dimer crystal structure of the ef-tu.ef-ts complex from thermus thermophilus structures of the extracellular regions of the group ii/ iii metabotropic glutamate receptors mutant sod instability: implications for toxicity in amyotrophic lateral sclerosis disulfide cross-linked protein represents a significant fraction of als-associated cu, zn-superoxide dismutase aggregates in spinal cords of model mice the redox-switch domain of hsp functions as dual stress sensor liver betainehomocysteine s-methyltransferase activity undergoes a redox switch at the active site zinc human betaine-homocysteine methyltransferase is a zinc metalloenzyme molten globule monomer to condensed dimer: role of disulfide bonds in platelet factor- folding and subunit association flavors of protein disorder determination of the pk a of the four zn þ -coordinating residues of the distal finger motif of the hiv- nucleocapsid protein: consequences on the binding of zn þ structure of influenza hemagglutinin at the ph of membrane-fusion interconversion between two unrelated protein folds in the lymphotactin native state the mad conformational dimer: structure and implications for the spindle assembly checkpoint metamorphic proteins protein structure: evolutionary bridges to new folds the two cysteine-rich head domains of minicollagen from hydra nematocysts differ in their cystine framework and overall fold despite an identical cysteine sequence pattern disulfide isomerization after membrane release of its sar domain activates p lysozyme dictionary of protein secondary structure-pattern-recognition of hydrogenbonded and geometrical features a novel database of disulfide patterns and its application to the discovery of distantly related homologs basic local alignment search tool ontological analysis of gene expression data: current tools, limitations, and open problems supervised neural networks for clustering conditions in dna array data after reducing noise by clustering gene expression profiles ca-based torsion angles: a simple tool to analyze protein conformational changes scop-a structural classification of proteins database for the investigation of sequences and structures the authors thank dr. kieran scott for interesting discussions. disulfide torsional energy calculations were performed at the nci national facility. key: cord- -nu ckeud authors: nieto-rabiela, f.; suzán, g.; wiratsudakul, a.; rico-chávez, o. title: viral metacommunities associated to bats and rodents at different spatial scales date: - - journal: community ecol doi: . / . . . . sha: doc_id: cord_uid: nu ckeud one of the main goals of community ecology is to measure the relative importance of environmental filters to understand patterns of species distribution at different temporal and spatial scales. likewise, the identification of factors that shape symbiont metacommunity structures is important in disease ecology because resulting structures drive disease transmission. we tested the hypothesis that distributions of virus species and viral families from rodents and bats are defined by shared responses to host phylogeny and host functional characteristics, shaping the viral metacommunity structures at four spatial scales (continental, biogeographical, zoogeographical, and regional). the contribution of host phylogeny and host traits to the metacommunity of viruses at each spatial scale was calculated using a redundant analysis of canonical ordering (rda). for rodents, at american continental scale the coherence of viral species metacommunity increased while the spatial scale decreased and quasi-clementsian structures were observed. this pattern suggests a restricted distribution of viruses through their hosts, while in the big mass (europe, africa, and asia), the coherence decreased as spatial scale decreased. viral species metacommunities associated with bats was dominated by random structures along all spatial scales. we suggest that this random pattern is a result of the presence of viruses with high occupancy range such as rabies ( %) and coronavirus ( %), that disrupt such structures. at viral family scale, viral metacommunities associated with bats showed coherent structures, with the emergence of quasi- clementsian and checkerboard structures. rda analysis indicates that the assemblage of viral diversity associated with rodents and bats responds to phylogenetic and functional characteristics, which alternate between spatial scales. several of these variations could be subject to the spatial scale, in spite of this, we could identify patterns at macro ecological scale. the application of metacommunity theory at symbiont scales is particularly useful for large-scale ecological analysis. understanding the rules of host-virus association can be useful to take better decisions in epidemiological surveillance, control and even predictions of viral distribution and dissemination. electronic supplementary material: supplementary material is available for this article at . / . . . . and is accessible for authorized users. the distribution of species can either be explained by random processes, as neutral theory explains (chave , hubbell , gonzalez ), or by environmental filters, as niche theory proposes (leibold et al. , lorencio , morand and krasnov . however, species distributions are influenced by the spatial and temporal contexts in which they occur, thus, neutral theory and niche theory should be tested across a different scale of analysis. there is evidence for the influence of different abiotic factors like temperature and elevation that can determine the organized distribution of species diversity, depending on the spatial scale studied (rahbek and graves ) . the differences in species distributions between scales depend on three factors: physical barriers, the ability to co-occur, and their ability to disperse (chase and myers ) . the diversity of symbionts has not been excluded from this type of analysis (guernier et al. ) . a large body of evidence suggest that the distribution of symbionts, defined as all organisms that must infect or inhabit hosts for at least part of their life cycle (mihaljevic ) , also responds to environmental filters determined by their hosts (morand and krasnov , mihaljevic , dallas and presley . host phylogeny can act as an environmental filter to symbionts communites due to their interaction in the evolutionary history, co-adaptation and ecosystem process in the community (streicker et al. , krasnov et al. , córdova-tapia and zambrano . similarly, the host functional characteristics can play a role as an environmental filter due to shared life histories or by spillover events pedersen , morand and krasnov ) . we used viral communities associated with rodents and bats to analyse the effect of environmental filters. rodents and bats are the most diverse orders of mammals thus they constitute suitable model taxa to explore the viral diversity providing relevant information to understand the dynamics of virus-host distribution (lorencio ) . these taxa have been recognized as the main reservoirs of a high number of zoonotic viruses, some of them with an enormous impact in public and animal health (luis et al. (luis et al. , . besides the individual approach, the study of the association between viral metacommunities associated to bats and rodents at different spatial scales f. nieto-rabiela , g. suzán , a. wiratsudakul and o. rico-chávez , host and virus requires a clear understanding of the ecological context of infection and transmission be required (woolhouse , suzán et al. , johnson et al. . because the host-virus system is intimately embedded within the communities, it is possible to recognize the existence of an organization in the distribution of viral diversity and later recognize the filters that allow or not to associate with a host (suzán et al. ) . the dispersion of a virus within a host community is accomplished through transmission events and may depend on the viral richness present in the community, so it is common to have multi-pathogen systems that can be considered as metacommunity (suzán et al. ) . metacommunity theory implemented in viral communities at different spatial scales in combination with a redundancy analysis allows identifying the factors that facilitate virus distribution among hosts (mihaljevic , dallas and presley , suzán et al. . based on the metacommunity structures proposed by leibold and mikkelson ( ) , and mechanisms for infection and prevalence proposed by suzan et al. ( ) , we can expect random, checkerboard and clemenstian viral structures. a widespread distribution of abundant reservoir species are related with random structures while a limited viral distribution or a high viral specificity are related with checkerboard or clementsian structures (suzán et al. ) . the factors that shape viral communities and their distribution through their host at different spatial scales have not been studied. to measure the influence of the host phylogeny and functional characteristics of the host on viral community structure we hypothesized that both the expression of clementsian structures based on the niche theory would prevail at different macroecological scales, and the host phylogeny will explain the viral metacommunity distribution as response of the shared host evolutionary histories and ecological relationships. we analyzed the contribution of phylogenetic and functional factors to the structure of viral metacommunities associated with rodents and bats. in our model, the viral community is defined as all viruses detected in each host species inside the geographic scale to analyze. in this analysis, the metacommunities are composed by viral communities linked by processes of dispersion and transmission between hosts. to consider the spatial scale, the first analysis of viral metacommunities was performed on a continental scale considering the ocean as the main geographic barrier for viral distribution. the subsequent macroecological scales were selected by their similarity on diversity composition as biogeographical scales, recognized by their similarity in plant diversity (cox ) . zoogeographical scales by their similarity in animal diversity (holt et al. ), and regional scales that contain local shared evolutionary histories (holt et al. ) . we constructed a database based on reports of viruses isolated or detected by molecular techniques in bats and rodents. we recorded the host species, the virus species, and viral family according to the international committee of the taxonomy of viruses (ictv) (https://talk.ictvonline.org/). the bat viruses information was collected from dbatvir database (http://www.mgc.ac.cn/dbatvir/), and data from rodents were collected by a literature search in web of science (https://webofknowledge.com), elsevier (https://www.elsevier.com/advanced-search) and world wide science (https:// worldwidescience.org/) with the keywords: "rodent", "virus", "pcr" and "wild". the registered information was collected from years to and only studies with molecular techniques were considered. the geographic location was registered and classified into four spatial scales ( afrotropical, indomalayan and australian (cox ) ) zoogeographical scale: nearctic, panamian, neotropical, palearctic, afrotropical, oriental, saharo-arabian, sino-japaness, madagascan and australian (holt et al. ) . ) regional scales: north american, mexican, panamian, south american, amazonian, palearctic, african, guine-congolian, madagascan, indomalayan, oriental, saharo-arabian, sino-japaness, novozelandic, and australian (holt et al. ). a presence-absence data matrix was constructed for each spatial scale and for each viral taxonomic level (species and family), where the virus was the column and the host the row. we obtained rodent and bat matrices, but due to the measures and the capacity of the algorithm performed in the metacommunity structure analysis, we could only obtain results of and matrices respectively. to evaluate the metacommunity structure of virus-host species form each matrix, we measure three properties. the coherence as the degree to which pattern can be collapsed into a single dimension, turnover counting the number of species replacements along the matrix and boundary cumpling measuring how the edges of species boundaries are distributed along this dimension (leibold and mikkelson ) . the analysis of metacommunity structure was performed with the metacom package (dallas ) implemented in r (r core team ), and the detection of metacommunity structure was using the presley's decisions tree (presley et al. ). we measured the influence of the host phylogeny and functional characteristics of the host on viral community structure. the phylogenetic component was estimated taking the two first components of the pcoa analysis on the phylogenetic distance matrix, which was obtained by extracting the host species from the mammalian super-tree hosts (bininda-emonds et al. ) with the "picante" package. we include the body mass, litter size, number of litters per year and trophic guild, as host functional characteristics that may explain variation among hosts in viral community composition and their influence on the viral transmission. the variables were obtained from pantheria database (jones et al. ), animal diversity web (http://animaldiversity.org) and encyclopedia of life (http://eol.org/). a redundant analysis of canonical ordination (rda) has been performed to detect the relationship between the host phylogenetic component and host functional characteristics in the metacommunities from each spatial scale to obtain the explaining percentage on each one of detected metacommunity structures. the rda was calculated with the algorithm varpart (peres-neto et al. ) of "vegan" package (oksanen et al. ) implemented in r. this function performs a partition of the variation in community data for the explanatory variables. the rodents' database has records, and includes rodent host species, virus species distributed in viral families, of which species and families are zoonotic. the bats' database has , records and includes host bats species, virus species and viral families of which virus species and nine viral families are zoonotic. rodents. we detected a quasi-clementsian structure in the metacommunities of viruses in the american continent, and the distribution of these virus species was partly explained by host phylogeny ( %). the big mass scale showed a clementsian structure explained by a low percentage of host functional characteristics ( . %) ( table ). oceania continent was not included in the analysis because only four records were obtained. at biogeographical level, a clemenstian structure was detected in the nearctic region, a quasi-clementsian structure in the neotropic and palearctic, and a random structure was found in the afrotropical region. at zoogeographical scale, only four regions were analyzed including afrotropical with quasi-clemenstian structure, nearctic with clementsian and palearctic and oriental with random structures. at regional scale, three regions were analyzed including north amercian, with clementsian structure, african and palearctic with random structures. no relevant results were observed at metacommunities of viral families, where the random structures dominated in all scales except for nearctic biogeographic and zoogeographic scale where a quasi-clementsian structure was detected. chiropterans. except for the big mass scale where a quasi-clementsian structure was detected, the rest of the viral species metacommunities analyzed showed random structures. oceania continent with records was not possible to analyze. at biogeographic and zogeographical scale, afrotropical and palearctic regions presented a quasi-clemenstian structure, while we detected a checkerboard structure in the neotropical zoogeographical region. only the quasi-clementsian structure of the palearctic zone is maintained up to the regional scale. at all scales, the host phylogeny and host functional characteristic explain the distribution of the viral families (table ). we observed different patterns at different scales of analysis. for example, in the american continent the coherence increases as the geographic scale decreases, as shown in regional scales. contrarily, in the big mass an opposite pattern was found; the coherence decrease with a decreasing geographic scale (fig. ) . in the american continent these pat- terns may be explaining by the high influence of the latitudinal gradient on the host distribution and by the island biogeography and the edge effect because the american continent has a higher border surface (lovejoy et al. ). instead, the big mass surface allows a homogeneous host distribution (buckley et al. ) . at the big mass scale there are not strict physical barriers between bioregions and viruses are widely shared at the edges, mainly at the palearctic-afrotropical edge. these boundaries merged many years ago, forming a large area that allowed this exchange of viral diversity, so they are now arbitrary limits (morand and krasnov ) . therefore, it was possible to detect a clementsian structure in the big mass metacommunity, but when the spatial scale decreased we observed a dominance of random structures. at smaller communities, the characteristics of the host species are more widely shared without delimiting niches, and therefore the viral distribution depends on its capacity of dispersion rather than local filters. in the nearctic biogeographic region, the clementsian structure was explained by host phylogeny in % (table ) , suggesting a phylogenetic signal and therefore, a higher specificity for host clades. the quasi-clementsian structure detected in the neotropical biogeographical region showed a weak response to environmental host filters ( . %). when we compare these two regions, they show a response to a latitudinal diversity gradient (kaufman , gaston , guernier et al. , suggesting a greater diversity of host and viruses in the tropics probably influenced by a constant temperature (morand and krasnov ) . this property facilitates the survival and viral mutations, facilitated by vectors proliferation, incrementing the chance of spillover, giving rise to generalist symbionts (harvell et al. ) . the coherence in the structure of the afrotropical zoogeographical region increases by the loss of the middle east region, which was included at biogeographical scale and prevented the potential viral dispersion. functional and phylogenetic characteristics explained the random structure detected in the african region by % (table ) . this percentage can be explained by the absence of thryonomys swinderianus and xerus erythropus, species in the african region who contained extreme values in their phylogenetic characteristics. it also shows a structure with a clementsian tendency that is disturbed by mastomys natalensis, a rodent associated with seven of the viruses in this scale, in comparison with the remaining rodents that host - virus species. also, it is the only species in this level with reports of banzi virus, gairo virus, mopeia and morongo virus. probably because of its anthropism and the most considerable sampling effort. in rodents we can assume that coherence decrease in viral families metacommunities may emerge due to the loss of information of viral species characteristics when viral families data was analyzed. the metacommunity structure of viral species associated with bats was dominated by a random structure, however, the distribution of the viruses is not aleatory because most of the metacommunities were explained by the host phylogeny and functional characteristics. besides some viruses associated with chiropterans are cosmopolitan, like rabies virus, which have a wide range distribution within the metacommunitiy preventing the detection of a coherent structure. in chiropterans, contrary to rodents, higher coherence at viral family scale suggests a clustering pattern of viral families. besides, their classification of viral species is more specific, they even take the name of the host in which they were isolated. the coherence of viral families metacommunities was only detectable at biogeographical and zoogeographical scales, while in the continental and regional scales the coherence cannot be observed. this result can be explained by removal of the host and viral families with a wide range of distribution, due that our model assumes a homogeneous distribution of host and viruses. at america continent scale, we detected several random and checkerboard structures in viral families metacommunities due to the presence of cosmopolitan viruses with a high sampling effort due to their public health relevance, such as the rhabdoviridae and coronaviridae families, found in % and % of the host species distributed in america respectively. in the palearctic and afrotropical biogeographic regions a quasi-clementsian structure was detected. when analyzing the palearctic region at zoogeographical scale, we can observe a coherence increase due to a separation of random structures which belong to the zoogeographical regions saharo-arabica and sino-japanese (fig. ) , with a low sampling effort. despite this, the sino-japanese region is explained by phylogenetic ( . %) and functional ( . %) characteristics but separately, because the region contains extreme climates (urteaga ) that could generate divergence of host and viruses (gorman et al. ) . the random structure observed in oriental regional scale was explained by a high value ( . %) of environmental host filter (phylogeny + functional characteristics). this result suggests that the absence of a coherent structure is resulted by the poor sampling effort in the area. the virus families' distribution in the quasi-clementsian structure detected in the afrotropical biogeographic region is explained by environmental host filters ( . %), however, when the geographic scale decreases the coherence increases with environmental host filter ( . %). this increase of coherence can be explained by the absence of madagascan region due to the geographic barrier that prohibits host migration. meanwhile, the host migration between african and guineo-congolian regions could be possible, explaining the decreases of coherence and the random structures detected in these two regions. in general, a higher number of clementsian and quasi-clementsian structures was observed in response to environmental host filters. the viral distribution responds primarily to dispersion filter by the geographic scale and secondly to the host characteristics, being affected by two types of simultaneous filters, at different spatial scales. random structures are explained by taking into account the information biases and the dynamism in which the ecosystems are involved, undergoing constant changes, of which we only manage to capture moments of their history. in spite of random spatial structures, host-virus relationships can still be highly specific, suggesting coevolution between hosts and viruses (drexler et al. ) including coronaviruses (cov, however our framework addressed to community scale cannot measure these events. even so, the rules of community assembly are not a law, and they are only one of several mechanisms that alternate, so it proposes the predominance of clements superorganism at macroecological level (jaisson ) . thus, the study of such viral community assembly rules must be deepened to understand these processes. clarifying the influence of these environmental host filters, enables us to address the effect of differential study efforts and also to plan surveillance systems and responds to situations like emergent diseases. to understand and predict viral transmission dynamics it is important to identify those variables that explain viral distribution through their hosts. if the viral distribution is explained by the phylogenetic component we will be able to predict new hosts based on the phylogenetic similarity. instead, if viral distribution is explained by the functional component we could predict new hosts based on functional traits similarity. we must not forget that multiple factors interact and affect the direction of changes in viral metacommunities so that their dynamism must be monitored and understood by multidisciplinary approaches. in general, it is more feasible to analyze the viral metacommunities associated with rodents at viral species scale by overlapping families, showing a weak phylogenetic signal between the host and the virus species. bats, on the other hand, showed more order at the viral family level due to viral taxonomic classification, but also present more cosmopolitan viruses. these inferences were based on the currently available data. unfortunately, the present data set is insufficient to analyze the virus assemblages of small regions like the australian and madagascan due to the scarcity of data. our data set is also likely to be biased because synanthropic species and viruses of public health relevance were sampled heavily, and this likely interferes with the influence of natural structures. however, at the macroecological level, viral metacommunities associated with rodentia and chiropterans showed clementsian structures or at least tended to them (quasi-clementsians). the viral metacommunities mainly respond to spatial abiotic constraints, and secondarily to host environmental filters, which offer us an approach for the understanding of these clusters that explain a part of the set. the delayed rise of present-day mammals phylogeny, niche conservatism and the latitudinal diversity gradient in mammals disentangling the importance of ecological niches from stochastic processes across scales neutral theory and community ecology la diversidad funcional en la ecología de comunidades the biogeographic regions reconsidered metacom: an r package for the analysis of metacommunity structure relative importance of host environment, transmission potential and host phylogeny to the structure of parasite metacommunities phylogeny and geography predict pathogen community similarity in wild primates and humans genomic characterization of severe acute respiratory syndrome-related coronavirus in european bats and classification of coronaviruses based on partial rna-dependent rna polymerase gene sequences global patterns in biodiversity evolutionary processes in influenza viruses: divergence, rapid evolution, and stasis ecology drives the worldwide distribution of human diseases climate warming and disease risks for terrestrial and marine biota response to comment on "an update of wallace's zoogeographic regions of the world neutral theory in community ecology and the hypothesis of functional equivalence la hormiga y el sociobiólogo. (no. . j ) why infectious disease research needs community ecology pantheria: a species-level database of life history, ecology, and geography of extant and recently extinct mammals diversity of new world mammals: universality of the latitudinal gradients of species and bauplans co-occurrence and phylogenetic distance in communities of mammalian ectoparasites: limiting similarity versus environmental filtering the metacommunity concept: a framework for multi-scale community ecology coherence, species turnover, and boundary clumping: elements of meta-community structure avances en ecología: hacia un mejor conocimiento de la naturaleza edge and other effects of isolation on amazon forest fragments a comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? network analysis of host-virus communities in bats and rodents reveals determinants of cross-species transmission linking metacommunity theory and symbiont evolutionary ecology the biogeography of host-parasite interactions variation partitioning of species data matrices: estimation and comparison of fractions a comprehensive framework for the evaluation of metacommunity structure r: a language and environment for statistical computing multiscale assessment of patterns of avian species richness host phylogeny constrains cross-species emergence and establishment of rabies virus in bats metacommunity and phylogenetic structure determine wildlife and zoonotic infectious disease patterns in time and space la teoría de los climas y los orígenes del ambientalismo population biology of multihost pathogens we are very grateful to papiit (project ia ), programa de apoyo de los estudios de posgrado, unam, conacyt, and laboratorio de ecología de enfermedades y una salud, fmvz, unam, especially to m. lópez santana and d. mendizabal castillo for their contribution in databases construction. supplementary table . results of the analysis of coherence, range turnover, and boundary clumping for the viral families metacommunities of rodents and results of rda analysis. abs, embedded absences; sd, standard deviation; df, degree freedom. results of the analysis of coherence, range turnover, and boundary clumping for the viral species metacommunities of bats and results of rda analysis. abs, embedded absences; sd, standard deviation; df, degree freedom.the file may be dowloaded from www.akademiai.com.open access. this article is distributed under the terms of the creative commons attribution . international license (https://creativecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, you give a link to the creative commons license, and indicate if changes were made. key: cord- -p sgaypq authors: west, christopher m. title: current ideas on the significance of protein glycosylation date: journal: mol cell biochem doi: . /bf sha: doc_id: cord_uid: p sgaypq carbohydrate has been removed from a number of glycoproteins without major effect on the structure or enzyme activity of the protein. thus carbohydrate has been suggested to underly a non-primary function for proteins, such as in relatively non-specific interactions with other carbohydrates or macromolecules, stabilization of protein conformation, or protection from proteolysis. this non-specific concept is consistent with both the general similarity in carbohydrate structure on very diverse glycoproteins and the frequent structural microheterogeneity of carbohydrate chains at given sites. the concept is supported in a general sense by the viability of cells whose glycosylation processes have been globally disrupted by mutation or pharmacological inhibitors. in contrast to the above observations, other studies have revealed the existence of specific, selective receptors for discrete oligosaccharide structures on glycoproteins which seem to be important for compartmentalization of the glycoprotein, or the positioning of cells on which the glycoprotein is concentrated. sometimes multivalency in the carbohydrate-receptor interaction is crucial. there are additional possible roles for carbohydrate in the transduction of information upon binding to a receptor. the possibility of specific roles for carbohydrate is supported by the existence of numerous unique carbohydrate structures, many of which have been detected as glycoantigens by monoclonal antibodies, with unique distributions in developing and differentiated cells. this article attempts to summarize and rationalize the contradictory results. it appears that in general carbohydrate does in fact underlie only roles secondary to a protein's primary function. these secondary roles are simple non-specific ones of protection and stabilization, but often also satisfy the more sophisticated needs of spatial position control and compartmentalization in multicellular eukaryotic organisms. it is suggested that there are advantages, evolutionarily speaking, for the shared use of carbohydrate for non-specific roles and for specific roles primarily as luxury functions to be executed during the processes of cell differentiation and morphogenesis. new information about the significance of protein glycosylation is rapidly accumulating. this can be attributed to advancements in methods to detect and isolate new structures, to determine structure, and to modify carbohydrate structures experimentally. nevertheless, the field continues to experience profound unrest. many studies have failed to un-cover significant cellular or molecular consequences for dramatic changes in carbohydrate structures. other studies have pointed to isolated specific roles but no sweeping generalizations of function have been possible. interpreters of the former studies would attribute apparent specific effects to secondary consequences of changes in carbohydrate on protein structure. interpreters of the latter studies would argue that the former studies simply failed to ask the right questions of function. it is clear that there is much to be done to extend our knowledge for this particular type of posttranslational modification. the purpose of this article is to outline the scope of investigations presently being carried out in this subject area and to generalize the possible significance of these new findings. in particular, this article will attempt to compare and contrast the two prevailing views in the field. as intimated above, one view is that glycosylation plays a relatively non-specific role in maintaining and preserving polypeptide function. the alternate view is that carbohydrate structures participate in numerous specific interactions with discrete protein receptors, and that these interactions lead to predictable modifications in the localization or activity of the glycoprotein. these opposing views will be simply referred to as the non-specific and specific models. the importance of this issue is dramatized by the fact that nearly all secretory, extracellular matrix, cell surface and lysosomal proteins are glycosylated. there are many published comprehensive reviews on aspects of this subject ( - , , , , , , , , ) and the reader is referred to these for more detailed summaries and documentation. since this article will in part serve as an introduction and a background to the others in this volume, an overview of general features of the glycosylation process has been provided to precede the summary of existing and new ideas in the field which follows. oligosaccharides linked to asparagine occur in cellular slime molds, yeast, higher plants, insects, and humans, and several essential features of these structures are highly conserved throughout this phylogenetic range ( ) . typically though not invariably, a glc man glcnac structure is synthesized on a polyisoprenoid derivative (dolichol-p-p) and transferred en bloc to asparagine residues on polypeptide acceptors forming an n-glycosidic linkage (hence n-linked). the three glcs are usually trimmed by a tandem pair of a-glucosidases to yield a high mannose n-linked structure. the highmannose structure is variably modified, beginning with removal of mannoses in a typical sequence by a tandem pair of o~-mannosidases. one pathway retains most mannoses but permits additional substitutions of e.g., sulfate and/or phosphate. a second pathway trims down to mansglcnac and makes limited additions such as glcnac, and sometimes gal and core-linked fuc. these are referred to as hybrid structures. the third pathway continues the second pathway to build complex structures usually terminated by sialic acid. there can be two, three or four chains emanating from the branching mannose (and secondary branching mans) and these structures are accordingly referred to as biantennary, triantennary or tetraantennary complex nlinked oligosaccharides. the sialic acids, which appear not to precede echinoderms in their evolutionary appearance, comprise a diverse family of sugars ( ) . numerous additional substituents including sulfate, phosphate, methyl groups and others have been documented ( , , ) . each step in the glycosylation pathway is catalyzed enzymatically; there is usually a unique enzyme for each linkage position formed as monosaccharides are accreted or deleted ( ) . n-linked glycosylation is not template driven. only the first step might be considered so, inasmuch there is a necessary but not sufficient requirement of an xasn-x-ser/thr-x sequence for the oligosaccharide acceptor. protein structure or corfformation has also been shown to influence whether a site becomes glycosylated as well as whether it becomes high mannose, hybrid, or complex ( , ) or whether it receives a phosphodiester-linked glcnac-po ( ) or galnac-so ( ). a relationship between position in the polypeptide chain (distance from n-to c-terminus) and whether an oligosaccharide becomes high mannose or complex has been observed ( ) . finally these relations are not absolutely determinate, for spontaneous heterogeneity of carbohydrate structures at particular positions has been documented ( , , ) . changes in structure also result from extracellular influences (see below). the other major structural class of oligosaccharides is o-linked on ser on thr ( , ) . a largely distinct set of enzymes catalyzes the synthesis of these structures. these oligosaccharides are built by monomer addition from sugar-nucleotide donors directly on the polypeptide rather than on a lipid precursor. trimming does not appear to play a major role. gal and galnac are typical proximal sugars and man is rarely found. o-linked structures are found both in branched and straight chain forms. o-linked structures often resemble the oligosaccharides of glycolipids ( ) , and in fact these may be built from shared enzymes. an experimental consideration of the role of these oligosaccharides on protein must inevitably involve an examination of homologous glycolipids. although the n-linked and o-linked structures discussed above account for - % of incorporation of labelled sugar precursors into cellular glycoproteins, numerous additional small classes of oligosaccharides are also found. for example, galglc is found on hydroxylysine and hydroxyproline of collagens. glcnac-pon-ser is found on a lysosomal enzyme in d. discoideum ( ) and glcnac-asn is found on certain glycoproteins of what is probably the nuclear envelope ( ) . archeobacterial proteins are also glycosylated. a major class of these glycosylations shares some homology with n-glycosylation and may be evolutionarily related ( , , ) . a polyisoprenoid lipid carrier is utilized prior to en bloc transfer. the ancient character of the mammalian n-linked glycan suggests important functional attributes for his posttranslational modification. several physical methods have been applied to ascertain the conformation of oligosaccharides on proteins ( , ) . studies in model systems have implied that there is a preferred conformation at least for the core sugars of n-linked oligosaccharide chains. this conformation is stabilized by hydrogen bonding with the polypeptide chain, which orients the structure, and limited intrachain hydrogenbonding. the existence of preferred conformations implies that shape may be important for specific interactions of protein-linked carbohydrate with receptors. n-linked glycosylation appears to occur only in the rough endoplasmic reticulum (rer) and hence only to proteins which possess a signal sequence ( ) . the process is co-or post-translational ( ) . mannose trimming begins in the rer but can be concluded in golgi. modification of high-mannose forms or conversion to hybrid or complex forms occurs in the golgi. nearly all glycosylation is believed to be restricted to these two compartments. certain steps in glycosylation have, however, been postulated to occur in the ser, secretory vesicles, nuclear envelope and cell surface (e.g., , , ) . of course, deglycosylation occurs in lysosomes as a part of protein turnover ( ) . o-linked glycosylation appears to be initiated and concluded in the golgi ( , , but see ). as for n-linked glycosylation, there has been some resolution of processes between the cis, medial and trans elements of the golgi apparatus. nearly all proteins which pass through the rer and golgi become glycosylated in some form. one exception is the secretory protein serum albumin ( ) , which is synthesized in hepatocytes. there is no evidence for a glycosylated precursor. it should be recognized that at least some glycoproteins, e.g., cell surface transferrin receptor, can probably be reexposed to the golgi compartment subsequent to primary passage through this organelle ( ) . this provides new opportunities for glycosylation subsequent to initial synthesis. occasional reports have appeared in the literature regarding the presence of glycoproteins in cytoplasmic compartments topologically discontinuous with the lumen of the rer and golgi. histones and other nucleoplasmic proteins ( , , ) , a ribosomal protein ( ) and certain mitochondrial proteins ( , ) have been suggested to be glycoproteins. these findings contradict the dogma that glycosylation is strictly an rer and golgi dependent event unless it is postulated that modified proteins subsequently translocate across the membrane back to the cytoplasmic space. further work remains to be performed to confirm the existence of oligosaccharides on these proteins and to characterize their structure as a means for understanding their enzymatic basis. it must be appreciated that the description of the glycosylation pathway as we now understand it suggests many interesting kinds of regulatory mechanisms. for example, each step of the pathway can be influenced by enzyme synthesis and turnover, acceptor accessibility, as well as the supply of donor sugar, cofactors such as lipids, divalent cations, etc. the question of accessibility is particularly interesting because sequentially acting enzymes tend not to be mixed but are distributed vectorially through the rer and the multiple compartments of the golgi. movement of polypeptides through these compartments appears to be mediated by atp-dependent dissociative movement of vesicles ( ) . thus glycosylation may conceivably be regulated by the pathway of movement of the acceptor protein. there is also an indication that some glycosyltransferases form supramolecular complexes. thus, it is possible that certain carbohydrate structures are dictated not only by vesicle movement, but also by which enzyme cluster initially captures the acceptor protein ( ) . ideas about the role of protein glycosylation may be divided into two general groups: ) those which imply specificity associated with particular carbohydrate structures, and ) those which stress the general similarities of oligosaccharide structures, and the presence of microheterogeneity in the structure of particular oligosaccharides, in suggesting that combined carbohydrate bulk creates a common microenvironment for resident polypeptides much as the lipid bilayer provides an environment for integral membrane proteins. most effort has been devoted to the former category of ideas because of the greater ease of formulating hypotheses of this type. a non-specific role for oligosaccharides that is dependent on their generalized chemical properties has been suggested in several contexts. it was appreciated early on that carbohydrate afforded a stabilizing effect on protein solubility and against heat denaturation (see, e.g. , and ) . it was also realized that oligosaccharides afford resistance to proteolytic degradation ( ) , presumably by a mechanism of steric hindrance, and these ideas have been reinforced in numerous studies up to the present (e.g., , , ) . there are in addition several instances where carbohydrate is known to interfere sterically with protein-protein interaction not involving proteolysis ( , ) . predating this understanding, it was known that carbohydrate is a major constituent of the extracellular matrix in the form of polysaccharides known as glycosaminoglycans (gags) ( ). in the past years, it has been discovered that most gags are covalently attached to polypeptides, resulting in the formation of glycoproteins which, due to the specialized nature of the attached carbohydrate, are known as proteoglycans. gags are anionic polymers which hydrate to form enlarged domains which can interact with one another, as well as with polycationic polymers such as collagen and with other glycoproteins ( ) . a three dimensional space is thus defined by polysaccharide, protein, and the bound water which provides an environment for cells. convection and diffusion are modified, and fluctuations in ionic strength and ph are minimized. these structures depend on the polyanionic nature of the carbohydrate, and the general chemistry rather than the specific monosaccharide identities and sequences may be dominant in governing the properties of the system. carbohydrate oligomers are also known to selfassociate in the formation of mucous gels and in yeast and plant cell walls ( ) ( ) ( ) ) . in the former, the anionic nature of the carbohydrate appears to be important although in the algal and yeast gels the interactions are non-coulombic. the collective oligosaccharide density on cell surface proteins is sufficiently great so as to invite comparison with the carbohydrate density of the matrix space. this observation has led to the suggestion that cell surface carbohydrate might also establish a convection and diffusionqimited region around the cell in an area thick enough to be named, in certain cell types, the glycocalyx or fuzz. the polyanionic nature of the carbohydrate found on some cell surfaces probably results in marked cation and ph differences in this region relative to the surrounding medium ( ). it has recently been proposed that the heterogeneity of carbohydrate structure found on proteins of this region might result in minor differences in protein function, leading to a broader range in the ionic strength and ph values optimal for protein function ( , ) . as such, protein oligosaccharides might subserve a homeostatic process used by cells to maintain their viability in potentially changing milieus. it has also been suggested that a layer of non-immunogenic protein-associated carbohydrate may play an immuno-protective role for underlying cell surface antigens, especially on neoplastic cells ( ) . plasma membrane carbohydrate, owing to intercarbohydrate associations, might also influence the lateral diffusion of certain glycoproteins. inter-molecular association at the cell surface has been suggested to be so extensive in an archeobacterial system that it can influence the shape of the included cell ( ) . an extension of intermolecular association to contact between two cells in close mutual proximity leads to a possible mechanism for cell adhesion. interaction of oligosaccharides might be due to an apposition of the hydrophobic faces of two monosaccharides such as mannose ( ) . implicit in this model is the idea of a large number of weak interactions equalling in strength a smaller number of strong interactions. this mechanism is feasible in polymeric systems with repreating similar subunits. cell surface oligosaccharides could simulate a polymer inasmuch as they are coanchored in the same membrane. the interactions involved in these phenomena may be relatively nonspecific. the notion of a 'non-specific' role for carbohydrate has received perhaps its strongest support from studies on cells whose glycosylation processes have been globally altered by mutation or drugs. the most dramatic studies have involved mutant cells selected on the basis of resistance to the toxic effects of certain lectins. many mutations leading to loss of lectin recognition have been identified in cho and other cells ( ) . some have been characterized and in fact most steps in the pathway to complex, n-linked glycan formation are mutable. the striking finding is that mutant cells are largely viable in culture. most mutant cho cells remain adherent under appropriate conditions, proliferate, and are tumorigenic. mutants defective in early steps leading to the formation of high-mannose glycans are also viable ( ) . this result was not, however, found in a yeast mutant ( ) . selected activities of cells have also been investigated in some mutant strains. for example, the half-life of cell surface proteins was found not to be dramatically affected ( ) . similar results were found following treatment of cells with sialidase ( ) . a considerable literature details the consequences of carbohydrate modification on the secretory process. though several examples of apparently specific effects will be enumerated in a later section, the number of cases where little or no effect is found is noteworthy ( , , , ) . in some cases detailed kinetic analysis has found only small effects on secretory rates ( ) . similar results have been found regarding accxumulation of receptors in the plasma membrane and in their function at that site ( , , , ) . genetic modification of the gene for the ldl receptor has deleted a sequence required for a cluster of o-linked glycans and even for this poorlystudied glycan type no consequence on function could be detected ( ) . mutants in the enzymatic pathway of o-glycosylation still place the ldl receptor, albeit in a less stable form, on the cell surface ( ) . these negative results have suggested that carbohydrate cannot underlie essential housekeeping functions in cells. this implies a non-specificity of function in the sense that obvious functions cannot specifically be attributed to the carbohydrate when it is present. this represents a shift in meaning from the above definition of nonspecificity inasmuch as specific receptors may be involved in functions non-essential in the tissue culture environment. thus it has alternatively been suggested that carbohydrate may be more important for particular differentiated functions, which are not expressed in cell culture systems such as the cho cell line. there are but few investigations into this possibility. for example, in the cellular slime mold d. discoideum, mutations have eliminated two developmentally regulated glycoantigens ( , ) . nevertheless, the cells develop reasonably normally to produce fruiting bodies and viable spores. the result may imply that carbohydrate is either nonspecific or subserves subtle modulatory roles or that their roles are redundant with alternate (or back-up) mechanisms. alternatively, the value of these carbohydrate structures may only become evident under the competitive conditions of natural survival outside of the laboratory. although the above ideas have been grouped together under 'non-specific mechanisms', this is not meant to imply that carbohydrate structure per se is completely irrelevant. 'non-specific' should be interpreted relative to the types to be discussed below, were specific, saturable recognition mechanisms related in principle to enzyme-substrate or hormone-receptor interactions are apperently involved. in any case, the non-specific mechanisms tend to rely on the bulk chemical properties of the participating oligo(poly)saccharides. this phrase implies interactions between receptors and ligands (usually the oligosaccharide) of saturability and high selectivity and avidity. selectivity can be achieved by high affinity monovalent interactions or multiple weaker interactions. selective interaction is often equated with molecular or, on a more complex level, cellular recognition. the phrase also implies that a specific function can be attributed to the carbohydrate structure. perhaps the strongest indication for specific roles are the variety of structures which have been detected on protein carbohydrate. the following sections outline how several characterized structures are thought to function in specialized processes. in some cases, a receptor protein capable of recognizing a given structure is known. in eylar ( ) presented the idea that proteil' glycans constitute a 'chemical passport' signalling export of a protein to the cell surface or for secretion. though this idea has since been supplanted by the 'signal hypothesis' of blobel and sabbatini ( ) , it has served as a precursor to more refined ideas of chemical signals leading to the accumulation of glycoproteins in specific compartments ( ) . for example, a mannose- -phosphate moiety has been suggested to be responsible for associating proteins with receptors which in turn lead to protein accumulation in lysosomes ( ) . the key to this understanding was the finding of mutant cells which appeared to secrete constitutively certain lysosomal enzymes that were distinct in their oligosaccharide structures: namely that normal enzymes possessed multiple phosphate residues - inked to non-peripheral mans in high-mannose structures, and that mutant enzymes lacked these phosphate moieties. this suggested that there was a man- -po receptor in membranes of the golgi, and plasma membrane (since lysosomal enzymes can also be absorbed from the medium and translocated to lysosomes), which was responsible for binding the man- -po recognition marker on certain glycoproteins. two such receptors, one of kd and the other of kd, have been discovered ( , ) . these are located in the golgi and/or on the cell surface in concordance with their proposed functions. from studies on receptor movement and the effects of inhibitors there is fairly good evidence that the man- -po moiety, together with one or both of these receptors, plays a recognition role in the intracellular routing. however, not all lysosomal enzymes seem to be involved because some copies of the altered enzymes still accumulate in mutant cells ( ) , and some lysosomal enzymes are not affected. it has been suggested that there is lysosomal heterogeneity with only one class of lysosome employing this recognition marker. it is nevertheless apparent that for some lysosomal enzymes in some cell types the man- -po receptor does not function alone, even as an intermediate signal, for lysosomal routing. there must be other recognition signals on the enzyme polypeptide which lead to its potential lysosomal accumulation. it is interesting to note that the distinctive carbohydrate structure plays a compartmentalization role separate from the enzyme's function in catalytic degradation. furthermore, carbohydrate is employed to perform a common role shared by multiple proteins. a posttranslational modification such as glycosylation is well-suited to the demands of a mechanism for compartmentalization. the rer, golgi, smooth endoplasmic reticulum, and nuclear envelope are also intracellular destinations for proteins which traverse the rer cotranslationally. these compartments contain numerous characteristic and uniquely distributed glycoproteins, but there is not enough information to evaluate the possibility that carbohydrate structure is involved in routing or maintaining the positions of the glycoproteins. several cases of rer-specific glycoproteins have been considered. cells infected with rotavirus synthesize, under control of the virus, several proteins including vp , an integral membrane glycoprotein whose distribution is restricted to the rer ( ). this protein together with the membrane of the rer form a temporary coat for the virus. in a genetically modified form of vp , a hydrophobic region, which presumably serves as a transmembrane anchor defining the proteins as an integral membrane glycoprotein, was deleted. the truncated gene encoding vp was introduced to cells by transfection. truncated vp was synthesized, but was then secreted despite this fact that glycosylation at its single site toward the c-terminus retained its high-mannose character. these results show that the key factor which retained the glycoprotein in the rer was the transmembrane sequence and was not related to the glycosylation site. the results would also imply that glycosylation is not essential to the process of secretion. several nuclear envelope glycoproteins have been found to express an unusual carbohydrate structure consisting of glcnac-asn ( ) , which is an expected product of endo-glycosidic degradation. it is at present unknown where this structure is synthesized and thus whether glycosylation precedes or follows arrival into the nuclear envelope. the secretion of protein appears to involve at least two pathways: a constitutive pathway and a pathway which includes a secretory vesicle which is stored cytoplasmically ( ) . a range of high mannose and complex bi-, tri-and tetra-antennary nlinked structures has been observed on both secretory and plasma membrane associated glycoproteins. most studies have been directed, however, toward n-linked glycans since these structures are easier to modify and detect than are olinked and other, unknown structures. one approach to evaluate the potential role of nlinked glycans has involved the use of pharmacological inhibitors of several steps, namely the initial steps of synthesis, and deglucosylation and demannosylation. tunicamycin blocks n-linked glycosylation completely (as well, possibly, as other types of glycosylation ( )), whereas deoxynojirimycin and swainsonine block certain steps leading from high mannose structures to the complex type. though the effects are not simple it tentatively appears that lack of n-linked glycosylation has a measureable affect on the rate of secretion of some proteins from hepatocytes or hepatoma cells ( , , ) . secretion from these cells appears not to involve a stored secretory vesicle. modification of glycosylation by deoxynojirimycin or swainsonine appears in some cases to affect the rate of movement from the rer to the golgi and, in other cases, from the golgi to the extracellular space ( , , ) . some glycoproteins and albumin are not affected at all. this result has led to speculation that there is a receptor which can recognize and retain glycoproteins bearing the high mannose marker. this mechanism would not be universal for all proteins. alternatively, these differences may reflect spurious differences in rates of dissociation from glycosylation enzymes. it seems reasonable to consider that glycans may be designed to play a modulatory role in the kinetics of secretion for only selected proteins. since secretion of these proteins is not regulated by storage in a secretory vesicle, this mechanism may be used by the cell as a finetuning mechanism for controlling release of protein. it is plausible that the passive constitutive pathway for secretion ( , ) does not involve carbohydrate per se, but that modifications of this pathway or its kinetics do. further work remains to be done to compare carbohydrate structure on different glycoproteins and to consider the possible involvement of neighboring polypeptide regions in the interaction with a hypothetical receptor. entry of protein into stored secretory vesicles requires a specific signal as shown by the observation that some hormone polypeptides produced by pituitary cells are stored whereas others are constitutively secreted. it has been observed that peptide hormones which are stored in pituitary gonadotropes and thyrotropes are sulfated whereas related peptide hormones released from placental cells, but not stored there, are not sulfated. this sulfate has subsequently been shown to reside at one or more non-reducing termini of n-linked complex oligosaccharides in the position usually occupied by sialic acid. this substitution may be directed by a substitution of the galnac for underlying gal ( ) . the functional significance of the relationship between galnac-so and delivery to a vesicle which is stored cytoplasmically prior to release by a secretagogue remains to be explored. an exception to this structure-function correlation is posed by human fsh, which is stored in human pituitary gonadotropes but, unlike the lh stored in these cells, is not sulfated. possibilities consistent with a role for galnac-so are that fsh may segregate to a discrete vesicle population relative to lh, or that the two homones colocalize and that some intermolecular association substitutes for the lack of this carbohydrate modification. there is no concensus oligosaccharide structure discovered thus far which distinguishes plasma membrane from secreted glycoproteins. although this may suggest the possibility that plasma membrane and secretory glycoproteins may in fact share a common signal, on account of their topologically equivalent destinations, inhibitor experiments have shown that an absence of n-linked glycans does not consistently block the appearance of glycoproteins on the cell surface ( , ) . there are, however, some instances of inhibition ( , , , ( ) ( ) ( ) , which allow for the possibility that nlinked glycans may act as a signal or modulator for the export of specific proteins to the plasma membrane. alternatively, the failure of a few altered glycoproteins to be exported may be explained by enhanced proteolysis, by a secondary effect on conformation, or by the display of another signal elsewhere on the polypeptide ( ) . a special class of extrinsic plasma membrane glycoproteins is known, however, for which a glycan structure appears to be significant for localization. this includes glycoproteins which are recognized by the cell surface protein ligatin, which recognizes a man- -po - -glc, linked to a highmannose backbone ( ) . this structure appears to be important for the cell surface association of these glycoproteins since glc-i-po can elute them from cells and a high concentration of ca + +, which can elute ligatin, also elutes the associated glycoproteins. glycoproteins associated by this mechanism were first recognized ultrastructurally on the surface of the intestinal mucosa of the suckling rat, but have since been discovered on the surfaces of a variety of cell types in sea urchin, chicken and mouse. it is unclear whether the gic-i-po marker serves as a routing signal for export to the cell surface, using ligatin as an intermediate carrier, or whether it serves a recognition role for secondary association with the cell surface once secretion in soluble form has already occurred. though the identity of many of the ligatin associated glycoproteins is unknown, the first to be discovered in the intestinal mucosa was j -nacetylhexosaminidase. in this case, the primary function of the glycoprotein can be viewed as an enzymatic one. the appended carbohydrate appears to serve a role in localization, contributing to the spatial organization of the system. consideration of the possible role of carbohydrate in other molecular associations with the plasma membrane will be considered below in sections on hormone-receptor interactions and cellsubstratum (e.g., extracellular matrix) and cell-cell adhesion. it has been proposed that specific carbohydrate/protein interactions might designate the localization of secreted glycoproteins to specific sites in the extracellular matrix ( ). this idea has received support recently from the detection of animal lectins in matrices, basement membranes, and mucous secretions ( ) . lectins, which are multivalent carbohydrate binding proteins with oligosaccharide specificity, are believed to crosslink other molecules to contribute to, e.g., the viscosity of certain mucous structures ( ) . binding of matrix proteins to carbohydrate of gags has been reported ( , ) . a recent report suggests that the neural adhesion protein n-cam has a binding site for heparan sulfate-like gags ( ) . in the early s, ashwell and morrell demonstrated that desialylation of serum glycoproteins, resulting in the exposure of a penultimate galactose in mammalian species, caused rapid removal of proteins by hepatocytes once these altered proteins were restored to the blood (reviewed in , ) . this suggested that normal turnover of serum proteins is regulated by the structure of their glycans, which would be regulated by interaction with sialidases. though a carbohydrate binding receptor for (non-reducing) terminal gal/galnac has been characterized in hepatocytes, it has not been possible to prove this hypothesis directly. this mechanism is not universal because not all serum proteins, albumin being a notable example, are enzymatically glycosylated. further work must be done to determine whether this system actually functions in serum protein turnover. it is interesting to note that a family of serum proteins (iga ) binds this receptor by way of o-linked oligosaccharides, but only upon denaturation ( ). it has also been recently shown that liver endothelial cells can desialate a serum protein, thus rendering it susceptible to uptake by hepatocytes via the gal/galnac receptor ( ) . additional studies have revealed the presence of a distinct carbohydrate binding protein in kupffer and other macrophage-like cells ( , , , ) . following recognition, these and other carbo-hs~drate binding proteins become internalized together with their ligands, which carry a man/glcnac/fuc specificity, but later return to the cell surface free of ligand. in addition, cell surface localized man- -po receptors have been suggested to be responsible for hepatic uptake of enzymes supplied to the fetal blood from placental fluids ( ). it has been suggested that receptors on the kupffer cell and other types of macrophages may also recognize cells which present the appropriate configuration of carbohydrate residues ( ) . binding in these circumstances may lead to phagocytosis. this kind of binding has been detected in model systems ( ) . several examples of a role for protein glycan in other forms of intermolecular recognition have been offered. these include glycans on the fc portion of igg being involved in the recognition by complement proteins and cell surface receptors ( , ) , glycans of human chorionic gonadotropin being involved in activation of its receptor subsequent to the actual binding event ( ) ( ) ( ) , and interaction between anti-thrombin iii and heparin leading to activation of inhibition of thrombin protease activity ( ). as early as , burnett ( ) and his colleagues observed the importance of cell surface proteinbound sialic acid in virus binding and infection. this phenomenon is sufficiently complex, however, that it is necessary to discuss the kinds of evidence which are involved in concluding that carbohydrate plays a direct, rather than a regulatory, role in this and other examples of adhesion. any demonstration that carbohydrate comprises part of a physical bridge in cell adhesion (or any molecular binding) requires that four general criteria be satisfied: ) removal or alteration of the carbohydrate diminishes cell adhesion; ) reconstitution of the carbohydrate either on the cell or on an artificial surface re- stores cell adhesion; ) a specific receptor for the carbohydrate is identified on the cell (complementary) surface; ) inactivation of the receptor diminishes cell adhesion. satisfaction of these criteria can be complicated because cells appear to have multiple adhesion systems, rendering it difficult to assay one independently of the others. removal of carbohydrate can have other effects on cells. for example, cell viability can be reduced by drugs or mutations affecting glycosylation, and transport of polypeptides to the cell surface and other mechanisms of turnover and placement of glycoproteins can be altered. specific receptors may be difficult to detect because individual receptor-ligand interactions may have low affinity (e.g., ). a rationale for this generalization is that, because of the large area of cell surfaces, cells can theoretically interact with other surfaces using a large number of low affinity associations. this may in fact be desirable for cell interactions which would need to be reversible. the results of several approaches where cell reassociation has been competed with model sugars have shown that the competing sugars must be crosslinked to form multivalent structures in order to be competitive ( ) . a multivalency requirement has also been implicated in the man- -po receptor system ( ) . examination of cell-ceu and cell-substratum adhesion in several systems has identified candidate molecules which participate in the formation of the physical bridge ( , ) . in some cases independent approaches have converged on the same candidate molecules. the candidate molecules are usually glycoproteins and in some cases the relative roles of carbohydrate and protein have yet to be resolved. in other studies, candidates have not yet been identified. the simplest adhesion systems involve parasite/host interaction. considerable evidence has been adduced that various parasites such as viruses, bacteria, protozoa, etc., possess carbohydrate binding proteins which can interact with sugar structures on the surfaces of cells with which the parasites associate ( , , ( ) ( ) ( ) ( ) . in some cases it is suspected that this sugar is associated with glycolipid but in other cases man is believed to be involved on the basis of competition studies (e.g., ). this would imply the involvement of glycoprotein because this sugar is rare in glycolipid. the use of carbohydrate as attachment points on cells presumably reflects the stability of these structures on cell surfaces and their specific associations with target cells suitable for the parasite. these would of course be useful features for any mechanism of specific cell-cell interaction. species-specific cell-cell adhesion (the so-called secondary type) in the phylogenetically-primitive marine sponge is suspected to involve carbohydrate ligands ( ) . ca-free sea water elutes a fraction from cells of microcionaprolifera which is required for their reaggregation. this fraction is the source of a purified proteoglycan which is referred to as aggregation factor. hypotonic shock elutes another molecular species (the so-caued baseplate or aggregation factor receptor) which is also required for reaggregation and may recognize the proteoglycan by a mechanism involving glucuronic acid on the proteoglycan. these preliminary findings were ascertained by competition studies with model sugars and enzymatic treatments, and it will be interesting to confirm these findings using purified molecules ( ) . results of comparable studies in another marine sponge, geodia cydonium, are even more intriguing ( ) . bound t -glucuronic acid is also believed to be important in aggregation factor/baseplate recognition but its position is reversed so that the key sugar is on the baseplate glycoprotein. beta-glucuronidase and j -glucuronyltransferase can be eluted from extracellular material suggesting a mechanism for the dynamic regulation of cell recognition. an investigation into the tissue specificity of cell recognition showed that ~-galactosidase treatment of nonaggregating cells permitted their aggregation in the presence of aggregation factor. a protein was then isolated which could inhibit aggregation of aggregation-competent cells in a fl-galactosidase sensitive fashion. finally, a t -galactoside binding protein was isolated from extracellular material which rendered an effect similar to that of the glycosidase. though the significance of these observations is as yet unclear, it has been suggested that carbohydrate structures may underlie specific mechanisms of cell recognition in multiple ways in these species. cell-substratum adhesion in another primitive multicellular system, aggregating cellular slime mold cells, appears to involve a multivalent carbohydrate binding protein termed discoidin i. the involvement of discoidin i was initially recognized based on sequence homology between this protein and an oligopeptide sequence of vertebrate fibronectin which has been shown to contain its cell binding site. this oligopeptide, which blocks fibronectin binding to cells, also affects association of dictyosteliurn cells with their substrata ( ) . the ceils become incapable of migrating properly on a surface. the effect can be mimicked by antidiscoidin i. the effect is also replicated if the cells are transformed with a gene which encodes an mrna which hybridizes with authentic discoidin i mrna and blocks its translation ( ) . the evidence is very strong that this carbohydrate binding protein is involved in cell-substratum association. what is unclear, however, is the role of the carbohydrate binding activity. there is evidence that it does not associate with the cell via this site ( ) . it is unclear whether the carbohydrate-binding site might associate the protein with elements of certain substrata, but it is apparent that this is not necessary. there is recent evidence that food bacteria are recognized by this lectin and hence discoidin i might assist in the adhesion of ceils to their food source ( ) . perhaps the best documented instance of a direct role for carbohydrate in cell-cell adhesion occurs in higher organisms and involves the protein galactosyltransferase and bound glcnac, sometimes associated with oligolactosamine ( , , , ) . this pair of molecules has been implicated in adhesion between mouse sperm and egg, neural crest cells and substrated, and embryonal carcinoma ceils. each of the four criteria cited above has been addressed by experimental approaches using antibodies, enzymes and drugs to inactivate the receptor and the ligand. a genetic approach to modify receptor or ligand has yet to be applied. though the latter approach with its inherent specificity is very important, the evidence using the approaches already employed is strong. the system is interesting because the receptor-ligand interaction can be readily reversed by supplying substrate for the galactosyltransferase enzyme activity. an independent approach to the problem of mouse sperm-egg recognition has resulted in the isolation, from the jelly coat of the egg, of a glycoprotein, zp , which can, upon presentation to the sperm, block its ability to recognize eggs ( ) . it was found that careful alkaline borohydride treatment to strip o-linked glycans without altering the polypeptide chain neutralized the inhibitory activity of zp . finally, it was shown that the released glycans possessed inhibitory activity and in addition bound to sperm, suggesting that the carbohydrate itself served as the recognition site in the jelly coat for sperm. further work remains to be done to identify the sperm receptor ( ) as well as to relate the findings to the above results implicating the galactosyltransferase. carbohydrate of a different type has also been implicated in cell-cell adhesion between mature mammalian cells. for example, it has been suggested that cell surface carbohydrate may be important for recognition between certain lymphocytes and certain high endothelial venule surfaces (hev) in lymphatic tissues ( ) . pretreatment of lymphocytes with specific sugars renders them incapable of binding to hevs in an in vitro assay, and glycosidase treatment of hevs renders them incapable of being recognized by lymphocytes. there is conflicting evidence from these studies regarding the nature of the carbohydrate structure involved. additional results have indicated that one lymphocyte subclass recognition site involves a previously studied homing receptor, the ubiquitin-conjugated glycoprotein defined by the mel- antigen. recent work has found carbohydrate-derivatized polyacrylamide surfaces to which lymphocytes will specifically bind ( ) . an effort is underway to compare the specificity of this interaction with that to hevs. the exciting prospect is that the manipulability of the artificial surface ( ) will allow dissection of multiple adhesion mechanisms and permit more rapid investigation of the nature of the carbohydrate recognition system believed to be responsible for lymphocyte recognition preparatory to extravasation. carbohydrate has been suggested to perform an opposite kind of role in the function of the protein n-cam in associating neural cells ( ) . fab fragments from anti-n-cam inhibit reassociation of dissociated neural cells and isolated n-cam can self associate. there are several forms of n-cam which differ in their degree of sialylation. the more heavily sialylated forms show less affinity for one another in the self association assays. thus it has been postulated that sialylation is a posttranslational modification which modulates the strength of adhesion mediated by this glycoprotein and in vivo evidence has now been provided ( ) . recent-ly it has alo been suggested that n-cam can bind carbohydrate in the heparan sulfate proteoglycan ( ) and that this recognition is involved in intercellular ( ) and cell-substratum adhesion. it is too soon to speculate whether binding to a carbohydrate ligand may have secondary effects on cells as as been suggested for, e.g., binding of human chorionic gonadotropin ( ) ( ) ( ) . this hormone does not depend upon its carbohydrate for binding to its receptor but the carbohydrate is important for activating the cellular response. the selected examples discussed here indicate that protein-linked (or possibly lipid-linked) carbohydrate may play a fundamental role in multicellular organization in a wide phylogenetic range of organisms. though in all of the examples cited there is a specificity in the adhesion, in some cases this is due to multiple possibly low-affinity binding interactions. in the extreme, each single binding event may approach non-specificity, with specificity accruing through multivalency. perhaps this implies that the formalization of non-specific carbohydrate-mediated events obscures an actual continuum of specificity in these events between these two extremes. it is unclear whether the involvement of carbohydrate in adhesion is universal. several other adhesion molecules, including glycoproteins such as gp in d. discoideurn ( ) , cam / in mammalian cells, etc. and glycolipids ( ) , are glycosylated but the role of carbohydrate is not known. if the use of carbohydrate is universal, then the specificity inherent in the examples discussed would suggest an underlying structural code which may be important for the affiliated processes of cell sorting and morphogenesis, to which adhesion makes a very important contribution. this may explain some of the interesting morphogenetic effects circumstantially suggested by many studies involving carbohydrate structure perturbations on developing cells. a role for carbohydrate in cell adhesion choice or sorting correlates with its postulated role in intracellular or extracellular compartmentalization of proteins. cell sorting and molecular compartmentalization can be regarded as sorting at different levels. it will be interesting to learn whether the proteins on which these carbohydrates are located exist only to place the carbohydrate or whether the carbohydrate piggybacks on another essential function of the protein. were the latter true, this would strengthen the analogy with the role of carbohydrate in routing or molecular compartmentalization. it may then become feasible to generalize carbohydrate's role as important for the three-dimensional compartmentalization of proteins, and the cells, with which they are associated. as such, carbohydrate may be the chemical structure cells used to address and organize space as they evolved from prokaryotes to unicellular eukaryotes, and continued to exploit as they evolved to form multicellular tissues and organs. numerous studies have implicated protein-linked carbohydrate in more complicated cellular phenomena ( , ) . glycosylation is modulated coincidentally with numerous developmental processes ( , - , , , ) . carbohydrate interactions have been implicated in mammalian morula compaction ( , , ) . proliferation has been reported to require glycosylation ( ) or to be inhibited by glycopeptides and a defined fragment of heparan sulfate ( , ) . oligosaccharides released from plant cell walls can stimulate cell proliferation and alter cell differentiation in plant cells ( ) . myogenesis and chondrogenesis have been speculated to be affected by gags linked to proteoglycans (e.g., ), but conclusions in this field are very controversial. attachment of cells to substratum-associated carbohydrate can result in its covalent modification ( , ). neuritogenesis has been shown to be inhibited in vitro by tunicamycin ( ) , though the specificity of the effect is difficult to assess, as tunicamycin-sensitivity does not convincingly demonstrate the involvement of protein-linked carbohydrate ( , ) . a recent report suggests that alteration of processing of nlinked oligosaccharides with swainsonine, without affecting cell viablility, inhibits the ability of a line of melanoma cells to metastasize to the lung ( ). this finding is consistent with another report documenting carbohydrate differences between metastatic and non-metastatic cell lines ( ) . there is much new territory to explore in the field. the use of monoclonal antibodies and lectins which recognize carbohydrate-dependent epitopes continues to reveal an unexpected diversity of carbohydrate structures with surprisingly intricate distributions in embryonic and adult tissues. this has recently been appreciated in amphibian ( ) and mouse ( , ) embryos and in the simpler model system dictyostelium discoideum ( , ) . tumor cells also differ in many cases in specific carbohydrate structures on both lipid and protein ( - , - , ) . several of the antibodies employed recognize carbohydrate epitopes whose corresponding structures have been determined. the antibodies provide unrivaled sensitivity and specificity relative to standard methods for detection of unique carbohydrate structures. caution must be applied, however, because ) it is theoretically possible that unique structures at the monosaccharide level may present structures similar enough at the atomic level to be immunologically cross-reactive and ) identical or similar structures may be immunologically distinguished by conformational restrictions imposed by the surrounding polypeptide environment. thus inference of structure based on antibody data must be confirmed directly by structural studies. in any case, antibodies and lectins may model potential receptors in cells which might specifically recognize carbohydrate-associated structures. the significance of the association of distinct carbohydrate structures with distinct embryonic or tumor cell types is absolutely unclear at present. ultrastructural localization has placed some epitopes intracellularly in specific compartments and others on the cell surface or extracellularly in the surrounding matrix. a cell surface localization is a necessary condition for a direct role in cell recognition and, on the strength of suggestions from other systems, this has been promiscuously interpreted to imply a role in cell recognition. this reflects the primitive status of our general understanding of the significance of protein glycosylation. thus the potential developmental roles of protein-linked carbohydrate in morphogenesis, differentiation, adherence and proliferation will likely be a primary target of investigation in this field in the future. interest in developmental involvement of carbohydrate is based not only on a large bank of indirect data but also on an intrigue in the special kinds of epigenetic regulatory mechanisms with which carbohydrate might be associated. for example, glycosylation offers the cell a mechanism to regulate the structure of multiple proteins without altering gene expression. in addition, monosaccharide residues may be processed independently of the associated polypeptide ( , ) either by recycling through the golgi ( ) or at locations near the site of funtion ( , ) , far from the point of origin in the rer and golgi. it can be envisaged that the cell responded to the evolutionary requirements for cell differentiation by using epigenetic pathways such as glycosylation and other posttranslational modifications ( ) . consistent with these speculations, there are, as cited above, numerous examples of glycosylation steps modified by differentiation ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ) , as well as by the anchorage state of the cell and the ph and composition of the surrounding medium ( , ) . an example of what the future may bear is exemplified by a group of developmental arrest mutants recently characterized in d. discoideum ( ) . isolated on the basis of failure to accumulate an early regulated enzyme, many of these mutants appear to be altered in posttranslational modifications, including glycosylation. thus many potentially necessary roles played by glycosylation in development remain unexplored. been shown that carbohydrate interaction is multivalent, composed of multiple weak associations which only in aggregate are strong ( , , ) . it would appear that most protein-linked carbohydrate is not solely responsible for the essential housekeeping functions of cells required for life in the tissue culture environment. perhaps this is because specification of its structure, which is nontemplate-directed, is not precise enough. glycosylation presumably has instead afforded the cell, during the evolution of developmental mechanisms, an economy in allowing the structural modification of many preexisting proteins using the same enzymatic mechanism. it provides the cell with another control point over structure which can be exerted on many proteins at the site of function (away from the site of synthesis) without necessitating a change in gene expression. change in structure by glycosylation may be very suitable for the regulation of the 'luxury' functions of cell differentiation and morphogenesis. an economy of shared non-specifc and specific functions of carbohydrate would thus offer an unifying explanation for the often contradictory conclusions yielded from the varied experimental approaches summarized in this article. the nearly ubiquitous glycosylation of extracellular and cell surface proteins and proteins from certain organelles may reflect a primitive, nonspecific function supporting protein structure and protection. this carbohydrate carries out a shared function, however, when it becomes modified in subtle ways without affecting non-specific functions, to allow discrimination by receptors. recognition by discriminate receptors allows the carbohydrate to subserve specific functions. carbohydrate would appear to be used by cells to carry out secondary functions (both specific and non-specific) for proteins. these include specialized instances of compartmentalization, rate of transport through the cell, intermolecular association, conformation control and general protection. interaction of the cell with the environment, which is in a sense secondary to the life of the cell, would also appear to involve protein-linked carbohydrate. this might include compartmentalization or sorting of cells into tissues. in several instances it has structure, biosynthesis and functions of glycoprotein glycans carbohydrate moieties of glycoproteins complex carbohydrates of the extracellnlar matrix. structures, interactions and biological roles glycoprotein biosynthesis in yeast control of asparagine-linked oligosaccharide chain processing: studies on bovine pancreatic ribonuclease b correlation of glycosylation forms with position in amino acid sequence lysosomal enzyme phosphorylation occurrence of n-acetylglucosamine -phosphate in complex carbohydrates - -methylation of mannose residues transient metbylation of dolichyl oligosaccharides is an obligatory step in halobacterial sulfated glycoprotein biosynthesis glycoproteins as ceil-surface components either high-mannose-type or hybrid type oligosaccharide is linked to the same asparagine residue in ovalbumin posttranslational covalent modification of proteins assembly of asparagine-linked oligosaccharides tunicamycin inhibits ganglioside biosynthesis in neuronal cells tunicamycin blocks neuritogenesis and glucosamine labeling of gangliosides in developing cerebral neuron cultures suppression of placental alkaline phosphatase biosynthesis by tunicamycin occurrence of nacetylglucosamine-l-phosphate in proteinase i from dictyostelium discoideum glycosylation mutants of animal cells laminin, proteoglycan, nidogen and collagen iv: structural models and molecular interactions purification, composition, molecular weight and subunit structure of ovine submaxillary mucin effect of altered oligosaccharide structure onithe cell surface number, distribution and turnover of the high molecular weight acidic glycoproteins of cho cells preliminary characterization of a chinese hamster ovary cell glycosylation mutant isolated by screening for low intracellular lysosomal enzyme activity temporal aspects of the n-and o-glycosylation of human chorionic gonadotropin o-linked oligosaccharides are acquired by herpes simplex virus glycoproteins in the golgi apparatus structural and functional aspects of tumor cell sialomucins antigenic determinants shared by lysosomal proteins of dictyostelium discoideum adhesion mutants of dictyostelium disclideum lacking the saccharide determinant recognized by two adhesion blocking monoclonal antibodies conformation of the glycopeptide linkage in asparagine-linked glycoproteins conformation of the complex oligosaccharides of glycoproteins multiple classes of heparan sulfate proteoglycans from fibroblast substratum adhesion sites effects of deglycosylation on the architecture of ovine submaxillary mucin glycoprotein preparation and properties of serum and plasma proteins. xxix. separation from human plasma of polysaccharides, peptides and proteins of low molecular weight. crystallization of an acid glycoprotein studies on fetuin, a glycoprotein of fetal serum. i. isolation, chemical composition, and physicochemical properties variation of the carbohydrates of glycoproteins of cells growing on different surfaces the function of protein-bound carbohydrates in normal and pathological cells asparaginyl-nacetylgalactosamine olden k: retinoic acid alters the proportion of high mannose to complex type oligosaccharides on fibronectin secreted by cultured chondrocytes a temperature-sensitive n-glycosylation mutant of s. cerevisiae that behaves like a ceil-cycle mutant nglycosylation of nascent proteins early in the prereplicative phase constitutes a process for controlling animal cell proliferation studies on the regulation of the biosynthesis of glucose-containing oligosaccharide-lipids carbohydrate structure, biological recognition, and immune function. in: ivatt rj (ed). the biology of glycoproteins glybopeptide changes and malignant transformation. a possible role for carbohydrate in maligant behavior demonstration by monoclonal antibodies that carbohydrate structures of glycoproteins and glycolipids are onco-developmental antigens glycosphingolipids as differentiationdependent, tumor-associated markers and as regulators of cell proliferation biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharides aggregation-deficient embryonal carcinoma cells: defects in peanut agglutinin (pna) receptors asparagine-linked oligosaccharides in murine tumor cells: comparison of a wga-resistant (wga r) nonmetastatic mutant and a related wga-sensitive (wga s) metastatic line wp: carbohydrates of the tumor cell surface oligosaccharide modification by swainsonine treatment inhibits pulmonary colonization by b -f murine melanoma cells regional specificity of glycoconjugates in xenopus and axolotl embryos saccharide structures of the mouse embryo during the first eight days of development reactivity of five nacetylgalactosamine-recognizing lectins with preimplantation embryos, early postimplantation embryos, and teratocarcinoma cells of the mouse inhibition of glycoprotein synthesis in saccharomyces cerevisiae by mating pheromones ssea- , a stagespecific antigen of the mouse, is carried by the glycoprotein-bound large carbohydrate in embryonal carcinoma cells trypanosoma cruzi cells undergo an alteration in protein n-glycosylation upon differentiation appearance of a class of cell-surface fucosyl-glyeopeptides in differentiated muscle cells in culture changes in protein glycosylation during chick embryo development changes in the expression of blood-group carbohydrates during oral mucosal development in human fetuses cell surface glycoconjugates as oncodifferentiation markers in hematopoietic cells glycoprotein synthesis and embryonic development decreased transfer of oligosaccharide from oligosaccharideqipid to protein acceptors in regenerating rat liver stimulation of lipid-linked oligosaccharide assembly duing oviduct differentiation cell-type-related segregation of surface galactosyl-containing components at an early developmental stage in hemopoietic bone marrow cells in the rabbit changes in asparagine-linked sugar chains of human promyelocytic leukemic cells (hl- ) during monocytoid differentiation and myeloid differentiation are glycoproteins and glycosaminoglycans components of the eukaryotic genome? n-linked glycoprotein biosynthesis in the developing mouse embryo biological significance of carbohydrate chains on monoclonal antibodies a role for glycosylation of the c~-subunit in transduction of biological signal in glycoprotein hormones role of carbohydrate in human chorionic gonadotropin antibodies against human chorionic gonadotropin convert the deglycosylated hormone from an antagonist to an agonist cell surface galactosyltransferase as a recognition molecule during development nelsestuen gl: deglycosylated prothrombin fragment polylactosamine glycosylation on human fetal placental fibronectin weakens the binding affinity of fibronectin to gelatin investigations of the possible functions for glycosylation in the high mobility group proteins preferential association of glycoproteins to the euchromatin regions of crossfractured nuclei is revealed by fracture-label eukaryotic ribosomes possess a binding site for concanavalin a on the biological role of glycoproteins the significance of glycosylated proteins effect of tunicamycin on the secretion of serum proteins by primary cultures of rat and chick hepatocytes differing requirements for glycosylation in the secretion of related glycoproteins is determined neither by the producing cell nor by the relative number of oligosaccharide units effect of the threonine analog/ -hydroxynorvaline on the glycosylation and secretion of c~l-acid glycoprotein by rat hepatocytes differential effects of -deoxynojirimycin on the intracellular transport of secretory glycoproteins of human hepatoma cells in culture the effects of processing inhibitors of nlinked oligosaccharides on the intracellular migration of glycoprotein e of mouse hepatitis virus and the maturation of coronavirus particles role of glycosylation in processing of newly translated insulin proreceptor in t -l adipocytes influence of the nlinked oligosaccharides on the biosynthesis, intracellular routing, and function of the human asialoglycoprotein receptor glycosylation of the epidermal growth factor receptor in a- cells inhibition of n-linked igosaccharide trimming does not interfere with surface expression of certain integral membrane proteins evidence for a glycoprotein 'signal' involved in transport between subcellular organelles a single nlinked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus g protein to the cell surface the role of protein glycosylation in the compartmentalization and processing of mouse mammary tumor virus glycoproteins in mouse mammary tumor virus-infected rat hepatoma cells the formation of vesicular stomatitis virus (san juan strain) becomes temperature-sensitive when glucose residues are retained on the oligosaccharides of the glycoprotein deletions into a ni-i -terrninal hydrophobic domain result in secretion of rotavirus vp , a resident endoplasmic reticulum membrane glycoprotein glycoantigen expression is regulated both temporally and spatially during development in the cellular slime molds dictyostelium discoideum and d. mucoroides the identification of n-linked oligosaccharides on the human cr /epstein-barr virus receptor and their function in receptor metabolism, plasma membrane expression, and ligand binding glycosylation and secretion of surfactant-associated glycoprotein a complete glycosylation of the insulin and insulin-like growth factor receptors is not necessary for their biosynthesis and function chemistry, metabolism, and biological functions of sialic acids effect of size and location of the oligosaccharide chain on protease degradation of bovine pancreatic ribonuclease swainsonine inhibits glycoprotein degradation by isolated rat liver lysosomes slime mold lectins bacterial glycoconjugates are natural ligands for the carbohydrate binding site of discoidin i and influence its cellular compartmentalization carbohydrate-specific receptors of the liver hepatic clearance of serum glycoproteins the repair of the surface structure of animal cells carbohydrate-mediated clearance of immune complexes from the circulation carbohydrate-mediated clearance of antibody-antigen complexes from the circulation surface carbohydrates and surface lectins are recognition determinants in phagocytosis the carbohydrate structure of porcine uteroferrin and the role of the high mannose chains in promoting uptake by the reticuloendothelial cells of the fetal liver intracellular movement of cell surface receptors after endocytosis: resialylation of asialotransferrin receptor in human erythroleukemia cells hyaluronic acid bonded to cell culture surfaces stimulated chondrogenesis in stage limb mesenchyme cell cultures lectin activation in giardia lamblia by host protease: a novel hostparasite interaction carbohydrates as recognition determinants in phagocytosis and in lectin-mediated killing of target cells specificity of binding of a strain of uropathogenic escherichia coli to galcd- gal-containing glycosphingolipids evidence for a malarial parasite interaction site on the major transmembrane protein of the human erythrocyte adherent bacterial colonization in the pathogenesis of osteomyelitis mucoproteins in relation to virus action the synthesis of complex carbohydrates by multiglycosyltransferase systems and their potential function in intercellular adhesion the receptor function of galactosyltransferase during cellular interactions the biology of glycoproteins a multivalent lacto-n-fucopentaose iii-lysyllysine conjugate decompacts preimplantation mouse embryos, while the free oligosaccharide is ineffective receptor function of mouse sperm surface galactosyltransferase during fertilization o-linked oligosaccharides of mouse egg zp account for its sperm receptor activity differential cell adhesion may result from nonspecific interactions between cell surface glycoproteins specific alteration of n-cam-mediated cell adhesion by an endoneuraminidase a heparin-bindifig domain from n-cam is involved in neural cell-substratum adhesion recognition of igg by fc receptor and complement: effects of glycosidase digestion cell surface carbohydrates in cell recognition and response feizi t: cell interactions in preimplantation embryos: evidence for involvement of saccharides of the poly-nacetyllactosamine series mitochondrial synthesis of glycoproteins and surface properties of mitochondrial membranes cell membranes in sponges the phosphomannosyl recognition system for intracellular and intercellular transport of lysosomal enzymes cell-cell recognition in yeast, characterization of the sexual agglutination factors from saccharomyces kluyveri lymphocyte attachement to high endothelial venules during recirculation: a possible role for carbohydrates as recognition determinants lymphocyte adhesion to immobolized polysaccharides suggests multiple carbohydrate receptors for recirculation spontaneous glycosylation of glycosaminoglycan substrates by adherent fibroblasts immunocytochemical demonstration of ecto-galactosyltransferase in absorptive intestinal cells soluble lectins: a new class of extraeellular proteins effect of choroquine on the degradation of l-fucose and the polypeptide moiety of plasma membrane glycoproteins different half-lives of the carbohydrate and protein moieties of a dalton glycoprotein isolated from plasma membraned of rat liver structural determinants of the capacity of heparin to inhibit the proliferation of vascular smooth muscle cells. ii. evidence for a pentasaccharide sequence that contains a - -sulfate group pathways of protein secretion in eukaryotes structure and function of plant cell wall polysaccharides gangliosides support neural retina cell adhesion ~-mannosidase- mutants of dictyostelium discoideum: early aggregationessential genes regulate enzyme precursor synthesis, modification, and processing discoidin i is implicated in cell-substratum attachment and ordered cell migration of dictyostelium discoideum phenocopy of discoidin i-minus mutants by antisense transformation in dictyostelium mechanisms for the incorporation of proteins in membranes and organelles glycopeptides prepared from mouse cerebrum inhibit protein synthesis and cell division in baby hamster kidney ceils, but not in their polyoma virus-transformed analogs stereochemistry and binding behavior of carbohydrate chains associations of like and unlike polysaccharides: mechanism and specificity in galactomannans, intreacting bacterial polysaccharides, and related systems model for the structure of the gastric mucous gel freeze hh: interaction of dictyostelium discoideum lysosomal enzymes with the mammalian phosphomannosyl receptor differential processing of asn-linked oligisaccharides on pituitary glycoprotein hormones: implications for biologic function lysosomal enzyme bind!ng to mouse p d l macrophage membranes lacking the -kda mannose -phosphate receptor: evidence for the existence of a second mannose -phosphate receptor identification and characterization of cells deficient in the mannose -phosphate receptor: evidence for an alternate pathway for lysosomal enzyme targetting variability in transport rates of secretory glycoproteins through the endoplasmic reticulum and golgi in human hepatoma cells glucose phosphotransferase and intracellular protein trafficking freeze hh: modifications of lysosomal enzymes in dictyostelium discoideum glucose removal from n-linked oligosaccharides is required for efficient maturation of cerain secretory glycoproteins from the rough endoplasmic reticulum to the golgi complex deletion of clustered o-linked carbohydrates does not impair function of low density lipoprotein receptor in transfected fibroblasts absence of a carbohydrate modification does not affect the level or the subcellular . . . localization of three membrane glycoproteins in modb mutants of dictyostelium discoideum reversible defects in o-linked glycosylation and ldl receptor expression in a udp-gal/udp-galnac -epimerase deficient mutant groth sf: studies on mucoproteins. iii. the accesibility to trypsin of the susceptible bonds in ovine submaxillary land mucoproteins influenza c virus uses - -acetyl-n-acetylneuraminic acid as a high affinity receptor determinant for attachment to cells carbohydrate-binding sites of the mannose-specific fimbrial lectins of enterobacteria the mouse egg's receptor for sperm; what is it and how does it work? cell adhesion molecules neuronal cell-cell adhesion depends on interactions of n-cam with heparin-like molecules reconstitution of high cell binding affinity of a marine sponge aggregation factor by cross-linking of small low affinity fragments into a large polyvalent polymer localization of galactosyl-and sialyltransferase by immunofluorescence: evidence for different sites outer membrane terminal saccharide of bovine liver nuclei and mitochondria hendil kb: ion exchange properties of the glycocalyx of the amoeba chaos chaos and its relation to pinocytosis liver endothelium mediates the hepatocyte's uptake of ceruloplasmin the molecular basis of species specific cell-cell recognition in marine sponges, and a study on organogenesis during metamorphosis rous sarcoma virus-transformed baby hamster kidney cells express higher levels of asparagine-linked tri-and tetraantennary glycopeptides containing [glcnac- ( , )man-a( , )man] and poly-nacetyllactosamine sequences than baby hamster kidney cells i am grateful to drs. c. feldherr and g. erdos for their critisms of the manuscript during preparation. key: cord- - nqrrnqr authors: haslinger, christian; stadler, peter f. title: rna structures with pseudo-knots: graph-theoretical, combinatorial, and statistical properties date: journal: bull math biol doi: . /bulm. . sha: doc_id: cord_uid: nqrrnqr the secondary structures of nucleic acids form a particularly important class of contact structures. many important rna molecules, however, contain pseudo-knots, a structural feature that is excluded explicitly from the conventional definition of secondary structures. we propose here a generalization of secondary structures incorporating ‘non-nested’ pseudo-knots, which we call bi-secondary structures, and discuss measures for the complexity of more general contact structures based on their graph-theoretical properties. bi-secondary structures are planar trivalent graphs that are characterized by special embedding properties. we derive exact upper bounds on their number (as a function of the chain length n) implying that there are fewer different structures than sequences. computational results show that the number of bi-secondary structures grows approximately like . (n). numerical studies based on kinetic folding and a simple extension of the standard energy model show that the global features of the sequence-structure map of rna do not change when pseudo-knots are introduced into the secondary structure picture. we find a large fraction of neutral mutations and, in particular, networks of sequences that fold into the same shape. these neutral networks percolate through the entire sequence space. the secondary structures of nucleic acids form a particularly important class of contact structures. many important rna molecules, however, contain pseudoknots, a structural feature that is excluded explicitly from the conventional definition of secondary structures. we propose here a generalization of secondary structures incorporating 'non-nested' pseudo-knots, which we call bi-secondary structures, and discuss measures for the complexity of more general contact structures based on their graph-theoretical properties. bi-secondary structures are planar trivalent graphs that are characterized by special embedding properties. we derive exact upper bounds on their number (as a function of the chain length n) implying that there are fewer different structures than sequences. computational results show that the number of bi-secondary structures grows approximately like . n . numerical studies based on kinetic folding and a simple extension of the standard energy model show that the global features of the sequence-structure map of rna do not change when pseudo-knots are introduced into the secondary structure picture. we find a large fraction of neutral mutations and, in particular, networks of sequences that fold into the same shape. these neutral networks percolate through the entire sequence space. c society for mathematical biology presumably the most important problem and the greatest challenge in present day theoretical biophysics is deciphering the code that transforms sequences of biopolymers into spatial molecular structures. a sequence is properly visualized as a string of symbols which together with the environment encodes the molecular architecture of the biopolymer. in case of one particular class of biopolymers, the ribonucleic acid (rna) molecules, decoding of information stored in the sequence can be properly decomposed into two steps: (i) formation of the secondary structure, that is, of the pattern of watson-crick (and gu) base pairs, and (ii) the embedding of the contact structure in three-dimensional space. the sequence structure relation of rna was studied in detail in a series of papers (fontana et al., (fontana et al., , a bonhoeffer et al., ; tacker et al., ; grüner et al., a,b; tacker et al., ) at the level of secondary structures. the most salient findings of these investigations are: (i) there are many more sequences than (secondary) structures. (ii) there are few frequent and many rare structures. almost all sequences fold into frequent or 'common' structures. (ii) sequences that fold into a 'common' structure are distributed nearly uniformly in sequence space. (iv) a sequence folding into a 'common' structure has a large number of neutral neighbors (folding into the same structure) and a large number of neighboring sequences that fold into very different secondary structures. (v) neutral paths percolate sequence space along which all sequences fold into the same secondary structure. in fact, there are extended neutral networks of sequences folding into the same 'common' structure (grüner et al., b; . (vi) almost all 'common' structures can be found close to any point in sequence space. this property is called shape space covering. the impact of these features on evolutionary dynamics is discussed in schuster ( ) and : a population explores sequence space in a diffusion-like manner along the neutral network of a viable structure. along the fringes of the population novel structures are produced by mutation at a constant rate (huynen, ) . fast diffusion together with perpetual innovation makes these landscapes ideal for evolutionary adaptation (fontana and schuster, ) . the 'classical' definition of secondary structures incorporates a quite restrictive condition on the set of base pairs that implies a tree-like arrangement of the doublehelical regions, see fig. . additional interactions between different branches of this tree are referred to as pseudo-knots (for an exact definition see section ). pseudoknots are excluded from many studies for a mostly technical reason (waterman and smith, a, b) : the folding problem for rna can be solved efficiently by dynamic programming (waterman and smith, b; zuker and sankoff, ) in their absence. on the other hand, an increasing number of experimental findings, as well as results from comparative sequence analysis, suggest that pseudo-knots are important structural elements in many rna molecules (westhof and jaeger, ) . notably, figure . the contact structure of escherichia coli rnase p rna contains two pseudoknots [http://jwbrown.mbio.ncsu.edu/rnasep/home.html]. the conventional secondary structure is drawn on the l.h.s., the (four) regions forming the pseudo-knots are marked by braces, interaction regions are connected. the arc diagram of the same structure is obtained by arranging the backbone along a line and indicating base pairs by arcs connecting the corresponding bases. the base pairs of the conventional secondary structure are drawn above the line, the two pseudo-knot stems are shown below the back-bone. for details see section . functional rnas such as rnasep rna (loria and pan, ) and ribosomal rna (konings and gutell, ) contain pseudo-knots. the diversity of molecular biological functions performed by pseudo-knots can be subdivided into three groups. pseudo-knots at the -end of mrnas appear to adopt a role in the control of mrna translation. for instance, the expression of replicase is controlled in several viruses either by ribosomal frame shifting (ten dam et al., ; brierley et al., ; dinman et al., ; chamorro et al., ; tzeng et al., ) or by in-frame readthrough of stop codons (wills et al., ) . both mechanisms involve pseudo-knots. core pseudo-knots are necessary to form the reaction center of ribozymes. most of the enzymatic rnas with core pseudo-knots, such as rnasep, are involved in cleavage or self-cleavage reactions (michel and westhof, ; forster and altman, ; brown, ; haas et al., ) . pseudo-knots in the trna-like motifs at the -end of the genomic rna mediate replication control in several groups of plant viral rna (mans et al., ) . it is important, therefore, to include pseudo-knotted structures into investigations of rna sequence-structure relationships. in particular, we need to know whether the findings (i) through (vi) described above remain true when pseudo-knots are taken into account. assertion (i), the existence of more sequences than structures, is a necessary prerequisite for all subsequent statements concerning the sequencestructure map of rna. it is necessary therefore to estimate the number of rna structures with pseudo-knots in order to decide whether the results quoted above can in fact be true for 'real' rna molecules. in the following two sections we give a detailed mathematical analysis of what we call bi-secondary structures. in a nutshell, bi-secondary structures generalize to the notion of secondary structures to include pseudo-knots without allowing overly involved knotted structures or nested pseudo-knots. in fact, almost all known pseudo-knotted structures, with the notable exception of the e. coli αmrna, fall into this class. in section we review a variety of equivalent graph-theoretical characterizations of bi-secondary structures and provide a way of efficiently determining whether a list of base pairs corresponds to a bi-secondary structure. then we briefly review a few graph invariants that might be useful for determining the complexity of higher-order structures beyond the realm of bi-secondary structures. at the end of section we show that a convenient distance measure for comparing secondary structures can be used also in the presence of pseudo-knots (section . ). in subsection . we argue that the intersection theorem is valid for general nucleic acid contact structures. we say that an rna sequence is compatible with a structure s if it can in principle form this structure irrespective of energetic constraints. this means that for each base pair (i, j) in s the sequence positions x i and x j are one of the six possible rna base pairs au, ua, gc, cg, gu, or ug. the set of sequences that actually fold into a given structure s is therefore a subset of the set of compatible sequences. the intersection theorem (reidys et al., ) now states that for any two structures s and s there are sequences which are compatible with both of them. this result is the reason why very different structures with very closely related sequences can exist. the fact that the intersection theorem holds for structures with pseudo-knots means that we have to expect shape space covering provided the fraction of neutral mutations is large enough (reidys et al., ) . in section we determine the number of different structures with pseudo-knots. combinatorial aspects of rna secondary structures have been studied in detail by waterman and co-workers (stein and waterman, ; waterman, ; waterman and smith, a, b; penner and waterman, ; schmitt and waterman, ; waterman, ) and hofacker et al. ( ) . using different techniques we give analytical upper bounds on the number of different bi-secondary structures showing that their number does not increase much faster than the number of secondary structures. the analytical results are complemented by numerical data (see table at the end of section ) indicating that the number s n of 'reasonable' bi-secondary structures with chain length n grows approximately as s n ∼ . n . 'reasonable' means here that the structures have no isolated base pairs (i.e., the minimum stack size is l = ) and that hairpin loops contain at least m = unpaired bases. for comparison, the number of secondary structures without pseudo-knots grows like . n . exhaustive enumeration for short sequences suggest that only . n different secondary structures appear as minimum energy structures of sequences of length n (grüner et al., a) . hence the number n of rna sequences of length n, is much larger than the number of possible structures, independent of whether or not one takes pseudo-knots into account. this observation poses the question how the sequences that fold into a given structure are distributed in sequence space. in section we describe a set of numerical experiments strongly suggesting that the inclusion of pseudo-knots does not alter the qualitative picture [properties (i) through (vi) above] of the rna sequence-structure map. a short discussion (section ) concludes this contribution. readers who are not interested in the mathematical details of defining, characterizing, and counting contact structures of various types might want to skip sections and . the three-dimensional structure of a linear biopolymer, such as rna, dna, or a protein can be approximated by its contact structure, i.e., by the list of all pairs of monomers that are spatial neighbors. contact structures of polypeptides have been introduced by ken dill and co-workers in the context of lattice models of protein folding (chan and dill, ; chen and dill, ) . they arise implicitly in knowledge-based potentials for polypeptides such as the delauney-tesselation potential described in singh et al. ( ) . last but not least, rna secondary structures form a special class of contact structures. the purpose of this section is to bring together different mathematical approaches that can be used to describe biopolymer structures: contact graphs, linked diagrams, book embeddings, and graph colorings. a contact structure is represented by the contact matrix c with the entries c i j = if the monomers i and j are spatial neighbors without being adjacent along the backbone, and c i j = otherwise. hence c i j = if |i − j| ≤ . we shall use the notation [n] = { , . . . , n}. we define a diagram ([n] , ) to consists of n vertices labeled to n and a set of arcs that connect non-consecutive vertices. a closely related class of diagrams which also allow arcs between consecutive vertices are the linked diagrams introduced by touchard ( ). these are studied in some detail in hsieh ( ), kleitman ( ), stein ( and stein and everett ( ) . it is customary to arrange the vertices along the x-axis and to draw the vertices in such a way that they are confined in either the upper or the lower half-plane. the diagram of a contact structure with contact matrix c has the set of arcs = {{i, j}|c i j = }. ( ) the contact matrix is thus the adjacency matrix of the corresponding diagram. with each diagram we may associate a diagram graph with the following properties: let b be the adjacency matrix of the backbone, i.e., the matrix with the entries b i,i+ = b i+ ,i = , i = , . . . , n − , and b n = b n = . then the adjacency matrix of a diagram graph with n + vertices is of the form equation ( ) establishes the - correspondence of diagrams and the associated diagram graphs. essentially the same construction can be used for contact structures of molecules with a circular backbone, i.e., for circular ssrna or ssdna. the only restriction is that { , n} cannot be an arc in the case of a circular molecule. it is convenient in this case to define the corresponding diagram graph without the artificial root . each graph with a hamiltonian cycle is then the diagram graph of a contact structure with a circular backbone. the results in the following discussion hold for both linear and circular nucleic acids. a diagram is a -diagram if for any two arcs α, β ∈ holds α ∩ β = ∅ or α = β. a diagram is a -diagram if and only if associated diagram graph ( ) has vertex degrees less or equal to . such graphs are often called sub-cubic or trivalent. the diagram graphs of -diagrams are closely related to cubic hamiltonian graphs. the latter are studied in detail in section . of wagner and bodendiek ( ): a graph s is homeomorphic from a graph if s can be produced from by inserting vertices of degree into some edges of . s is also called a subdivision of . obviously each cubic hamiltonian graph gives rise to a diagram graph on n vertices by subdividing the edges of a hamiltonian cycle. on the other hand, not all diagram graphs are homeomorphic from a cubic hamiltonian graph: suppose { , } is an arc and is an unpaired vertex. the corresponding diagram graph cannot be cubic because the triangle , , cannot be obtained from a cubic graph. the classical definition of secondary structures (waterman, ) requires that each base interacts with at most one other nucleotide. thus nucleic acid secondary structures are special types of -diagrams. the second defining condition is that arcs do not cross. in terms of the contact matrix this means: if c i j = c kl = and i < k < j then i < l < j. with the following notation we will find a simpler formulation of condition : let α = {i, j} with i < j be an arc of a diagram. we writeᾱ = [i, j] ⊂ r for the associated interval. two arcs of a diagram are consistent if they can be drawn in the same half-plane without crossing each other. equivalently, two arcs α, β ∈ of a diagram are consistent if either one of the following four conditions is satisfied: case (iv) is ruled out by definition in -diagrams. the non-crossing condition thus may be expressed as follows: whenever the intervals of two arcs {i, j} and {k, l} have non-empty intersection then one is contained in the other (schmitt and waterman, ) . equivalently, we may simply define that a secondary structure is a -diagram in which any two arcs are consistent. as a consequence, each secondary structure can be encoded as a string s of length n in the following way: if the vertex i is unpaired, then s i = '.'. each arc α = {p, q} with p < q translates to s p = '(' and s q = ')'. as the arcs are consistent their corresponding parentheses are either nested, (( )), or next to each other, ()(). as there are no arcs between neighboring vertices in a diagram there is at least one dot contained within each parenthesis. a variant of this notation is the mountain representation of rna secondary structures (hogeweg and hesper, ) . the 'dot-parenthesis' notation is used as a convenient notation in input and output of the vienna rna package, a piece of public domain software for folding and comparing rna molecules . a graph that can be embedded in the plane (or, equivalently on the sphere) is called planar. if it can be embedded in the plane in such a way that all its vertices lie on the exterior region it is called outer-planar. this class of graphs was introduced and characterized in terms of subgraphs in chartrand and harary ( ) and sys lo ( ). clearly, a -diagram is a secondary structure if and only if its diagram graph ( ) is outer-planar. the outer-planar embedding corresponds to the 'circle representation' of secondary structures. a similar procedure leads to book-embeddings. a p-book is a set of p distinct half-planes (the pages of the book) that share a common boundary line , called the spine of the book. an embedding of a graph into a book b consists of an ordering of the vertices along the spine of the book together with an assignment of each edge to a page of the book, in which edges assigned to the same page do not cross. the book-thickness (sometimes also called the page-number) bt( ) of a graph is the minimal number p of pages of a book into which it can be embedded (bernhart and kainen, ) . book-embeddings have a practical application in the context of vlsi design. for an overview see chung et al. ( ) and heath et al. ( ) . not surprisingly, the book thickness is closely related to other embedding properties of graphs. below we list a few important results: (v) bt(k n ) = n/ , where k n is the complete graph with n vertices (bernhart and kainen, ) . n + for sub-cubic graphs (chung et al., ) . of a graph is the minimum number of 'handles' one needs to add to a sphere so that the graph can be embedded on the resulting surface without crossing edges.) the book thickness of a variety of other graph classes has been studied in detail, among them hypercubes (chung et al., ) , de bruijn graphs (obrenić, ) , and various types of network graphs of practical interest (games, ). essentially the same construction is used for the investigation of cubic hamiltonian graphs in wagner and bodendiek ( ) . we shall see that the inconsistency graph is a useful construction for characterizing embedding properties of diagram graphs. theorem . let be a diagram. then the following statements are equivalent. proof. (i ⇐⇒ ii) can be drawn without intersection arcs if and only if ( ) is planar because the hamiltonian cycle h of ( ) divides the plane into the interior and the exterior of h which correspond to the upper and lower half-plane of the diagram , respectively. (ii ⇐⇒ iii) can be shown in the same way as the analogous result for cubic hamiltonian graphs in wagner and bodendiek ( ) , see also even and itai ( ) . (ii ⇐⇒ iv) follows immediate from bernhart and kainen ( , theorem . ) as a planar diagram graph is by construction hamiltonian. as noted in even and itai ( ) , the determination of the book thickness of a is equivalent to finding a minimal vertex-coloring of a certain circle graph, which in our case is the intersection graph ( ). this problem is in general np-complete. the following observation simplifies the task by reducing the number of arcs that have to be considered. two it is easy to show that the arcs of a stem of a -diagram are either all isolated vertices or they are contained in the same component of the inconsistency graph ( ). furthermore, all arcs of a stem have the same adjacent vertices in ( ). we may therefore use a reduced intersection graphˆ ( ), the vertices of which are the stems. (in addition, we may recursively remove vertices of degree that are not contained in a triangle before forming the intersection graph. this has the effect of removing bulges and interior loops that interrupt stems.) examples of reduced intersection graphs are given in figs and . most of the literature on linked diagrams deals with complete diagrams, that is, each vertex x ∈ [n] is incident with an arc (touchard, ; kleitman, ; stein, ) . it is straightforward to extend touchard's definition of reducible diagrams to the incomplete diagrams considered here: the following equivalence is proved in haslinger ( ): reducible diagrams can therefore be viewed as being composed of substructures. these substructures do not in general conform to the conventional decomposition into stems and loops of an rna that forms the basis of the standard energy model of nucleic acid secondary structures (freier et al., ) . the contact structure of the proposed srv- frameshift signal contains a pseudoknot, see ref. ten dam et al. ( ) . pseudo-knots such as this one belong to the class of bi-secondary structures. knots such as the one in the lower part of the figure do not belong to the class of bi-secondary structures. knots, in contrast to pseudo-knots, may contain parallel stranded helices which so far have not been described for rna. we may draw the arcs in the upper or lower half-plane, but they are not allowed to intersect the x-axis. in other words, it can be embedded in -page book. bi-secondary structures are therefore 'superpositions' of two secondary structures. the virtue of bi-secondary structures is that they capture a wide variety of rna pseudo-knots, [figs and (upper part)], while at the same time they exclude true knots. knotted rnas could in principle arise either from parallel stranded helices (fig. ) , or in very large molecules from sufficiently complicated crosslinking patterns. parallel-stranded rna has not been observed (so far), see, however, fortsch et al. ( ) on parallel-stranded dna. wollenzien cantor et al. ( ) have searched unsuccessfully for knots in large rnas. the definition of bi-secondary structures, by allowing a planar drawing of the structure, rules out both possibilities. among the rna structures with pseudo-knots, almost all are bi-secondary structures. our examples include several viral rnas such as coronavirus (brierley et al., ) , luteovirus (ten dam et al., ) , and retrovirus rna (chamorro et al., ) , as well as catalytic rnas such as rnasep rna (loria and pan, ), tmrna (vlassov et al., ; felden et al., ) , and ribosomal rnas (gutell et al., ) . we have encountered only a single exception, namely αmrna (tang and draper, ) . theorem . let be a -diagram. then the following statements are equivalent: (v) among any three arcs of at least two are consistent. proof. the equivalence of (i), through (iv) is proved in theorem for all diagrams. the equivalence of (v) and (vi) follows immediately from the definition of ( ). the implication (iii ⇒v) is obvious. finally, it is possible to show that ¬(ii) implies ¬(v) based on kuratowski's ( ) theorem. for the details we refer to haslinger ( ). the practical importance of theorem lies in the fact that existence or nonexistence of triangles in ( ) can be checked very easily, and hence we have a very efficient (polynomial time) method for deciding whether a diagram is a bi-secondary structure or not. note that the equivalence of (iii) and (vi) does not hold for general diagrams. a counterexample is shown in fig. . being the union of the two secondary structures ([n], u ) and ([n], l ) we can represent each bi-secondary structure as a string s using two types of parentheses: as in a secondary structure we write a dot '.' for all unpaired vertices. a pair { p, q} ∈ u becomes s p = '(' and s q = ')', while an arc { p, q} ∈ l becomes s p = '[' and s q = ']'. unfortunately, the decomposition of a bi-secondary structure into two secondary structures is in general not unique, see fig. . the fact that ( ) is bipartite allows us to define a normal form for this representation by means of the following rule: the leftmost arc of each connected component of ( ) belongs to u . in particular, all isolated vertices of ( ) are contained in u . the normal form of a secondary structure therefore contains only dots and (round) parentheses. within each non-trivial connected component of ( ) the distribution of arcs between u and l is unique because the component is bipartite. all arcs in a stack have a common neighboring vertex in ( ), hence they all belong to the same class of the partition. therefore, in normal form, all arcs belonging to the same stack are written with the same type of brackets. the following example shows that there are natural rna structures that are more complicated than bi-secondary structures. the escherichia coli α-operon mrna folds into a structure that is required for allosteric control of translational initiation (tang and draper, ) . compensatory mutations have defined an unusual pseudo-knotted structure (tang and draper, ) , the thermodynamics of which were subsequently investigated in detail (gluick and draper, ) . the diagram of its contact structure cannot be drawn without intersections, see fig. . to our knowledge it is the only known rna structure that cannot be embedded in a -page book. in this subsection we briefly discuss a few graph properties that could be used for a classification of polymer structure complexity beyond the realm of bi-secondary structures. clearly, one may use its book thickness. a closely related quantity is the chromatic number of the intersection graph: a color partition of a graph is partition v = v ∪ v ∪ · · · ∪ v c of its vertex set into c subsets v i such that no two vertices in v i are adjacent. the chromatic number χ( ) is the smallest number c of colors for which a color partition of can be found. an arbitrary diagram can be decomposed into substructures by means of the following obvious result: let = ([n], ) be a diagram and let v : = ∪ figure . diagram of the contact structure of e. coli α-mrna. the structure contains five stems, labeled by uppercase greek letters. we may choose the color partition if ( ) such that all arcs in a stem have the same color. it therefore suffices to draw the inconsistency graph for stems (r.h.s. of the figure) . it contains triangles, thus the diagram of this rna structure is not a bi-secondary structure. it is easy to check that χ( ( )) = . the inconsistency graph ( ). noticing that χ( ) = if contains no edges and χ( ) = if is bipartite with non-empty edge set, the following characterization follows immediately: is a bi-secondary structure iff χ( ( )) ≤ . clearly, χ ( ( )) equals the minimum number of pages of all book embeddings in which the the ordering of the vertices along the spine coincided with the natural ordering along the backbone. in general, we have bt( ( )) ≤ χ( ( )) for all diagrams. we remark that graphs with moderate chromatic numbers can be characterized by results similar to kuratowski's theorem for planar graphs. for instance, one can show for k ≤ , that a graph with chromatic number χ( ) ≥ k contains a subdivision of the complete graph k k (dirac, ) . the generalization of this proposition to k > is known as hajós' conjecture. it is false for k ≥ and unsolved for k = and k = (holton and sheehan, ). it seems that χ( ( )) is in fact the more useful quantity, as there are no efficient algorithms to determine the book-thickness of a given graph, and χ( ( )) accounts for the immutable ordering of the backbone vertices, whereas the book-thickness might decrease by changing this ordering. a quite different algebraic graph invariant µ, introduced by de verdière ( ), leads to the same classification of structures for small µ: µ = ( ) is a circle, has no arcs. µ ≤ ( ) is outer-planar, is a secondary structure. µ ≤ ( ) is planar, is a bi-secondary structure. the graphs with µ ≤ have recently been identified as the flat or linklessly embeddable graphs (lovász and schrijver, ) . a useful characterization of this class of graphs is proved in robertson et al. ( , b) : 'a graph is non-flat if and only if it has no minor in the so-called petersen family'. the graph v * , fig. , is a valid diagram graph. it is easy to check that v * is flat and that its inconsistency graph is γ δ α β γ δ α β figure . the graph v * and its inconsistency graph. (v * ) = k . hence there are flat diagram graphs for which χ( ( )) ≥ . thus there is no direct correspondence between χ( ( )) and µ, not even for -diagrams. . interpreting each arc {i, j} as a transposition (i, j) on [n] we may assign the permutation to each diagram . one observes: (i) if a -diagram then π( ) is an involution. (ii) an involution π is the permutation representation of a -diagram if and only if its cycle decomposition does not contain a canonical transposition, i.e., a transposition of the form (i, i + ). (iii) different -diagrams give rise to different involutions. a natural set of generators for the symmetric group s n is the set t of all transpositions. the corresponding length function is where cyc(π ) is the number of cycles into which π decomposes. we have (τ ) = if and only if τ ∈ t is a transposition. the associated metric is the canonical metric on the cayley graph (s n , t ), see reidys and stadler ( ) for a detailed discussion. as the involutions form a subset of s n we have theorem . the function where π( ) denotes the permutation representation of a diagram , is a metric on the set of all -diagrams with n vertices. in particular, two -diagrams and have distance d( , ) = if and only if they differ by a single arc. metrics on 'shape space' are necessary for a detailed quantitative study of sequence-structure maps. applications to rna secondary structures are reported for instance in fontana et al. ( a) and . ( ) is not limited to defining a metric on the set of structures. suppose we are given an alphabet of monomers (for instance {a, u, g, c} in the case of rna) and a rule that determines which pairs of monomers may form a base pair (au, ua, gc, cg, gu, ug in the case of rna). the proof of this result in reidys et al. ( ) is valid for all -diagrams, not only for secondary structures. the intersection theorem sets the stage for shape space covering: it allows close-by sequences to fold into structures that are as different as desired -given a suitable folding potential. further applications of equation ( ) can be found in weber ( ). the number x n of all diagrams on n vertices is x n = (n− )(n− )/ as there are (n − )(n − )/ possible arcs (söler and jankowski, ), which can be arbitrarily combined to form a diagram. in section . we have shown that all -diagrams correspond to involutions, therefore the number t n of involutions on [n] is an upper bound for the number d n of -diagrams on [n]. the combinatorics of involutions is discussed for instance in the book by wilf ( ): t n = t n− + (n − )t n− n ≥ and t = t = , and has the asymptotic form the number of involutions t n therefore grows faster than exponential in the sense that n √ t n → ∞. -diagrams can be counted by a very similar recursion as the following result shows: proof. the first few values of d n are obvious, d = is a convenient definition. the recursion is derived as follows: a -diagram on n + vertices can be formed either by adding a lone vertex to a -diagram on n + vertices or by adding an arc { , k} to a -diagram on n vertices by inserting the vertex labeled k between the k − st and the kth vertex of . note, however, that must be a -diagram, but in addition it might have an arc {k − , k} in , as these vertices are separated by the endpoint of the newly introduced arc in the new structure. viewing this differently, we may either add the arc { , k} or the -like structure consisting of the arcs { , k} and {k − , k + }, which leaves us with a -diagram on n − vertices and the same problem. repeating this argument we arrive at the following expansion: hence we have d n+ = d n+ + n d n + (n − )d n− + (n − )d n− + · · · . observing that d n+ can of course be written in the same form and substituting into the above equations yields d n+ = (n + )d n +n d n− +(n − )d n− +(n − )d n− +· · ·+ d + d − d n− . subtracting the corresponding expansion for d n+ yields a simple rearrangement now completes the proof. proof. the series d n is obviously monotonically increasing. hence the series a n+ = (n + )a n , a = a = is a lower bound. it is well known that a n = (n − )!! grows faster than exponentially. a very similar formula is obtained for the case of a circular backbone. there are d n− diagrams with arc { , n} on n vertices. thus the number of -diagrams with circular backbone is d n = d n − d n− . an exponential upper bound can be found, however, on the numbers d n (c) of -diagrams whose inconsistency graph has chromatic χ( ( )) ≤ c. we find theorem . d n (c) ≤ ( c + ) n . then there is a color partition of with c colors. as ([n] , i ) is a secondary structure, it can be encoded in dot-parenthesis notation. coloring the parenthesis with a different color for each class i of the color partition hence yields a unique representation of . this representation can be interpreted as a string of length n over an alphabet consisting of '.' and c different pairs of brackets, i.e., with c + letters. theorem is not a very good estimate as we shall see in section . . a secondary structure on n+ digits may be obtained from a structure on n digits either by adding a free end at the right-hand end or by inserting a base pair ≡ (k + ). in the second case the substructure enclosed by this pair is an arbitrary structure on k digits, and the remaining part of length n − k − is also an arbitrary valid secondary structure. therefore, we obtain the following recursion formula for the number s n of secondary structures: this expression has first been derived by waterman ( ) the most important numbers are collected in table . a more detailed table can be found in hofacker et al. ( ) . detailed combinatorial studies on various aspects of secondary structure graphs are based on equation ( ), see for instance penner and waterman ( ) , stein and waterman ( ), waterman ( , smith ( a, b) and hofacker et al. ( ) . in the following we shall make use of the number of secondary structures of length n with k base pairs. this closed formula was recently derived in schmitt and waterman ( ). a first naive upper bound is d n ( ) ≤ s n , because on each side of the x-axis we have a secondary structure on n vertices. theorem implies d n ( ) ≤ n . a slightly better bound can be derived using the enumeration of secondary structures: proof. we start with the s(n, k) secondary structures with k arcs. in order to produce a bi-secondary structure we use l of the n − k unpaired positions for introducing l additional arcs. there are n− k l possible choices for these additional pairs, which may form any of the c l = l+ l l possible configurations of l matched parentheses. c l is a catalan number. without losing generality we may assume that l ≤ k, i.e., the partial secondary structure with the larger number of pairs is drawn above the x-axis. thus replacing the sums by appropriate multiples of the maximum entry is trivial. note that this bound is still a gross overestimate: (i) it contains all the redundancy of the ().[]-representation. (ii) the number c l also counts conformations of square brackets of the form [], which do not correspond to a graph at all, and it counts conformations in which not all square brackets are inconsistent with an arc that is represented by a round bracket. these latter configurations are counted more than once. proof. let a n (k, l) denote argument of the maximum in lemma . it is straightforward to compute solving the optimization problem that defines a is straightforward. a short computation shows thatŷ = / √ andx = ( − √ )/ is the only local maximum with x, y ≤ / . it violates the condition y ≤ x, however. the solution thus lies on the boundary of the triangle ( , ), ( / , ) and ( / , / ). setting y = one obtains the maximumx = / − / √ . along the edge x + y = / we findŷ = / √ violating the condition y ≤ x. with x = y we arrive at the cubic equation x − x + x − = which has a single real solution x ≈ . . we find a(x,x) ≈ . = a, because this value is much larger than the values of a(x, y) at the three corners of the triangle. more sophisticated models of rna take into account that (i) base pairs must enclose at least m = other bases, and (ii) that isolated base pairs are energetically disfavored. in hofacker et al. ( ) the numbers (m,l) n of secondary structures with stack size at least l base pairs and separation of the vertices incident with an arc at least m is derived. we define (m,l;κ) n to be the number of -diagrams with χ ( ( )) ≤ κ and with the same restrictions, and set clearly we have (m,l; ) n ≤ [ (m,l) n ] κ because the -diagram is a superposition of at most κ secondary structures. in particular, we find the upper bound a ( ) , ≤ . for the biophysical case. we have not been able to derive an exact counting series for bi-secondary structures. hence we resorted to a numerical survey. we pursued three different strategies for estimating the number of bi-secondary structures: ( ) complete enumeration is feasible only for very small values of n because the number of structures grows faster than n . ( ) as an alternative we produce random strings from the alphabet ().[] and check each string if it is the normal form of a bi-secondary structure. the number of secondary structures is then estimated by n × n nf /n sample , where n sample is the size of the random sample and n nf is the number of detected normal forms in the sample. our best estimates are compiled in table . in the biologically interesting case, m = and l = , we find a ( ) , ≈ . . judging from the exhaustive enumeration data (grüner et al., a) we should expect that the number of structures that actually occur as minimum energy structures is still smaller. in order to incorporate pseudoknots into secondary structure computations we first have to devise an energy model. naturally, we require that this energy function extends the standard model for rna secondary structures without pseudo-knots. the standard energy model is based on decomposing a secondary structure into its 'loops' (zuker and sankoff, ) . for secondary structures without pseudo-knots this decomposition is unique and coincides with the so-called minimum cycle basis of the secondary structure graph (leydold and stadler, ) . the free energy of a particular secondary structure is computed as the sum of the contributions of the individual loops. these contributions depend on the type of the loop (stacked base pairs, hairpin loop, bulge, interior loop, or multi-branch loop), its size, and on the sequence of nucleotides, see e.g., walter et al. ( ) . we emphasize that the energy model for pseudo-knotted structures introduced in this section is not intended as an accurate potential for predicting pseudo-knots in particular (biologically relevant) sequences. it is intended as a simplified model that allows us to investigate the likelihood of pseudo-knots in an ensemble of sequences and the stability of pseudo-knots against point mutations of the sequence. it is shown in tacker et al. ( ) for (pseudo-knot-free) rna secondary structures that such statistical properties are surprisingly robust against changes in the parameter set and the choice of the folding algorithm. for instance, most global properties of rna folding are already present in the 'maximum matching' model, which, instead of an elaborate energy model, simply seeks to maximize the number of base pairs (tacker et al., ) . a potential function that captures the most salient features of pseudo-knots is therefore sufficient for our purposes. very little experimental information is available on the thermodynamics of pseudoknots, see, however, wyatt et al. ( ) . on the other hand, the geometric constraints of rna structures are well understood (saenger, ; pleij et al., ) . hence we start from the following three principles: (i) loops that are not involved in pseudo-knots have the same energy contributions as in pseudo-knot-free rna secondary structures. (ii) the stacking energies of base pairs are not affected by pseudo-knot formation even in stems that are part of pseudo-knots. (iii) steric hindrance is the major contribution to the pseudo-knot energies. the energy parameters detailed in walter et al. ( ) , and implemented in release . of the vienna rna package , are used in this study for . schematic drawing of an rna structure with pseudo-knots. the three loops a, b, c and the four stems , , , and are involved in pseudo-knots. the evaluation of loops a and c is straightforward as they contain only a single paired region, namely stack . three stems are contained in loop b; we assume that stack is the longest one. the ν-parameters of the three pseudo-knotted loops are listed on the r.h.s. the energy contributions of base pair stacking and the contributions of all unmarked loops are evaluated according to the standard model. the non-pseudo-knot contributions. the basic idea for parameterizing the pseudoknot contributions rests on two simplifications: (i) rna stacks are viewed as stiff rods and (ii) unpaired regions are assumed to be very flexible. within a loop that is involved in pseudo-knot formation, we assume that each of the stacks formed by the pseudo-knotted base pairs is a stiff helix. this reasoning leads to an ansatz based upon the following quantities: u = number of unpaired bases in the loop. l max = number of base pairs in the longest pseudo-knot stack. l i = number of bases in pseudo-knot stack i. k = number of stacked base pairs that can be bridged by one unpaired base. first we define a measure for the sterical hindrance in the pseudo-knotted loop: this expression assumes that all other parts of a loop can be used to meet the constraint introduced by the longest stacked region l max within the loop, see fig. . the free energy contributions of the unpaired regions can be estimated from a theory by jacobson and stockmeyer ( ) . the same approach is used for long loops in the standard energy model for rna secondary structures. if the free energy needed to join the ends of an unrestricted, zero volume polymer is known, the theory predicts the free energy needed to form a similar but larger loop. the minimum length of an rna loop that behaves according to the jacobson-stockmayer theory is not known. we therefore introduce a parameterν and define the energy function as follows: our energy model therefore has four free parameters that need to be estimated from the available experimental data, namely k ,ν, e ps and α. for simplicity we fixed α at the same value that is used for all non-pseudo-knotted loops: α = . cal mol − (at • c). given the sequence, one can compute the secondary structure with the minimum energy by means of dynamic programming (waterman, ; zuker and sankoff, ) . in the presence of pseudo-knots this is no longer true. in the present study we use tacker's kinetic folding algorithm (tacker et al., ) which is based on (martinez, ) . it first produces a list of all possible stems of a given sequence and then determines the free energies of the loops and stacks. the most stable stem is the first one added to the folding structure. using this as a constraint, we compile a list of the remaining possible stems and add the most stable one to the growing structure. this procedure is repeated until the free energy of the structure cannot be decreased anymore. the parameters k ,ν, and e ps are adjusted by predicting the structures of a sample of sequences that are known to form pseudo-knots. this set includes seven fragments with about nt from bacteriophages that form h-type pseudo-knots, e. coli tmrna containing five pseudo-knots, and rnase p sequences from several different species [for details see haslinger ( ) ]. the best results were obtained using k = ,ν = , e ps = . kcal mol − . the same value of e ps was used in abrahams et al. ( ) . in order to check the influence of these parameters on the sequence-structure relation of rna we also used a parameter set leading to an unrealistically large number of predicted pseudo-knots in the test sequences (k = ,ν = , e ps = . kcal mol − ). the average number of base pairs and related statistical properties of the predicted structures depend very little on the inclusion of pseudo-knots and the choice of the pseudo-knot parameters. this is not surprising as the relative stability of base pairs and unpaired regions remains essentially unchanged. the average loop size decreases with the 'unrealistic' pseudo-knot potential because loop regions may take part in pseudo-knots at very little entropic cost. the frequency of pseudo-knots in random sequences is tabulated in table . for the realistic potential we find a pseudo-knot every ∼ bases, while with the exaggerated potential one would expect one pseudo-knot in every random sequence of length n = . as we have seen in the previous section, there are still many more sequences than structures. in order to obtain a better impression of the relationship between the numbers of sequences and structures that arise through folding, we determine the rank order statistics of folded structures. to this end we compute the structures of a large number of randomly chosen sequences and rank them according to their frequency f of occurrence in the sample. a plot of log f versus the logarithm of the rank reveals a generalized zipf's law (zipf, ) , fig. . while the inclusion of pseudo-knots somewhat increases the fraction and the diversity of rare structures (large ranks) it does not change the general shape of the distribution. as for 'pure' secondary structures there is only a small number of common structures into which almost all sequences fold. naturally, we ask how sequences folding into the same (common) secondary structure are distributed in sequence space. we call the set s(ψ) of all sequences (genotypes) folding into phenotype (contact structure) ψ the neutral set of ψ. more precisely, s(ψ) is the pre-image of ψ w.r.t. the folding map algorithm. as for 'pure' secondary structures, a large fraction λ of point mutations is neutral, i.e., does not change the structure. on the other hand, rna sequences folding into a particular structure are not significantly clustered: they form a percolating network spanning the entire sequence. the fraction λ of neutral point mutations was estimated from independently generated random sequences, see table . as observed in grüner et al. ( a, b) , we find that λ decreases somewhat with chain length (the large values for n = being caused in part by the large number of short sequences that 'fold' into the open structure). the fraction of neutral neighbors approaches an asymptotic value slightly above . . surprisingly, this value is almost independent of the potential function: even a potential leading to a large fraction of pseudo-knotted structures decreases λ only by a few percent. a random graph theory (reidys et al., ; reidys, ) shows that there is threshold value of about λ * = . (for a -letter alphabet). if the fraction of neutral neighbors exceeds this threshold, then the set of all sequences folding into a given structure s forms a single connected network, which has been termed the neutral network of s. these neutral networks can be conveniently detected by means of a simple computer experiment. a neutral path starts at a randomly chosen sequence. then we construct a series of subsequent mutants such that each sequence along the path folds into the same structure as the initial sequence, and such that each step increases the hamming distance from the starting point. the strict logic on base pairing in rna makes it necessary to consider two types of mutations: (i) point mutations in the unpaired regions of the molecules, and (ii) the substitution of one possible base pair (gc, cg, gu, ug, au, ua). all other mutations in paired regions necessarily change the structure, for instance by changing a gu pair into a gg mismatch. if there are neutral networks in sequence space the neutral path will reach a length l close to n before there is no neutral mutant further away from the starting point (n is the maximal hamming distance between sequences of length n). on the other hand, if the neutral sets s(ψ) form isolated clusters we will find l n. when interpreting the lengths of neutral paths we have to keep in mind that (i) the search procedure only produced lower bounds on the diameter of neutral networks, and (ii) that a pair of random sequences has an expected distance of . n for a -letter alphabet. the data in fig. are therefore a clear indication for the existence of percolating neutral networks in the presence of pseudo-knots. secondary structures form a particular class of contact structures. in this contribution we have considered a natural generalization of this class. indeed, most known rna structures with pseudo-knots are bi-secondary structures (which do not involve nested pseudo-knots). bi-secondary structures correspond to planar graphs while secondary structures form the sub-class of outer-planar graphs. the inconsistency graph introduced in section . is a useful construction capturing most of the geometrical features of nucleic acid structure. its chromatic number may serve as a measure of structural complexity. it seems possible that an analogous construction will be useful for classifying and comparing protein structures as well. the analysis of graph-theoretical properties of classes of contact structures might also be useful for designing energy models that are more realistic and/or algorithmically easier than pair potentials. the standard folding potential for rna and dna secondary structures, for instance, is based on loops, that is, induced subgraphs of the diagram graph that are circles. the total energy of a secondary structure is defined as the sum of the sequence-dependent energy contributions of all loops [see, e.g., freier et al. ( ) ]. it is by no means obvious how this energy function should be generalized to include non-secondary structure features such as pseudo-knots, g-quartets, or knots, because in general there is no unique decomposition of a graph into loops. in order to understand the sequence-structure mapping of a class of biopolymers it is necessary to have bounds on the number of structures that can possibly be formed for a given set of sequences. we can expect the existence of neutral networks and shape space covering only if the number of sequences by far exceeds the number of structures. while the number of possible contact structures grows faster than exponentially with the length of the molecules we find exponential upper bounds when the structural complexity is limited. in particular, there are not more than some . n possible bi-secondary structures. if we enforce in addition the sterical (looplength at least ) and thermodynamic (no isolated base pairs) constraints of natural rna sequences, then this bound drops to . n . exhaustive enumeration indicates that the actual number of bi-secondary structures with biophysical constraints grows roughly as . n . therefore the number of rna sequences, n , exceeds by far the number of possible bi-secondary structures. we have then devised a simple energy function extending the standard model to incorporate pseudo-knots. our ansatz assumes that steric hindrance is the major contribution to pseudo-knot energies counteracting the stabilizing effect of the additional base pairings. based on this approach we used a kinetic folding procedure to show that the inclusion of pseudo-knots does not significantly change the global features of the sequence structure map of rna: there are many more sequences than structures, and almost all sequences fold into one of a small number of common structures. common structures are uniformly distributed over sequence space. neutral networks in sequence space can therefore be modeled as random graphs (reidys et al., ) . this ansatz generalizes from secondary structures to -diagrams without modifications. the only input parameter in this model, namely the fraction λ of neutral neighbors, has been determined computationally. computer simulations agree with the prediction of a random graph theory: the fraction of neutral mutations, λ > . , is well above the threshold value of λ * ≈ . , hence all sequences folding into a given common structure form a single percolating network that spans the entire sequence space. this is verified by the detection of neutral paths that extend through the entire sequence space. the intersection theorem is valid for bi-secondary structures, hence the random graph approach (reidys et al., ) , can be used to predict the relative locations of the neutral networks of two different common structures. in particular, we have to expect shape space covering, i.e., the neutral networks of any two common structure come very close to each other at least in some parts of the sequence space. this sets the stage for the evolutionary transitions between different structures described in detail in weber ( ) and fontana and schuster ( ) . in summary, the mathematical results and the computer simulations presented in this contribution indicate that pseudo-knots do not change the qualitative picture of the rna sequence-structure map as it was obtained from studying secondary structures. prediction of rna secondary structure, including pseudoknotting, by computer simulation the book thickness of a graph rna multi-structure landscapes. a study based on temperature dependent partition functions mutational analysis of the rna pseudoknot component of a coronavirus ribosomal frameshifting signal structure and evolution of ribonuclease p rna structure and topology of s ribosomal rna. an analysis of the pattern of psoralen crosslinking an rna pseudoknot and an optimal heptameric shift site are required for highly efficient ribosomal frameshifting on a 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human behaviour and the principle of least effort rna secondary structures and their prediction stimulating discussions with ivo l. hofacker and christoph flamm are gratefully acknowledged. key: cord- - eoio uk authors: bassetto, marcella; massarotti, alberto; coluccia, antonio; brancale, andrea title: structural biology in antiviral drug discovery date: - - journal: curr opin pharmacol doi: . /j.coph. . . sha: doc_id: cord_uid: eoio uk structural biology has emerged during the last thirty years as a powerful tool for rational drug discovery. crystal structures of biological targets alone and in complex with ligands and inhibitors provide essential insights into the mechanisms of actions of enzymes, their conformational changes upon ligand binding, the architectures and interactions of binding pockets. structure-based methods such as crystallographic fragment screening represent nowadays invaluable instruments for the identification of new biologically active compounds. in this context, three-dimensional protein structures have played essential roles for the understanding of the activity and for the design of novel antiviral agents against several different viruses. in this review, the evolution in the resolution of viral structures is analysed, along with the role of crystal structures in the discovery and optimisation of new antivirals. marcella bassetto , alberto massarotti , antonio coluccia and andrea brancale structural biology has emerged during the last thirty years as a powerful tool for rational drug discovery. crystal structures of biological targets alone and in complex with ligands and inhibitors provide essential insights into the mechanisms of actions of enzymes, their conformational changes upon ligand binding, the architectures and interactions of binding pockets. structure-based methods such as crystallographic fragment screening represent nowadays invaluable instruments for the identification of new biologically active compounds. in this context, three-dimensional protein structures have played essential roles for the understanding of the activity and for the design of novel antiviral agents against several different viruses. in this review, the evolution in the resolution of viral structures is analysed, along with the role of crystal structures in the discovery and optimisation of new antivirals. knowledge of the three-dimensional structures of proteins has been long recognised as a powerful tool to accelerate drug discovery, providing information about target shape, hydrophobic and hydrophilic behaviours of macromolecules and interactions with substrates [ ] . since , when the first x-ray diffraction structure was reported for pepsin, this method emerged as an invaluable source of detailed and reliable information about protein structure, representing a significant step forward in comparison with previously used physical or chemical methods [ ] . the idea that three-dimensional structural information could be useful in defining topographies of the complementary surfaces of ligands and their protein targets raised in the early s, when scientists started to use this information to optimise potency and selectivity of lead compounds [ ] . structural details gathered from a target structure in the presence of unique ligands can provide fundamental insights on the geometric fit of these compounds into the binding site, on the binding of active conformations, on molecular electrostatic potentials and on hydrophobic interactions [ ] . from to nowadays, the applicability of structural biology was extended to the assessment of target druggability, to the identification of hits by virtual screening with structure-based virtual screening methods, to target identification by structuresequence homology recognition [ ] . during the last three decades, the application of structural biology to drug discovery followed a classical path. structure-based became extremely fashionable during the s, as a consequence of the publication of the structures of the first important drug targets. later on, due the limited number of protein structures available and to the cost and time required to set up the crystallization process, the use of these rational approaches dropped in favour of high-throughput screening methods and combinatorial chemistry [ ] . the successful story of hiv protease inhibitors [ , ] and influenza antiviral drug relenza [ ] led to a renewed interest in target structure-driven drug discovery. furthermore, the numerous advances in science and technology promoted a faster and less expensive application of structural biology, increasing the speed of macromolecular structure determination, increasing the resolution of new crystal structures and allowing smaller amounts of protein and fewer crystals to be required to solve a structure [ ] . one of the most recent applications of structural biology is crystallographic fragment screening [ ] . the low affinity for the target that characterises chemical fragments has made them unsuitable for classical high throughput screening, but the advances in high throughput nmr and crystallography have allowed the use of structural information of protein-fragment complexes, which provide reliable proof of binding pockets and hit binding mode, and give clear indications on how the fragment structures can be optimised into potent lead compounds [ ] . these characteristics make fragments very attractive starting points for iterative medicinal chemistry optimisation [ ] . several biologically active compounds discovered by structure-based design are now drugs in the market, confirming the crucial role played by structural biology in drug development [ ] , while protein structure determination has become one of the earliest and most crucial steps for many drug-discovery programs of pharmaceutical companies [ ] . the protein data bank (pdb) was established in as a curated archive that evolves with new developments in structural biology [ ] . the holdings in the pdb continue to grow and the usage of pdb data is also growing. in , million downloads (ftp and website) of data occurred from the pdb sites, compared to million downloads in . download statistics for the overall archive and for individual entries are available from the wwpdb website (http://www. wwpdb.org/stats/download). the first atomic viral structure was published less than years ago [ ] , while now about viral related structures are available in the pdb from about different virus, more than % of them containing viruses only (figure a,b) . most viral structures in the pdb (around %), have been determined using x-ray crystallography ( figure b) . since the establishment of this archive, the number of structure depositions has grown steadily. the average resolution of x-ray structures has remained constant at about . Å (figure c ). however, with the large volume of data available, there are now substantial numbers of structures determined to a high resolution (below . Å ), including at least one viral structure [ ] . at the same time, as more large macromolecular machines are being studied using x-ray methods, there are many examples of low resolution structures (above . Å ) [ ] [ ] [ ] . over the years the number of viral pdb entries has increased at an increasingly faster rate (figure a ). analysis of the taxonomy in the pdb shows that the most studied viruses are, respectively, human immunodeficiency virus (hiv- ), enterobacteria phage, influenza a virus, hepatitis c virus (hcv), human herpesvirus, sars-cov, dengue virus, norwalk virus, vaccinia virus, enterovirus c, enterovirus a, influenza b virus, murine leukemia virus (mlv), rsv-sra and simian foamy virus(foamv) (figure b ). this is most likely due to the important roles that these viruses play in biomedical research. for each top virus a sequence cluster analysis (table ) was preformed using a sequence identity of % as cutoff. more clusters than expected were obtained, as their number is bigger than the number of proteins of each viral proteome. biomedical research on viruses does not involve only structural biology, as highlighted in figure , where the general interest of scientists is compared with the entries of viruses in the pdb. a search in the pubmed database using each virus name revealed the number of original research papers published per virus. surprisingly, the growth rate of publications in pubmed is not always correlated with the number of pdb entries. the number of ligand/viral pdb entries continues to increase; there are now more than complexes with ligands, including different marketed drugs (table ). one of the most striking examples of successful application of structural biology for antiviral drug discovery is represented by hiv- protease and reverse-transcriptase structural biology in antiviral drug discovery bassetto et al. viruses only % inhibitors. hiv- was recognised as the responsible for the acquired immune deficiency syndrome (aids) in the early s [ ] , and the discovery of the first selective hiv- protease inhibitors is still one of the most popular examples of the use of x-ray crystallography in the development of a drug in clinical use. the hiv protease was validated as a potential drug target in [ , ] , the first x-ray crystal structures of the enzyme began appearing in [ ] [ ] [ ] [ ] and the first hiv protease inhibitor saquinavir was licensed only six years later, followed by the approval of ritonavir four months later [ ] . three fundamental steps led to saquinavir discovery, the first one being the classification of hiv protease as a member of the aspartate protease family, which comprises also pepsin and renin [ ] . homology with renin, already a target in the design of anti-hypertensive agents, suggested a potential approach for the development of selective inhibitors of this enzyme [ ] , and this research interest was reinforced by the resolution of the first crystal structures for hiv [ , ] , along with the protease structure of the related rous sarcoma virus [ ] . finally, useful information on protease inhibition by transition-state analogue inhibitors [ ] [ ] [ ] guided a series of investigations on the minimum size required for a small molecule to inhibit this enzyme. extensive structure-activity relationship studies and x-ray experiments directed to address this aspect resulted in the discovery of saquinavir. a key step in hiv- life cycle is reverse transcription (rt), therefore the rt/dna polymerisation has been immediately considered as a prime drug target, with the first approved anti-aids drug being the nucleoside analogue azt (zidovudine, zdv) in [ ] . the hiv reverse transcriptase (rt) is a heterodimer consisting of two polypeptide chains, p and p ( figure ). the p chain contains an n-terminal polymerase domain and a c-terminal rnase h domain [ , ] . the subdomains in p are flexible and can rearrange to different conformational states, required to carry out the enzyme essential functions for the viral replication. sequencing of the complete rt from clinical isolates have shown that mutations in the remote connection subdomain and the rnase h domain enhance resistance to both nucleoside (nrtis) and non-nucleoside (nnrtis) inhibitors [ , ] , following indirect mechanisms that are not well understood. since the publication of the first crystal structure of the hiv reverse-transcriptase in complex with the non-nucleoside inhibitor nevirapine in [ ] , the resolution of the structures of several conformations of the complex with different inhibitors has provided an extremely powerful tool for gaining essential insights into the mechanism of action of this enzyme, for understanding the importance of its flexibility and its different conformational states, for rationalising the occurrence of resistance, for elucidating the binding of nucleoside and non-nucleoside inhibitors and for the design and optimisation of new chemical agents targeting this protein. among the several examples available on how structural biology has been essential for the design and optimisation of new non-nucleoside inhibitors of the hiv- rt, the resolution of a crystal structure of the enzyme in complex with the rnase inhibitor dihydroxy benzoyl naphthyl hydrazone in (dhbnh, figure ) has led to the discovery of a novel site of the protein, near both the anti-infectives polymerase active site and the nnrti binding pocket [ ] . structure-based modifications on the dhbnh scaffold resulted in the identification of dual inhibitors of both the polymerase and the rnh activities of the hiv- rt (exemplified by compound in figure ). more recently, following the resolution of a crystal structure of hiv- rt containing the nnrti tmc [ ] in the dna polymerase domain and a-hydroxytropolone manicol in the rnase h active site, the structure of manicol was rationally modified to obtain a new series of a-hydroxytropolones, which show antiviral activities at non-cytotoxic concentrations and occupy an additional site surrounding the dna polymerase catalytic centre (compound in figure ) [ ] . in , with the application of an x-ray crystallographic fragment screening methodology to evaluate the intrinsic flexibility of the rt for the discovery of new allosteric sites, seven new sites were identified within this protein [ ] . three of these sites (named the knuckles, the nnrti adjacent and the incoming nucleotide binding sites) were proven inhibitory in an enzymatic assay, while the co-crystallised fragments ( a-c in figure ) were found to be novel scaffolds in comparison with previously reported rt inhibitors, thus providing the basis for the development of novel leads [ ] . after the resolution of a crystal structure of hiv- rt in complex with potent pyrimidine-based nnrti a, structure-based modifications on its chemical scaffold directed to the occupation of the entrance channel to the nnrti binding site resulted in the identification of much more soluble analogues such as b ( figure ) , with which the solubility issues associated with a were significantly improved [ ] . in , the study of several reported structures of the enzyme in complex with efavirenz and other nonnucleoside inhibitors, and the inspection of the ligand structural biology in antiviral drug discovery bassetto et al. table diversity of pdb entries. all viral pdb entries were analysed using a sequence identity of % as cut-off to identify clusters. geometries required for the interaction with the enzyme revealed in these structures, guided the design of a new series of aryl-phospho-indoles as potent inhibitors of the enzyme (exemplified by compound a in figure ) [ ] . subsequent rational optimisation of the original lead a resulted in the identification of b, a nanomolar inhibitor of the y c/k n double mutant (both mutations are clinically relevant) that reached phase iib clinical trials [ ] . in the same year, multiple rt crystal structures have been used for the in silico screening of a library of more than two million compounds using molecular docking methods, taking into account different protein conformations in order to overcome resistance [ ] . one hit was found with . mm potency against wt hiv- (compound a in figure ). computational analyses and rational modifications on the structure of a led to the discovery of catechol diether b, a -pm anti-hiv agent that retains nanomolar activity against the y c mutant [ ] . structural biology has often been extremely helpful for the identification or optimisation of novel antiviral agents also in the case of hcv, in particular for the discovery of novel inhibitors of the ns - a protein and the ns b polymerase. in , a fragment-based screening of fragments against the full length hcv ns -ns a genotype b enzyme led to the discovery of a new allosteric pocket at the interface between the protease and helicase domains [ ] . structure-based optimisation of a first hit found to bind this new pocket (compound a in figure ) guided the identification of potent inhibitor b (figure ), which also inhibits the viral replication with an ec value in the nanomolar range [ ] . crystallographic fragment-based screening methodologies have proven successful also in the identification of non-nucleoside inhibitors of the hcv ns b polymerase. in , a small bromo-aryl fragment ( a) was found to bind the thumb domain of the protein with an initial binding affinity in the millimolar range [ ] . a series of structure-based optimisation cycles on the fragment scaffold led to the identification of a family of structures with high affinity for the enzyme and low micromolar activities in the hcv replicon assay (compound b in figure ). more recently, the application of a similar fragment-based approach guided the identification of sulfonamide fragment a (figure ), which binds the polymerase allosteric thumb pocket [ ] . the scaffold of this small fragment was the starting point for different structure-based modifications, which resulted in the identification of phenoxyantranilic acid sulfonamide derivative b as a -fold more potent inhibitor of the hcv ns b polymerase [ ] . further structure-based optimisation attempts on the scaffold of b led to the discovery of derivative c, which is slightly more potent in inhibiting the enzyme and shows a much more potent inhibition of the viral replication in cell culture, with an ec < nm [ ] . along with fragment-based screening methods, structurebased design and optimisation techniques have played an important role in the discovery of novel non-nucleoside anti-infectives inhibitors of the hcv ns b polymerase. one of the first of these studies was reported in , when the structure of the non-nucleoside inhibitor a, a hit identified by a biochemical hts assay, was resolved in complex with the enzyme, revealing that it binds to an allosteric region between the thumb and palm domains [ ] . starting from the examination of this crystal structure, a series of rational modifications on the hit scaffold, directed to a better occupation of the allosteric pocket, resulted in the discovery of potent inhibitor b ( figure ), with ic < nm and activities against the viral replication in cellular systems in the low micromolar range [ ] . other recent examples of structure-based lead optimisation include the identification of a novel quinazolinone chemotype as thumb pocket allosteric inhibitor anti-infectives current opinion in pharmacology chemical structures of hiv- rt inhibitors discovered or optimised using structure-based methods. (compound a in figure ), which has been rationally designed following inspection of the enzyme crystal structures in complex with previously reported allosteric inhibitors [ ] . after the crystal structure of the newly designed quinazolinone a in complex with the enzyme was resolved, further structure-based optimisation attempts aiming to improve the key interactions within the protein binding channel were carried out, resulting in the identification of derivative b, which shows improved potency in both the biochemical and the cellular antiviral assays ( figure ) [ ] . finally, starting from the crystal structure of non-nucleoside inhibitor a in complex with the polymerase, synthetic efforts directed towards the optimisation of the interactions with different sub-pockets of the enzyme resulted in the identification of several new analogues with potent antiviral activities in vitro against both genotype a and b, high metabolic stability and good oral bioavailability (represented by b in figure ) [ ] . another interesting case of application of structural biology to the identification of hcv ns b non-nucleoside inhibitors has been reported in , when the structure of compound a, an attractive hit deriving from a biochemical hts assay of a large compound collection, was sequentially optimised using a combination of x-ray crystallography, nmr analyses and molecular modelling studies, along with binding-site resistance mutant experiments structural biology in antiviral drug discovery bassetto et al. current opinion in pharmacology chemical structures of non-nucleoside inhibitors of the hcv ns b polymerase discovered or optimised with structure-based methods. and photoaffinity labelling studies [ ] . this approach resulted in the identification of different series of new analogues with significantly improved potency in both the enzymatic assay and a cellular antiviral assay (exemplified by compound b figure ). as mentioned above, there are many cases in which structural biology has been key to the identification of novel and improved antivirals, involving many of the viruses for which structural information has become available in the last decades, and including examples of antiviral drugs on the market, such as the neuraminidase inhibitor zanamivir for influenza a and b [ ] . another example of successful application of a structurebased approach has been recently reported for the identification of a new class of influenza endonuclease inhibitors ( figure ) [ ] . an engineered high-resolution crystal form of pandemic influenza polymerase acidic protein n-terminal endonuclease domain was used for the crystallographic fragment screening of fragments, leading to the identification of hit fragment a, which showed a binding affinity to the enzyme in the range of mm and also revealed the presence of a third metal ion in the active site cleft, previously unknown. different cycles of rational modifications on the hit scaffold were performed in order to maximise the interactions with the active site of the enzyme. this structure-based optimisation approach resulted in the identification of compound b, which shows an antiviral ec of mm (figure ) . structural biology has been fundamental also to understand the mechanism of action and the importance of conformational flexibility of the dengue virus polymerase [ , , ] , and structure-based methods have been extremely useful for the discovery of non-nucleoside inhibitors of this enzyme. an x-ray-based screen of the novartis fragment collection against dengue virus- (denv- ) polymerase resulted in the identification of biphenyl acetic acid a (figure ) bound to a novel pocket in the palm subdomain of the protein [ ] . growing and optimisation of this first hit through crystallography and computer-aided structure-based design led to the development of b, an antiviral agent active against all four dengue serotypes with ec values in the low micromolar range. among other examples, structure-based methods have been particularly useful for the discovery of novel chemical agents against enterovirus and norovirus. in , the analysis of the crystal structures of the ev complete viral particle in complex with uncoating inhibitors (exemplified by derivative a in figure ), in combination with in silico docking-based methods, guided the design of an improved and potent inhibitor of the virus-induced cytophatic effect in cells (compound b), which inhibits the full range of ev subtypes [ ] . more recently, the analysis of the crystal structure of the ev c proteinase in complex with moderate inhibitor a, and the subsequent structurebased modification of its scaffold, guided the design of b, an improved inhibitor in both the enzymatic and a virus cell-based assay [ ] . finally, a structure-based fragmentwise design of new inhibitors of the same enzyme led to the discovery of the new chemical scaffold as a potent inhibitor of both the enzyme activity and the virus-induced cytopathic effect in cells [ ] . in the case of norovirus, an iterative process of structureguided design and optimisation on dipeptidyl inhibitors of the viral c-like protease has guided the discovery of potent derivative , which displays in vivo efficacy in a murine model of norovirus infection [ ] . for the study of the same viral target, different crystal structures in complex with peptidyl inhibitors have recently been used to design novel triazole-based macrocyclic inhibitors (represented by compound in figure ), which also inhibit the viral replication in cells with ec values in the low micromolar range [ ]. finally, structural biology has been essential also for the identification of novel inhibitors of norovirus rna-dependent rna-polymerase. a docking-based in silico search method on the polymerase structure using commercially available compounds led to the discovery of suramin and its analogues [ ] and of ppnds [ , ] as novel inhibitors of the norovirus rdrp activity. the resolution of the crystal structure of ppnds in complex with the enzyme has also indicated the possibility to target a new binding sub-pocket in the thumb domain of the enzyme for the design of new norovirus inhibitors [ ] . following the increasing number of viral target structures deposited in the pdb, structural biology has become a fundamental tool in antiviral research, not only providing essential insights into the mechanisms of actions of viral enzymes and their interactions with substrates and antiviral agents, but representing in several cases the very basis for the rational discovery and optimisation of new antivirals, as exemplified by the striking case of drugs in the market such as saquinavir and zanamivir. used in combination with computational techniques, structure-based structural biology in antiviral drug discovery bassetto et al. methods are often among the earliest and most important steps of drug-discovery campaigns of most pharmaceutical companies, and their fundamental application for the discovery of new antivirals can only be expected to increase over the next years, supported by the continuous evolution of associated technologies. high-throughput crystallography for lead discovery in drug design dorothy hodgkin and her contributions to biochemistry structural biology of viruses by the combination of electron cryomicroscopy and x-ray crystallography successful applications of computer aided drug discovery: moving drugs from concept to the clinic structural biology and drug discovery a structural model for the retroviral proteases knowledge-based protein modelling and design development of neuraminidase inhibitors as antiinfluenza virus drugs new roles for structure in biology and drug discovery discovering novel ligands for macromolecules using x-ray crystallographic screening the impact of structure-guided drug design on clinical agents the protein data bank at : reflecting on the past to prepare for the future tomato bushy stunt virus at . Å resolution construction and crystal structure of recombinant stnv capsids improving diffraction by humidity control: a novel device compatible with x-ray beamlines crystal structures of a group ii chaperonin reveal the open and closed states associated with the protein folding cycle room temperature femtosecond x-ray diffraction of photosystem ii microcrystals isolation of a t-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (aids) murine leukemia virus maturation: protease region required for conversion from ''immature'' to ''mature'' core form and for virus infectivity a deletion mutation in the part of the pol gene of moloney murine leukemia virus blocks proteolytic processing of the gag and pol polyproteins x-ray analysis of hiv- proteinase at . a resolution confirms structural homology among retroviral enzymes molecular modeling of the hiv- protease and its substrate binding site conserved folding in retroviral proteases: crystal structure of a synthetic hiv- protease rational design of peptide-based hiv proteinase inhibitors structure-based design of aids drugs and the development of resistance inhibition of retroviral protease activity by an aspartyl proteinase inhibitor potent new inhibitors of human renin novel renin inhibitors containing the amino acid statine renin inhibitors. synthesis of transition-state analogue inhibitors containing phosphorus acid derivatives at the scissile bond the efficacy of azidothymidine (azt) in the treatment of patients with aids and aids-related complex. a double-blind, placebo-controlled trial crystal structure at . a resolution of hiv- reverse transcriptase complexed with an inhibitor crystal structure of human immunodeficiency virus type reverse transcriptase complexed with double-stranded dna at . a resolution shows bent dna mechanism for nucleoside analog-mediated abrogation of hiv- replication: balance between rnase h activity and nucleotide excision n i in the connection domain of hiv- reverse transcriptase confers zidovudine and nevirapine resistance structure of the binding site for nonnucleoside inhibitors of the reverse transcriptase of human immunodeficiency virus type hiv- reverse transcriptase structure with rnase h inhibitor dihydroxy benzoyl naphthyl hydrazone bound at a novel site next-generation hiv- non-nucleoside reverse transcriptase inhibitors sfj: synthesis, activity and structural analysis of novel a-hydroxytropolone inhibitors of human immunodeficiency virus reverse transcriptase-associated ribonuclease h detecting allosteric sites of hiv- reverse transcriptase by xray crystallographic fragment screening extension into the entrance channel of hiv- reverse transcriptasecrystallography and enhanced solubility synthesis and biological evaluation of aryl-phospho-indole as novel hiv- non-nucleoside reverse transcriptase inhibitors discovery of the aryl-phospho-indole idx , a highly potent anti-hiv nonnucleoside reverse transcriptase inhibitor computationally-guided optimization of a docking hit to yield catechol diethers as potent anti-hiv agents discovery of an allosteric mechanism for the regulation of hcv ns protein function fragment-based discovery of hepatitis c virus ns b rna polymerase inhibitors discovery of a novel series of non-nucleoside thumb pocket hcv ns b polymerase inhibitors anthranilic acid-based thumb pocket hcv ns b polymerase inhibitors with sub-micromolar potency in the cell-based replicon assay identification of anthranilic acid derivatives as a novel class of allosteric inhibitors of hepatitis c ns b polymerase structurebased design of novel hcv ns b thumb pocket allosteric inhibitors with submicromolar gt replicon potency: discovery of a quinazolinone chemotype discovery of tricyclic , -dihydro- h-pyridin- -ones as novel, potent, and orally bioavailable inhibitors of hcv ns b polymerase importance of ligand bioactive conformation in the discovery of potent indole-diamide inhibitors of the hepatitis c virus ns b crystallographic fragment screening and structure-based optimization yields a new class of influenza endonuclease inhibitors the flavivirus polymerase as a target for drug discovery conformational flexibility of the dengue virus rna-dependent rna polymerase revealed by a complex with an inhibitor a crystal structure of the dengue virus ns protein reveals a novel inter-domain interface essential for protein flexibility and virus replication this paper represents a significant example of the use of structural biology for understanding the mechanism of action of viral enzymes and the importance of their conformational flexibility, and for discovering new inhibitors discovery of potent nonnucleoside inhibitors of dengue viral rna dependent rna polymerase from a fragment hit using structure-based drug design this paper represents an excellent example of the importance of structural biology for the discovery of novel antivirals more powerful virus inhibitors from structure-based analysis of hev capsid-binding molecules this paper represents a significant example of the importance of structural biology for the rational optimisation of antiviral compounds optimize the interactions at s with efficient inhibitors targeting c proteinase from enterovirus fragment-wise design of inhibitors to c proteinase from enterovirus this paper represents a significant example of the importance of structural methods for identifying novel antivirals structure-guided design and optimization of dipeptidyl inhibitors of norovirus structural biology in antiviral drug discovery bassetto et al. www.sciencedirect structure-activity relationships and biochemical, x-ray crystallographic, cell-based, and in vivo studies structure-based design and synthesis of triazole-based macrocyclic inhibitors of norovirus protease: structural, biochemical, spectroscopic, and antiviral studies this paper represents a significant example of the use of structure-based methods for the discovery of novel antivirals structurebased inhibition of norovirus rna-dependent rnapolymerase ppnds inhibits murine norovirus rna-dependent rna-polymerase mimicking two rna stacking bases naphthalene-sulfonate inhibitors of human norovirus rna-dependent rnapolymerase nothing declared. papers of particular interest, published within the period of review, have been highlighted as:of special interest of outstanding interest key: cord- - bfchw u authors: rollinger, judith m.; stuppner, hermann; langer, thierry title: virtual screening for the discovery of bioactive natural products date: journal: natural compounds as drugs volume i doi: . / - - - - _ sha: doc_id: cord_uid: bfchw u in this survey the impact of the virtual screening concept is discussed in the field of drug discovery from nature. confronted by a steadily increasing number of secondary metabolites and a growing number of molecular targets relevant in the therapy of human disorders, the huge amount of information needs to be handled. virtual screening filtering experiments already showed great promise for dealing with large libraries of potential bioactive molecules. it can be utilized for browsing databases for molecules fitting either an established pharmacophore model or a three dimensional ( d) structure of a macromolecular target. however, for the discovery of natural lead candidates the application of this in silico tool has so far almost been neglected. there are several reasons for that. one concerns the scarce availability of natural product (np) d databases in contrast to synthetic libraries; another reason is the problematic compatibility of nps with modern robotized high throughput screening (hts) technologies. further arguments deal with the incalculable availability of pure natural compounds and their often too complex chemistry. thus research in this field is time-consuming, highly complex, expensive and ineffective. nevertheless, naturally derived compounds are among the most favorable source of drug candidates. a more rational and economic search for new lead structures from nature must therefore be a priority in order to overcome these problems. here we demonstrate some basic principles, requirements and limitations of virtual screening strategies and support their applicability in np research with already performed studies. a sensible exploitation of the molecular diversity of secondary metabolites however asks for virtual screening concepts that are interfaced with well-established strategies from classical pharmacognosy that are used in an effort to maximize their efficacy in drug discovery. such integrated virtual screening workflows are outlined here and shall help to motivate np researchers to dare a step towards this powerful in silico tool. in the field of drug discovery we are confronted by a paradox situation: highly efficient tools and advanced technological and molecular knowhow, e.g., in the area of genomics, combinatorial chemistry, high throughput screening (hts), robotized and miniaturized process cycles, could find entrance in big pharmaceutical industries. these costly procedures were expected to raise the number of launched drug substances; however the results were disappointing [ , ] . in , adam smith, the chief-editor of nature presented the sobering data of research and development expenses of the leading pharma companies versus new drugs on the market. they have steadily fallen in recent years despite the increasing financial efforts [ ] . on the other side we are faced by a high traditional impact of naturally derived medicines and incredible success stories of natural products (nps) as potent remedies from the beginnings of human therapeutic activity to modern research and drug development. nevertheless, most large pharmaceutical companies scaled down or terminated their work in nps operations. the reasons behind this are that the drug discovery process starting from natural sources is hardly compatible with the today's highly automated drug discovery technologies. thus, the pre-eminence of combinatorial chemistry as the preferred method for generating new drug leads has led to the comparative neglect of this valuable resource. william strohl from merck research laboratories summarized the difficulties of np programs versus synthetic chemicals in his editorial remarks in drug discovery today [ ] . these include (i) the existence of already found potent antimicrobic and antitumor nps and the lack of sufficient dereplication programs which prevent their repeated discovery; (ii) the fact that -in contrast to the highly sophisticated molecular targets -np extracts are generally regarded as too 'dirty', too difficult to assay and too time-consuming; (iii) obtaining an assay hit resulting from a bio-guided fractionation, the nps' structure still has to be elucidated compared with synthetic chemicals; (iv) nps are often deemed as too structurally complex, possessing multiple hydroxyl moieties, ketones and chiral centers. strohl nevertheless concluded by listing a number of advantages applying an active np program, which he finally described as an 'expensive endeavor' which, however, is 'well worth the cost'. the use of nps has been the single-most successful strategy for the discovery of new drug leads, which is clearly shown by different statistics [ , ] . with increased calls in recent years for further research on nps [ , ] there are again signs that they may play a more active role in the future drug discovery process, since their reintroducing may help to re-discover the sweet spot in drug discovery [ ] . to date some , natural compounds [ - ] have been published. the terrestrial flora has been intensively investigated over the last decades; the potential in finding new nps slumbering in untapped biota is however nearly inconceivable. it is estimated that only - % of the approximately , described high plant species have ever been in the focus of phytochemical and pharmacological investigations [ ] . more sobering is the percentage in the field of bacterial (less than %) and fungal species (less than %) [ ] . the main part of known nps belongs to secondary metabolites. these compounds provide living systems with their characteristic features mandatory for surviving. they contain an inherently large-scale of structural diversity. about % of the chemical scaffolds of published nps are unique and have not been made by any chemist [ ] . in the past years researchers have discovered many potential therapeutic targets. since the completion of the human genome, , to , genes and at least the same number of proteins are assumed [ ] . thus, we are up against an increasing number of macromolecular targets, like proteins, receptors, enzymes, and ion channels -that might be of pathological concern for humankind. among them, proteins continue to attract significant attention from pharmaceutical technology as a valuable source of drugable targets [ ] . proteins provide the critical link between genes and disease, and thus are the key to understanding the basic biological processes. up to now drug discovery has been performed against only approximately targets [ ] , though the number of potential targets are estimated to be in the range of , to , [ , ] . taken together, it can be assumed that a large number of drug leads and hits are conserved in the inexhaustible pool of nps pre-screened by evolution. but how to dig out and to recognize the respective drug leads is a challenging task for both industry and academia, for medicinal chemists, pharmacognosists and pharmacologists. np research is affected with a wealth of time-consuming and cost intensive investigations. collection of the natural material, phytochemical analysis, isolation and identification of the constituents is just the basic procedure. a biological screening of extracts or even the arbitrary testing of isolated metabolites is feasible and often performed, though is not at all a focused procedure, thus unpractical and too expensive. the nps' diversity has to be accessed in a more rational way. holistic versus molecular approaches in drug discovery from nature during the last century and even today the discovery of bioactive nps and their development into potential drug candidates are mainly covered by a holistic approach. a characteristic workflow of this procedure is given in figure . starting from the knowledge or assumption about a biological effect the natural material is selected and adequately extracted. if a positive effect in the obtained multi-component extract is recorded, it is attempted to trace it back to the active principle/s by intense phytochemical and analytical investigations (fig. ). this can for instance be achieved by a bioactivity-guided fractionation. a more targeted approach focuses on innovative technological tools combining analytical and biological information. an overview of recent developments in this area and successful examples thereof are presented by potterat and hamburger [ , ] . as soon as the constituent regarded to be responsible for the overall effect is isolated, further research focuses on a molecular level including structure elucidation and pharmacological profiling. synthesis and testing of series of derivatives enable an insight into a structure-activity-relationship and pharmacokinetic aspects. finally, potential drug leads become drug candidates after some intense toxicological studies and after the verified effectiveness in vivo (fig. ) . recent advances in lead identification from nature work on a molecular base more than on a holistic one. a first prerequisite for that is on bioin- traditional early drug development of a nature based drug candidate formatics comprising d structures from genes and proteins (bioinformatics), substantial knowledge on molecular target functions with accurate structural information and protein-ligand interactions. secondly, it is essential to refer to unambiguously characterized structures of secondary metabolites preferably with some information to their biological effect. based on available structural as well as biological knowledge from both sides, information can be deduced from chemoinformatics to bridge the gap between known ligands and the discovery of new lead structures (fig. ). the increasing understanding of fundamental principles of protein-ligand interactions and the steadily growing number of d-structures of potential and experimentally proved ligands provide undreamed of possibilities towards more rationalized concepts in drug discovery. however, too much is expected of the human brain to profit from the already published information. thus, efficient and effective approaches benefit from today's knowledge about nps. in the area of medicinal chemistry, computational methods, like virtual screening experiments, have already proved to satisfy these requirements. they are needed to exploit the available structural information, to understand specific molecular recognition events, and to clarify the function of the target macromolecule. though rationalized procedures in the search for bioactive natural products are in great demand to find the 'needles in a haystack', computational assistance could hardly break into natural product research. the common idea of all computational approaches within the early drug discovery process is to mine more or less large compound databases in silico and to select a limited number of candidates proposed to have the desired biological activity. for this process the term 'data mining' was coined in [ ] , which was concisely defined by gasteiger and coauthors: 'to extract knowledge from a large set of data in order to make predictions of new events' [ ] . within the lead discovery process, virtual screening technologies have largely enhanced the impact of computational chemistry and nowadays chemoinformatics plays a predominant role in early phase drug research [ , ] . the key goal of the use of such methods is to reduce the overall cost associated to the discovery and development of a new drug, by identifying the most promising candidates to focus the experimental efforts on. recently published books and reviews on the impact of computational chemistry for lead structure determination highlight these efforts [ ] [ ] [ ] [ ] . if the d structure of the biological target is known, high throughput docking turned out to be a valuable structure-based virtual screening method to be used [ ] [ ] [ ] [ ] . within this context, the scoring of hits retrieved still remains a question that is often discussed. in fact, currently the major weakness of docking programs lies not in the docking algorithms themselves but still in the inaccuracy of the functions that are used to estimate the affinity between ligand and target, the so-called scoring functions. previously, stahl and rarey analyzed scoring functions for virtual screening [ ] , giving valuable insight into strengths and weaknesses of currently used models for affinity estimation. the combination of several different scoring functions termed as consensus scoring turns out to be one of the possible answers to the question raised previously. in fact, several authors recently described their efforts in this area; an example is given in reference [ ] . in a theoretical study, other authors demonstrate that consensus scoring outperforms any single scoring for simple statistical reasons and that a moderate number of scoring functions (i.e., three or four) are sufficient for the purpose of consensus scoring [ ] . however, it has been shown that consensus scoring alone is not suitable for all cases of docking, and, as highlighted in a recent review by krovat and co-authors, considerable efforts are still devoted to the optimization of scoring functions [ ] . because of the restricted free access to np d libraries (see below), the number of virtual screening studies published for the rational access to bioactive nps is limited. some examples using high throughput docking as a structure-based virtual screening tool will be given here: liu and zhou applied a theoretical approach to find natural ligands as potential inhibitors of the sars-cov protease, a virus target of the severe acute respiratory syndrome [ ] . they used a docking-based virtual screening cycle and applied drug-like filters to finally propose drug candidates out of two d databases comprising metabolites from marine organism and compounds from traditional chinese medicine. the same virus organism was the main interest in the study performed by toney et al., who focused on its main proteinase, clpro. the crystal structure of this attractive target was used as the starting point for the virtual docking screening of the nci database. searching for non-peptidyl inhibitors, the authors identified the naturally occurring terpenoid alkaloid sabadinine (i.e., cevine; ) as potential anti-sars agent [ ] . the author group around stefano moro could identify ellagic acid ( ) as inhibitor of the protein kinase ck screening an in-house generated database with almost , structures of natural compounds [ ] . a combination of four docking protocols and five scoring functions has been utilized to dock and rank the molecules in the database. the consensus docking suggested ellagic acid to be one of the most promising candidates. this assumption could be verified by experimental studies revealing this np as highly potent ck inhibitor (k i = nm). estrogen receptor-plays a key role in regulating brain development and estrogen-induced promotion of neurogenesis and memory. using the d coordinates of the co-crystal structure of human estrogen receptorbound with genistein as starting point, zhao and brinton pursued a receptor-based molecular docking approach [ ] . they focused on the search for natural estrogen receptor--selective ligands. twelve candidate molecules, which had been suggested by the database screening, were selected. the authors determined their binding affinity and selectivity; three of the com-pounds belonging to the flavanoid family ( ) ( ) ( ) displayed over -fold binding selectivity to the estrogen receptor-over . a similar approach was employed by liu and co-authors. applying a docking virtual screening filtering experiment, the authors discovered potent inhibitors of the potassium ion channel from a chinese np database [ ] . the pharmacophore concept has proven to be extremely successful, not only in rationalizing structure-activity relationships, but also by its large impact in developing the appropriate d-tools for efficient virtual screening [ ] . profiling of combinatorial libraries and compound classification are other often-used applications of this concept. although well established in combinatorial chemistry, it has to be pointed out that the tools described in this section have likewise a considerable impact on the rational finding of new potential lead compounds originating from the immense source of secondary metabolites. the prior use of pharmacophore models in biological screening of nps is an efficient procedure since it quickly eliminates molecules that do not possess the required features thus leading to a dramatic increase of enrichment, when compared to a purely random screening experiment. in a previous study conducted by doman and co-authors [ ] , only molecules or . % revealed as protein tyrosine phosphatase- b inhibitors (ic < µm) by a hts of approximately , compounds. on the other hand, of molecules suggested by molecular docking, or . % were found to be active. thus, dockingbased virtual screening enriched the hit rate by almost , -fold over random screening. one should not forget, however, that additional molecular characteristics not reflected by pharmacophore models (physicochemical properties relevant for adme and toxicological properties) must be taken into account when deciding upon which compounds should be further developed [ ] . a rapid identification and elimination of compounds with unsuitable physicochemical and pharmacokinetic properties is a pivotal step in the early drug discovery process [ , ] . they can be evaluated traditionally or by high throughput screening, which are discussed in detail by avdeef and testa [ ] . this must be considered for synthetics as well as nps, though studies revealed secondary metabolites not only high scaffold diversity; biosynthesized molecules also show structural and spatial characteristics that are closer to drug leads than those of synthetic molecules [ , ] . typically, nps include more chiral centers and their stereochemical architecture is much more complex than that of synthetic molecules. furthermore, they usually contain more carbons, hydrogen and oxygen, however, less nitrogen and other atoms compared to synthetics. surprisingly, nps often show a molecular weight higher than da combined with a high polarity [ ] , which is in clear contrast to lipinski's rule of five [ ] . nevertheless only about % of nps contain two or more violations of lipinski's rules [ ] . in summary, natural chemistry can be seen as highly diverse scaffolds endowed with potential drugable pharmacophores. structure-based pharmacophore model an inevitable prerequisite for generating a structure-based model is the knowledge about the ligand-target interaction [ ] including the availability of the d structure of the target either by x-ray crystallography or nmr or constructed on the basis of the structure of homologous proteins. a unique platform containing d coordinates of experimentally solved protein structures is the brookhaven protein data bank (pdb [ ] ). a crystalline complex with a ligand bound to a protein's active site is the best requirement to start the construction of a structure-based d model. in this case, one may profit from the exact information of the ligand's bioactive conformation which is preserved in the binding site of the crystalline complex. the building of a structure-based pharmacophore is depicted in a step by step way in figure . a new software tool has recently been described for the successful generation of such chemical features-based models: the software ligand-scout [ ] is a program for ligand interpretation and data mining in the pdb. the performance of this program allows the detection of relevant interaction points between ligand and protein. the binding mode of the ligand in the active site of a protein can be visualized in a sophisticated way. ligandscout's algorithms perform a stepwise interpretation of the ligand molecules: planar ring detection, assignment of functional group patterns, determination of the hybridization state and finally the assignment of kekulé pattern. the interpretation of the ligand molecules is the basis for the next step, an automated generation of pharmacophore models, derived from the data provided by a crystalline complex of the pdb. an automatic detection and classification of protein-ligand interactions into hydrogen bonds, charge transfer, and lipophilic regions leads to a collection of chemical features in a pharmacophore model. the graphical user-interface can provide an integrated view of protein, ligand, pharmacophore model, and interaction lines. in a previously published study, ligandscout was used for the detection and interpretation of crucial interaction patterns between ligands and the factor xa protein structure [ ] . in a second step, the program catalyst, a state of the art virtual screening platform, was used for rapid virtual screening of multiconformational d structure databases. the information for the pharmacophore pattern (i.e., d coordinates of interaction points) was obtained by the interpretation of ligandscout pharmacophore definitions and resulted in specific interaction models that were able to map the ligand in their bioactive conformation and to retrieve selectively a % fraction of the known factor xa inhibitors within a small subset of the large derwent world drug index library. a further application of the ligandscout pharmacophore definitions covers the rationalized search for angiotensin converting enzyme (ace)- inhibitors by virtual screening of approximately . million compounds from various commercial databases [ ] . hit reduction and selection was achieved using a five feature hypothesis based on a recently resolved inhibitor-bound ace crystal structure. seventeen virtual hits were selected for their experimental validation in a bioassay; the concept was confirmed since all of them were revealed as ace- inhibitors. barreca and co-authors developed a d structure-based pharmacophore model with ligandscout for the discovery of new scaffolds acting as hiv- non-nucleoside reverse transcriptase inhibitors by virtual screening of large chemical databases. six virtual hits were finally selected for determination of their inhibitory effects. those belonging to the new scaffold class of the quinolin- ( h)-one family exhibited reverse transcriptase inhibitory activity at sub-micromolar concentrations [ ] . in a recently published work, schuster et al. presented a so-called cytochrome p profiler [ ] . several structure-based (generated with ligandscout) and ligand-based pharmacophore models (using cata-lyst) for substrates and inhibitors of five cytochrome p isoenzymes ( a , p c , p c , p d , and p a ) were created and validated by the authors' group. their results showed that the models were suitable for fast pharmacokinetic profiling of large drug-like databases. in this context the parallel screening is of particular interest. whereas in usual virtual screening cycles interactions of thousands or even millions of d database entries are browsed against one pharmacophore model, it is contrary in the case of parallel screening; low-energetic conformers of one structure are screened for their potential interactions against numerous models. the basics of parallel screening have just recently been presented by steindl and co-authors [ , ] . furthermore, the authors exemplified this strategy for the activity profiling using a set of hiv protease pharmacophore models [ ] . this in silico concept is of particular interest to virtually scrutinize drug candidates for their preliminary activity profiling relevant to putative side effects and toxicity [ ] . according to the obtained interactions to virtually screened antitargets (e.g., herg, sigma- , sigma- , alpha- a, alpha- b, alpha- d, alpha- a, alpha- b, alpha- c, d l, d , d . , -ht a , -ht a , -ht , h , i , a a, a b, cytochrome p ) a first insight to potentially risky affinities is provided before time and cost intensive toxicological studies are performed. the virtual screening approach using a structure-based pharmacophore model has revealed some first application examples in np research: niko-lovska-coleska and co-authors successfully pursued this in silico strategy in the area of x-linked inhibitors of apoptosis (xiap) [ ] . a high resolution d structure of the xiap bir domain complexed with the n-terminal end of the smac/diablo protein [ ] , which is an endogenous ligand of the respective xiap binding pocket, was used as the starting point to virtually screen an in-house d-np database. embelin ( ) from the japanese ardisia herb emerged as virtual small molecule weight hit, which was found to be a fairly potent inhibitor of xiap using a fluorescence polarization binding assay. in our group, we previously focused on acetylcholinesterase (ache) [ ] ; according to the cholinergic hypothesis of the pathogenesis of alzheimer's disease, inhibitors of the ache are successfully used as therapeutic strategy. based on the co-crystal structure of ache with its ligand galanthamine, a structure-based pharmacophore model was generated and used for an in silico screening of a multi-conformational database consisting of more than , nps. from the obtained hit list, promising, virtually active candidates were selected, namely scopoletin ( ) and its glucoside scopolin ( ) . their ache inhibitory effect was first verified from the crude extract of scopolia carniolica roots using a bioautographic tlc assay. the isolated coumarins showed a significant and dose-dependent inhibition of the ache in the microplate enzyme assay as well as in the in vivo test. the i.c.v. application of both coumarins on rats resulted in a long-lasting, pronounced and -in case of the glucoside -even in a two-fold higher increase of the neurotransmitter's concentration than the one caused by the positive control galanthamine. ligand-based pharmacophore model very often, however, lead discovery projects have reached a well-advanced stage before detailed structural data on the protein target has become available, even though it is well recognized that modern methods of molecular biology together with biophysics and computational approaches enhance the likelihood of successfully obtaining detailed atomic structure information. a possible consequence is that often scientists identify and develop novel compounds for a target using preliminary structure-activity information, together with theoretical models of interaction. only responses that are consistent with the working hypotheses contribute to an evolution of the used models. within this framework, the chemical feature-based pharmacophore approach has proven to be successful [ ] allowing the perception and understanding of key interactions between a receptor and a ligand on a generalized level. a function-based pharmacophore represents the common ensemble of steric and electrostatic features of different compounds which are necessary for their interaction with a specific biological target structure (fig. ) . such pharmacophore models together with large d structure databases originating either from in-house compound collections, from commercial vendors, or from natural products databases have proven to be extremely useful in silico screening experiments. when using ligand-based pharmacophore models as screening filters instead of protein d structures, affinity estimation is only based on geometric fit of compound atoms or groups to features of the model. in these cases, the values calculated are often far away from reality, however, still are useful for filtering possible hits from non-binding molecules. additionally, in pharmacophore fitting procedures, calculation demands are considerably lower than in docking algorithms allowing the number of compounds to be processed in the same time to be by far higher than even in high throughput docking. since in most of the studies no experimental information on either the biological conformation of the ligand or the target protein are currently available, the ligand-based chemical feature pharmacophore approach can provide essential information for medicinal chemists. several successful applications within this subject have been performed using the cata-lyst program, one of the leading software packages in chemical featurebased pharmacophore modeling. schuster and co-workers succeeded in the identification of -hydroxysteroid dehydrogenase type inhibitors applying a common feature-based pharmacophore model for their virtual screening filtering experiments [ ] . similarly, the authors preceded by suggesting compounds with a proposed inhibition to the cytochrome p isoenzyme [ ] . several reviews covering successful applications of such feature-based methods have been published by kurogi et al. [ ] , by krovat et al. [ ] and by güner et al. [ ] . they outline the theoretical background and describe several significant studies including d database search strategies. in the field of nps only a very limited number of studies report from the rationalized access to bioactive compounds via ligand-based virtual screening. for example, this method was pursued for the discovery of inhibitors of the cop signalosome (cns) associated kinases ck and pkd [ ] . using nps curcumin and emodin as lead structures, a virtual screening of an in-house database was carried out. among the virtual hits seven nps, e.g., anthraquinone ( ) and piceatannol ( ), were found to significantly induce apoptosis by inhibition of the csn-associated kinases using in vitro and cell culture experiments. a further study has demonstrated the power of the ligand-based approach applied to pharmacophore modeling of sigma- ligands [ ] . therein, some reliable pharmacophore models could be extracted solely from ligand structure information. compounds with potent affinities to the sigma- receptor known from literature were structurally aligned to derive distinct common features. their d arrangement in combination with a spatial restriction was then used for the generation of a pharmacophore model, which was able to retrieve compounds with high affinity values, among them also nps, like solanidine ( ). further ligand-based approaches use various forms of discriminant analysis, e.g., artificial neural network simulations. they are based on collections of mathematical models that are interconnected and organized in different layers. they are analogous to an adaptive human learning process and usually trained with learning sets applying one or more molecular descriptors in order to form clusters that enable to distinguish between different objects and their properties. the resulting models are then applied to make predictions on test sets, until the validated models may be used to derive a qsar of chemically related structures or to mine larger datasets. one may distinguish between supervised and unsupervised (e.g., kohonen network) learning methods as discussed in detail by zupan and gasteiger [ ] . a successful application example within the field of nps was published by wagner et al. [ ] . the authors used a dataset of structurally diverse sesquiterpene lactones with known nf-b inhibitory activity to derive a qsar. by the application of multiple d structure representations as descriptors, a single model was achieved which provided detailed information on the structural influence of the investigated biological activity. sangma and co-authors pursued a combination of two approaches to predict new inhibitors of the hiv- rt and hiv- pr from a np database comprising metabolites from thai medicinal plants. after a high throughput docking of the molecules into the target enzymes, self-organizing maps were generated to reduce the number of promising candidates to be tested [ ] . a set of different in silico methodologies was previously applied by cherkasov and co-authors to aid in the discovery of natural non-steroidal ligands for human sex hormone binding globulin [ ] . therein, a rigorously cross-validated neural network based qsar model identified prospective compounds from a structure collection of , commercial natural substances. this stringent qsar ranking was combined with docking studies and pharmacophore-aided database search. the integrated computational methods resulted in a convincing predictive tool which identified a set of structurally diverse nps, of which every fourth compound was able to inhibit the target protein in a micromolar range. compounds of arbitrary structural diversity and with known activity against a target are particularly suitable not only for generating a ligand-based pharmacophore model (as described before), but also for structure similarity studies using a decision tree. the object is to find as good a distinction as possible on the basis of a set of molecular descriptors, which identify molecular features shared by different subsets of active compounds and accordingly filter out compounds within the dataset in which these combinations are lacking. using not only a simple logical description of one model, but an ensemble of decision trees tend to be the preferred option, since the consensus voting among trees give the approach higher predictive accuracy. one form of multiple decision trees well performed to virtually screen large d databases is random forest [ ] . this chemoinformatic method was recently applied in a theoretical work performed by ehrman and co-workers to predict ligands of multiple targets, like cyclooxygenase (cox), lipoxygenase (lox), aldose reductase, hiv- enzymes etc., from a large dataset of chinese herbs [ ] . the advent of structure databases has provided a basis for the development and feasibility of automatic methods for the search of new lead structures. conceptually, all the virtual screening concepts presented above have their origins in synthetic chemistry. their application, however, is just as well adaptable to nps' chemistry. prior to the in silico filtering experiment, a d structure database requires an efficient generation of reasonable, energetically minimized conformations assumed to meet approximately those conformations that might be of biological relevance [ ] . the underlying algorithms for d structure generation and conformation analysis are implemented in commercial software tools, e.g., in corina [ ] or the catalyst program (catalyst, available from accelrys inc., san diego, ca, usa; www.accelrys.com). in the field of nps the virtual screening application is mainly restricted due to the lack of searchable resources for structurally well defined natural compounds. in general, molecular databases with free access on the internet may comprise a high number of molecules, e.g., chembank (> , , , http://chembank.broad.harvard.edu) or pubchem (> , , ; http:// pubchem.ncbi.nlm.nih.gov); however, information about the number of contained natural molecules is rarely available. the library of the national cancer institute (nci) stores structural information of more than half a million compounds from both synthetic and natural origin that have been collected and tested by the nci since . about half of the synthetic compounds, which represent the large majority of the samples, may be used for free and are thus in the public domain. it is called the 'open nci database' (development therapeutics program nci/nih; http://dtp.nci. nih.gov/webdata.html). an interesting property prediction approach to the more than , compounds contained in this open database was provided by poroikov and co-authors [ ] . by use of the program pass (prediction of activity spectra for substances) an in silico tool for complex searches of different types of activities is provided; e.g., in the case of antineoplastic effects, the authors could demonstrate a substantial dataset enrichment over random selection by the use of pass-predicted probabilities. libraries covering a major part of entities from nature (at least some thousands) or consisting of structural information exclusively from natural origin are not free of charge, e.g., the traditional chinese medicinal database (tcmd; http://tcm d.com/services.htm [ ] ) or the dictionary of natural product database launched by chapman & hall (dnp; http:// www.chemnetbase.com) providing chemical and physical data on some , natural compounds gathered from the world's chemical literature. an excellent survey of public and commercial databases focusing on nps has recently been published by füllbeck and co-authors [ ] . the authors provide information as to storing characteristics of the databases, web-addresses, total number of compounds and -if given -number of natural ones. in addition, a selection of suppliers and manufacturers of natural compounds and extracts are given. a new database is introduced by the authors (super natural database [ ] ) storing information on available nps, thus allowing the selection of compounds that can be purchased. moreover a number of non-commercial in-house created databases have been used from different groups for their virtual screening studies on nps, e.g., a marine natural product database (mndp [ ] ), a natural product database (npd [ , ] ), a database based on the antique source 'de materia medica' by pedanius dioscurides (dios [ ] ), or a database fed with metabolites of ethnopharmacologically known plants [ ] . recently, ehrman and co-authors generated a d multiconformational database of chinese herbal constituents containing a total of more than , compounds from chinese herbs [ ] . integrated strategies for the discovery of bioactive nps the more or less accurate prediction of potentially active compounds by virtual screening has doubtlessly rationalized the early drug discovery process. these filtering experiments definitely assist in saving costly and time-intensive pharmacological assays, since the pool of predicted ligands (i.e., virtual hits) is usually drastically reduced compared to the initial amount of compounds (i.e., d-database). demands to be made on a good model are selectivity and target-specificity on the one hand, but it is also seminal not to lose too many valuable ligands during the filtering process. how far all of these demands can be fulfilled strongly depends on the quality of information used as the basis for generating the model and the algorithm underlying the virtual screening process. in medicinal chemistry, an activity prediction of - % is usually regarded as satisfying enrichment. in np research, however, this percentage may be too scarce. it is rarely found that a large set of natural compounds can be acquired so easily. only a minority of secondary metabolites are commercially available -usually at incredibly high prices. thus, extraordinary charges and efforts are typically necessary before a virtual hit from nature is available for pharmacological testing. this process embraces the acquisition of the natural material described to contain the desired metabolite to the point of phytochemical analysis and isolation. though advanced separation techniques, analytical instrumentation, and innovative tools for structure identification are at the phytochemists' disposal, it remains a complex and sometimes uncertain endeavor. this is why the results obtained from in silico predictions may nevertheless be too vague for a np researcher. methods are asked to further increase the probability of following the straight tip. there is the possibility to hyphenate sundry computational approaches, e.g., pharmacophore-based virtual screening combined with docking of the resulting virtual hits, or to consider only the consensus hits applying two or more screening concepts. nevertheless all these strate-gies remain virtual and speculative. the combination of two approaches, which are completely divergent in nature, like a computational and an empirical one may however offer a more deepened access to bioactive nps and may sometimes help to avoid a distorted view. thus, the computer-aided molecular selection is best combined with further discovery methods, labeled as integrated approaches, to increase the probability in finding a real hit. in traditional pharmacognosy there are some well established methods in targeting this aim starting from a holistic level. these include (i) hints from ethnopharmacology, (ii) phenomenological effects registered after application of naturally derived preparations, (iii) guidance of chemotaxonomy, (iv) phylogenetic selection criteria, or (v) simply information gathered from a high/medium throughput screening of extracts. in a recently published review from our group, different strategies in the field of nps have been presented with special emphasis on anti-inflammatory nps interacting within the arachidonic cascade [ ] . integrated computational strategies for the discovery of natural bioactive compounds have been introduced elsewhere concentrating on their scope, strengths and limits [ ] . some strategies and examples from literature combining virtual screening approaches and classical methods for activity exploitation are outlined below. as soon as a sensitive data-mining tool has been developed and has proved itself by more or less selectively finding the active compounds within a test set, it can be applied for screening a d multi-conformational database. the subsequent procedure consists of the evaluation of the virtual hits considering physicochemical properties, toxicity and pharmacokinetics. in this stage additional virtual filtering tools for the profiling of adme parameters [ ] might have an invaluable impact to aid a refined selection of compounds. then, a sensible choice of natural materials known to contain the focused metabolites and worth investigating in detail is a crucial step. it requires a comprehensive study in literature considering the hit content in the natural source, its availability and maybe hints from ethnopharmacology. once some natural materials are selected, it is advisable to perform a preliminary assay with those crude extracts and fractions assumed to contain the promising metabolite/s. though being aware that in case of small hit amounts present in the natural material the activity may be overseen. therefore, it is advisable to first identify the promising constituent and to possibly enrich it in the extract to be tested. those samples that scored well are then subjected to phytochemical investigations. in this way, the tricky selection of the natural material turns from a bold venture to a more rationalized endeavor. as soon as a promising (i.e., active) starting material is found, there are in principle two possible strategies to embark on: the first one relies more on the in silico approach and focuses directly on . strategy a for the discovery of bioactive nps using an integrated virtual screening approach the identification of the initially obtained virtual hits within the natural matrix applying analytical tools, like lc-ms or lc-nmr, gc-ms etc. in a straightforward manner the hits are isolated using different chromatographic separation steps. after structural confirmation the compounds are then tested to hopefully verify the predicted activities. this strategy is very goal-oriented, since only pharmacological assays for the finally isolated virtual hits are necessary. on the other side, one may run the risk of ignoring further active nps not necessarily fitting into the pharmacophore model. the second strategy focuses on a bioactivity-guided fractionation irrespective of the virtual hits used for the selection of the starting material. following the concept, the finally isolated active ingredients should correspond to the predicted virtual hits. this approach is usually associated with higher phytochemical efforts and costs, because it requires an iterative testing of all arising fractions and sub-fractions. for the evaluation of all the bioactive constituents in detail and for the discovery of possibly unknown metabolites this procedure is however indispensable. the decision, which of the presented ways is the more appropriate for the investigation at hand, strongly depends on the reliability and selectivity of the used pharmacophore model, and the costliness of the used assay. the strategy schematized in figure was recently applied to a medicinal plant with anti-inflammatory potential known from ethnopharmacological sources [ ] . from the pharmacophore based virtual screening filtering experiment a number of secondary metabolites known from the mulberry tree complied with all the models' requirements, thus revealed as virtual hits. indeed, in vitro tests attested extracts of morus root bark a distinct cox inhibitory potential. the objective was to find the active principles from this plant material applying both different methods for their discovery. first, the computer-aided approach was used to identify the virtually active compounds able to interact with the pharmacophore models for cox- and - . second, the bioactivity-guided fractionation was conducted for the isolation of the cox-inhibiting constituents. this resulted in the isolation of nine compounds belonging to the chemical classes of sanggenons and moracins. in the enzyme assay, all the isolates showed moderate to potent inhibitory effects on cox- and - . when comparing the hits of the virtual screening with the experimental data, a good correlation between predictions provided by the computer assisted method and in vitro data could be obtained in the case of the isolated sanggenons (e.g., sanggenon c; ). however, this agreement could not be achieved with the moracins (e.g., moracin m; ). in any case the virtual screening was particularly helpful for the decision regarding which plant material is worth extensive study. furthermore, the disclosed interactions of the sanggenons with the pharmacophore model -miming the binding site of the target -provided us with some essential information about the molecular requirements of cox-ligands. a different integrated procedure is schematized in figure . applying this approach, the pre-selection of the natural material is not guided by virtual prediction; but a number of extracts is roughly screened with a bioassay to identify the active ones. a similar strategy is to collect information about the traditional application of natural preparations in the field of the focused pharmacological target. a d database is then generated consisting of all the metabolites known from literature to be included in that extract/s that came off well. likewise, ethnopharmacological knowledge about useful preparations from nature may guide the selection of nps. the resulting biased database is virtually screened with an established pharmacophore model of the aiming target. the impact of ethnopharmacology has been analyzed in a previous study from our group; there we investigated the statistical evidence considering hints from folk medicine for the discovery of anti-inflammatory nps utilizing pharmacophore-based virtual screening techniques [ ] . cox- and - were used as preferential targets, since they are key enzymes in the inflammation process. dioscorides' de materia medica, which was written in the st century ad, was used as the ethno-pharmacological source. secondary metabolites of those medicinal plants, which dioscorides described as active against fever, rheumatism, pain and pus were stored in a multiconformational d database. this was virtually screened against the validated pharmacophore models. the resulted hit list was analyzed and compared with those obtained by screening unbiased databases of natural as well as of synthetic origin. the effectiveness of an ethnopharmacological approach could be statistically demonstrated by obtaining a significantly higher hit rate compared to the hit rates of the unbiased natural as well as synthetic databases. following this strategy the putative hits may then be identified by modern analytical tools like lc-ms or lc-nmr to isolate them from the natural matrix in a target-oriented way for pharmacological testing. this approach is especially helpful for intricate pharmacological assays, which would turn a bioguided fractionation into an unrealistic endeavor. a combination of an ethnopharmacologically based pre-selection of plant material and a computational approach was reported by bernard and co-workers, who used this strategy to rationalize a phytochemical lead discovery [ ] . starting with an in vitro screening on phospholipase a performed with traditionally used anti-inflammatory plant extracts, a focused structural database was generated and virtually screened on an established ligand-based pharmacophore model for human non-pancreatic phospholipase a . the combination of experimental data with database exploitation and molecular modeling resulted in the efficient identification of betulin ( ) and betulinic acid ( ) as extract ingredients with distinct anti-inflammatory in vitro effects. the combination of the two different, but complementary strategies consisting of in vitro screens and in silico assessment has recently been described by van de waterbeemd [ ] . he labeled this method as 'in combo' approach and used it for the straight forward access of various adme properties. the application of the 'in combo' approach for the discovery of nps has recently been tested in our group by the search of natural acetylcholinesterase inhibitors [ ] . in a medium-sized throughput screening about plant extracts were investigated using an acetylcholinesterase enzyme test. from the sample showing the best inhibitory activity, all the known secondary metabolites were fed into a small d multiconformational database and subsequently subjected to a virtual screening on a generated pharmacophore model. the efficacy of this procedure could be confirmed by the isolation of the obtained virtual hits, i.e., -deoxylactucin ( ) and lactucopicrine ( ) . they showed a significant and dose-dependent inhibitory effect in the enzyme assay. methods and expectations of this integrated virtual screening concept have previously been discussed in detail by j. bajorath [ , ] with the author's final statement that 'a meaningful integration of virtual and experimental screening programs, together with lessons to be learned from structural genomics, holds great promise for more rapid and consistent identification of high quality hits or leads across divers classes of therapeutic targets'. though this conclusion was not particularly coined to nps, it comes especially true in the rich world of secondary metabolites. further hybridized computational strategies are quite sensible to get an improved understanding of ligand-target interactions. in the following two examples docking protocols helped enlighten the molecular mechanism of bioactive natural compounds. chimenti and co-authors isolated quercetin ( ) among other secondary metabolites from the mediterra-nean shrub hypericum hircinum and identified this flavonol as selective inhibitor of the mao-a with an activity in the nanomolar range (ic = nm) [ ] . for a more comprehensive understanding of the underlying molecular selectivity, conformation analysis and docking simulations were performed using the most recent crystallographic structures of both human isoforms mao-a and mao-b. this enabled the authors to identify the most important interactions between the residues and the cofactor within the enzymatic cleft. the estimated free energies of complexation were in agreement with experimental data and confirmed the distinct preference for the mao-a cleft with more intermolecular hydrogen bonds and -interactions. the goal of a recent in-house study was to rationalize the binding interaction of the protoalkaloid taspine ( ) within acetylcholinesterase. taspine was isolated in a bioactivity-guided manner from magnolia x soulangiana and revealed as selective inhibitor of acetylcholinesterase with a significantly higher effect than the positive control galanthamine ( ; ic = . ± . µm). extensive molecular docking studies were performed with human and torpedo californica-acetylcholinesterase employing gold software (vers. . ; www.ccdc.cam.ac.uk/products/life_sciences/gold/). the results suggested taspine to bind in an alternative binding orientation than galanthamine [ ] . while this is located in close vicinity to the catalytic amino acid triad, taspine was found to be mainly stabilized by sandwichlike -stacking interactions in the aromatic gorge of the enzyme. in both case studies the active natural compound was already identified. thus, the in silico tool was not employed for data mining, but to elicit the putative binding mode in the macromolecular target. docking simulations turned out to be excellent tools to get an idea about the assumed molecular ligand target interaction. another approach capitalizes exactly on the just-mentioned observation that computational predictions may reveal an idea about the interaction to a specific target's binding site. thus, it is possible to start with one compound of unknown activity and to mine it against a number of structurally disclosed targets in terms of elaborated pharmacophore models (fig. ) , i.e., parallel in silico screening (see previous). as soon as the orphaned molecule is able to comply with all the requirements and restrictions imposed by any model, it can be assessed as rational hint. consequently, the focused compound will be subjected to a pharmacological testing on the predicted target/s. in this way, the parallel screening is not only helpful to estimate the interactions of a drug candidate with diverse antitargets; or to canvass its interactions to related targets as is performed for an activity profiling. in this approach, the parallel screening is a computational tool for target fishing to get a rational idea about any potential target interaction and to prioritize a few targets for experimental evaluation by applying simple ligand-based or target-based queries. the potential of virtual screening of target libraries was recently discussed by didier rognan [ ] . in his group a structure-based method for target screening was pursued applying inverse docking [ ] . the authors used , structurally well-defined pdb entries to build a d protein library. the virtual screening of this protein library with four unrelated ligands was suitable for recovering the true targets of specific ligands and may as well be used for virtual selectivity profiling of any ligand of interest. nettles and co-authors performed the target fishing approach using a ligand-based procedure [ ] . the potential of both d and d chemical descriptors were compared as tools for predicting the biological targets of ligand probes on the basis of their similarity to reference molecules in a chemical database comprising , biologically annotated compounds. the ligand-based d tool fepops (feature point pharmacophores), which provides pharmacophoric alignment of the small molecules' chemical features consistent with those seen in experimental ligand/receptor complexes, was used for scaffold jumping within the screened database. using atp the authors were able to identify the natural compound balanol ( ) as ligand of cdk . the highest effort applying this strategy is the availability of a representative amount of reliable pharmacophore models covering a wide range of relevant targets (fig. ) . thus, it may be of particular interest to focus on one pathological syndrome, e.g., obesity, inflammation, apoptosis etc., where a phenomenological activity of a np is already evident. applying this approach the disposition of pharmacophore models for targets involved in the respective pathological complex is easier to manage. in this way, a goal-oriented strategy may help to bridge the gap between a phenomenological effect and the underlying molecular mode of action. pertaining to the drug discovery from nature we are facing two facts: (i) statistics show that the myriad of structurally diverse natural compounds are the most favored source of new drugs for clinical use [ ] ; (ii) the drug discovery process has moved towards more rational concepts based on the increasing understanding of the molecular principles of protein-ligand interactions. spurred on by economic interest fundamental advances have been made in research applying data mining strategies, like virtual screening. though being aware of both potentials, their combined benefit could only rudimentary be savored. only limited attempts applying innovative in silico tools in np research are pursued so far, because the search for bioactive compounds is a complex and multidisciplinary challenge. thus, a sensible adaptation of computational strategies is in demand to profit in an economic way from the unique chemical and biological diversity associated with nps. virtual screening techniques, however, must not be used exclusively as activity-predicting tools, since the results provide merely an indication for a putative activity: it is only by the creation of interfaces between computational tools and well-established methods from pharmacognosy that a reasonable standard of success can be achieved. the search for the most effective strategy is best performed by a drug discovery process that involves the exploitation of all the information which can be gathered from bioactivity-guided fractionation, on-line analytical activity profiling, ethnopharmacological screening, chemoinformatics, virtual and in vitro screening studies. in the first instance it behoves modern pharmacognosy to skillfully exploit knowledge from all these fields because it is of paramount importance to sift through the enormous wealth of nps. examples underlining the impact of virtual screening on the identification of active nps have been presented in this survey. though the full potential in this field is by far untapped, these early results indicate that the integrated virtual screening approaches are target-oriented and trendsetting strategies. however, as any computer-based technique, the successful use of virtual screening will entirely depend on the way it is utilized and the quality of its underlying experimental data. the advantages implemented to a virtual screening cycle compared to a conventional in vitro screening are obvious: (i) higher capacity, (ii) no need for isolated compounds, (iii) less experimental efforts for testing; (iv) theoretically, interactions of all known nps to all structurally defined targets can be calculated and predicted, (v) the quality of hit compounds can be increased by additional drug-like filters and virtually restricted adme properties; thus diminishing failures in the early drug development. nevertheless experimental investigations are seminal, but can be focused in a more effective fashion. a cautious handling of virtual hits together with lessons learned from traditional pharmacognosy seems to be crucial for a successful exploitation of treasures from nature. in this area, virtual screening will most likely play an essential role in accelerating the early stage of drug discovery by efficiently digging out lead compounds from nature. rediscovering the sweet spot in drug discovery strategic trends in the drug industry screening for drug discovery: the leading question the role of natural products in a modern drug discovery program natural products as sources of new drugs over the last years strategies for discovering drugs from previously unexplored natural products lessons from natural molecules the role of pharmacognosy in modern medicine and pharmacy rediscovery of known natural compounds: nuisance or goldmine strasburger -lehrbuch der botanik, . auflage, spektrum akademischer verlag plant-derived natural products in drug discovery and development: an overview natural products in drug discovery and development statistical investigation into the structural complementarity of natural products and 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-hydroxysteroid dehydrogenase type inhibitors by common feature pharmacophore modeling and virtual screening pharmacophore modeling and in silico screening for new p (aromatase) inhibitors pharmacophore modeling and three-dimensional database searching for drug design using catalyst pharmacophore modeling and three dimensional database searching for drug design using catalyst: recent advances novel curcumin-and emodin-related compounds identified by in silico d/ d conformer screening induce apoptosis in tumor cells feature based pharmacophore models for sigma receptor, erg and ebp neural networks in chemistry and drug design development of a structural model for nf-b inhibition of sesquiterpene lactones using self-organizing neural networks virtual screening for anti-hiv- rt and anti-hiv- pr inhibitors from the thai medicinal plants database: a combined docking with neural networks approach successful in silico discovery of novel nonsteroidal ligands for human sex hormone binding 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silico tools to exploit the natural products' bioactivity admet in silico modelling: towards prediction paradise? discovering cox-inhibiting constituents of morus root bark: activity-guided versus computer-aided methods ethnopharmacology and bioinformatic combination for leads discovery: application to phospholipase a inhibitors which in vitro screens guide the prediction of oral absorption and volume of distribution? application of the in combo screening approach for the discovery of non-alkaloid acetylcholinesterase inhibitors from cichorium intybus virtual screening in drug discovery: methods, expectations and reality integration of virtual and high-throughput screening quercetin as the active principle of hypericum hircinum exerts a selective inhibitory activity against mao-a: extraction, biological analysis, and computational study taspine: bioactivity-guided isolation and molecular ligand-target insight of a potent acetylcholinesterase inhibitor from magnolia x soulangiana in silico screening of the protein structure repertoire and of protein families recovering the true targets of specific ligands by virtual screening of the protein data bank bridging chemical and biological space: "target fishing" using d and d molecular descriptors key: cord- - k ll authors: bastos, paulo; trindade, fábio; da costa, joão; ferreira, rita; vitorino, rui title: human antimicrobial peptides in bodily fluids: current knowledge and therapeutic perspectives in the postantibiotic era date: - - journal: med res rev doi: . /med. sha: doc_id: cord_uid: k ll antimicrobial peptides (amps) are an integral part of the innate immune defense mechanism of many organisms. due to the alarming increase of resistance to antimicrobial therapeutics, a growing interest in alternative antimicrobial agents has led to the exploitation of amps, both synthetic and isolated from natural sources. thus, many peptide‐based drugs have been the focus of increasing attention by many researchers not only in identifying novel amps, but in defining mechanisms of antimicrobial peptide activity as well. herein, we review the available strategies for the identification of amps in human body fluids and their mechanism(s) of action. in addition, an overview of the distribution of amps across different human body fluids is provided, as well as its relation with microorganisms and infectious conditions. human antimicrobial peptides (amps) represent approximately % of all curated amps catalogued to date. human host defense peptides are an intrinsic part of the innate immune system and exhibit a broad activity spectrum against bacteria, fungi, viruses, and parasites while amps can be antibacterial (abps), antifungal, antiprotist, antiviral, anticancer, antiparasitic, insecticidal, spermicidal, chemotactic, antioxidant, protease inhibitors, or even exhibit wound healing properties (supporting information table s ), their scope of action overlaps considerably and some peptides show activity at several levels (fig. ). [ ] [ ] [ ] [ ] [ ] for instance, on the one hand, human plasma adrenomedullin-derived peptides are active against multiple bacteria species, human urinary and gingival fluid calcitonin gene-related peptide displays antimicrobial activity against several bacteria and fungi species, and human neutrophil peptides (hnps)/defensins isolated from most biological fluids display activity against several bacterial, fungal, and viral species (fig. ) . on the other hand, dozens of human amps known to date display antimicrobial activity against the same human colonizers or pathogens, including escherichia coli, pseudomonas aeruginosa, and staphylococcus aureus (figs. and ) . also, it is intriguing to observe that even though some amps display broad activity spectra, others seem to be anywhere from species-to kingdom-specific (fig. ) . while likely to be confounded global network depicting the distribution of human antimicrobial peptides across biological fluids and their microbial targets. rectangular nodes correspond to biofluids, circular nodes to target species, and each edge correspond to a given gene encoding one or more antimicrobial peptides. the thicker the edge, the stronger is the association of a biofluid to a pathogen, representing increased number of antimicrobial peptides defending against such pathogen in such biofluid. also, pathogens represented by bigger nodes (e.g., escherichia coli, candida albicans, staphylococcus aureus) represent those that are targeted by more antimicrobial peptides across different biofluids. by observational bias (whereby some species, e.g., e. coli, tend to be tested more frequently than others), this observation also reflects that the activity spectra of amps depend not only on their physicochemical properties (which group them according to unique families) but also on target properties and on the environment/biological fluid in which they are found. as the large majority of amps identified to date are abps (ß %, figs. - ) , most concepts and examples provided in this review will be based on results from studies focusing on abps, closely followed by those derived from studies on antiviral and antifungal peptides. however, the same principles tend to apply across all amps. amps are very potent cationic molecules displaying minimum inhibitory concentrations (the minimum concentration that prevents bacterial growth) as low as - μg/ml, which highlights their promise as broad-spectrum antimicrobial agents. furthermore, amps act more rapidly than conventional antibiotics and are not affected by the typical resistance mechanisms involving conventional drugs, making them very attractive for therapeutic purposes. in addition, amps are not mere microbicide agents, as their scope of action may encompass other functions, such as cellular development and immune system modulation. for instance, fetal keratinocytes express significantly more amps compared to neonatal and adult keratinocytes, despite the lower degree of exposition to the environment or to pathogenic agents. these a rough estimate of all amps catalogued to date (supporting information table s ) indicates . % to be α-helices, followed by β-sheets ( . %), and peptides presenting with both helices and β-sheets ( . %). however, approximately % of all amps currently identified do not have a known d structure, while only . % have a known structure that is neither a helix nor a β-sheet. excluding those that have unknown structures, . % of all amps are either α-helices and/or β-sheets, suggesting a highly conserved structure for a highly primordial biological function. secondary structures of human amps may include α-helices (e.g., ll- ), β-sheets (e.g., defensin ), and extended peptides (e.g., indolicidin). considering that β-sheets and α-helices in polypeptide chains typically contain three to ten amino acids per strand and . residues per turn, respectively, and that amino acid sequences can be virtually unlimited, one can easily envision that a given peptide can display a multitude of secondary structures and, thus, many diverse conformations (see for protein secondary structures predictive approaches). among secondary structures, α-helices (in addition to being the most frequently encountered in nature) are the most regular and predictable, and, consequently, the most extensively studied. amps with linear α-helices are notably positively charged and amphipathic, but only adopt such secondary conformation upon binding to bacterial membranes. therefore, their antimicrobial activity is directly dependent on the secondary structure. in contrast, β-sheet amps possess well-defined conformations due to the presence of disulfide bridges formed between thiol groups in cysteines. in these peptides, such bridges are often required for antimicrobial activity. contrariwise, bovine lactoferricin was shown to be effective against e. coli even when such bridges were blocked by pyridylethylation. other human amps, such as human β-defensin (hbd- ), present both α-helices and β-sheets. in the specific case of hbd- , disrupting the three stabilizing disulfide bonds does not compromise its antimicrobial activity, but it does diminish its chemotactic properties. furthermore, the correlation of the conformation with peptides' antimicrobial activity is not always as straightforward as one could imagine. for example, disruption of hbd- 's disulfide bonds results in peptides with free cysteines at the carboxy terminal that show increased antimicrobial activity against the pathogenic candida albicans and the gram-positive commensals bifidobacterium and lactobacillus. moreover, linear extended peptides do not keep a fixed conformation in solution, and are enriched in one or more amino acids, particularly arginine, proline, and tryptophan. for instance, the amp histatin found in human saliva is enriched in histidine residues, while prophenin is very rich in proline and phenylalanine residues. when it comes to loop rich peptides, the presence of many prolines makes an amp much less likely to form amphipathic structures, adopting what is frequently referred to as polyproline helical type-ii (ppii) structures. these structures are specifically bound by sh domains, which are usually found in proteins that interact with other proteins and mediate assembly of specific protein complexes (upon proline-rich peptides binding). human antibacterial peptides (abps), which compose the largest group of human amps, exhibit a weighted average net charge of + . per peptide, which is considerably above the average net charge of all amps identified to date. also, despite being intuitive that the more positively charged an amp is, the more potency it will present, human amps do not show greater potency or efficacy when compared to those of other species, suggesting that the environment and the presence of different amino acids (d and rare amino acids) also play a significant role. notwithstanding the large diversity of amps across human biologic fluids (figs. and , table i ), it should be noted that amps are much more diverse across all species, than those identified in humans, which present rather conserved properties. in fact, some amps from other species are characterized by more intriguing sequences, structures, and physicochemical growth-regulated alpha protein properties. case in point, bacillus subtilis is known to produce several extremely potent lipopeptides (active at concentrations as low as . - . μm) with antimicrobial activity against several species. one of these, gageotetrin a is a very unique anionic amp that consists of only leucine and glutamic acid residues conjugated with a unique fatty acid, -hydroxy- -methyltridecanoic acid. another example is that of sonorensin from bacillus sonorensis, a broad-spectrum amp from the heterocycloanthracin subfamily, active against both gram-negative and gram-positive bacteria, including listeria monocytogenes and s. aureus. sonorensin displays an amino acid sequence with multiple copies of the same motif, making this peptide rather unique. baceridin is a circular peptide from a plant-associated bacillus that is synthesized by ribosome-independent machinery and bears only six amino acids, % in the d configuration. moreover, baceridin is a proteasome inhibitor, compromising cell cycle progression and inducing apoptosis in tumor cells by a p -independent pathway. copsin from coprinopsis cinerea is bactericidal against enterococcus faecium and l. monocytogenes by interacting with the peptidoglycan precursor lipid ii and interfering with the cell wall biosynthesis. curiously, it is modified with a pyroglutamate, which confers a higher degree of thermal stability and resistance toward protease digestion. even though some display fungicidal and virucidal properties, the chief activity of human amps is against gram-positive and gram-negative bacteria (figs. - ). furthermore, while amps represent an extremely diverse group of biological active molecules, most share common properties that contribute for their membranolytic or otherwise antimicrobial activity: positive charge, hydrophobicity, and amphiphilicity. therefore, the mechanism of action of most amps involves membrane disruption (fig. ) , a process that is also required when their translocation is warranted and the respective targets are localized intracellularly. the interaction of amps with bacterial membranes depends on the establishment of attractive electrostatic forces between these. bacterial species are known to carry negatively charged outer membranes due to phosphate groups of the lipopolysaccharides in gram-negative bacteria and to the lipoteichoic acids in gram-positive bacteria. the fact that all gramnegative and gram-positive bacteria display these type of negatively charged lipids accounts for the lack of specificity of most abps, and promotes the attraction between amps and bacterial membranes while preventing their binding to most host cells membranes. moreover, the inherent hydrophobicity and amphiphilicity allow abps to form clusters at the membrane's surface once attached. , at low concentrations (low peptide-to-lipid ratio), abps tend to adsorb onto the surface, adopting an orientation parallel to the membrane bilayer. however, clustering increases the peptide-to-lipid ratio, which subsequently promotes additional clustering. once a certain peptide-to-lipid ratio threshold is reached, abps adopt a perpendicular orientation relative to the bacterial membrane, which allows them to insert themselves into the membrane, as will be further discussed (fig. ) . peptides can adopt many different secondary structures and conformations, which depend on their amino acids sequence, as well as on the environment. consequently, different peptides and different environments favor the formation of unique structures, such as barrel staves, carpets, and toroidal pores. , , when the peptides attach and are inserted into the membrane bilayer so that the hydrophobic regions align with the phospholipids' acyl groups and the hydrophilic regions create the central region of a pore, a barrel stave is formed. in addition, these structures act as a primer for the aggregation of new peptides, thus expanding the pore's diameter. due to abps' cationic sites, membrane crossover would be very energetically unfavorable in their original parallel orientation. however, this orientation changes to a ( ) cluster at the cell surface and cause membrane disruption by several different mechanisms (e.g., barrel staves, carpets, toroidal pores), ( ) translocate into cells and impair intracellular organelle machineries, ( ) impair protein-protein interactions, enzymatic cascades, and cytosolic signaling pathways, ( ) interact with nucleic (not in the case of bacteria) acids, trap replication forks, and compromise nucleic acids as well as protein synthesis, ( ) preclude several steps of viral replication, ( ) inhibit genetic material trafficking, reverse transcriptase, and viral proteases, ( ) block the interaction between virus and host cells (e.g., viral envelope glycoproteins gp and gp or coreceptor cxcr ), compromising virus binding and entry, and ( ) cause membrane lysis on enveloped viruses. see text for detailed description. designed using servier medical art. perpendicular one upon abps' clustering, and the peptides are reoriented with their hydrophobic portions facing the membrane acyl groups and their cationic sites facing the interior of the pores. thus, clustering and pore formation provides a means for membrane disruption and the leakage of intracellular molecules from bacterial cells. in contrast, if peptides remain parallel to the membrane surface with their hydrophobic residues facing the membrane while their hydrophilic residues face the surrounding milieu, a "carpet"-like structure is achieved. with such organization, membrane's rigidity weakens progressively as new peptides are added, culminating in its dissolution and breakup. mechanistically, this mode of action is less stringent because peptides do not need to adopt a specific structure. however, the necessary concentration of abps appears be much higher than that required for the formation of barrel staves. [ ] [ ] [ ] for toroidal pores to form, peptides need to aggregate onto the membrane's surface and insert themselves in a perpendicular fashion. , subsequently, abps are reoriented parallel to the membrane so that polar residues interact with the polar heads of the membrane lipids, thus causing membranes to bend. this induced bending of the bilayer causes the upper and lower leaflets to meet, and allows a mixture of peptides and lipid head groups to line with the interior of the pore, forming toroidal or wormhole structures. , , abps may also inhibit bacterial growth and have a bactericidal effect by promoting the clustering of anionic lipids, such as phosphatidylglycerols (pgs) and cardiolipin. for instance, for ll- , initially believed to act according to the "carpet" model, there is evidence that its fragments, namely kr- , act as a magnet that competes for the negatively charged pgs, promoting their redistribution into "pg-rich domains." such phospholipidic reorganization may disturb cell signaling and compromise the activity of membrane-bound proteins, namely, the voltage-dependent potassium channels, which can seriously jeopardize bacteria survival. likewise, n-acylated peptides derived from lactoferricin were shown to induce defects in e. coli cell division by rearranging the distribution of inner membrane phospholipids, essentially due to the clustering of cardiolipin and other anionic lipids. the bias toward positive charges confers amps the ability to easily interact not only with biological membranes, but also with other negatively charged molecules (fig. ) such as nucleic acids and those bearing phosphate groups. , in addition, because this type of interaction is largely unspecific, amps can exhibit not only a broad-spectrum activity, but also multiple mechanisms of action (fig. ) . , for instance, lactoferricin is known to inhibit atp-dependent multidrug efflux pumps as well as dna, rna, and protein synthesis. ten human amps derived from lactoferricin and lysozyme are thought (by sequence homology) to act by trapping the replication fork and preventing base recombination and dna repair. furthermore, the presence of many prolines in some peptides (also known as ppii structures as aforementioned) hampers the organization of amphipathic structures. these structures favor interactions between peptidic structures and between these and nucleic acids, thus playing a major role in signal transduction and complexes assembly. however, ppii structures do not necessarily contain prolines, and abps rich in arginine residues are also able to translocate across membranes and to interact with nucleic acids and proteins. hence, both proline and/or arginine-rich abps may be capable of inhibiting prosynthetic signaling pathways and enzymatic activity (fig. ) , shifting the conformation of cellular structures and activating autolysins. however, this is a speculation that warrants further studying. in addition to targeting bacterial membranes, human defensins also bind to the peptidoglycan precursor lipid ii and inhibit cell wall biosynthetic enzymes in staphylococci species. moreover, when stabilizing disulfide bridges between conserved cysteine residues in human amps with β-hairpin or β-sheet conformations are disrupted, the resulting linear peptides still maintain their antimicrobial properties despite losing membranolytic activity. , the antifungal histatins act at multiple levels by mechanisms of action conserved across the histatin family of amps, with histatin the most potent amp in this family. this amp binds the yeast transmembrane receptor heat-shock protein ssa / p, is internalized, and then targets primarily yeast mitochondria. , by doing so, it leads to the formation of reactive oxygen species (ros), causes atp efflux, and inhibits oxidative phosphorylation. , simultaneously, histatin also interacts with the potassium transporter trk p, causing release of k + ions and further atp efflux. when the functional domain of histatin was synthetically multiplied, its antifungal activities were significantly potentiated and it became significantly faster-acting compared to the normal histatin . such an observation might prove itself very useful, because it indicates that modulating the number of functional domains per amp may enhance its antimicrobial activity without requiring the administration of increased quantities of amp. histatins may also exert their antimicrobial activities by inhibiting host and microorganismal proteolytic enzymes. histatin is a strong inhibitor of human matrix metalloproteases / and metalloproteases from porphyromonas gingivalis. thus, its inhibitory activity over proteolytic enzymes may attenuate tissue damage and microbial propagation during the onset of periodontal inflammatory disease. also, by inhibiting the cysteine protease clostripain from clostridium histolyticum, histatin mitigates the virulence factors produced by this clostridium species and may therefore alleviate the complications associated with gas gangrene syndrome. so far, the above-mentioned mechanisms of action have been discussed in the context of abps or antifungal peptides, and sometimes the same peptide is active against both bacteria and fungi. in turn, the action of antiviral peptides can diverge from these mechanisms and should thus be independently commented (fig. ) , although some overlap does exist. extracellularly, most native antiviral peptides often present as α-helical structures, have primarily lytic activity on enveloped viruses, and can directly inactivate cell-free virions (fig. ) . human lysozyme fragments are this group's prototype, capable of aggregating on the viral surface and causing disruption of membrane envelopes. , similarly, cap peptide appears to exert its antiviral activity by destructing the virus envelope of type i herpes simplex virus (hsv) or even by disrupting the capsids of adenoviruses. however, because membrane lipids of enveloped viruses derive from host cell membranes, special attention is required when considering the exploitation of this feature, as antiviral peptides may disrupt host cell membranes indiscriminately. alternatively, antiviral peptides may block the interaction between the virus and the host cell (fig. ) . that is believed to be, at least in part, the mechanism of action of human defensins, θ -defensin, human α -antitrypsin virip, cyclic lactoferricin, and serine protease inhibitor elafin. for instance, it has been demonstrated that all α-defensins [hnp- , hnp- , hnp- , and hnp- and human α-defensin (hd)- and hd- ] and hbd- prevented hsv infection by blocking virus binding and entry. such antiviral activity was attributed to hnp- - , hd- , and hbd- 's high affinity to hsv's glycoprotein b and hnp- , hd- , and hbd- 's avidity toward endogenous heparan sulfate. similarly, protection against hiv is conferred by peptides with lectin properties that can bind viral envelope glycoproteins gp and gp , crucial for viral attachment, fusion of envelopes with host membranes, and subsequent entry into host cells. hnp- that binds and blocks the action of gp , and θ -defensins (e.g., the pseudogene retrocyclin) together with the human α -antitrypsin-derived -residue viral-inhibitory peptide both targeting gp represent examples of such peptides. [ ] [ ] [ ] likewise, hsv- infection can also be blocked by retrocyclin, which binds tightly to glycoprotein b . the cyclic peptide lactoferricin seems to compete for the ligation to extracellular heparan sulfate or chondroitin sulfate, thus preventing the very first event in cytomegalovirus, hsv, and papillomavirus infection. [ ] [ ] [ ] finally, elafin is thought to act indirectly in host cells by hampering viral attachment and probably by modulating host's antiviral and inflammatory responses toward hsv- infection. synergistically with this mechanism, antiviral peptides can also bind and inhibit the activation of viral co-receptors, leading to further inhibition of glycoproteins-mediated viral attachment. not only that, hbd- and - inhibit viral infection by promoting the internalization of its co-receptor cxcr . , virucidal peptides may also preclude several steps of viral replication by interacting with intracellular targets (fig. ) . this is the case of hnp- and hd- , which may prevent hsv replication by binding directly to its dna. also, hnp- arrests influenza virus replication by inhibiting protein kinase c phosphorylation, an essential step for nuclear trafficking events. human cathelicidin (ll- ) derived peptides, in turn, inhibit reverse transcriptase and viral proteases, thus preventing integration of the viral genetic load into human dna. , the few resistance mechanisms against amps demonstrated so far among microorganisms have been observed almost exclusively in bacteria. with very few exceptions, bacteria do not seem to develop complete resistance against cationic abps, though some strategies do exist among bacterial species for diminishing their potency and efficacy. the difficulty in acquiring resistance may stem mainly from the fact that abps target an essential cellular component for bacteria survival, which cannot be easily modulated by the cell without causing significant adverse effects. therefore, resistance mechanisms are not likely to be a limitation in the therapeutic utilization of abps. in light of this scarce resistance mechanisms de novo acquisition, what mechanisms do bacteria naturally develop/possess against abps? first, one should consider that the primary target of abps consists of bacterial cell membranes and bear in mind how abps depend on the electrostatic interactions between membrane negative charges and positive peptidic residues. accordingly, bacteria have learned how to modulate cell membrane charges and composition. gram-positive bacteria can partially modify the peptidoglycan's teichoic acid and gram-negative bacteria can partly change its lipopolysaccharide lipid a moiety, thus compromising this interaction. for instance, d-alanine-activating enzyme and d-alanine transfer protein are known to transport positively charged d-alanines to the surface anionic teichoic acids in gram-positive s. aureus, reducing the negative net charge of its outer membrane. also in s. aureus, the five-component system graxsr-vrafg increases the expression of mprf and dltabcd genes upon exposure to cationic amps. when s. aureus strains are mutated so that the regulation of these genes is disturbed, these strains become more susceptible to daptomycin, polymyxin b, hnp, and platelet factor -derived peptide, and less infectious in vivo in a nonclinical endocarditis model. [ ] [ ] [ ] the gene product mprf attaches lysine residues, which bear a positively charged ε-amino group, to the anionic phophatidylglycerols, making these less negatively charged and thus less susceptible to interactions with cationic amps. , the gram-negative vibrio cholerae can increase its resistance to polymyxins more than -fold by modifying its lipopolysaccharides in a process mediated by almf. the lpta gene product found in gonococcal species, such as the gram-negative bacteria neisseria gonorrhoeae, protects lipopolysaccharide lipid a moieties by attaching phosphoethanolamine, making these bacteria less susceptible to amps and increasing their capacity to modulate host's immune response by evading complement-mediated cellular death. , moreover, in n. gonorrhoeae and neisseria meningitides, the presence of phosphoryl and phosphoethanolamine in lipid a moieties enhances the activation of the nf-κb pathway and the production of proinflammatory cytokines in human cells, which can amplify their virulence. despite this modification-dependent virulence, the absence of such modifications in commensal neisseriae species allow these to colonize and coexist with the human host without inducing bactericidal host immune responses. in addition, the adaptation of the composition and, hence, the electrostatic properties of the membrane in order to circumvent the host immune system is not an exclusive phenomenon of pathogenic bacteria. commensal bacteria of the intestinal tract can remove phosphate groups from their membrane's lipopolysaccharides, decreasing their negative charge and allowing greater survival and proliferation in the gut. similarly to the above-mentioned paradoxical modification-dependent virulence, mutated commensal gut microbiota bacteroides that fail to remove phosphate groups from their lipopolysaccharides are killed during inflammation while normal strains keep their resilience. therefore, there appears to be an inverse relationship between acquired resistance and bacterial virulence, so that induced/acquired modifications render cells less virulent while simultaneously allowing these to more easily colonize and coexist within the human host environment. second, bacteria are also able to adapt the fluidity of the membrane by increasing the hydrophobic interactions within their outer membrane, thus hindering pore formation. third, bacteria can express proteases against abps or transporters and efflux pumps like atp-binding cassette (abc) transporters to circumvent such innate defense barriers. , glu-c protease produced by s. aureus induces the loss of antibacterial activity of neuroendocrine peptides, and chromofungin, procatestatin, and human catestatin are enzymatically degraded when treated with bacterial supernatants. in e. faecalis, bacitracin binds the abc transporter ef - , which interacts with the regulatory domain of the two-component system ef - , increasing its expression and conferring resistance against bacitracin. two-component systems regulate the expression of abc transporters and are constituted by a transport permease and a sensor kinase. however, at least in b. subtilis, the abc transporter itself is required for both sensing of and resistance to bacitracin, suggesting these transporters to also function as environmental sensors. lastly, bacteria may also synthesize molecules capable of directly neutralizing abps, which can provide a way for bacteria to bypass amps. however, such resistance mechanisms are very limited and resistance to amps does not take place to a large extent. r bastos et al. in humans, endogenous amps have been found in several fluids throughout the body, as depicted in figures and as well as in table i . as noted, multiple amps have been found in tears, saliva, milk, blood, urine, sweat (and others not-so-well-explored biofluids), reflecting not only a remarkable versatility but also a huge conservation that reinforces its essential role. the robust knowledge of amps on some fluids (e.g., tears, urine, saliva) over others (e.g., amniotic fluid, cerebrospinal fluid) most likely reflects their availability, but different exposure to pathogens from the environment (figs. and ) should also account for these discrepancies to a large extent. additionally, saliva and urine screening is of special interest, since both the oral cavity and the urinary tract represent the main doorways for microbial invasion and colonization, and are thus enriched with host defense peptides. likewise, milk screening is of high relevance due to the presence of particular proteins and amps that will boost neonate's immunity. despite underrepresented, amps from other fluids are equally relevant (figs. and ). for instance, amps collected from airway secretions, bronchoalveloar lavages, and nasopharyngeal aspirates can be valuable sources of information regarding the physiological response to pathogens of the respiratory tract and may, themselves, constitute raw models for new therapeutic drugs. also, other than circulating immune cells derived, blood has not been regarded as a rich source of amps, but this view can be questioned (figs. and ), most likely due to tissue-derived amps' passage to the blood circulation. however, it should be noted that human fluid reservoirs are not independent/isolated, and, consequently, peptides may be present throughout and cross between, possibly undergoing modifications between such reservoirs. amps may be present in fluids as a result of many different processes, including exocytosis, in situ proteolysis, tissue necrosis, and cellular lysis. therefore, in this section, we will characterize naturally occurring amps based on structural and functional similarities rather than their source of collection. defensins are cationic peptides rich in cysteine residues, presenting as β-sheet structures stabilized by disulfide bridges. according to the alignment of these stabilizing disulfide bounds, mammalian defensins are classified into three subclasses, α-defensins, β-defensins, and θdefensins. disulfide bounds of α-defensins occur between cysteines - , - , and - . these peptides consist of - amino acids and adopt a triple-stranded β-sheet conformation. human neutrophils express four distinct α-defensins, known as hnp- to hnp- . despite having been initially isolated from peripheral blood leukocytes, hnp- to hnp- can also be found in bone marrow, spleen, and thymus. the remaining two already identified human α-defensins, hd- and hd- , are found only in paneth cells of the small intestine and in epithelial cells, thus being tissue specific. expressed in the aforementioned cell types, amps can also be found in bodily fluids in direct contact with these cells. among the most recent discoveries, hd- has been found to be active against bifidobacterium adolescentis and other anaerobic gut commensals, but not (directly) bacteriostatic or bactericidal against most pathogenic bacteria, and may therefore play an important role in maintaining a balance among the microbiota of the gut. similarly, human hd- is particularly potent against the gram-positive bacillus clostridium difficile, one of the most common pathogens responsible for nosocomial infections, whose highly virulent strains often attack small intestine mucosa. in addition, α-defensins may show both cellular and regional specificity throughout the gastrointestinal tract, either secreted or active inside intracellular granules and constitutively expressed or amenable to induction. , , it should also be emphasized that the activity spectrum of α-defensins is not limited to bacteria and fungi, exhibiting activity against several virus, including hiv, hsv, adenovirus, and human papillomavirus (hpv), and displaying several unique mechanisms of action (please refer to section ). it should be noted that the antimicrobial activity of α-defensins is influenced even by the difference in one amino acid, such as for hnp- to hnp- . in fact, an additional acidic residue in hnp- lowers its effectiveness (most accurately, its potency) against s. aureus, p. aeruginosa, and e. coli. this is explained by the easily donated proton, which makes this defensin less positively charged (at least when considering interaction domains only), thus reinforcing the importance of a positive net charge for the antimicrobial activity of amps, , as evidenced in section . in terms of disease, α-defensins are known to have a paramount role, particularly when the demands for defense mechanisms are increased. accordingly, a remarkable -to fold increase in the concentration of hnp- and hnp- (and hnp- to a smaller extent) in human tears as been reported after ocular surgery, an increase that allowed these peptides to reach the required concentrations for antimicrobial activity but that is followed by a decrease once healing has been completed and the demands for immune defenses have returned to baseline. nonetheless, the goal of achieving this increased α-defensin concentration in tears is not restricted to the enhancement of the immune defenses, as it is also known to promote local cellular proliferation and tissue repair. in parallel, salivary α-defensins are active against streptococcus mutans, but their secretion has been found deregulated in caries-positive human subjects. in contrast, prolonged physical exercise has been shown to significantly increase the levels of salivary α-defensins, showing that lifestyles may have a considerable impact on our amp-based defenses against microorganisms and it is possible to modulated the activity thereof. disulfide bounds of β-defensins occur between cysteines - , - , and - . these defensins are longer than α-defensins, spanning from to amino acids, but also share a triple-stranded βsheet conformation. the first hbd was isolated from plasma samples and kidneys constitute the major source of hbd- . since then, peptides derived from human hbd- have been identified in tears, urine, vaginal lavage, sweat, and blood plasma, and these are effective primarily against e. coli. [ ] [ ] [ ] [ ] in turn, hbd- was isolated from psoriatic skin samples, displaying bactericidal activity toward gram-negative e. coli, p. aeruginosa, and c. albicans, and bacteriostatic activity toward gram-positive s. aureus. despite very effective in vitro, both hbd- and hbd- present attenuated activities in vivo as a result of their sensitivity to physiological salinity. in contrast, hbd- is active against s. aureus and vancomycin-resistant e. faecium even at physiological salt concentrations. the hbd- defensin has been found in seminal plasma, and it has been reported as potential efficacious against e. coli, s. aureus, and c. albicans. , furthermore, hbd- has been identified in epididymal fluid and has been shown to regulate lipopolysaccharide-mediated inflammation toward e. coli, s. aureus, and c. albicans, protecting sperm from motility loss. in disease conditions, increased concentrations of hbds in plasma and bronchoalveolar lavage fluid were reported in patients with diffuse panbronchiolitis. in particular, β-defensin a was suggested to play a role against p. aeruginosa. also, while β-defensins are expressed throughout the gastrointestinal tract, these seem to have evolved together with changes in human gut bacteria, displaying characteristic expression patterns in response to certain microorganisms. their expression may be significantly altered in human diseases, most notably in inflammatory bowel diseases (for insightful reviews on this topic, please refer to , and enhanced in inflammatory conditions of the ocular surface ). with α-defensins, the role of the ph for amps' activity is considered. here, β-defensins serve to illustrate the role of salt concentration. in fact, tears contain a remarkable concentration of sodium chloride, which inhibits the activity of human amps (by ß % in the case of hbd- , an inhibition that increases up to % in the presence whole tear fluid, suggesting the presence of other inhibiting factors). therefore, whenever there is an increase in the demand of amps activity, (e.g., upon infection or tissue damage), their concentrations must surge to reach the required protective levels. however, an excessive increase in the concentration of any amp may lead to tissue damage and local functions may be compromised. for this reason, a complex interplay of amps is required at surfaces like the ocular epithelia, which is achieved by a qualitative enrichment, in addition to the expected quantitative increase (for a depiction of the multitude of amps found in human tears, please refer to ). similarly to α-defensins, the activity spectrum of β-defensins is not limited to bacteria and fungi, and they are active against several virus, namely, hiv, influenza a virus (iav), respiratory syncytial virus, and vaccinia virus, and display several unique mechanisms of action, depending on their target (see above section on mechanisms of action). lastly, genomic studies have predicted additional β-defensins to be expressed in human cells, but their presence in human bodily fluids and their antimicrobial activity in vivo is yet to be demonstrated. while αand β-defensins adopt triple-stranded β-sheet conformations, θ -defensins have shorter sequences and adopt a complete circular conformation, with stabilizing disulfide bounds between cysteines - , - , and - . the peptide rhesus θ -defensin is the prototype of this more recently discovered family of amps. like αand β-defensins, θ -defensins possess broadspectrum microbial activity against bacteria and fungi, while also being capable of protecting mononuclear cells from infection by hiv- . , moreover, these peptides display antiviral activity against hsv, hiv, iav, and severe acute respiratory syndrome (sars) coronavirus, acting by several mechanisms, depending on their target. structurally, θ -defensin peptides are very interesting and very odd, representing the first cyclic peptide family discovered in animals, albeit not in humans. moreover, their circular structure appears to be required for antimicrobial activity. these peptides were first isolated as antimicrobial octadecapeptides expressed in leukocytes of rhesus monkeys, and their formation results from the head-to-tail joining of two independent nine-amino acid peptides derived from truncated pro-α-defensins. in contrast to the previously described classes of defensins, human θ -defensins are thought to be completely inactivated, despite the existence of mrnas encoding at least two θ -defensins expressed in the bone marrow. while genes encoding θ -defensins in humans also encode premature stop codons (thus hampering θ -defensin expression), synthetic human θ -defensins (called retrocyclins) have been shown as promising antiviral agents. in fact, retrocyclin- inhibits the entry of hiv, hsv, and iav into host cells. in other mammals, retrocyclin- also protects from infection by bacillus anthracis spores and the rhesus θ -defensin protects mice from sars coronavirus infection. cathelicidins are a very diverse group of cationic α-helical and amphipathic amps that exhibit a broad-spectrum activity against bacteria, fungi, and virus. the term "cathelicidin" originally referred only to the entire precursor protein, though currently is commonly used for the whole family, including the resulting peptides with antimicrobial activity. in contrast to the mammalian trend in which cathelicidins and defensins are the main amps, there is only one human gene encoding multiple cathelicidin-related peptides, , located on chromosome and expressed in the airways, mouth, tongue, esophagus, epididymis, and small intestine. , despite this tissue expression pattern, ll- is constitutively formed in spleen, liver, stomach, intestine, and bone marrow. ll- is highly positively charged (+ charge at physiological ph . ), due to the high content of arginine and lysine amino acids, and adopts an α-helical structure in solutions with ionic composition similar to that of human plasma. while defensins share common structural features, cathelicidin-related peptides are highly heterogeneous despite deriving from the same precursor. cathelicidins are characterized by a highly conserved n-terminal signal peptide (the so-called "cathelin domain") and a highly variable c-terminal antimicrobial domain that can be released after cleavage by proteinases. , cathelicidin is cleaved into the antimicrobial peptide ll- by both kallikrein and kallikrein serine proteases, it is upregulated by vitamin d, and has been shown to significantly reduce the risk of death from infection dialysis patients. ll- was shown to disrupt bacterial membranes through the formation of toroidal pores and carpet structures, and to exhibit chemotactic properties, attracting leukocytes and activating secretion of chemokines. in addition, ll- is internalized by cells, acidified in endosomes, and activates the signaling pathway downstream to toll-like receptor by interacting with double-stranded rna. profiling of airway fluids collected from premature infants' tracheal aspirates during infection evidenced the production of ll- even at an early stage of development. in these samples, ll- was found in significantly increased concentrations during pulmonary or systemic infections, suggesting this peptide to be as important to avoid infectious processes as to fight already established infections. the concentration of ll- has been measured in seminal plasma samples from healthy donors and found to be up to -fold higher than in blood plasma. furthermore, it was found to be attached to spermatozoa, eliciting a possible role in human fertilization or its precursor to be extensively cleaved by the high concentration of serine proteases present in seminal plasma. ll- , an alternative form of human cathelicidin peptides, contains one more alanine at the n-terminus than ll- and was initially found to be produced in female vaginal secretions due to the action of gastricsin on sperm precursor cathelicidin. both ll- and ll- are active against bacteria such as e. coli, s. aureus, p. aeruginosa, and bacillus megaterium, and synergistically with peptides from seminal plasma play an essential defense role protecting the human reproductive system. , interestingly, both citrullination and adp (adenosine diphosphate)-ribosylation of arginines can compromise the ability of ll- to prevent endotoxin-induced sepsis, perhaps because the attachment of these negatively charged molecules reduces the peptide's cationicity or by raising steric hindrance. , histatins are a normal component of human saliva and part of the innate immune system, protecting against a broad array of infectious agents, especially against candida species, saccharomyces cerevisiae, cryptococcus neoformans, and neurospora crassa. [ ] [ ] [ ] there are only two genes enconding histatins (both located on chromosome ), and they are exclusively expressed in human salivary glands. however, other active amps resulting from the cleavage of histatins and also exist. histatins and are encoded by genes htn and htn , respectively. histatins , , and - are the products of posttranslational proteolytic cleavage of histatins and , though histatin can also result from posttranscriptional modification of histatin mrna. , peptides derived from histatin bear distinct domains associated with specific functions, namely an n-terminal domain with antimicrobial activity and a c-terminal domain with wound-healing properties. , histatin has two important metal-binding motifs, consisting of an amino-terminal copper (ii)/nickel (ii)-binding motif and a zinc (ii)-binding motif, although they can also bind to cobalt (ii) ions. , the ability to coordinate metal ions appears to be important for the stabilization of the secondary structure, particularly in the presence of negatively charged membranes. nevertheless, histatins lack any defined secondary structure and are unordered when in aqueous solutions, assuming α-helical structures only in organic solutions. , in addition, histatin binding to copper (ii) and nickel (ii) ions induces the formation of ros, which contributes for its fungicidal activity. , histatins can bind to a receptor on fungal cell membranes and induce cell death by disrupting the cell cycle and causing nonlytic loss of atp, as discussed above. while histatins are effective, potent, and fast-acting amps against several antimicrobial species, these peptides are nontoxic to human cells and human microflora, which may be due to the fact that their translocation across the membrane is site and species specific. , both parotid, submandibular, and sublingual glands secret histatins, and their concentration and secretion have been shown to decrease with age, even when accounted for total protein concentration. moreover, oral candidal infections increase with age, suggesting a link between age-associated deficiency of human histatins and an increased risk of oral infections. dermcidins are broad-spectrum anionic amps with no apparent homology to other known amps. peptides in this group comprise amino acid residues, which are subsequently processed into smaller but still active ones. dermcidins are constitutively expressed in human eccrine sweat glands and secreted in sweat, exhibiting antimicrobial activity against common human pathogenic microorganisms such as s. aureus, staphylococcus epidermidis, e. coli, e. faecalis, and c. albicans. [ ] [ ] [ ] this constitutive production contrasts with human defensins and cathelicidins, which are induced during inflammatory and stressful conditions. a small percentage of human breast cancer cells displays rna encoding dermcidin, and after oxidative stress induction different types of tumor cells leads to the production of diverse and biologically active proteolytically processed dermcidin peptides. , therefore, the upregulation of dermicidins may confer human breast cancer cells a selective advantage compared to nonmalignant cells. in contrast, reduced expression of dermcidins in sweat from atopic dermatitis patients contributes to skin infections and altered skin colonization. hence, the downregulation of dermicidins in atopic dermatitis patients may represents a suppression of an innate and fundamental defense mechanism, which, in turn, could confer an advantage to infectious microorganisms. unlike histatins, which are nontoxic to human cells and human microflora (see above), dermicidins can have serious deleterious consequences to the human host. dermcidin isoform is known as a stress-induced oxidative protein capable of inhibiting the synthesis of nitric oxide in endothelial cells, insulin in pancreatic islet β-cells, and hepatic hormone synthesis. also, hypertensive patients show significantly higher levels of plasma dermcidin, but salicylic acid has been shown to reduce dermcidin levels while simultaneously attenuating dermcidin-mediated insulin inhibition in patients with acute myocardial infarction. therefore, the exploitation of dermcidin isoform for antimicrobial therapeutic purposes is most likely not possible due to severe side effects. despite this, it may be a promising therapeutic target for hypertension and diabetes management. in animal models, dermcidin has been shown to induce platelet aggregation and coronary artery disease by activating platelet cyclooxygenase and inhibiting the constitutive form of nitric oxide synthase, respectively, with an efficiency times higher than that of adp at activating cyclooxygenase. notably, dermcidin was able to stimulate the development of coronary artery disease in one animal model, even at suboptimal concentrations of adp within min. together, these observations suggest that the ability of amps to fight infections depends on their induction by infectious microorganisms, the particular peptides generated, and the local environment/tissue in which they act. also, biological roles performed by human amps are more diverse than immediately expected, playing essential functions other than fighting infectious microorganism(s). in fact, dermcidin-mediated inhibition of insulin is unique in the sense that no other protein is known to arrest pancreatic insulin synthesis in such way, and similar observations might result in novel and unexpected applications of human amps. amps expressed by human liver (leap and ) were initially discovered in human blood, and subsequently found in urine. these peptides are known as hepcidins and are characterized by a high content in cysteine residues (approximately % of all residues, with eight disulfide bounds). leap- or hepcidin- is essential for maintaining iron homeostasis, and, when mutated, leads to severe iron overload and juvenile hemochromatosis. hepcidins are the main regulators of plasma iron concentration and its secretion is promoted during inflammatory responses, particularly by the action of interleukin , which is one of the main mediators of the acute phase response. as iron bioavailability is a limiting factor for bacterial growth, hepcidins represent an organic mechanism for sequestering iron from invasive pathogens. however, when several iron sources are available, as it happens within the human host, eliminating a single reservoir may not be sufficient to attenuate microorganisms' virulence. moreover, bacteria are known to exploit almost every host iron-binding protein and reservoir and, consequently, human laeps/hepcidins are a rather limited source of antimicrobial activity. for these reasons, the antimicrobial role of iron sequestration depends not only on hepcidins, but also on several other mediators, such as transferrin, lactoferrin, ceruloplasmin, haptoglobin, and hemopexin. mutations responsible for decreasing the levels of hepcidins or compromising the function of other regulators of iron homeostasis, such as the major histocompatibility complex class i like hereditary hemochromatosis protein (hfe), transferrin receptor , and ferroportin, lead to increased iron blood levels. when levels are high enough to produce hemochromatosis, individuals are at an increased risk of infectious complications, including bacteremia and meningitis caused by e. coli, bacteremic cellulitis, and hemorrhagic bullous skin lesions caused by v. cholera, multiple pyogenic abscesses in the liver parenchyma as a result of yersinia enterocolitica infection, meningitis, endocarditis, and pericarditis resulting from infection with l. monocytogenes, and fatal septicemia during infection with vibrio vulnificus. in contrast, hemochromatosis can also be associated with decreased macrophage iron storages in the case of hfe-associated hemochromatosis. this depletion makes these cells more efficient in fighting salmonella enterica subsp. enterica, serovar typhimurium, and mycobacterium tuberculosis infections. , however, these protective effects of macrophages iron storage depletion have only been demonstrated in animal models and appear to be mediated not by hepcidins, but rather by lipocalin- , which reduces the availability of iron for salmonella, and transferrin and lactoferrin, both of which compromise iron acquisition by m. tuberculosis. , neuropeptides and neuroendocrine peptides may exert mitogenic actions through which innate barriers are reinforced and may display neurotransmitter, immunoregulatory, and direct antimicrobial activity. vasostatin- was the first natural antifungal n-terminal chromogranin a derived fragment peptide, having been first isolated from chromaffin secretory granules from bovine adrenal medulla. however, its sequence is highly conserved in humans ( % identity homology) and its microbial activity also overlaps, to some extent, with that of bovine origin. irrespectively, human vasostatin- displays antimicrobial activities against gram-positive bacteria (micrococcus luteus, b. megaterium) and a large variety of filamentous fungi (n. crassa, aspergillus fumigatus, alternaria brassicola, nectria haematococca, fusarium culmorum, fusarium oxysporum, trichophyton mentagrophytes) and yeast cells (s. cerevisiae, c. albicans). moreover, many autocrine and paracrine biological activities of chromogranin a, its precursor protein, have been attributed to peptides located along its sequence, which are co-released with catecholamines by chromaffin cells upon stimulation and can be found in human bodily fluids. , therefore, the antimicrobial activity spectrum of vasostatin- may be extended by that of co-released peptides also formed by chromogranin a proteolytic cleavage, and some of the coproduced peptides are even more potent than vasostatin- itself. in addition to the structural requirements for the antimicrobial activity of chromogranin a derived peptides, posttranslational modifications may also modulate their activity. for instance, s-pyridylethylation (an alkylating modification consisting of the addition of pyridylethyl moieties to the -sh functional groups of cysteine residues that disrupts disulfide bridges) renders it selectively active against b. megaterium but not m. luteus, while oxidation seems to render it inactive against bacteria but still active against fungi. in addition, chromogranin a derived peptides also undergo phosphorylation and glycosylation, which can modulate their physicochemical properties. currently, vasostatin- is known to be stored in endocrine, neuroendocrine, and neuronal cells, and to be released from stimulated chromaffin and immune cells upon stress. moreover, the stimulation of polymorphonuclear immune cells induces the processing of chromogranin a and the secretion of vasostatin- and other chromogranin a derived peptides, which may account for local inflammation. lastly, recombinant and synthetic human vasostatinderived fragments have been shown to inhibit vascular contraction induced by endothelin- , parathyroid hormonal secretion, neuronal survival, expression of neurofilaments, and neuronal gaba uptake. , adrenomedullin was first isolated from a human adrenal pheochromocytoma and its plasma concentration is raised under specific physiological conditions, namely, renal failure, hypertension, and sepsis. this peptide has antibacterial activity against several grampositive and gram-negative bacteria, including propionibacterium acnes, s. aureus, m. luteus, p. gingivalis, actinomyces naeslundii, s. mutans, c. albicans, eikenella corrodens, actinobacillus actinomycetemcomitans, streptococcus pneumoniae, haemophilus influenzae, streptococcus pyogenes, bacteroides fragilis, e. coli, and helicobacter pylori. human neuropeptide y displays antimicrobial activity against bacteria and fungi, but truncated fragments are tenfold more potent than the intact precursor neuropeptide y, perhaps because the net charge of the fragments is more positive than that of the intact neuropeptide. encephalins and their derived peptides may either enhance or inhibit the immune response and the corresponding circulating immune cells/mediators in both humans and animal models. , as well as their subsequent antimicrobial effects, may thus take place indirectly in immune system modulation. simultaneously, antibacterial assays have revealed that peptide b/enkelytin (a c-terminal fragment of proenkephalin a) specifically targets gram-positive bacteria, including m. luteus, b. megaterium, m. luteus, and s. aureus, while having no effect over gram-negative bacteria. , classically, proenkephalin a has been ascribed to the secretory granules from adrenal medullary chromaffin cells and various brain regions, such as the striatum and the pituitary, according to some animal studies. , despite this, it has also been demonstrated to be expressed in and secreted by polymorphonuclear neutrophils (accounting for its particular enrichment in wound fluids), t and b lymphocytes, macrophages, and mast cells. , however, it should be noted that the antimicrobial effects of encephalins and their derived peptides result mostly from animal studies and have not been adequately studied in human secretions, despite the high conservation of their sequences across species, which most likely contribute for the similar activity spectrum. even so, phosphorylation and glycosylation are characteristic of these peptides, , and it is possible to envision that differences at the level of posttranslational modifications or as a result of gene divergence may result in distinct activity spectra. interestingly, peptide b/enkelytin is metabolized in vivo to opioid peptides, thus revealing an intricate relationship between innate immunity and pain modulation, and its plasma concentration is increased after coronary artery bypass grafting in humans. because it is expressed in both immune and neuronal cells, it is possible that the same stimuli may act on both cells populations, thereby accounting for immune system modulation at the periphery and alterations in central nervous system signaling. however, the exact roles and mechanisms of this interplay are not clear. in addition, other amps found in epithelial secretions from the gut and the skin, for example, include peptides derived from alpha-melanocyte-stimulating hormone, which is active against s. aureus and c. albicans. accordingly, evidences suggest that amps like these act by inhibiting mrna and protein synthesis rather than inducing direct membrane disruption, as is the case for most amps. several cationic proteins display nonenzymatic antimicrobial activity, which is sustained by the resulting cleavage peptides after protein fragmentation. for example, lactoferrin is a glycoprotein enriched in milk and neutrophilic granules, which has the ability to sequester iron and thus prevent bacterial overgrowth. when digested by pepsin, lactoferrin yields lactoferricin h (human), an amp able to neutralize bacterial toxins, regulate gene transcription, inhibit complement activation, viral infection, and tumor overgrowth, thus further extending the antimicrobial properties of its precursor. human chemokines are secreted by macrophages and polymorphonuclear immune cells and, therefore, found in the blood circulation. moreover, during allergic inflammatory responses to bacterial infections of the airways, the eosinophil-recruiting chemokines eotaxin- /ccl , eotaxin- /ccl , and eotaxin- /ccl are generated by mast cell proteases, active against several airway pathogens, including s. pneumoniae, s. aureus, h. aeruginosa. peptides resulting from the cleavage of pepsinogen a and c prosequences have also been shown to exert antimicrobial activity. although these were initially identified from the stomach of bullfrogs, synthesized human pepsinogen c prosequences with similar structural characteristics (amphipathic and helical structures) to these cleavage peptides have also exhibited antimicrobial activity. calcitermin is an amp that results from the cleavage of a amino acids long sequence from the c-terminal region of protein calgranulin c, isolated from human airway secretions. by adopting an alpha-helical conformation in bacterial membranes, calcitermin is able to target gram-negative bacteria (e.g., e. coli and p. aeruginosa) and fungi (e.g., c. albicans). furthermore, the precursor protein and fragment peptide may act synergistically to protect the human host against foreign microorganisms present in the airways. on the one hand, the cleavage peptide calcitermin inserts into membranes of microorganisms. on the other hand, the precursor protein calgranulin c has a proinflammatory activity. by acting as a dangerassociated molecular pattern molecule and binding to the receptor for advanced glycation end products in innate immune cells, it activates the map-kinase and nf-κb signaling pathways, leading to the production of proinflammatory cytokines and histamine, upregulation of cell adhesion molecules, recruitment of leukocytes, and degranulation of granulocytes. , dermcidin-derived peptides such as dcds, ssls, and leks (the letters correspond to the first three amino acids of the corresponding precursor dermcidin protein) are also formed in eccrine sweat glands (but not in apocrine sweat glands) at these body sites with high probability for contact with pathogens (e.g., palms, face, arms) and exhibit antimicrobial activity against a large number of microorganisms, including s. aureus, e. faecalis, e. coli, s. epidermidis, pseudomonas putida, methicillin-resistant s. aureus, and rifampicin/isoniazid-resistant m. tuberculosis. [ ] [ ] [ ] such dermcidin-derived amps are regulated by proteolytic processing at several levels (see section ) and are differentially expressed during inflammatory conditions. these peptides are resistant to proteolytic degradation in sweat up to at least hr and, interestingly, most dermcidin-derived peptides are anionic in nature. if such distinct properties may lead to different antimicrobial activity spectrum is unclear, though not surprising, as skin represents a harsh and particularly unique environment. as of specific interest, it should be noted that the consensual average skin surface ph has been decreasing in the last few years and is now generally accepted to be below . while showering and cosmetic products decrease skin's surface ph, plain tap water can increase the skin ph up to hr. noticeably, negative charges seem to be important for dermcidin-derived peptides' antimicrobial activity, in contrast with what would be expected for amps. in fact, most dermcidin-derived peptides are naturally anionic, but low ph (high h + concentration) leads to an increase in their net charge (toward positivity). therefore, while an acidic skin actually keeps the resident bacterial flora attached to it, a more alkaline skin compromises such attachment, suggesting that the more negatively charged such skin peptides are (alkaline skin), the more efficient these become. despite the average length of . residues and net charge of + . for the close to amps already identified, human amps tend to be longer and more positively charged. amps with more than amino acids have been identified across the six kingdoms of life. curiously, most (approximately half) appear to be of human origin, presenting as amino acid sequences of residues on average and suggesting large amps to be a human feature. moreover, compared to all the remaining amps, large human amps tend to be biased toward leucine ( . %) and lysine ( . %) residues, displaying nonpolar aliphatic and positively charged side groups, respectively (table i) . therefore, large human amps are enriched with amino acids that promote distinguishing features of amps: high overall positive charge, hydrophobicity domains, and amphiphilicity. nevertheless, this bias toward lysine residues is neither unique to human amps nor more pronounced among these. for instance, the amphibian cathelicidin rc- is composed by % lysine residues, and its homologous snake crotalicidin contains % lysine residues, showing high potency against s. pyogenes, acinetobacter baumannii, e. faecalis, s. aureus, e. coli, klebsiella pneumoniae, and p. aeruginosa. as evidenced in table i , large human amps have been found in a variety of bodily fluids, including tears, saliva, urine, airways secretions, breast milk, blood, sweat, and epididymal fluids. in addition, other large amps are also expected to exist despite not constitutively found in biologic fluids, due to their inducible nature. for instance, paneth cells in the small intestine release amps-rich granules upon stimulation by cholinergic or bacterial stimuli, and granulocytes are known to contain amps in granules destined for extracellular secretion. , therefore, as the synthesis and secretion of large amps may not be constitutive but amenable to induction by microbial macromolecules and inflammatory cytokine stimuli, several other large amps may yet to be catalogued. hence, exploration of novel large antimicrobial human peptides is an ongoing work that may yield novel therapeutic agents. peptidomics is the complete study or global analysis of the peptides present in a biological sample, at a given time, under a particular condition. , the identification of naturally occurring amps in human biological fluids is a laborious, daunting, and sometimes unsuccessful task. such naturally occurring peptides can directly result from gene encoding, from passage to bodily fluids as a result of cellular damage and renewal, or from extracellular proteolytic processing of precursor proteins. the latter, considered to be the most prevalent, hampers the identification and interpretation of biologically active amps because precursor proteins may themselves display antimicrobial activity, but may also be cleaved into multiple functional and nonfunctional peptides. , moreover, owing to the large dependency of amps on their sequence and d structure, these peptides should not be subject to mass spectrometry (ms) based approaches employing enzymatic or chemical digestion, neither to any in vitro step that may cause their fragmentation. instead, top-down proteomic strategies are preferred. classically, different peptides fractions have been separated and tested for their biological activity, often without ever addressing the peptides' biological/antimicrobial activity, their sequence, target, or hypothetical mechanism(s) of action. however, current strategies are more focused, precise, and informative, capable of addressing all these parameters, sometimes in a single study (see below). , , human biological fluids consist of complex samples, containing lipids, salts, proteins, carbohydrates, and other molecules that make the study of endogenous peptides extremely difficult. consequently, enrichment and separation steps are almost always required. of these biomolecules, proteins and larger peptides as well as salts are the most complicated to separate from the target peptides. luckily, salts can sometimes be removed in the same procedures applied for protein removal. first and foremost, proteases/peptidases have to be inactivated, which can be done by denaturing procedures (e.g., microwaving, boiling) or by adding protease/peptidase inhibitors. , however, both of these can modify the peptide structure and, thus, these steps can be avoided when samples are resistant enough to further protease/peptidase activity. this is the case for urine samples, for example, as, when collected, have long been subject to proteolytic processing in the bladder. proteins are frequently removed by selective precipitation, most commonly by organic solvent (e.g., acetone) or acidic (e.g., trichloroacetic acid) precipitation, which also removes salts (left in the supernatant). , however, precipitation-based proteins removal is not complete and may lead to the formation of peptide aggregates, which are consequently lost in the precipitate. since peptides can present a great variety of sizes, sequences, structures, hydrophobicity properties, net charges, and other characteristics, their isolation is complex. still, such complexity allows for isolation steps to take place at multiple dimensions, increasing peptides isolation efficiency and accuracy. for instance, peptides can first be isolated based on their size and charge, , bias toward specific amino acids (e.g., cysteines, tryptophans, methionines), [ ] [ ] [ ] or on the presence of post-translational modifications (ptms) (e.g., phosphopeptides, glycopeptides). , still, because of the aforementioned limitations, the search for endogenous amps in human bodily fluids has mostly relied on the identification of candidate amps by chromatographic techniques (e.g., high-performance liquid chromatography, hplc) and sequencing (e.g., edman sequencing), followed by their de novo chemical synthesis and in vitro antimicrobial activity testing. for these reasons, the identification and in vivo testing of novel amps is often time consuming and requires multiple studies/phases and resources. in order to mitigate this problem, candidate human amps have come to be first predicted in multiple ways, including mining of the entire human genome for particular motifs known to confer antimicrobial activity, and screening of human peptidomes (e.g., salivary, urinary) in order to find homologous peptides to other known amps. accordingly, several libraries and databases are currently available for data comparison, and some search programs can even combine both previously obtained data and de novo information. even so, the identification of amps benefits the most from combinations of chromatographic and immunological techniques, genomics and proteomics. while genomics and proteomics both allow the prediction of novel amps and have the potential for contributing with a larger number of putative amps, peptides with proven in vivo antimicrobial activity have to be more accurately identified by combinations of chromatographic and immunological techniques, chemical sequencing and antimicrobial assays. though immunoassays can detect the intended target in a variety of complex biological samples, these techniques do not provide an integrative view of the peptidome, can suffer from cross-reactivity or lack of specificity issues and require previous knowledge of the target structure. , moreover, this issue becomes a particular problem when considering that shorter or longer peptides resulting from the same precursor protein may all contain the same epitopes and thus be recognized as the same peptide fragment despite exhibiting distinct biological activities. electrospray ionization (esi) and matrix-assisted laser desorption ionization (maldi) have been the most frequently and successfully applied ionization techniques in the field of bodily fluids peptidomics. in line with these, peptides are most frequently previously separated by lc-based and capillary electrophoresis (ce) based approaches, allowing different peptide fractions to be independently separated. from this point onwards, almost any mass analyzer can be used, although in practice quadrupole time-of-flight (q-tof) and ion traps (it) instruments are the most frequently utilized ones in peptidomics, especially in exploratory studies. [ ] [ ] [ ] in turn, fourier transform-ion cyclotron resonance-ms is more suitable for targeted/confirmatory analysis when dealing with fewer peptides. because these apparatuses allow tandem ms to be performed, peptides can be readily sequenced in addition to their identification, circumventing the above-mentioned limitations concerning immunological assays and largely simplifying peptidomics workflows. in summary, when aiming at the isolation of a particular peptide, with predicted or known charge and molecular weight, an ms-based procedure can theoretically be very straightforward. first, fluid samples require (sometimes multiple) centrifugation steps to remove cells, cellular debris, wastes, and possible contaminants. then, proteins and peptides in the supernatant are separated and fractionated by multidimensional chromatographic techniques. within these procedures, proteins and peptides can be initially fractionated by gel filtration (size dimension), followed by cation-exchange (charge dimension) fractionation. cation-exchange chromatography uses a negatively charged ion exchange resin with affinity for molecules with net positive charge. a salt gradient is used to separate the peptides of interest from other bound peptides, based on their predicted isoelectric point. finally, peptides can be subject to ms analysis. they are typically ionized by esi or maldi and subsequently analyzed in a q-tof, it, or hybrid ltq. as amps tend to be highly basic peptides due to a bias toward arginine and lysine residues, these have a propensity to be particularly enriched when applying cation exchange chromatography based techniques. however, because biological fluids are complex matrices, these sometimes require multiple fractionation, isolation, and extraction steps. these are laborious optimization techniques for the detection and quantification of amps and, despite their inherent complexity, they are often based in not always accurate functional and structural predictions. a recent approach has proved itself capable for the isolation of peptides present in bodily fluids that can bind directly to certain bacterial membrane components, such as lipopolysaccharide, and induce an inflammatory response against these. , after obtaining a bacterial homogenate and extracting membrane components with an organic solvent such as n-butanol, functionalized beads with n-hydroxysuccinimidyl-sepharose (an agarose used in affinity and protein chromatography) are conjugated with these membrane bacterial components and incubated with a human biological fluid (e.g., saliva, urine). then, peptides bound to the conjugated beads are eluted and analyzed in a ltq-orbitrap hybrid fourier transform apparatus. moreover, functionalized beads conjugated with bacterial membrane components can be tested in vivo for their capacity to induce the production of inflammatory mediators. thus, this exploratory approach could result in the identification of potential human amps with selective properties capable of binding bacterial membranes and inducing an inflammatory response. recently, dallas and colleagues have attained the most complete and prolific analysis of naturally occurring peptides with potential antimicrobial activity in human milk by nano-lc quadrupole-tof tandem mass spectrometry (ms/ms), while searching against libraries of human milk peptides and known amps. in order to remove milk peptides produced by in vitro proteolysis, fresh milk derived peptides were compared to control samples of the same origin but immediately subject to boiling. as boiling denatures proteases, in vitro proteolysis does not take place and peptides present in these controls could then be confidently assigned in the original analysis. moreover, masses fragmented in the first round of ms/ms were excluded from subsequent rounds of ms. the authors realized that more than % of the + unique naturally occurring peptides identified with % confidence have yet unknown functions, which serves to illustrate how much there is still to discover regarding the biological roles of human endogenous peptides in biological fluids. nevertheless, the antimicrobial activity of the milk peptides ( predicted amps) was confirmed in vivo by radial diffusion assays against the gram-negative e. coli and the gram-positive s. aureus. furthermore, this study reinforced the observation that the background against which mass spectra are searched seems to be a more critical factor than the mass analyzer or the ionization technique used, when it comes to both the number of peptides identified and the confidence by which these can be assigned. in the same study, authors have emphasized and demonstrated the importance of avoiding in vitro hydrolysis of amps in human bodily fluids, which can be attained by adding protease inhibitors after sample collection or by adequately accounting for this phenomenon, as the authors did by using an appropriate control. in contrast, not all bodily fluids seem to be susceptible to this degree of hydrolysis, at least not to the same extent. urine, for instance, is thought to be more stable and not to require treatment with protease inhibitors. however, by the time urine is collected it has already remained stagnant for considerably longer periods of time, and, hence, probably already extensively exposed to the action of proteases/peptidases inherently present in urine. therefore, how intact urinary amps actually are should always be questionable and these should ideally be interpreted considering the background of hydrolytic enzymes present in the same urine sample. also questionable is the inhibition of proteolysis in seminal plasma samples. in fact, the liquefaction process of human semen performed by seminin and other proteolytic enzymes is physiological, and, therefore, naturally occurring amps should be analyzed in samples that were allowed to liquefy without the addition of protease inhibitors. curiously, seminin, which is the primary agent responsible for liquefaction of the seminal coagulum, is partially destroyed by freezing at − °c, reinforcing the vitality of analyzing fresh samples. similarly, the degradation of peptide fragments derived from semenogelins in seminal plasma samples is time dependent and responsible for the decline in hiv-enhancing activity of sperm, once again underscoring the importance of timely performing analyses of peptides in bodily fluids. as previously stated, amps activity depends largely on their d structures, and their correct characterization becomes of paramount importance. the best experimental techniques have been and currently are x-ray crystallography and nuclear magnetic resonance, both of which allow for the study of structure-function relationships, though very expensive. therefore, currently, these are commonly replaced or complemented by bioinformatics prediction tools whenever possible. using such tools, the sequence of amino acids of a peptide allows for the accurate and immediate prediction of the presence of secondary structures (e.g., αhelices, β-sheets). then, the overall d structure is devised by algorithms employing energy minimization principles and molecular dynamics, sometimes resulting in multiple and not mutually exclusive d conformations, which is in accordance with peptides dynamic structure in biological fluids. , the most common identification software referenced in the literature for peptide/precursor protein identification are sequest and mascot. , currently, however, there is a considerable need for algorithms and software capable of correctly identifying amps and predicting their structure-function relationship. amps are a promising replacement for conventional antibiotics owing to their effectiveness against multiresistant bacteria and over multiple bacterial species simultaneously, invoking a diverse set of mechanisms that cannot be easily shortcut by bacteria, fungi, and/or virus. an ideal amp would be (i) highly selective against its target while leaving human host cells unaffected, (ii) not prone to resistance mechanisms, (iii) relatively easy to produce at low costs, and (iv) stable during storage or upon administration. drug design of such amps focuses on peptidic and nonpeptidic mimetic drugs, modulating the hydrophobicity, positivity, and amphiphilicity of endogenous amps to increase their antimicrobial potency. however, exploiting amps for therapeutic purposes is actually more complex than initially envisioned. while linear amps present a simpler design and a more predictable structure-function relationship, circular peptides may allow the design of longer acting and more resistant amps. such peptides have been designed by inserting the intended amp into other circular peptide (cyclotides) and have been proven efficacious against hiv and inflammatory diseases by circumventing their instability and poor bioavailability. , similarly, while humans produce amps with l-amino acids and their antimicrobial activity may depend on the action of endogenous enzymes, which can only act upon l-type amino acids, producing amps with d-amino acids may render these more resistant to hydrolysis. rather than administering exogenous amps, it is possible and perhaps more secure and controllable to pharmacologically stimulate the production of endogenous amps. for instance, , -dihydroxycalcypherol (active vitamin d ) has been successfully used to induce the expression of defb (defensin, beta ) and camp (cationic antimicrobial peptide) genes, the production of both ll- and human β-defensin in cell cultures of keratinocytes from diabetic foot ulcers and to promote wound healing. accordingly, the persistence of functional milk human κ-caseinoglycopeptides in plasma of human infants after human milk feeding serves the purpose of exogenously administering amps without the requirement for their de novo synthesis. amps can help preventing biofilm formation and microorganisms' growth. that is the case of ll- and its fragments, which were already shown to have antibiofilm formation properties against several methicillin-resistant s. aureus strains and p. aeruginosa, common pathogens of hospital-acquired infections, as well as against the melioidoisis' etiological agent burkholderia pseudomallei and the uropathogenic e. coli. [ ] [ ] [ ] [ ] another amp with antibiofilm formation activity is hbd- , found to prevent the development of methicillin-resistant s. epidermidis and s. aureus biofilms, largely responsible for orthopedic implants associated infections. likewise, amps may also inhibit the formation of fungal biofilms. for instance, histatin has been demonstrated to successfully inhibit the growth of c. albicans biofilm on denture acrylic. these examples may broaden the applications of amps in the clinical and biomedical fields by allowing functionalization of catheters. alternatively, implants and other medical devices can also reduce bacterial colonization, biofilm formation, and infection rates, presenting long lasting functionality, broad-spectrum activity, and minimal cytotoxicity against human cells. , human amps could, theoretically, also be used as biosensors immobilized onto microchips for identification purposes, allowing the identification of microorganisms. however, such applications have only been exploited with animal or synthetic amps. these biosensors on electrical impedance alterations resulting from the presence of bacterial or fungal surface elements, or on the specificity of peptide nucleic acid probes against their targets, presenting higher versatility, lower costs, and lower sensitivity limits compared to other conventional methodologies. [ ] [ ] [ ] alternatively, amps could be used as drug delivery systems in the form of conjugates in order to accurately target drugs and other agents to tumor sites or intended organs, as typified by monodisperse "endosomolytic" nanoparticles for in vivo gene delivery using intravenous injection, or by functionalized nanoparticles with higher membrane penetration targeted against gliomas. , such functionalization may allow for deeper tissue penetration, increased antimicrobial potency, sustained release, and novel routes of administration to be achieved. ever since the isolation of the first peptide from the frog skin in , several breakthroughs were made in the isolation, synthesis, and application of amps. still, many challenges are yet to overcome in the field of amps peptidomics, reflecting mainly their huge diversity and how little is known about their behavior in vivo and their structure-function relationships. in terms of costs, producing amps can be several hundred times more expensive than the production of conventional antibiotics. moreover, amps' design can be very complex and the quest for the perfect amp a never-ending search. in fact, considering the possibility of working with only amino acids, a small peptide with ten amino acids could have up to = . × different sequences, a larger peptide with amino acids could present with up to = . × different sequences, and one with amino acids could adopt any one of = . × different sequences. such possibilities do not even account for the presence of posttranslational modifications. then, each one of these peptides could shape into several different secondary structures and fall into a wide spectrum of activity, depending on the milieu conditions. furthermore, in a way to overcome the challenge of peptide stability, one has to consider the possibility to synthesize and to administer amps containing d-amino acids, thus decreasing their susceptibility to hydrolysis by human enzymes. to deal with such large amount of amino acid combinations one should mind using peptide libraries and computational models (such as by quantitative structure-activity relationship analysis) in order to narrow large collections to few surrogate amps (for a review please see blondelle and lohner's paper. another challenge to overcome is the lower amp's activity in vivo when compared to that observed in vitro. this has largely hampered the development of amps as therapeutic agents, as this decrease in activity stems from differences in ph and salt concentrations, the presence of proteases and corresponding inhibitors, and other interacting molecules hindering antimicrobial activity. accordingly, several amps (e.g., pexiganan, iseganan, neuprex have reached phase ii clinical trials only to fail after approval for marketing because they did not evidence superior activity over already marketed conventional antibiotics. in early , clinicaltrials.gov (a registry and result database of publicly and privately supported clinical studies of human participants conducted around the world) listed few clinical trials involving amps. for instance, ll is currently being studied for its efficacy in melanoma patients due to its ability to stimulate the immune system, while c g and chromogranin a derived peptides are under scrutiny for their therapeutic potential against dental diseases. simultaneously, therapeutic strategies are also being studied to augment amps' expression in amp-deficient patients. this deficiency is believed to be due to an increase in th lymphocytes derived cytokines, il- , il- , and il- , and a decrease in tnf-α, il- , il- , and interferonγ . one example of such strategy is pimecrolimus, a calcineurin inhibitor that binds with high affinity to macrophilin- , preventing the translocation of nuclear factor of activated t cells to the nucleus and the transcription and release of such inhibitory cytokines (for more up-to-date trials, readers can access clinicaltrials.gov). another possible caveat concerns the cytotoxicity to mammalian cells when in concentrations above naturally occurring values. when bacteria produce amps, they also develop means to avoid their targeting by such peptides, such as efflux pumps and the sequestering of enzymes. however, eukaryotic cells are equally prone to damage if the administration of exogenous amps is carried out in high enough concentrations. therefore, administering amps not constitutively produced or at concentrations above those normally found in the human host environment can be toxic and very detrimental to human cells. furthermore, rapid metabolism and low bioavailability are characteristic of amps because these peptides can suffer extensive proteolysis in vivo, thereby accounting for their inactivation and short half-life. , , similarly, for peptides to be available at sites distant from their origin, these would have to cross many cellular membranes. however, due to amps' polarity, membrane hydrophobicity may prevent them from doing so. moreover, in contrast to other highly polar mediators (e.g., ionic salts and a few metabolites), endogenous peptides tend not to have a transporter, further compromising their bioavailability in different compartments and the subsequent impossibility to be absorbed through the gastrointestinal tract. therefore, the number of possibilities rapidly escalades, and each amp-based therapy can be further optimized based on the modulation of chemical properties and engineering of delivery systems. even acknowledging all these factors, the human host is most certainly an unpredictable environment and the same peptide may behave differently, displaying unique individual pharmacokinetics and pharmacodynamics. on a final note, due to amps' interaction with and dependence on other immune mediators, variability will most likely result from immune status alterations as those frequently found in human diseases, including infectious ones. taken together, the above discussed results support amps as constituting a paramount group of defense molecules in human biological fluids, being present in most fluids, contributing for the sterility of some of these, but being most notably enriched in biological fluids bathing tissues with the largest load of microorganisms (fig. ) , such as saliva and urine. also, while some amps seem the display activity spectra against only a handful of microorganisms, others display broad-spectrum effects. thus, the expression and activity patterns of human amps enriched in biofluids may have suffered selective pressures resulting from exposers to microorganisms specifically present in each fluid (fig. ) . this work was financed by national funding from fct (fundação para a ciência e a tecnologia) through the project uid/bim/ / , uid/ic/ / . rv thanks fct for if/ / fellowship. the authors report no declarations of interest. with saliva proteomics research. since then, he extended the application of proteomics to the characterization of other biofluids' proteome and peptidome in order to identify the biological pathways modulated by diseases such as diabetes mellitus and bladder cancer. currently, he works to a large extent with bioinformatics tools for the identification of enzymes involved in the regulation of proteome remodeling. additional supporting information may be found in the online version of this article at the publisher's web site: apd : the antimicrobial peptide database as a tool for research and education anticancer efficacy of magainin and analogue peptides structure-activity analysis of thanatin, a -residue inducible insect defense peptide with sequence homology to frog skin antimicrobial peptides cationic host defence peptides: potential as antiviral therapeutics the n-and c-terminal fragments of ubiquitin are important for the antimicrobial activities cationic peptides: effectors in innate immunity and novel antimicrobials fetal human keratinocytes produce large amounts of antimicrobial peptides: 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antimicrobial peptide (hcap ) predicts increased infectious disease mortality in patients undergoing hemodialysis ll- peptide enhancement of signal transduction by toll-like receptor is regulated by ph identification of a peptide antagonist of ll- increased levels of antimicrobial peptides in tracheal aspirates of newborn infants during infection identification of novel semenogelin iderived antimicrobial peptide from liquefied human seminal plasma processing of seminal plasma hcap- to all- by gastricsin. a novel mechanism of generating antimicrobial peptides in vagina citrullination alters immunomodulatory function of ll- essential for prevention of endotoxin-induced sepsis nad-dependent adp-ribosylation of the human antimicrobial and immune-modulatory peptide ll- by adpribosyltransferase- human salivary histatin- exerts potent fungicidal activity against cryptococcus neoformans the effect of histatin , adsorbed on pmma and hydroxyapatite, on candida albicans colonization anticandidal activity of major human salivary histatins localization of the genes for histatins to human chromosome q and tissue distribution of the mrnas histatins, a novel family of histidine-rich proteins in human parotid secretion. isolation, characterization, primary structure, and fungistatic effects on candida albicans histatins, a family of salivary histidine-rich proteins, are encoded by at least two loci (his and his ) structural relationship between human salivary histatins zinc and copper bind to unique sites of histatin is salivary histatin a metallopeptide? amino terminal cu(ii)-and ni(ii)-binding (atcun) motif of proteins and peptides: metal binding, dna cleavage, and other properties † zn( +) ions selectively induce antimicrobial salivary peptide histatin- to fuse negatively charged vesicles. identification and characterization of a zinc-binding motif present in the functional domain nmr studies of the antimicrobial salivary peptides histatin and histatin in aqueous and nonaqueous solutions salivary histatin : dependence of sequence, chain length, and helical conformation for candidacidal activity antimicrobial peptides with therapeutic potential the antimicrobial peptide histatin- causes a spatially restricted disruption on the candida albicans surface, allowing rapid entry of the peptide into the cytoplasm effect of donor age on the concentrations of histatins in human parotid and submandibular/sublingual saliva functional and structural characterization of recombinant dermcidin- l, a human antimicrobial peptide dermcidin: a novel human antibiotic peptide secreted by sweat glands polysaccharide intercellular adhesin (pia) protects staphylococcus epidermidis against major components of the human innate immune system proteolysis-inducing factor is expressed in tumours of patients with gastrointestinal cancers and correlates with weight loss identification of a survival-promoting peptide in medium conditioned by oxidatively stressed cell lines of nervous system origin deficiency of dermcidin-derived antimicrobial peptides in sweat of patients with atopic dermatitis correlates with an impaired innate defense of human skin in vivo the role of dermcidin isoform : a two-faceted atherosclerotic risk factor for coronary artery disease and the effect of acetyl salicylic acid on it the appearance of dermcidin isoform , a novel platelet aggregating agent in the circulation in acute myocardial infarction that inhibits insulin synthesis and the restoration by acetyl salicylic acid of its effects hepcidin revisited, disulfide connectivity, dynamics, and structure mutant antimicrobial peptide hepcidin is associated with severe juvenile hemochromatosis the gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation sequestration and scavenging of iron in infection escherichia coli bacteremia, meningitis, and hemochromatosis bacteremic cellulitis caused by non- , non- vibrio cholerae: report of a case in a patient with hemochromatosis yersinia enterocolitica infection with multiple liver abscesses uncovering a primary hemochromatosis fatal listeria meningitis, endocarditis and pericarditis in a patient with haemochromatosis hemochromatosis, iron and septicemia caused by vibrio vulnificus absence of functional hfe protects mice from invasive salmonella enterica serovar typhimurium infection via induction of lipocalin- hereditary hemochromatosis results in decreased iron acquisition and growth by mycobacterium tuberculosis within human macrophages antibacterial and antifungal activities of vasostatin- , the n-terminal fragment of chromogranin a the primary structure of human chromogranin a and pancreastatin phosphorylation and o-glycosylation sites of human chromogranin a (cga - ) from urine of patients with carcinoid tumors intracellular and extracellular processing of chromogranin a. determination of cleavage sites the vasoinhibitory activity of bovine chromogranin a fragment (vasostatin) and its independence of extracellular calcium in isolated segments of human blood vessels chromogranin a induces a neurotoxic phenotype in brain microglial cells matsuoka h. plasma levels of adrenomedullin, a newly identified hypotensive peptide, in patients with hypertension and renal failure elevation of circulating and ventricular adrenomedullin in human congestive heart failure increased circulating adrenomedullin, a novel vasodilatory peptide, in sepsis an investigation into the antimicrobial effects of adrenomedullin on members of the skin, oral, respiratory tract and gut microflora enhancement of antimicrobial activity of neuropeptide y by n-terminal truncation inhibition of pulmonary metastases and enhancement of natural killer cell activity by methionine-enkephalin methionine-enkephalin secreted by human colorectal cancer cells suppresses t lymphocytes characterization of antibacterial cooh-terminal proenkephalin-a-derived peptides (peap) in infectious fluids: importance of enkelytin, the antibacterial peap - secreted by stimulated chromaffin cells two adrenal opioid polypeptides: proposed intermediates in the processing of proenkephalin quantitation of proenkephalin a messenger rna in bovine brain, pituitary and adrenal medulla: correlation between mrna and peptide levels differential expression of preproenkephalin and preprodynorphin mrnas in striatal neurons: high levels of preproenkephalin expression depend on cerebral cortical afferents preproenkephalin mrna in t-cells, macrophages, and mast cells regulated expression of proenkephalin a in normal lymphocytes polymorphism and absence of leu-enkephalin sequences in proenkephalin genes in xenopus laevis the presence of antibacterial and opioid peptides in human plasma during coronary artery bypass surgery antimicrobial effects of alpha-msh peptides effect of cyclic amp on rna and protein synthesis in candida albicans human lactoferrin and peptides derived from its n terminus are highly effective against infections with antibiotic-resistant bacteria many chemokines including ccl /mip- alpha display antimicrobial activity eotaxin- (ccl ) exerts innate host defense activities that are modulated by mast cell proteases antimicrobial peptides derived from pepsinogens in the stomach of the bullfrog, rana catesbeiana calcitermin, a novel antimicrobial peptide isolated from human airway secretions mast cell and monocyte recruitment by s a and its hinge domain s a provokes mast cell activation: a potential amplification pathway in asthma and innate immunity generation of multiple stable dermcidin-derived antimicrobial peptides in sweat of different body sites natural skin surface ph is on average below , which is beneficial for its resident flora vipericidins: a novel family of cathelicidin-related peptides from the venom gland of south american pit vipers paneth cell defensins: endogenous peptide components of intestinal host defense defensin-rich dense granules of human neutrophils the human antibacterial cathelicidin, hcap- , is synthesized in myelocytes and metamyelocytes and localized to specific granules in neutrophils peptides in body fluids and tissues as markers of disease peptidomics-based discovery of novel neuropeptides the glucagon-like peptides human cathelicidin, hcap- , is processed to the antimicrobial peptide ll- by extracellular cleavage with proteinase alpha-amylase is a human salivary protein with affinity to lipopolysaccharide of aggregatibacter actinomycetemcomitans extensive in vivo human milk peptidomics reveals specific proteolysis yielding protective antimicrobial peptides proteomic analysis of normal human urinary proteins isolated by acetone precipitation or ultracentrifugation antioxidant peptidomics reveals novel skin antioxidant system targeted protein degradation by salmonella under phagosome-mimicking culture conditions investigated using comparative peptidomics optimisation of protein precipitation based upon effectiveness of protein removal and ionisation effect in liquid chromatography-tandem mass spectrometry peptidomics technologies for human body fluids human cerebrospinal fluid peptidomics selective enrichment of tryptophan-containing peptides from protein digests employing a reversible derivatization with malondialdehyde and solid-phase capture on hydrazide beads selective solid-phase isolation of methioninecontaining peptides and subsequent matrix-assisted laser desorption/ionisation mass spectrometric detection of methionine-and of methionine-sulfoxide-containing peptides porous polymer monolithic column with surface-bound gold nanoparticles for the capture and separation of cysteine-containing peptides mass spectrometric identification of n-linked glycopeptides using lectin-mediated affinity capture and glycosylation site-specific stable isotope tagging highly selective enrichment of phosphorylated peptides using titanium dioxide identification of protein modifications using ms/ms de novo sequencing and the opensea alignment algorithm immunoassays for the incretin hormones gip and glp- mass-spectrometric identification of a novel angiotensin peptide in human plasma salivary peptidomic as a tool to disclose new potential antimicrobial peptides capillary electrophoresis-electrospray-mass spectrometry in peptide analysis and peptidomics the endogenous peptides of normal human serum extracted from the acetonitrile-insoluble precipitate using modified aqueous buffer with analysis by lc-esi-paul ion trap and qq-tof endogenous peptides from biophysical and biochemical fractionation of serum analyzed by matrix-assisted laser desorption/ionization and electrospray ionization hybrid quadrupole time-offlight matrixassisted laser desorption/ionization quadrupole time-of-flight mass spectrometry: an elegant tool for peptidomics the differential diagnostic model for serous peptidomics in hbv carriers established by maldi-tof-ms analysis peptidomics-based discovery of an antimicrobial peptide derived from insulin-like growth factor-binding protein naturally occurring human urinary peptides for use in diagnosis of chronic kidney disease biochemical aspects of the coagulation and liquefaction of human semen liquefaction of semen generates and later degrades a conserved semenogelin peptide that enhances hiv infection x-ray crystallography of peptides: the contributions of the italian laboratories chapter nmr of antimicrobial peptides pep-fold: an updated de novo structure prediction server for both linear and disulfide bonded cyclic peptides prediction of peptide structure: how far are we? comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations large-scale database searching using tandem mass spectra: looking up the answer in the back of the book molecular grafting onto a stable framework yields novel cyclic peptides for the treatment of multiple sclerosis -dihydroxyvitamin d induces ll- and hbd- production in keratinocytes from diabetic foot ulcers promoting wound healing: an in vitro model characterization of an antithrombotic peptide from kappa-casein in newborn plasma after milk ingestion uropathogenic escherichia coli modulates immune responses and its curli fimbriae interact with the antimicrobial peptide ll- antimicrobial and antibiofilm activity of ll- and its truncated variants against burkholderia pseudomallei anti-staphylococcal biofilm effects of human cathelicidin peptides human host defense peptide ll- prevents bacterial biofilm formation human β-defensin inhibits antibiotic-resistant staphylococcus biofilm formation sensitivity of candida albicans biofilm cells grown on denture acrylic to antifungal proteins and chlorhexidine biocompatibility of antimicrobial melimine lenses: rabbit and human studies development of a catheter functionalized by a polydopamine peptide coating with antimicrobial and antibiofilm properties impedimetric detection of pathogenic gram-positive bacteria using an antimicrobial peptide from class iia bacteriocins design and evaluation of peptide nucleic acid probes for specific identification of candida albicans electrical impedance detection of bacterial pathogens using immobilized antimicrobial peptides novel endosomolytic peptides for enhancing gene delivery in nanoparticles silicon microfluidic flow focusing devices for the production of size-controlled plga based drug loaded microparticles a novel peptide designated pyla and its precursor as predicted from cloned mrna of xenopus laevis skin therapeutic peptides under the spotlight antibacterial peptides for therapeutic use: obstacles and realistic outlook optimization and high-throughput screening of antimicrobial peptides pexiganan acetate a multinational, randomized phase iii trial of iseganan hcl oral solution for reducing the severity of oral mucositis in patients receiving radiotherapy for head-and-neck malignancy prolyl peptidases: a serine protease subfamily with high potential for drug discovery bioavailability and transport of peptides and peptide drugs into the brain key: cord- -k fze authors: scherbinina, sofya i.; toukach, philip v. title: three-dimensional structures of carbohydrates and where to find them date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: k fze analysis and systematization of accumulated data on carbohydrate structural diversity is a subject of great interest for structural glycobiology. despite being a challenging task, development of computational methods for efficient treatment and management of spatial ( d) structural features of carbohydrates breaks new ground in modern glycoscience. this review is dedicated to approaches of chemo- and glyco-informatics towards d structural data generation, deposition and processing in regard to carbohydrates and their derivatives. databases, molecular modeling and experimental data validation services, and structure visualization facilities developed for last five years are reviewed. knowledge of carbohydrate spatial ( d) structure is crucial for investigation of glycoconjugate biological activity [ , ] , vaccine development [ , ] , estimation of ligand-receptor interaction energy [ ] [ ] [ ] studies of conformational mobility of macromolecules [ ] , drug design [ ] , studies of cell wall construction aspects [ ] , glycosylation processes [ ] , and many other aspects of carbohydrate chemistry and biology. therefore, providing information support for carbohydrate d structure is vital for the development of modern glycomics and glycoproteomics. as result of growing interest to glycoprofiling, glycan microarrays, carbohydrate active enzymes (cazy) and glycan-binding proteins (gbp) which are involved in biological processes, several major international projects (e.g., glyspace [ ] , glycosmos [ ] , glycomics@expasy [ ] , glygen [ ] , jcggdb [ ] , glytoucan [ ] , mirage [ ] , cfg [ ] , rings [ ] , glic (https://glic.glycoinfo.org/), sysglyco (https://sysglyco.org/)) were launched to integrate variety of data produced by glycobiological research. the main goal of existing glycoinformatics projects is to provide versatile resources with user-friendly access helpful for disease diagnostics [ , ] , glycobioinformatics studies [ ] , glycosylation site prediction [ ] , cazy activity prognosis [ , ] and other applications. appending of structural repositories with d structural data opens the way for computational glycobiology and modeling of carbohydrate structures at atomic resolution. design of novel workflows and techniques to connect carbohydrate spatial structure modes and experimental data with verification, processing, analysis and deposition of associated data has gained increased popularity in glycoscience community [ ] . a carbohydrate structure database (csdb, [ ] ) module for carbohydrate d structure modeling is a demonstrative example of d structural data integration facilities (as a database) combined with dedicated interface (as a glycoinformatics project). further details on csdb d facilities are discussed below. herein we focus on the important aspects of carbohydrate d structure availability to researchers: structural repositories; glycoinformatics tools and workflows to assist structure building, modeling and erroneous molecular geometry data detection and remediation; carbohydrate d structure presentation and visualization methods. structural databases make significant contribution to bringing information technologies to glycoscience [ ] . with no focus on spatial structure, glycan databases and online tools have been recently reviewed [ ] [ ] [ ] . depositing huge number of carbohydrates with detailed data for each entry, databases are valuable sources of structural information, biological assignments, references and external links. structural data are often accompanied by original and sometimes assigned experimental observables: nmr spectra, hplc and ms profiles, etc. the services built on top of the databases can include d structure simulation, validation, and storage. a viewpoint of the authors at the ideal integration of data resources and services in glycoinformatics is summarized in figure . a subject of this review is databases providing theoretical or empirical d structures of carbohydrates and related data-mining tools. herein we focus on the important aspects of carbohydrate d structure availability to researchers: structural repositories; glycoinformatics tools and workflows to assist structure building, modeling and erroneous molecular geometry data detection and remediation; carbohydrate d structure presentation and visualization methods. structural databases make significant contribution to bringing information technologies to glycoscience [ ] . with no focus on spatial structure, glycan databases and online tools have been recently reviewed [ ] [ ] [ ] . depositing huge number of carbohydrates with detailed data for each entry, databases are valuable sources of structural information, biological assignments, references and external links. structural data are often accompanied by original and sometimes assigned experimental observables: nmr spectra, hplc and ms profiles, etc. the services built on top of the databases can include d structure simulation, validation, and storage. a viewpoint of the authors at the ideal integration of data resources and services in glycoinformatics is summarized in figure . a subject of this review is databases providing theoretical or empirical d structures of carbohydrates and related data-mining tools. networking between glycoinformatics projects and related services that promotes achievement of data integration in glycomics. reproduced with permission from [ ] , © wiley-vch verlag gmbh & co. kgaa, weinheim. the majority of existing repositories for carbohydrate d structures offer open-access data via web interface. deposited datasets can be represented by glycoproteins, protein-carbohydrate complexes, poly-and oligosaccharides with d structure experimentally resolved or specified by means of nmr, x-ray crystallography, cryoem, small angle x-ray scattering, etc. [ ] . several databases such as glycam-web, ek d, dsdscar, glycomapsdb contain data from molecular dynamics simulations. we have also mentioned databases featuring information on protein structures involving carbohydrate moiety in terms of glycosylation (as post-translational modification, dbptm), carbohydrate active enzymes (cazy) and homology modeling (swiss-model). table displays currently active structural databases maintaining three-dimensional data on carbohydrates. for table , we have selected carbohydrate and related databases using the following criteria: • database can be freely accessed through web user interface; • database must contain experimentally confirmed and/or predicted d structures (preprocessed and/or generated on-the-fly from a primary structure input) of glycans, glycoproteins, or protein-carbohydrate complexes; • stored d structures must be deposited as atomic coordinates in pdb, mol, or other format, and the structures must contain a saccharide moiety; • databases with records linked to other large d data collections (e.g., rcsb pdb, pdbe, pdbj, pdbsum, uniprotkb etc.) are included in table (as long as database entries contain carbohydrate moiety, e.g., as a part of a lectin or an antibody); • databases with derived carbohydrate d structural data (conformational maps, conformer energy minima, etc.) are included in table even if they provide no atomic coordinates (e.g., glycomapsdb and gfdb). despite no fit to the criteria above, assistance of large structure repositories offering only glycan primary structures (e.g., glytoucan [ ] (https://glytoucan.org/), unicarbkb [ ] (http://www.unicarbkb.org/)) can be useful for cross-referencing of existing carbohydrate resources and serve as supplementation to d modeling pipelines. some out-of-date projects, such as complex carbohydrate structural database (ccsd) [ , ] , eurocarbdb [ , ] , glycomedb [ ] [ ] [ ] , glycoconjugate data bank [ ] , glycosuite [ , ] are noteworthy as they had shaped the modern vision of structural glycoinformatics. networking between glycoinformatics projects and related services that promotes achievement of data integration in glycomics. reproduced with permission from [ ] , © wiley-vch verlag gmbh & co. kgaa, weinheim. the majority of existing repositories for carbohydrate d structures offer open-access data via web interface. deposited datasets can be represented by glycoproteins, protein-carbohydrate complexes, poly-and oligosaccharides with d structure experimentally resolved or specified by means of nmr, x-ray crystallography, cryoem, small angle x-ray scattering, etc. [ ] . several databases such as glycam-web, ek d, dsdscar, glycomapsdb contain data from molecular dynamics simulations. we have also mentioned databases featuring information on protein structures involving carbohydrate moiety in terms of glycosylation (as post-translational modification, dbptm), carbohydrate active enzymes (cazy) and homology modeling (swiss-model). table displays currently active structural databases maintaining three-dimensional data on carbohydrates. for table , we have selected carbohydrate and related databases using the following criteria: • database can be freely accessed through web user interface; • database must contain experimentally confirmed and/or predicted d structures (preprocessed and/or generated on-the-fly from a primary structure input) of glycans, glycoproteins, or protein-carbohydrate complexes; • stored d structures must be deposited as atomic coordinates in pdb, mol, or other format, and the structures must contain a saccharide moiety; • databases with records linked to other large d data collections (e.g., rcsb pdb, pdbe, pdbj, pdbsum, uniprotkb etc.) are included in table (as long as database entries contain carbohydrate moiety, e.g., as a part of a lectin or an antibody); • databases with derived carbohydrate d structural data (conformational maps, conformer energy minima, etc.) are included in table even if they provide no atomic coordinates (e.g., glycomapsdb and gfdb). despite no fit to the criteria above, assistance of large structure repositories offering only glycan primary structures (e.g., glytoucan [ ] (https://glytoucan.org/), unicarbkb [ ] (http://www. unicarbkb.org/)) can be useful for cross-referencing of existing carbohydrate resources and serve as supplementation to d modeling pipelines. some out-of-date projects, such as complex carbohydrate structural database (ccsd) [ , ] , eurocarbdb [ , ] , glycomedb [ ] [ ] [ ] , glycoconjugate data bank [ ] , glycosuite [ , ] are noteworthy as they had shaped the modern vision of structural glycoinformatics. • mammalian glycans • pre-built libraries of predicted d structures of common bioglycans • d structure models * • d-atomic coordinates generation (http://glycam.org/pre-builtlibraries.jsp) a where unknown, the year of the first publication is given. b database is marked as curated if manual verification of data was reported in the original publication or at the database web site. c published coverage data can be outdated; database interface provides no statistics on current coverage. * database provides no search facilities for indicated carbohydrate d structural data. methods to probe a d structure of carbohydrate-containing biomolecules has been developed for decades. nmr techniques (interatomic distances derived from noe, and torsion angles derived from coupling constants), x-ray crystallography, and electron cryo-microscopy (the two latter being atomic models built on the basis of electron density map) are among most demanded methods for d strucural elucidation. these methods have been reviewed [ ] [ ] [ ] [ ] and are beyond the scope of this review focused in information technologies. for use of instrumental methods for the validation of a simulated structure, please refer to section "experimental data validation". structural investigation of large biological systems involving protein-glycan interactions requires leveraging more resources and employing more complex experimental techniques compared to solely oligo-and polysaccharides studies. advances in nmr methods hold great potential for direct spatial structure determination of carbohydrate-protein complexes in solution based on intermolecular noes which affords estimation of atomic contacts between a protein and a carbohydrate ligand [ , ] . further extraction of noe-derived distance restraints for a saccharide molecule results in generation of representative conformational ensembles [ ] [ ] [ ] . support of experimental data with computer simulations can significantly improve quality of d structures. quantum mechanics [ , [ ] [ ] [ ] [ ] [ ] and molecular dynamics modeling [ ] [ ] [ ] [ ] [ ] are commonly applied to conformation search and nmr signal prediction. to date, the following theoretical models and methods are applied for in silico design of carbohydrate three-dimensional structure [ ] [ ] [ ] [ ] [ ] : based on scopus [ ] article count we estimated the application rate for quantum mechanics ( publications) and molecular mechanics ( publications) methods applied for carbohydrate structure modeling for the recent five years ( - ). search queries included abundant carbohydrate terms, typical glycan moieties, and common modeling approaches (query details are given in supplementary table s ). in spite of growing interest to qm approaches in carbohydrate structure simulation, the major contribution to the statistics for such resource-intensive calculations is application of qm to relatively simple model compounds. for complex bioglycans in solution predominance of mm methods is more pronounced [ , ] . molecular dynamics methods have achieved broad scope of application in terms of reasonable computer resource consumption. they fulfill advantageous compromise between calculation accuracy and performance, when applied to glycan molecules and their structural complexity (variety of known monomeric elements, presence of ionogenic groups), high bridge flexibility and stereo-electronic effects [ , , , ] . in molecular mechanics simulations, newtonian mechanics principles are applied to calculate potential energy of a system using parameter set specific for a class of compounds under study (force field). particular features of carbohydrate moiety, e.g., ring puckering, rotational barriers, hydrogen bonds, must be taken into account to perform precise analysis of molecular behavior in vacuo or in solution [ ] . molecular dynamics simulations consider newtonian motion equations to observe evolution of a system during a certain timespan. conformation ensemble generation occurs via calculation of molecular trajectory at given temperature. accuracy of calculation depends on the employed force field and sufficient conformational sampling. md simulations are commonly used for interpretation and analysis of the nmr and x-ray observables in the context of carbohydrate d structure [ ] . enhanced molecular dynamics sampling technologies, such as replica-exchange md (remd) [ , ] , hamiltonian replica-exchange md (hrex) [ ] [ ] [ ] , multidimensional swarm-enhanced sampling md (msesmd) [ , ] , gaussian accelerated md (gamd) [ , ] have been reported. density maps or energy maps built for a set of the glycosidic torsion angles (ϕ, ψ, ω) are a typical way to report conformational preferences of a glycan provided by population analysis of its md trajectory. as a representative example, conformational characteristics of highly flexible branched oligosaccharide glc man glcnac (gm ) were investigated by explicit-water remd study and validated using paramagnetism-assisted nmr spectroscopy [ ] (figure a,b) . due to the structural complexity of gm , adequate exploration of conformational space requires long-timescale simulations. regular md simulations of similar manno-oligosaccharides were reported to fail reproduction of experimental data [ ] . replica-exchange approach implies running periodically swapped parallel replicas of the system at different temperatures. ensemble of gm conformers sampled by this method was consistent with the nmr observables. populated areas of density maps built for glycosidic linkages of glc man branch of gm ( figure c ) were close to crystallographic conformations of a linear glc man tetrasaccharide (a gm determinant recognized by lectins) from pdb. molecular dynamics simulations consider newtonian motion equations to observe evolution of a system during a certain timespan. conformation ensemble generation occurs via calculation of molecular trajectory at given temperature. accuracy of calculation depends on the employed force field and sufficient conformational sampling. md simulations are commonly used for interpretation and analysis of the nmr and x-ray observables in the context of carbohydrate d structure [ ] . enhanced molecular dynamics sampling technologies, such as replica-exchange md (remd) [ , ] , hamiltonian replica-exchange md (hrex) [ ] [ ] [ ] , multidimensional swarm-enhanced sampling md (msesmd) [ , ] , gaussian accelerated md (gamd) [ , ] have been reported. density maps or energy maps built for a set of the glycosidic torsion angles (φ, ψ, ω) are a typical way to report conformational preferences of a glycan provided by population analysis of its md trajectory. as a representative example, conformational characteristics of highly flexible branched oligosaccharide glc man glcnac (gm ) were investigated by explicit-water remd study and validated using paramagnetism-assisted nmr spectroscopy [ ] (figure a,b) . due to the structural complexity of gm , adequate exploration of conformational space requires long-timescale simulations. regular md simulations of similar manno-oligosaccharides were reported to fail reproduction of experimental data [ ] . replica-exchange approach implies running periodically swapped parallel replicas of the system at different temperatures. ensemble of gm conformers sampled by this method was consistent with the nmr observables. populated areas of density maps built for glycosidic linkages of glc man branch of gm ( figure c ) were close to crystallographic conformations of a linear glc man tetrasaccharide (a gm determinant recognized by lectins) from pdb. force field (or potential energy function) is represented by atomistic parameter set obtained for a considered compound class. potential energy value can be calculated as a sum of interaction potentials for bonded (covalent bond stretching, angle bending, proper torsions) and non-bonded (electrostatic and van der waals interactions) terms, and can include other terms (e.g., improper torsions, solvation, hydrogen bonds [ ] , nonconventional hydrogen bonds [ ] , for protein-carbohydrate complexes-ch-π stacking interactions [ ] [ ] [ ] [ ] , chi carbohydrate intrinsic (chi) energy contribution [ , ] ). several force fields developed for general representation of wide range of organic compounds (e.g., allinger's mm , mm , mm ) can be applied to carbohydrate d modeling [ , , ] . of them, despite being a universal force field, mm [ , ] still exhibits good performance on glycans [ ] [ ] [ ] (reviews), [ , ] (exemplary articles). however, a number of force fields specially tuned for carbohydrates have been developed (figure ). in supplementary table s , we provided citation metrics of articles reporting carbohydrate-dedicated and selected general force fields that could be applied to carbohydrate structure modeling. unfortunately, usage of general force fields could not be adequately estimated via number of citations. automated full-text analysis and retrieval of data, needed to confirm employment of force fields for carbohydrate molecules, is beyond the scope of this review. nevertheless, statistical data obtained for general force fields supported in popular md software packages (e.g., amber, charmm, gromacs, tinker) shows obsolescence of modern force fields above allinger's ones, and mm in particular (see more detailed data, references to original publications and absolute values in supplementary table s ). force field (or potential energy function) is represented by atomistic parameter set obtained for a considered compound class. potential energy value can be calculated as a sum of interaction potentials for bonded (covalent bond stretching, angle bending, proper torsions) and non-bonded (electrostatic and van der waals interactions) terms, and can include other terms (e.g., improper torsions, solvation, hydrogen bonds [ ] , nonconventional hydrogen bonds [ ] , for protein-carbohydrate complexes-ch-π stacking interactions [ ] [ ] [ ] [ ] , chi carbohydrate intrinsic (chi) energy contribution [ , ] ). several force fields developed for general representation of wide range of organic compounds (e.g., allinger's mm , mm , mm ) can be applied to carbohydrate d modeling [ , , ] . of them, despite being a universal force field, mm [ , ] still exhibits good performance on glycans [ ] [ ] [ ] (reviews), [ , ] (exemplary articles). however, a number of force fields specially tuned for carbohydrates have been developed (figure ). in supplementary table s , we provided citation metrics of articles reporting carbohydrate-dedicated and selected general force fields that could be applied to carbohydrate structure modeling. unfortunately, usage of general force fields could not be adequately estimated via number of citations. automated full-text analysis and retrieval of data, needed to confirm employment of force fields for carbohydrate molecules, is beyond the scope of this review. nevertheless, statistical data obtained for general force fields supported in popular md software packages (e.g., amber, charmm, gromacs, tinker) shows obsolescence of modern force fields above allinger's ones, and mm in particular (see more detailed data, references to original publications and absolute values in supplementary table s ). detailed comparisons of all-chemical and dedicated force fields in a context of glycan modeling have been published [ , , , ] . charmm , glycam , gromos and opls-aa-sei were reported as commonly used force fields for handling carbohydrate or glycoconjugate molecules. more details are provided in figure . charmm force field with modern carbohydrate parameter table (c [ ] ) was derived from charmm all-atom biomolecular force field [ , ] . currently, charmm parameterization features include monosaccharides in furanose [ ] and pyranose [ ] forms, glycosidic linkages between monosaccharides [ , ] , complex carbohydrates and glycoproteins detailed comparisons of all-chemical and dedicated force fields in a context of glycan modeling have been published [ , , , ] . charmm , glycam , gromos and opls-aa-sei were reported as commonly used force fields for handling carbohydrate or glycoconjugate molecules. more details are provided in figure . charmm force field with modern carbohydrate parameter table (c [ ] ) was derived from charmm all-atom biomolecular force field [ , ] . currently, charmm parameterization features include monosaccharides in furanose [ ] and pyranose [ ] forms, glycosidic linkages between monosaccharides [ , ] , complex carbohydrates and glycoproteins [ ] , monosaccharide-linked sulfate and phosphate groups [ ] , acyclic carbohydrates and alditols [ ] , as well as carbohydrate simulations in aqueous solution [ ] . [ ] , monosaccharide-linked sulfate and phosphate groups [ ] , acyclic carbohydrates and alditols [ ] , as well as carbohydrate simulations in aqueous solution [ ] . glycam force field is compatible with carbohydrates of all ring sizes and conformations for both mono-and oligosaccharides built of residues common for mammalian glycans, such as widespread aldoses, n-acetylated amino-sugars, sialic, glucuronic and galacturonic acids [ ] . parameter set was extended to non-carbohydrate moieties such as lipids [ ] , glycolipids [ , ] , lipopolysaccharides [ ] , proteins and nucleic acids. parameterization of glycam for glycosaminoglycans was reported [ ] . gromos represents a broad family of carbohydrate force fields. having been a classic one since , gromos a [ ] parameter set is used for explicit-solvent simulation of hexopyranose-based saccharides. in the recent decade, several parameters of a were optimized in gromos acarbo [ ] including lipopolysaccharides [ ] . gromos a glyc was improved for explicit-solvent simulations [ ] and extended for glycoproteins [ ] . gromos acarbo_r [ ] was designed to improve description of ring conformational equilibria in hexopyranose-based saccharide chains as compared to the previous acarbo version. another modification of acarbo named acarbo_cht [ ] was developed for chitosan and its derivatives. recently, extensions of gromos acarbo/carbo_r parameter set were adapted towards charged, protonated and esterified urinates [ ] and furanose-based carbohydrates [ ] . gromos a was reported to have good performance on glycan structure simulation in glycoproteins [ , ] . opls-aa scaling of electrostatic interactions (sei) force field [ ] consists of improved parameters for conformational changes associated with φ-ψ dihedrals combined with enhanced accuracy of qm relative energy calculation in carbohydrate molecules refined for opls-aa biomolecular force field [ , ] . additionally opls force field was improved for explicit-water simulations [ ] . rapidly developing charmm drude polarizable force field for carbohydrates based on classical drude oscillator has to be mentioned. parameter sets obtained for hexapyranoses [ ] and their aqueous solutions [ ] , aldopentafuranoses and methyl-aldopentafuranosides [ ] , glycam force field is compatible with carbohydrates of all ring sizes and conformations for both mono-and oligosaccharides built of residues common for mammalian glycans, such as widespread aldoses, n-acetylated amino-sugars, sialic, glucuronic and galacturonic acids [ ] . parameter set was extended to non-carbohydrate moieties such as lipids [ ] , glycolipids [ , ] , lipopolysaccharides [ ] , proteins and nucleic acids. parameterization of glycam for glycosaminoglycans was reported [ ] . gromos represents a broad family of carbohydrate force fields. having been a classic one since , gromos a [ ] parameter set is used for explicit-solvent simulation of hexopyranose-based saccharides. in the recent decade, several parameters of a were optimized in gromos a carbo [ ] including lipopolysaccharides [ ] . gromos a glyc was improved for explicit-solvent simulations [ ] and extended for glycoproteins [ ] . gromos a carbo_r [ ] was designed to improve description of ring conformational equilibria in hexopyranose-based saccharide chains as compared to the previous a carbo version. another modification of a carbo named a carbo_cht [ ] was developed for chitosan and its derivatives. recently, extensions of gromos a carbo / carbo_r parameter set were adapted towards charged, protonated and esterified urinates [ ] and furanose-based carbohydrates [ ] . gromos a was reported to have good performance on glycan structure simulation in glycoproteins [ , ] . opls-aa scaling of electrostatic interactions (sei) force field [ ] consists of improved parameters for conformational changes associated with ϕ-ψ dihedrals combined with enhanced accuracy of qm relative energy calculation in carbohydrate molecules refined for opls-aa biomolecular force field [ , ] . additionally opls force field was improved for explicit-water simulations [ ] . rapidly developing charmm drude polarizable force field for carbohydrates based on classical drude oscillator has to be mentioned. parameter sets obtained for hexapyranoses [ ] and their aqueous solutions [ ] , aldopentafuranoses and methyl-aldopentafuranosides [ ] , carboxylate and n-acetylamine saccharide derivatives [ ] , alditols [ ] and glycosidic linkages [ ] demonstrated significant improvement of qm data reproduction compared to charmm additive force field. martini coarse-grained (cg) force field [ ] can be used alternatively to all-atom (aa) level simulations with advantage of modeling large carbohydrate systems (solutions of oligo-, polysaccharides, glycolipids [ ] [ ] [ ] ) on a long time scale at reasonable computational cost. blocked ring puckering (only c conformation is allowed) and restrictions on the anomeric effect and glycosidic bond flexibility cumulatively provide reduction of available degrees of freedom. another cg model pitomba [ ] for carbohydrate simulations was developed based on gromos a glyc force field. docking methods for carbohydrate ligands utilize molecular modeling approaches for protein-carbohydrate complexes for initial geometry generation, conformational sampling, grafting, active site mapping and binding affinity estimation [ , , [ ] [ ] [ ] . accurate reproduction of experimental data requires application of particular scoring function parameterization (empirical, force fields or knowledge-based [ ] ) and docking protocols, which depend on the interaction types present in a system (ch-π interactions, chi-energy, hydrogen bonding, solvent model, influence of solvent molecules inclusion effects, charged moiety etc.) [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . extension of several docking software packages to handle carbohydrate molecules was reported to improve modeling of biologically relevant systems such as lectin-glycan [ , ] , gag-protein [ ] [ ] [ ] , or antibody-carbohydrate [ ] . currently available web-based tools along with standalone software packages were developed to facilitate work with carbohydrate d structure. versatile online services for in silico molecular modeling allow users to start from a user-friendly structure input, and to automatize further procedures (see table for references). glycam-web provides tools for glycan structure prediction, glycosylated protein d model generation, grafting and docking. charmm-gui modeler offers options for d structure generation and modeling of glycans including n-/o-glycoproteins and glycolipids [ , ] . biological membranes can be simulated with the assistance of charmm-gui membrane builder (by combining features of lps and glycolipid charmm-gui modelers) and gnomm (a tool for building lipopolysaccharide-rich membranes). noteworthy standalone programming frameworks for structure modeling are glycosylated (modeling of glycans, glycoproteins and glycosylation) and rosetta carbohydrate (loop modeling [ ] , glycan-to-protein docking, and glycosylation modeling). to build diverse saccharide d models online, one can use such tools as restless and sweet-ii. doglycans standalone framework can be used for preparation of the atomistic models of glycopolymers, glycolipids and glycoproteins. complex polysaccharide d models can be generated via polys and carbbuilder. another special class of polysaccharide builders is dedicated to glycosaminoglycans (gags) which can be accessed using polys gag-builder and glycam-web gag-builder. recently, another approach for building gag molecules was reported [ ] (exemplary data pipeline only). unfortunately, application scope of the majority of the existing structure building and modeling services is limited to rigidly defined set of supported sugar residues, and lacks non-carbohydrate moiety support. tools for locating and identification of a carbohydrate moiety (e.g., pdb linucs, glyfinder, glycan reader) are useful for the atomic coordinate analysis and extraction of glycoproteins and protein-carbohydrate complexes deposited in protein data bank (pdb). automated molecular geometry processing facilities can be accessed via glycoinformatics tools designed for conformational data analysis (cat, bfmp), nuclear overhauser effect (noe) calculation (md noe, distance mapping) and d structural data analysis related to glycan moieties from pdb (glytorsion, glyvicinity, gs-align). in table , we summarized freely available tools for generation and processing carbohydrate d structural data and divided them into eight categories of application. a web-service implies an automated pipeline for running a specific software (e.g., molecular modeling, structure building, carbohydrate coordinate extraction, format conversion). it results in d structural data output starting from primary structure input or atomic coordinate file upload. web-tool is employed for d structural data processing and analysis without d structural data output; it is a simpler application designed primarily for statistics and visualization. other types are self-explanatory. vast variety of methods provide information about d structure of individual glycans and glycan moieties of glycoproteins and protein-carbohydrate complexes ( figure ) [ , ] . the following approaches are most utilized for d structural data validation [ ] [ ] [ ] : • ccombination of carbohydrate simulated geometry data with x-ray crystallographic data analysis [ , ] ; • analysis of inter-glycosidic nmr spin couplings, which depend on glycosidic bond torsions [ , , ] vast variety of methods provide information about d structure of individual glycans and glycan moieties of glycoproteins and protein-carbohydrate complexes ( figure ) [ , ] . the following approaches are most utilized for d structural data validation [ ] [ ] [ ] : • ccombination of carbohydrate simulated geometry data with x-ray crystallographic data analysis [ , ] ; • analysis of inter-glycosidic nmr spin couplings, which depend on glycosidic bond torsions [ , , ] ; • deriving nuclear overhauser effects (noes) from relative populations of the interatomic distances, with subsequent comparison to the experimental noes in solution [ , , ] ; • purely informatic detection of errors, such as incompatible atomic coordinates originating from incorrect processing or simulation [ ] [ ] [ ] [ ] ; • simulation by other computational methods at higher levels of theory [ , , , ] . unfortunately, most of the data obtained on the basis of crystallographic experiments can dramatically differ from glycan conformations in solution or have poor resolution which needs further adjustment [ , ] . moreover, not all of the objects of interest can be obtained as a single crystal. electron cryo-microscopy gains popularity for carbohydrate d structural research [ ] , however, this method requires additional refinement procedures due to resolution restrictions of the obtained density unfortunately, most of the data obtained on the basis of crystallographic experiments can dramatically differ from glycan conformations in solution or have poor resolution which needs further adjustment [ , ] . moreover, not all of the objects of interest can be obtained as a single crystal. electron cryo-microscopy gains popularity for carbohydrate d structural research [ ] , however, this method requires additional refinement procedures due to resolution restrictions of the obtained density maps [ ] [ ] [ ] . recently, cryo-em data were used for the refinement of sars-cov- spike glycoprotein stucture using privateer (see table for references) software [ , ] . van beusekom et al., illustrated [ ] quality improvement of the pdb glycan structure model with incorrect ( - )-linked fucose annotation, poor fit to the electron density, and missing ( - )-linked fucose (figure a ) with the help of pdb-redo ( figure b ) and carp (figure d ) tools (see table for references). structure model obtained by pdb-redo treatment was further manually inspected ( figure c ): corrections were made for acetylamino group geometry, distorted ( - )-linked fucose ring conformation, and ( - )-linked fucose residue was added. despite successful automated resolution of residue annotation problem and poor electron density refinement, complete revision could not be achieved without manual intervention. maps [ ] [ ] [ ] . recently, cryo-em data were used for the refinement of sars-cov- spike glycoprotein stucture using privateer (see table for references) software [ , ] . van beusekom et al., illustrated [ ] quality improvement of the pdb glycan structure model with incorrect ( - )-linked fucose annotation, poor fit to the electron density, and missing ( - )-linked fucose (figure a ) with the help of pdb-redo ( figure b ) and carp (figure d ) tools (see table for references). structure model obtained by pdb-redo treatment was further manually inspected (figure c ): corrections were made for acetylamino group geometry, distorted ( - )-linked fucose ring conformation, and ( - )-linked fucose residue was added. despite successful automated resolution of residue annotation problem and poor electron density refinement, complete revision could not be achieved without manual intervention. nmr techniques are a powerful approach to investigate conformational and dynamic behavior of carbohydrate moieties in biomolecules [ ] [ ] [ ] [ ] . however, the nature of noe enhancement factor has been hampering obtaining the sufficient number of distance restrains [ ] . in the case of saccharides with their multiple rotatable bonds, the stable d structure was difficult to define, making molecular modeling essential for this class of compounds. adjustment of experimental conditions helped to overcome the mentioned limitation and to reproduce crystal structures of oligosaccharides by modeling with noe-derived distance restraints [ , ] . nmr techniques are a powerful approach to investigate conformational and dynamic behavior of carbohydrate moieties in biomolecules [ ] [ ] [ ] [ ] . however, the nature of noe enhancement factor has been hampering obtaining the sufficient number of distance restrains [ ] . in the case of saccharides with their multiple rotatable bonds, the stable d structure was difficult to define, making molecular modeling essential for this class of compounds. adjustment of experimental conditions helped to overcome the mentioned limitation and to reproduce crystal structures of oligosaccharides by modeling with noe-derived distance restraints [ , ] . since there is no direct way to derive detailed three-dimensional representation from the observed noe intensities, additional molecular modeling protocols are required to establish comprehensive view of conformational space at the atomic level [ ] [ ] [ ] . frank et al., demonstrated conformation filtering based on the observed noe obtained by molecular dynamics in explicit solvent [ ] . as a representative example, figure depicts h- h spatial contacts and conformation selection criteria illustrated by moraxella catarrhalis lgt ∆ bacterium heptasaccharide, which adopts an unusual conformation. since there is no direct way to derive detailed three-dimensional representation from the observed noe intensities, additional molecular modeling protocols are required to establish comprehensive view of conformational space at the atomic level [ ] [ ] [ ] . frank et al., demonstrated conformation filtering based on the observed noe obtained by molecular dynamics in explicit solvent [ ] . as a representative example, figure depicts h- h spatial contacts and conformation selection criteria illustrated by moraxella catarrhalis lgt Δ bacterium heptasaccharide, which adopts an unusual conformation. protein data bank (pdb) [ ] and cambridge structural database (csd) [ ] are historically considered the main repositories of experimentally determined carbohydrate three-dimensional structures. csd is reported to deposit over crystal structures of oligosaccharides [ ] . unlike cambridge structural database, protein data bank provides open access to the entire structural archive. carbohydrate moieties deposited in pdb are usually represented as covalently bound to protein or imply non-covalently bound protein-carbohydrate complex formation [ ] . according to recent reports, as at november , protein data bank contained ~ carbohydrate structures representing ~ . % of total database records [ ] . despite being a valuable source of d structural data for glycoscientists, pdb lacks convenient search facilities for glycan structures. some projects have developed data-mining tools capable of retrieving bioglycan molecular geometry data from pdb: glycan reader (glycanstructure.org) [ , ] (http://www.glycanstructure.org/), pdb linucs (glycosciences.de) [ , , ] (http://www.glycosciences.de/database/start.php?action=form_pdb_data), glyconavi tcarp [ ] (https://glyconavi.org/tcarp/) (https://gitlab.com/glyconavi/pdb glycan) and glyfinder (glycam-web) [ , ] (https://dev.glycam.org/portal/gf_home/). another issue of concern related to protein data bank is large proportion of errors in deposited coordinates, leading to requirement for a thorough checkup and development of data remediation services [ ] . commonly occurring problems associated with nomenclature, poor glycan geometry, linkage errors, missing or surplus atoms can seriously decline the quality of the obtained d structures [ , , ] . using privateer software, it was discovered [ ] , [ ] that pdb deposits significant number of erroneous n-glycosylated structures with pyranose ring distortions, considering preferred adoption of c conformation for d-sugars and c conformation for l-sugars ( figure ). in most cases, poor electron density of carbohydrate moiety results in anomalous high-energy pyranose ring conformations (envelopes, half-chairs, boats, skew boats, etc.). to obtain a reasonable structure model, experimental data refinement programs should be applied to derive geometric restraints for sugar monomers. notably, despite a cryo-em method has a resolution limit protein data bank (pdb) [ ] and cambridge structural database (csd) [ ] are historically considered the main repositories of experimentally determined carbohydrate three-dimensional structures. csd is reported to deposit over crystal structures of oligosaccharides [ ] . unlike cambridge structural database, protein data bank provides open access to the entire structural archive. carbohydrate moieties deposited in pdb are usually represented as covalently bound to protein or imply non-covalently bound protein-carbohydrate complex formation [ ] . according to recent reports, as at november , protein data bank contained~ carbohydrate structures representing~ . % of total database records [ ] . despite being a valuable source of d structural data for glycoscientists, pdb lacks convenient search facilities for glycan structures. some projects have developed data-mining tools capable of retrieving bioglycan molecular geometry data from pdb: glycan reader (glycanstructure.org) [ , ] (http://www.glycanstructure.org/), pdb linucs (glycosciences.de) [ , , ] (http://www. glycosciences.de/database/start.php?action=form_pdb_data), glyconavi tcarp [ ] (https://glyconavi. org/tcarp/) (https://gitlab.com/glyconavi/pdb glycan) and glyfinder (glycam-web) [ , ] (https://dev.glycam.org/portal/gf_home/). another issue of concern related to protein data bank is large proportion of errors in deposited coordinates, leading to requirement for a thorough checkup and development of data remediation services [ ] . commonly occurring problems associated with nomenclature, poor glycan geometry, linkage errors, missing or surplus atoms can seriously decline the quality of the obtained d structures [ , , ] . using privateer software, it was discovered [ ] , [ ] that pdb deposits significant number of erroneous n-glycosylated structures with pyranose ring distortions, considering preferred adoption of c conformation for d-sugars and c conformation for l-sugars ( figure ). in most cases, poor electron density of carbohydrate moiety results in anomalous high-energy pyranose ring conformations (envelopes, half-chairs, boats, skew boats, etc.). to obtain a reasonable structure model, experimental data refinement programs should be applied to derive geometric restraints for sugar monomers. notably, despite a cryo-em method has a resolution limit disadvantage, observed results indicate larger content of atypical conformations solved by x-ray crystallography, as compared to cryo-em data. disadvantage, observed results indicate larger content of atypical conformations solved by x-ray crystallography, as compared to cryo-em data. exceptions for the relevancy of high-energy conformations were found in complexes involving carbohydrate-active enzymes, which force pyranose ring distortion enabling catalytic transformation of a carbohydrate substrate via transition states (e.g., glycosydic bond hydrolysis) [ ] . fushinobu has performed glycosidic torsion analysis for a set of pdb entries of crystal structure complexes bound to ligands bearing lacto-n-biose i (lnb, both α-and β-anomers) disaccharide unit presented in type- antigens. the study was supported by glycomaps db (see table for references) [ ] . obtained φ-ψ data for lnbs bound to various proteins was plotted against corresponding free energy maps. distortion of the energetically favored ring conformation strongly depended on substrate catalytic and recognition mechanisms. to date, existing tools for carbohydrate structural error detection and correction in pdb files (table ) cannot be used directly as an integral part of protein data bank. nevertheless, initiative aimed at improvement of quality at wwpdb was carried out via collaboration with glycoscience community in july [ ] (https://www.wwpdb.org/documentation/carbohydrate-remediation). it includes data annotation and validation of carbohydrate-containing records. exceptions for the relevancy of high-energy conformations were found in complexes involving carbohydrate-active enzymes, which force pyranose ring distortion enabling catalytic transformation of a carbohydrate substrate via transition states (e.g., glycosydic bond hydrolysis) [ ] . fushinobu has performed glycosidic torsion analysis for a set of pdb entries of crystal structure complexes bound to ligands bearing lacto-n-biose i (lnb, both αand β-anomers) disaccharide unit presented in type- antigens. the study was supported by glycomaps db (see table for references) [ ] . obtained ϕ-ψ data for lnbs bound to various proteins was plotted against corresponding free energy maps. distortion of the energetically favored ring conformation strongly depended on substrate catalytic and recognition mechanisms. to date, existing tools for carbohydrate structural error detection and correction in pdb files (table ) cannot be used directly as an integral part of protein data bank. nevertheless, initiative aimed at improvement of quality at wwpdb was carried out via collaboration with glycoscience community in july [ ] (https://www.wwpdb.org/documentation/carbohydrate-remediation). it includes data annotation and validation of carbohydrate-containing records. proportion of carbohydrate-containing structures in pdb has been recently reported in [ ] . figure presents our analysis of data published in the framework of protein data bank carbohydrate remediation project. pdb entries from carbohydrate remediation list (https://cdn.rcsb.org/ wwpdb/docs/documentation/carbohydrateremediation/pdb_carbohydrate_list.list) were sorted by release year and plotted against the growth of pdb structures released annually (https://www.rcsb.org/ stats/growth/growth-released-structures) (as on august , ; , pdb entries were available). obtained results indicated that~ . % of pdb records contained a carbohydrate moiety. additionally, each pdbx/mmcif file corresponding to pdb id from carbohydrate remediation list was parsed to reveal the presence of n-or o-glycosylation site annotations, which resulted in~ . % ( n-glycosylated entries) and . % ( o-glycosylated entries) of total database records. a few s-and c-glycans ( entries in total) were neglected. statistics on glycans in protein data bank was reported [ , , , ] , as well as tools that could facilitate collection of statistical data (glycan reader [ , , ] , glyfinder [ ] , pdb linucs and pdb-care [ ] proportion of carbohydrate-containing structures in pdb has been recently reported in [ ] . figure presents our analysis of data published in the framework of protein data bank carbohydrate remediation project. pdb entries from carbohydrate remediation list (https://cdn.rcsb.org/wwpdb/docs/documentation/carbohydrateremediation/pdb_carbohydrate_lis t.list) were sorted by release year and plotted against the growth of pdb structures released annually (https://www.rcsb.org/stats/growth/growth-released-structures) (as on august , ; , pdb entries were available). obtained results indicated that ~ . % of pdb records contained a carbohydrate moiety. additionally, each pdbx/mmcif file corresponding to pdb id from carbohydrate remediation list was parsed to reveal the presence of n-or o-glycosylation site annotations, which resulted in ~ . % ( n-glycosylated entries) and . % ( o-glycosylated entries) of total database records. a few s-and c-glycans ( entries in total) were neglected. statistics on glycans in protein data bank was reported [ , , , ] , as well as tools that could facilitate collection of statistical data (glycan reader [ , , ] , glyfinder [ ] , pdb linucs and pdb-care [ ] ). carbohydrate structure visualization in publications and computer interfaces is extremely important in terms of perception universality, unambiguity, and machine-readability. hence, carbohydrate input [ ] [ ] [ ] and visualization [ , ] tools are actively developing. feature comparison of glycan sketchers, builders and viewers (occasionally including d ones) was reported in a recently published review [ ] . in our review, we gave more emphasis to d visualization approaches. being informative to represent glycan primary structure, most of graphical input tools such as glycanbuilder [ ] , drawrings [ ] , sugarsketcher [ ] , drawglycan-snfg [ , ] and glycoglyph [ ] are inappropriate for obtaining d structural models and their visualization due to lack of underlying modeling and insufficient data conversion functionality. at present, glycan d molecular models can be built in user-friendly software allowing constructing glycans from individual saccharide components. free web-tools, such as glycam-web, charmm-gui, polys glycan builder, gag-builder, sweet-ii should be noted (more references are listed in table ). a few commercial molecular modeling software is equipped with special plugins for glycan d structure building based on a list of predefined monosaccharide templates, e.g., sugar builder tool in hyperchem (http://www.hyper.com/?tabid= ) software [ ] or azahar [ ] plugin in pymol package (schrödinger software) (https://pymol.org/ /) [ ] . to render d glycan structure and its conformational features, it should be recorded using a notation which includes atomic coordinates, such as mol [ ] or pdb [ ] . all-atom visualization based on atomic coordinates is supported by the majority of existing molecular modeling software. several carbohydrate structure databases utilize interactive d visualization using open-source software engines. as one of the pioneers, glycosciences.de portal developed pdb multigif [ ] (http: //www.glycosciences.de/modeling/pdb mgif/) visualization pipeline which generates an animated image of d model from a pdb file using rasmol [ ] (http://www.openrasmol.org/). rasmol visualization was included in w -sweet [ ] (ancestor of sweet-ii) pipeline developed by same project. nowadays, more advanced interactive visualization applications have been developed for carbohydrate d molecule presentation. jmol/jsmol [ ] (http://www.openrasmol.org/) visualization applet is useful to display d models of carbohydrate molecules applied in numerous projects, such as csdb, glycosciences.de, glycam-web and ek d (see references in table ). ngl [ , ] (http://nglviewer.org/), litemol [ ] (https://www.litemol.org/) and mol* [ ] (https://www.rcsb.org/ news?year= &article= efe f d f ) (https://molstar.org/) d viewers are handy for processing macromolecular pdb data stored in glycoproteomics databases (unilectin d, glycan binding site db, procarbdb, glyconavi, procaff, etc.; see references in table ) and general proteomics repositories such as pdb [ ] (http://www.wwpdb.org/), uniprotkb [ ] (https://www.uniprot.org/) or swiss-model [ ] (https://swissmodel.expasy.org/repository). ngl viewer was developed mainly for convenient protein macromolecule structure processing. it allows only ball-stick representation for small molecules or non-peptide fragments, such as saccharide residues. litemol (and its successor, mol*) viewer could be applied for the visualization of an arbitrary glycan with facility of highlighting carbohydrate fragments or displaying specific interactions in protein-carbohydrate complex structure. due to these features, it was implemented in multiple carbohydrate structure databases (e.g., csdb, glyco d, matrixdb, and eps-db). despite the absence of the experimental d structural data, a number of carbohydrate databases have opportunity to simulate d atomic coordinates for deposited or inputted compounds from primary structure owing to tools developed by glycoinformatics community. csdb (restless api [ ] ), glycosciences.de (sweet-ii [ , ] ) and glycam-web (http://glycam.org/) portals make it possible to generate d atomic coordinates recorded in pdb (all) and mol (csdb) file formats. polys developed by glyco d project enables the construction of polysaccharides in pdb format; it was introduced in matrixdb and eps-db databases. more details are provided in table . atomic coordinates and all-atom molecular models have not been popular in publications due to a lack of human readability. first attempts [ , ] of prof. kuttel et al., to visualize carbohydrate molecules in an efficient and simple way were made by developing paperchain and twister graphic algorithms as a part of carbohydra [ ] and visual molecular dynamics [ ] software packages. later, group of prof. pé rez suggested to restrict visualized molecule to skeletal atoms via conditional cycle plane coloring in accordance with the color code adopted in snfg [ ] visualization scheme (sweetunitymol software [ ] , figure a ). another unitymol visualization approach called umbrella visualization [ , ] was tailored for n-glycan structures. azahar plugin for pymol [ ] affords cartoon models with polygons and rods. several solutions for convenient visualization came up with the development of snfg notation [ ] . thus, group of prof. woods proposed to combine molecular structure elements with d snfg icons (figure a ). such convenient visualization technique was integrated in litemol (figure b ) [ ] and mol* (figure c) [ , ] . d snfg visualization plugins are available via visual molecular dynamics platform [ ] (http://glycam.org/docs/othertoolsservice/ / / / d-symbol-nomenclature-for-glycans- d-snfg/) and ucsf chimera [ ] visualization software tangram plugin (https://github.com/insilichem/tangram_snfg). designed as part of ccp mg [ ] molecular-graphics software, glycoblocks [ ] representation of monosacchrides uses shapes and colors, identical to those in snfg (figure d ). available as pymol plugin developed by widmalm group (http://www.organ.su.se/gw/doku.php?id= dcfg), d-cfg representation [ ] based on cfg notation [ ] (often referred to as a predecessor of snfg) should also be noted as earlier approach to interpretation of carbohydrate d structures based on a symbol library. considering efficiency and usability of d representation based on snfg concept, which grows popular among glycoscientists, the development of alternative solutions in carbohydrate d structure representations has a potential for application in glycoinformatics projects. support of colored residues in d structures implemented via jsmol on glycosciences.de portal was reported [ ] (figure b) . similarly, csdb project has developed a d viewer (http://csdb.glycoscience.ru/database/core/show_ d.php?csdb=- )admanp( - )[ac( - )?dglcpn( - )]bdgal?( -) with carbohydrate residue coloring according to the snfg notation in the framework of a modeling module based on restless api. in this tool, user can visualize input structure with help of sticks, balls and sticks, or van der waals spheres (figure c ). options for aglycone moiety (white) and pseudo-atoms (polymeric repeats, blue caps) are supported (figure d ). int. j. mol. sci. , , x for peer review of development of glycoinformatics resources makes great impact on treating enormous masses of data sets produced by glyco-related research. tools for carbohydrate d structural information retrieval provide a framework for experimental and computational data quality validation. data sources based on conformational ensemble generation and analysis assist structure-function and structure-activity relationship prediction of biologically relevant bioglycans and glycoconjugates. in this review, we have summarized existing facilities on working with glycan spatial features that can provide harmonious network of structural databases, web-services, tools and standalone software development of glycoinformatics resources makes great impact on treating enormous masses of data sets produced by glyco-related research. tools for carbohydrate d structural information retrieval provide a framework for experimental and computational data quality validation. data sources based on conformational ensemble generation and analysis assist structure-function and structure-activity relationship prediction of biologically relevant bioglycans and glycoconjugates. in this review, we have summarized existing facilities on working with glycan spatial features that can provide harmonious network of structural databases, web-services, tools and standalone software for modeling and processing structural data. further advances in this field will help building better understanding of glycan participation in biological processes and supply glycoscience community with user-friendly access to voluminous data collections. funding: the work with carbohydrate molecular modeling and pdb data was funded by russian foundation for basic research grant - - . the work with structural databases, glycoinformatic tools and visualization was funded by russian science foundation grant - - . the authors declare no conflict of interest. three structural aspects of carbohydrates and the relation 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alliance: toward a collaborative global glycoinformatics community the glycosmos portal: a unified and comprehensive web resource for the glycosciences glygen data model and processing workflow japan consortium for glycobiology and glycotechnology database an accessible glycan structure repository mirage: the minimum information required for a glycomics experiment a focused microarray approach to functional glycomics: transcriptional regulation of the glycome rings: a web resource of tools for analyzing glycomics data. in a practical guide to using glycomics databases representing glycophenotypes: semantic unification of glycobiology resources for disease discovery understanding the glycome: an interactive view of glycosylation from glycocompositions to glycoepitopes the glycome analytics platform: an integrative framework for glycobioinformatics glycosylation in health and disease glycoenzymes in glycan analysis and synthesis computational glycobiology: mechanistic studies of 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biological sources a curated relational database of glycoprotein glycan structures and their biological sources. update bacterial carbohydrate structure database : principles and realization a new curated database on glycosyltransferases. glycobiology new database of bacterial carbohydrate structures an internet portal to support glycomics and glycobiology research glycan data retrieval and analysis using glycosciences. de applications db: an annotated data collection linking glycomics and proteomics data glyco d: a portal for structural glycosciences glyco d: a suite of interlinked databases of d structures of complex carbohydrates, lectins, antibodies, and glycosyltransferases an annotated data base of dimensional structures of polysaccharides ek d: an e. coli k antigen -dimensional structure database dsdscar-a three dimensional structural database for sialic acid-containing carbohydrates through molecular dynamics simulation hema thanka christlet, t. three dimensional structures of carbohydrates and glycoinformatics: an overview the extracellular matrix interaction database the extracellular matrix interaction database: updated content, a new navigator and expanded functionalities integration of new data with a focus on glycosaminoglycan interactions the exopolysaccharide properties and structures database: eps-db. application to bacterial exopolysaccharides glycan microarray database and analysis toolset advancing glycomics: implementation strategies at the consortium for functional glycomics glycan array data management at consortium for functional glycomics databases for glycoconjugates (glycosmos glycoproteins and glycolipids, glycoprotdb, glyconavi:tcarp, glycopost). glycoforum integration of glycoscience data in glycosmos using semantic web technologies the glycosmos web portal: glycan structures, glycogenes, glycoproteins, pathways, diseases and more! in program and abstracts for annual meeting of the society for glycobiology sugarbinddb, a resource of glycan-mediated host-pathogen interactions glyconnect: glycoproteomics goes visual, interactive, and analytical proglycprot v . , a repository of experimentally validated glycoproteins and protein glycosyltransferases of prokaryotes proglycprot: a repository of experimentally characterized prokaryotic glycoproteins procarbdb: a database of carbohydrate-binding proteins procaff: protein-carbohydrate complex binding affinity database a systematic analysis of protein-carbohydrate interactions in the pdb a database of known and modelled carbohydrate-binding protein structures with sequence-based prediction tools imberty, a. unilectin d, a database of carbohydrate binding proteins with curated information on d structures and interacting ligands structural database for lectins and the unilectin web platform the lectin frontier database (lfdb), and data generation based on frontal affinity chromatography lectindb: a plant lectin database the integrated database of carbohydrate antigens and antibodies glycoepitope: a database of carbohydrate epitopes and antibodies using databases and web resources for glycomics research glycocd: a repository for carbohydrate-related cd antigens sacs-self-maintaining database of antibody crystal structure information the structural antibody database the carbohydrate-active enzymes database (cazy) in the carbohydrate-active enzymes database (cazy): an expert resource for glycogenomics the cazy database/the carbohydrate-active enzyme (cazy) database: principles and usage guidelines. in a practical guide to using glycomics databases an information repository of protein post-translational modification -year anniversary of a resource for post-translational modification of proteins dbptm in : exploring disease association and cross-talk of post-translational modifications the swiss-model repository of annotated three-dimensional protein structure homology models the swiss-model repository and associated resources the swiss-model repository-new features and functionality a database of the accessible conformational space of glycosidic linkages glycan fragment database: a database of pdb-based glycan d structures diffraction and crystallography of oligosaccharides and polysaccharides glycan structures and their interactions with proteins. a nmr view spin ballet for sweet encounters: saturation-transfer difference nmr and x-ray crystallography complement each other in the elucidation of protein-glycan interactions nmr of glycans: shedding new light on old problems insights into carbohydrate recognition by d structure determination of protein-carbohydrate complexes using nmr an excellent tool to understand rna and carbohydrate recognition by proteins molecular insights into dc-sign binding to self-antigens: the interaction with the blood group a/b antigens a secondary structural element in a wide range of fucosylated glycoepitopes stabilization of branched oligosaccharides: lewisx benefits from a nonconventional c-h···o hydrogen bond is n-acetyl-d-glucosamine a rigid c chair? glycobiology specific rotation of monosaccharides: a global property bringing local information conformational analysis of xylobiose by dft quantum mechanics stereoelectronic effects impact glycan recognition use of circular statistics to model αman-( → )-αman and αman-( → )-α/βman o-glycosidic linkage conformation in c-labeled disaccharides and high-mannose oligosaccharides the carbohydrate-binding site in galectin- is preorganized to recognize a sugarlike framework of oxygens: ultra-high-resolution structures and water dynamics an nmr and md study of complexes of bacteriophage lambda lysozyme with tetra-and hexa-n-acetylchitohexaose modeling of oligosaccharides within glycoproteins from free-energy landscapes conformational populations of β-( → ) o-glycosidic linkages using redundant nmr j-couplings and circular statistics shaping up for structural glycomics: a predictive protocol for oligosaccharide conformational analysis applied to n-linked glycans conformational analysis of carbohydrates-a historical overview predicting carbohydrate d structures using theoretical methods recent advances in computational predictions of nmr parameters for the structure elucidation of carbohydrates: methods and limitations computerized models of carbohydrates computerized molecular modeling of carbohydrates application of molecular dynamics simulation in food carbohydrate research-a review monte carlo-based searching as a tool to study carbohydrate structure combined monte carlo/torsion-angle molecular dynamics for ensemble modeling of proteins, nucleic acids and carbohydrates structures and energies of d-galactose and galabiose conformers as calculated by ab initio and semiempirical methods ring puckering: a metric for evaluating the accuracy of am , pm , pm carb- , and scc-dftb carbohydrate qm/mm simulations am /d-cb : a semiempirical model for qm/mm simulations of chemical glycobiology systems evaluating am /d-cb for chemical glycobiology qm/mm simulations correlated ab initio quantum chemical calculations of di-and trisaccharide conformations conformational analysis of cellobiose by electronic structure theories dft energy optimization of a large carbohydrate: cyclomaltohexaicosaose (ca- ) ab initio study of molecular interactions in cellulose iα aqueous-phase conformations of lactose, maltose, and sucrose and the assessment of low-cost dft methods with the dsconf set of conformers for the three disaccharides computational analysis of carbohydrate recognition based on hybrid qm/mm modeling: a case study of norovirus capsid protein in complex with lewis antigen structure and conformation of α-, β-and γ-cyclodextrin in solution: theoretical approaches and experimental validation reaction mechanisms in carbohydrate-active enzymes: glycoside hydrolases and glycosyltransferases. insights from ab initio quantum mechanics/molecular mechanics dynamic simulations atomistic insight into the catalytic mechanism of glycosyltransferases by combined quantum mechanics/molecular mechanics (qm/mm) methods twisting of glycosidic bonds by hydrolases the oniom method and its applications scopus database: a review bioinformatics and molecular modeling in glycobiology simulation of carbohydrates, from molecular docking to dynamics in water chapter -carbohydrate-protein interactions: molecular modeling insights molecular simulations of carbohydrates and protein-carbohydrate interactions: motivation, issues and prospects the conformational properties of methyl α-( , )-di/trisialosides and their n-acyl analogues: implications for anti-neisseria meningitidis b vaccine design conformational flexibility of n-glycans in solution studied by remd simulations conformational properties of α-or β-( → )-linked oligosaccharides: hamiltonian replica exchange md simulations and nmr experiments enhanced conformational sampling of carbohydrates by hamiltonian replica-exchange simulation influence of solvent and intramolecular hydrogen bonding on the conformational properties of o-linked glycopeptides identification of rare lewis oligosaccharide conformers in aqueous solution using enhanced sampling molecular dynamics ring puckering landscapes of glycosaminoglycan-related monosaccharides from molecular dynamics simulations the mechanism of high affinity pentasaccharide binding to antithrombin, insights from gaussian accelerated molecular dynamics simulations comparison of carbohydrate force fields using gaussian accelerated molecular dynamics simulations and development of force field parameters for heparin-analogue pentasaccharides conformational analysis of a high-mannose-type oligosaccharide displaying glucosyl determinant recognised by molecular chaperones using nmr-validated molecular dynamics simulation exploration of conformational spaces of high-mannose-type oligosaccharides by an nmr-validated simulation carbohydrate force fields dispersion interactions of carbohydrates with condensate aromatic moieties: theoretical study on the ch-π interaction additive properties carbohydrate-aromatic interactions in proteins the dependence of carbohydrate-aromatic interaction strengths on the structure of the carbohydrate carbohydrate-protein aromatic ring interactions beyond ch/π interactions: a protein data bank survey and quantum chemical calculations importance of ligand conformational energies in carbohydrate docking: sorting the wheat from the chaff improving glycosidic angles during carbohydrate docking -molecular modeling in glycoscience disaccharide conformational maps: adiabaticity in analogues with variable ring shapes molecular mechanics. the mm force field for hydrocarbons. a molecular mechanics force field (mm ) for alcohols and ethers comparative performance of mm ( ) and two tinker™ mm versions for the modeling of carbohydrates comparison of different force fields for the study of disaccharides conformational analysis of furanoside-containing mono-and oligosaccharides additive effects in the modeling of oligosaccharides with mm at high dielectric constants: an approach to the 'multiple minimum problem' mm potential energy surfaces of trisaccharide models of λ-, µ-, and ν-carrageenans force fields and scoring functions for carbohydrate simulation charmm force field files all-atom empirical potential for molecular modeling and dynamics studies of proteins extending the treatment of backbone energetics in protein force fields: limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations charmm additive all-atom force field for glycosidic linkages between hexopyranoses additive empirical force field for hexopyranose monosaccharides charmm additive all-atom force field for glycosidic linkages in carbohydrates involving furanoses charmm additive all-atom force field for carbohydrate derivatives and its utility in polysaccharide and carbohydrate-protein modeling charmm additive all-atom force field for phosphate and sulfate linked to carbohydrates kirkwood-buff-derived alcohol parameters for aqueous carbohydrates and their application to preferential interaction coefficient calculations of proteins glycam : a generalizable biomolecular force field. carbohydrates extension of the glycam biomolecular force field to lipids, lipid bilayers and glycolipids atomic-resolution conformational analysis of the gm ganglioside in a lipid bilayer and its implications for ganglioside-protein recognition at membrane surfaces molecular dynamics simulations of membrane-and protein-bound glycolipids using glycam a glycam-based force field for simulations of lipopolysaccharide membranes: parametrization and validation extension and validation of the glycam force field parameters for modeling glycosaminoglycans a new gromos force field for hexopyranose-based carbohydrates a reoptimized gromos force field for hexopyranose-based carbohydrates accounting for the relative free energies of ring conformers, anomers, epimers, hydroxymethyl rotamers, and glycosidic linkage conformers the effect of temperature, cations, and number of acyl chains on the lamellar to non-lamellar transition in lipid-a membranes: a microscopic view gromos a glyc, an improved gromos force field for hexopyranose-based carbohydrates extension and validation of the gromos a glyc parameter set for glycoproteins revision of the gromos a carbo force field: improving the description of ring-conformational equilibria in hexopyranose-based carbohydrates chains modification of acarbo force field for molecular dynamic calculations of chitosan and its derivatives extension of the gromos a carbo/carbo_r force field for charged, protonated, and esterified uronates a gromos force field for furanose-based carbohydrates gromos a performance on the characterization of glycoprotein conformational ensembles through molecular dynamics simulations gromos a performance in predicting oligosaccharide conformational ensembles within glycoproteins an improved opls-aa force field for carbohydrates opls all-atom force field for carbohydrates development and testing of the opls all-atom force field on conformational energetics and properties of organic liquids optimizing nonbonded interactions of the opls force field for aqueous solutions of carbohydrates: how to capture both thermodynamics and dynamics polarizable empirical force field for hexopyranose monosaccharides based on the classical drude oscillator proper balance of solvent-solute and solute-solute interactions in the treatment of the diffusion of glucose using the drude polarizable force field charmm drude polarizable force field for aldopentofuranoses and methyl-aldopentofuranosides drude polarizable force field parametrization of carboxylate and n-acetyl amine carbohydrate derivatives polarizable empirical force field for acyclic polyalcohols based on the classical drude oscillator charmm drude polarizable force field for glycosidic linkages involving pyranoses and furanoses martini coarse-grained force field: extension to carbohydrates overcoming the limitations of the martini force field in simulations of polysaccharides extending the martini coarse-grained forcefield to n-glycans martini force field parameters for glycolipids pitomba: parameter interface for oligosaccharide molecules based on atoms modelling of carbohydrate-aromatic interactions: ab initio energeticsand force field performance stacking interactions between carbohydrate and protein quantified by combination of theoretical and experimental methods applying pose clustering and md simulations to eliminate false positives in molecular docking scoring functions and their evaluation methods for protein-ligand docking: recent advances and future directions aromatic-carbohydrate interactions: an nmr and computational study of model systems a gibbs free energy correlation for automated docking of carbohydrates protein-carbohydrate interactions docking glycosaminoglycans to proteins: analysis of solvent inclusion flexibility and explicit solvent in molecular-dynamics-based docking of protein-glycosaminoglycan systems mannobiose binding induces changes in hydrogen bonding and protonation states of acidic residues in concanavalin a as revealed by neutron crystallography improvements, trends, and new ideas in molecular docking: - in review recognition of selected monosaccharides by pseudomonas aeruginosa lectin ii analyzed by molecular dynamics and free energy calculations in silico mutagenesis and docking study of ralstonia solanacearum rsl lectin: performance of docking software to predict saccharide binding finding a needle in a haystack: development of a combinatorial virtual screening approach for identifying high specificity heparin/heparan sulfate sequence(s) computational analysis of interactions in structurally available protein-glycosaminoglycan complexes identification and characterization of a glycosaminoglycan binding site on interleukin- via molecular simulation methods a. in silico analysis of antibody-carbohydrate interactions and its application to xenoreactive antibodies charmm-gui input generator for namd, gromacs, amber, openmm, and charmm/openmm simulations using the charmm additive force field charmm-gui supports the amber force fields residue-centric modeling and design of saccharide and glycoconjugate structures efficient construction of atomic-resolution models of non-sulfated chondroitin glycosaminoglycan using molecular dynamics data charmm-gui glycan modeler for modeling and simulation of carbohydrates and glycoconjugates glycosylator: a python framework for the rapid modeling of glycans the rosetta all-atom energy function for macromolecular modeling and design novel sampling strategies and a coarse-grained score function for docking homomers, flexible heteromers, and oligosaccharides using rosetta in capri rounds - macromolecular modeling and design in rosetta: recent methods and frameworks azahar: a pymol plugin for construction, visualization and analysis of glycan molecules automatic conformation prediction of carbohydrates using a genetic algorithm rapid generation of a representative ensemble of n-glycan conformations the use of a genetic algorithm search for molecular mechanics (mm )-based conformational analysis of oligosaccharides sugar folding: a novel structural prediction tool for oligosaccharides and polysaccharides sugar folding: a novel structural prediction tool for oligosaccharides and polysaccharides a tool for the prediction of structures of complex sugars computational study of the conformational structures of saccharides in solution based on j couplings and the "fast sugar structure prediction software glyprot: in silico glycosylation of proteins macromolecular structure determination using x-rays, neutrons and electrons: recent developments in phenix computational screening of the human tf-glycome provides a structural definition for the specificity of anti-tumor antibody jaa-f presentation, presentation, presentation! molecular-level insight into linker effects on glycan array screening data recent advances in employing molecular modelling to determine the specificity of glycan-binding proteins gly-spec: a webtool for predicting glycan specificity by integrating glycan array screening data and d structure combining d structure with glycan array data provides insight into the origin of glycan specificity automated builder and database of protein/membrane complexes for molecular dynamics simulations membrane builder for mixed bilayers and its application to yeast membranes charmm-gui membrane builder toward realistic biological membrane simulations modeling and simulation of bacterial outer membranes with lipopolysaccharides and enterobacterial common antigen the gram-negative outer membrane modeler: automated building of lipopolysaccharide-rich bacterial outer membranes in four force fields maker: an online tool for generating equilibrated micelles as direct input for molecular dynamics simulations probabilistic identification of saccharide moieties in biomolecules and their protein complexes new online tools for locating and curating carbohydrate structures in wwpdb tools to find glycoproteins in the protein data bank and generate realistic d structures for them data mining the protein data bank: automatic detection and assignment of carbohydrate structures glycan reader: automated sugar identification and simulation preparation for carbohydrates and glycoproteins glycan reader is improved to recognize most sugar types and chemical modifications in the protein data bank vattulainen, i. doglycans-tools for preparing carbohydrate structures for atomistic simulations of glycoproteins, glycolipids, and carbohydrate polymers for gromacs sweet-www-based rapid d construction of oligo-and polysaccharides automated translation of glycan sequences from residue-based notation to smiles and atomic coordinates a molecular builder for carbohydrates: application to polysaccharides and complex carbohydrates : an open source software package for building three-dimensional structures of polysaccharides an adjustable tool for building d molecular structures of carbohydrates for molecular simulation software for building molecular models of complex oligoand polysaccharide structures a pipeline to translate glycosaminoglycan sequences into d models. application to the exploration of glycosaminoglycan conformational space a web-tool for modeling d structures of glycosaminoglycans slick-scoring and energy functions for protein−carbohydrate interactions balldock/slick: a new method for protein-carbohydrate docking the haddock . web server: user-friendly integrative modeling of biomolecular complexes docking server for the identification of heparin binding sites on proteins so you think computational approaches to understanding glycosaminoglycan-protein interactions are too dry and too rigid? think again! predicting glycosaminoglycan surface protein interactions and implications for studying axonal growth improved docking of sulfated sugars using qm-derived scoring functions conformational analysis of oligosaccharides and polysaccharides using molecular dynamics simulations a method for discretizing and visualizing pyranose conformations direct noe simulation from long md trajectories gs-align for glycan structure alignment and similarity measurement analysis of carbohydrate d structures derived from the pdb statistical analysis of amino acids in the vicinity of carbohydrate residues performed by glyvicinity rules of engagement of protein-glycoconjugate interactions: a molecular view achievable by using nmr spectroscopy and molecular modeling conformational studies of oligosaccharides structure, conformation, and dynamics of bioactive oligosaccharides: theoretical approaches and experimental validations conformational studies of oligosaccharides and glycopeptides: complementarity of nmr, x-ray crystallography, and molecular modelling analysis and validation of carbohydrate three-dimensional structures an efficient use of x-ray information, homology modeling, molecular dynamics and knowledge-based docking techniques to predict protein-monosaccharide complexes chapter developments in the karplus equation as they relate to the nmr coupling constants of carbohydrates a perspective on the primary and three-dimensional structures of carbohydrates nmr structure determination of a segmentally labeled glycoprotein using in vitro glycosylation nmr structural biology of sulfated glycans making glycoproteins a little bit sweeter with pdb-redo automatically fixing errors in glycoprotein structures with rosetta leveraging glycomics data in glycoprotein d structure validation with privateer current developments in coot for macromolecular model building of electron cryo-microscopy and crystallographic data carbohydrate anomalies in the pdb numerous severely twisted n-acetylglucosamine conformations found in the protein databank structural glycobiology in the age of electron cryo-microscopy strategies for carbohydrate model building, refinement and validation structures of ebola virus gp and sgp in complex with therapeutic antibodies cryo-em structure of a native, fully glycosylated, cleaved hiv- envelope trimer cross-neutralization of sars-cov- by a human monoclonal sars-cov antibody structure, function, and antigenicity of the sars-cov- spike glycoprotein nmr spectroscopy in the study of carbohydrates: characterizing the structural complexity recent advances in the application of nmr methods to uncover the conformation and recognition features of glycans. in carbohydrate chemistry the recognition of glycans by protein receptors. insights from nmr spectroscopy novel nmr avenues to explore the conformation and interactions of glycans delineating the conformational flexibility of trisaccharides from nmr spectroscopy experiments and computer simulations conformational flexibility of the pentasaccharide lnf- deduced from nmr spectroscopy and molecular dynamics simulations molecular conformations of di-, tri-, and tetra-α-( → )-linked sialic acid from nmr spectroscopy and md simulations an unusual carbohydrate conformation is evident in moraxella catarrhalis oligosaccharides protein data bank (pdb): the single global macromolecular structure archive the cambridge structural database current status of carbohydrates information in the protein data bank data mining the pdb for glyco-related data carbohydrate structure: the rocky road to automation building meaningful models of glycoproteins carbohydrate d structure validation dissecting conformational contributions to glycosidase catalysis and inhibition conformations of the type- lacto-n-biose i unit in protein complex structures collaborating with glycoscience community to improve data representation of carbohydrates in the protein data bank building and rebuilding n-glycans in protein structure models pdb-care (pdb carbohydrate residue check): a program to support annotation of complex carbohydrate structures in pdb files crystallography & nmr system: a new software suite for macromolecular structure determination version . of the crystallography and nmr system tools to assist determination and validation of carbohydrate d structure data compatible topologies and parameters for nmr structure determination of carbohydrates by simulated annealing structural analysis of glycoproteins: building n-linked glycans with coot software for the conformational validation of carbohydrate structures motivevalidator: interactive web-based validation of ligand and residue structure in biomolecular complexes database of up-to-date validation results for ligands and non-standard residues from the protein data bank a practical guide to using glycomics databases implementation of glycanbuilder to draw a wide variety of ambiguous glycans a glycan visualizing, drawing and naming application symbol nomenclature for graphical representations of glycans updates to the symbol nomenclature for glycans (snfg) guidelines computational tools for drawing, building and displaying carbohydrates: a visual guide the glycanbuilder and glycoworkbench glycoinformatics tools: updates and new developments the rings resource for glycome informatics analysis and data mining on the web sugarsketcher: quick and intuitive online glycan drawing drawglycan-snfg: a robust tool to render glycans and glycopeptides with fragmentation information drawglycan-snfg and gpannotate: rendering glycans and annotating glycopeptide mass spectra the pymol molecular graphics system description of several chemical structure file formats used by computer programs developed at molecular design limited protein data bank contents guide: atomic coordinate entry format description. brookhaven natl. lab a web tool to create animated images of molecules biomolecular graphics for all fast and scriptable molecular graphics in web browsers without java d. nat. prec a web application for molecular visualization ngl viewer: web-based molecular graphics for large complexes litemol suite: interactive web-based visualization of large-scale macromolecular structure data mol: towards a common library and tools for web molecular graphics uniprot: a worldwide hub of protein knowledge eborn, i. techniques for visualization of carbohydrate molecules visualisation of cyclic and multi-branched molecules with vmd rendering carbohydrate cartoons vmd: visual molecular dynamics three-dimensional representations of complex carbohydrates and polysaccharides-sweetunitymol: a video game-based computer graphic software new visualization of dynamical flexibility of n-glycans: umbrella visualization in unitymol umbrella visualization: a method of analysis dedicated to glycan flexibility with unitymol rapidly display glycan symbols in d structures: d-snfg in litemol d implementation of the symbol nomenclature for graphical representation of glycans ucsf chimera-a visualization system for exploratory research and analysis presenting your structures: the ccp mg molecular-graphics software glycoblocks: a schematic three-dimensional representation for glycans and their interactions glycan synthesis, structure, and dynamics: a selection symbol nomenclature for glycan representation development of carbohydrate nomenclature and representation. in a practical guide to using glycomics databases key: cord- - vzwc d authors: nan title: proceedings of scanning /seems charleston, south carolina, usa date: - - journal: scanning doi: . /sca. sha: doc_id: cord_uid: vzwc d nan the widespread application of monte carlo electron trajectory modeling has never been realized in the electron microscopy community because of the computation-intensive nature of the monte carlo algorithm (e.g., many hours of computer time were required to run one simulation which might improve one's ability to interpret data if sufficient statistics were obtained). massively parallel supercomputers with multiple-instruction multiple-data (mimd) architecture are a good platform for monte carlo electron trajectory codes without the complications of vectorization of the computer code required on vector supercomputers (e.g., cray-ymp). the nist monte carlo electron trajectory simulation code has been adapted to run on a massively parallel computer. for the first time, the increased speed of the calculations has made monte carlo calculations a real-time tool for data interpretation. the increased speed achieved by the parallelization of the monte carlo code results in the ability to model small probability events due to the massively parallel monte carlo (mpmc) code's ability to simulate large numbers of electron trajectories quickly. applications include both thin-film and bulk x-ray microanalysis and examples of contrast in backscattered electron images obtained in the sem. this work was supported by the united states department of energy under contract #de-ac - al . in electron probe x-ray microanalysis, "secondary fluorescence" refers to the emission of characteristic x-rays following photoelectric absorption of the primary x-rays generated by the electron beam. while generally a minor contribution to the total x-ray emission, secondary x-ray fluorescence can become an important issue, when the spatial resolution of analysis is of interest, because of the significantly greater range of x-rays than of electrons. thus, for a nickel- % iron alloy excited with a kev electron beam, the range of excitation of the primary radiation will be a hemispheric volume with a diameter of about one micrometer, while the range of secondary fluorescence of iron k-shell radiation by nickel k-shell radiation will be a hemispheric volume with a diameter of approximately micrometers for % of the total secondary radiation. such long-range production of secondary radiation is important when the measurement of trace element distributions near phase boundaries is attempted in situations where an analyte appears at high concentration across the boundary. monte carlo electron trajectory simulation provides a powerful tool for the calculation of the three-dimensional distribution of the primary radiation. to calculate the spatial distribution of the secondary fluorescence due to the absorption of the primary radiation, a second calculation is needed that integrates the x-ray absorption equation in three dimensions. the resulting hybrid calculation can give the total secondary radiation induced for a given structure, such as a planar interface between two materials. the transport phenomenon of incident electrons in solid materials is an important subject in microscopy, microanalysis, and microlithography. it has been treated by either an analytical method, the transport equation, or monte carlo simulation. because of the flexibility of modeling, monte carlo simulation has been developed extensively. various models for monte carlo simulation proposed so far are classified into four groups from the aspect as to how inelastic interactions are treated. the most simple model is the one which uses the inelastic mean free path. this model is applied, for example, to spectroscopic analyses of elastically reflected electrons, auger electrons, and photoelectrons. the most popular model is the single scattering model in which the step length is taking the free path of elastic scattering of electrons, and an energy loss during their traveling is calculated by the continuous slowing-down approximation of bethe, [de/ds] bethe . the most complicated model is the direct simulation model in which all inelastic interactions are taken into consideration as the discrete process. this model requires all differential cross sections for inelastic scattering and, thus, a long computational time. the last one, a compromise model, is the hybrid model which introduces partially the discrete process of inelastic scattering. in the model the continuous energy loss [de/ds] ( ) where [de/ds] dis includes both energy losses due to core electron ionization and valence electron excitation. a variety of the cross sections has been used for inelastic scattering. the fast secondary production model uses the moller equation for the cross section, assuming that all atomic electrons are free (murata et al. ). the authors have published a hybrid model by using the differential inelastic cross sections of vriens for core electron ionization and the moller cross section for free electron excitation (murata et al. ). the mott cross section is used for elastic scattering. typical results for backscattering have shown fairly good agreement with experimental data. the present paper gives a brief survey of monte carlo modeling and discusses the validity of the hybrid model mentioned above. figure shows a typical example of the calculated depth distribution of generated x-rays in an au target in comparison with the experimental result of castaing and descamps ( ) . agreement is good. also shown is the result without the discrete processes of inelastic scattering. an appreciable deviation is seen in the vicinity of the peak. however, the effect of the energy straggling is not so significant. we have also done calculations for other x-rays and have obtained reasonable agreement with experimental data. the effect of the energy straggling will appear more significantly in electron scattering in a thin film. figure shows the energy distribution of transmitted electrons, with a comparison between two monte carlo results and the experimental data of shimizu et al. ( ) . the agreement is not as good; it seems that the disagreement comes from insufficient discrete processes because we still keep the continuous energy loss process. we also propose a new model to improve the discrepancy in figure , caused by energy straggling. the straggling is impor-tant in thin-film analysis, especially at initial energies near the ionization energy for x-ray production. discussions of ways to improve monte carlo simulations have usually concentrated on topics such as the effect of the choice of scattering cross section, or the appropriate model for electron stopping power at low energies. much less attention has been given to considering whether or not we actually have sufficient experimental data to make it possible to demonstrate by a comparison of simulation and experiment that one model, or a portion of that model, actually performs better than another alternative. the earliest experimental data on electron-solid interactions was published years ago (by starke in germany in , and by campbell-swinton in england in ), and numerous workers since then have contributed to the literature on this topic. unfortunately, there never seems to have been any attempt to collect and collate all of this material, and consequently workers seeking data such as the variation of the secondary electron yield with energy for silicon have been forced to conduct a random search of the literature to find what values are available. it is not surprising that such a search typically finishes as soon as a set of data plausibly matching the simulated values is found. in order to try and remedy this deficiency, a systematic search of published data has been carried out to generate material for a rudimentary database, with the hope that this will provide some of the necessary numbers against which monte carlo simulations can be tested. in its current form the database, derived from separate references, is divided into four segments arranged by atomic number (or compound) and comprising secondary electron yields, backscattered electron yields, x-ray ionization cross sections, and electron stopping power. only experimental values are included; thus interpolated, extrapolated, or normalized data sets, or values not specifically indicated by the author to be experimental, have been removed. this restriction unfortunately eliminates most of the voluminous secondary electron data, since such published values have invariably been normalized in order to facilitate fitting to a yield curve equation. no attempt has been made to judge the quality of any of the data since any such assessment would be premature until a sufficient number of independent values are available to permit obviously erroneous, or possibly dubious, results to be safely identified and eliminated. the database is available on request from the author both in printed form, or as a set of cricketgraph ™ files for the apple macintosh. while the quantity and quality of data available for a given element vary widely, they are mostly sparse and scattered. for about half the elements in the periodic table, no experimental values ever appear to have been published, and data are conspicuously absent for even the most common alloys and compounds. even for an element such as silicon ( fig. ) , the scatter in the data is too large to make it possible to be certain to better than a factor of what the se yield is at some energies. thus, unless a major source of quantitative results has been overlooked, it is fair to conclude that the experimental data presently available are not good enough to allow the merits of competing monte carlo models to be judged, or even to permit specific numerical comparisons (e.g., the backscattered yield of carbon, copper, and gold at kev) to be made with any satisfactory level of certainty. on the positive side, however, the database does give some indication of interesting trends (e.g., the variation of secondary yield with atomic number) that have not been easily accessible previously. school of electrical engineering and the national nanofabrication facility, cornell university, ithaca, new york, usa two simulation programs have been developed at cornell, seel [ ] [ ] [ ] (for simulation of electron energy loss) and pyra-mid. seel is a two-dimensional monte carlo program for the simulation of electron trajectories in complex, multi-material nanostructures; the program incorporates energy loss models that include quantum mechanical cross sections for energies down to < ev. the second program, pyramid, , is a very fast electron beam proximity correction program that can perform proximity correction (electron scattering correction) for complex ultra-large-scale integrated circuit patterns with nm minimum feature sizes (mfss). pyramid uses seel to generate the point spread function or radial exposure distribution (red) as the input. seel simulations have been tested against numerous published electron energy loss data and pyramid has been tested on ulsi density ( nm mfs) exposures using . µm pmma. simulation results are compared with experimental data to evaluate pyramid's performance. a more recent application of seel is the evaluation of signal-noise ratio (snr) for time-resolved microscopy of microelectromechanical structures (mems) . for this application, we are interested in estimating the snr for high-speed video recording , of moving high-aspect-ratio mems oscillating > mhz, particularly applications of nanoelectromechanical structures for metrology and time-resolved characterization. iv- scanning vol. , supplement iv ( ) simulation of image formation and detection systems in the sem is a vital link in performing image analysis to obtain precise measurements, to provide the necessary connection between image parameters and structural dimensions, and to reflect important microscope beam and detector parameters. monte carlo methods allow a wide degree of freedom in specifying simulation conditions for sample composition and geometry. published examples often limit the simulation to two-dimensional structures, with a zero-diameter electron beam and all secondary or backscattered electrons (bse) collected. a more useful and realistic approach will take into account the effect of beam diameter, and detector geometry and gain characteristics. these effects for simulated bse images for three-dimensional patterned structures of carbon on a silicon substrate are shown in figure . the monte carlo simulation used here is based on a single-scattering procedure with the rutherford scattering cross section and the bethe energy loss formula modified for low-energy primary electrons. the program is a further development of a single-scattering monte carlo program (in pascal) widely distributed by david joy and modified later by russ et al. the program is written in c language and runs on a sun workstation. the program can scan the beam position in x and y directions over an arbitrary multielement and multilayer sample with topographical features to produce images. using the monte carlo procedure, it is possible to extract information about the position, energy, and direction of the electron at each scattering point. this allows tracking bses as a function of angle. if the dependence of detector gain on electron energy is added, then the details of signal formation can be modeled. the effect of electron beam shape and diameter on the image can be added by convolution after an ideal image is generated. figure shows the matrix representing a gaussian electron beam used in this work. an example of a simulated bse-sem image is shown in figure for a structure with carbon features on a silicon substrate. fig. . contour plot of gaussian shape electron beam with standard deviation of Å was used to convolute the data obtained for a zero-width electron beam. fig. . bse image simulated using a zero-diameter electron beam (left) and after convolution using the gaussian beam width shown in figure (right). the simulation was performed for kev primary electrons with trajectories for each point. the image represents × pixels. monte carlo simulations of electron scattering in a target normally use one of two elastic cross sections, either the screened rutherford cross section or tabulated partial wave expansions of the mott cross section. the screened rutherford cross section gives acceptable results for high energies and low atomic numbers, but mott cross sections are required for low to medium incident energies ( . - kev) and high atomic number targets. however, computations tend to be slow using tabulated data due to the need to interpolate between data points. empirical equations for the total and differential electron/atom elastic scattering cross sections have been found that can be substituted for tabulated mott cross sections in predicting backscattering coefficients. the total elastic mott scattering cross section is fitted by similar form to the screened rutherford cross section but contains three terms in energy in the denominator. the empirical total elastic scattering cross section is valid for atomic numbers up to and for energies from ev to kev: ( ) the fit to the differential mott cross sections is decomposed into two parts, one part being of the same mathematical form as the screened rutherford cross section (σ r ), and the second part being an isotropic distribution (σ i ). the screened rutherford part of the differential scattering cross section is first fitted to the half angle of the mott cross sections. this fit of the differential screened rutherford is in turn reduced to a fit of the screening parameter alone over energy and atomic number. in marked contrast to the screened rutherford cross section, the tabulated mott cross sections show only a small overall downward trend in half angle with increasing atomic number(z). implying an average rutherford screening parameter for all z, with e the electron energy, of: the ratio of the total cross sections (σ r /σ i ) between the screened rutherford part of the differential scattering cross section and the isotropic part of the distribution is fitted to the backscattering coefficients calculated directly from tabulated mott cross sections. the ratio of rutherford to isotropic cross sections is: ( ) figure shows a comparison of the calculated backscattering factors using the present empirical fit (solid lines) with those calculated using mott cross sections. the fit for al, cu, and au is good over the entire energy range. the fit for ag is moderate and the fit for c is high. however, most deviations are similar to differences because of the use of different atomic models in the mott cross sections and are acceptable. there are two major reasons why the simple monotonic eqs. - work well. first, the scattering of the electrons in a solid is a multiple scattering process. thus, many of the complex quantum interference effects are averaged out. second, the elastic backscattering is monotonic with atomic number. these two factors serve to smooth out the effects of the complex multidimensional cross sectional surface that is being fitted over z, e, and θ. reference iv- scanning vol. , supplement iv ( ) σ rutherford σ isotropic = e − z / z + z × e σ t = . × − z . (e + . z . e . + . z / e . ) a scanning interference microscope may be constructed by allowing light which has probed the object and light which has not probed the object (the reference beam) to interfere on a suitable detector. if the detector is large in extent, a conventional scanning interference microscope results, whereas if a point detector is used we have a confocal scanning interference microscope. the image in both cases may be regarded as a superposition of three terms. the first represents a normal conventional or confocal image depending on the kind of interferometer used. the second we will call an interference term image, whereas the third represents a constant background. it is possible to design a system in which the interferometer term image is identical for both conventional and confocal scanning systems in all respects, including optical sectioning. a simple scheme permits the interference term image to be detected separately and then processed in a variety of ways. let us consider two specific geometries. the first is an almost common path confocal system based on a single mode optical fibre. the interferometer in this case is confocal. simple image processing then permits surface profilometry to be performed. the second implementation is a conventional scanning interference system. the interference term image from this conventional image may again be isolated and processed to give a confocal image. in biology, the description and precise representation of microstructural forms is of increasing importance for threedimensional ( -d) computer image understanding, in particular to enhance -d visualization and analysis. the intermediate level of computer vision, located between the bottom layer (signals) and the top layer (model) is best suited as a starting point to improve multidimensional image understanding. we implement the concept of -d topology embedded in the bottom-up structure of data processing to enhance subsequent rendering and specific quantitative analysis. segmented and contoured serial section images require sophisticated algorithms to generate correct surface-rendered views. we have, therefore, developed a method to evaluate connectivities between sections, based on bijective correspondance analysis of the center of gravities of contours. these connections indicate nods and branches of -d structures and can be interactively edited in exploded views of the contour stack. because of the strong data reduction, such procedure can be implemented even in computer graphic systems with entry graphics. the topological skeletons are the backbone to render correctly biological structures of free forms, in particular those featuring frequent branching patterns. examples where such an analysis is successful include microvessel networks, dendritic trees, lung anatomy, or dental root canals (baumann et al. ) . moreover, the automatic labeling of connected structures gives rise to the analysis of topological criteria, which was only available in stereological methods until now. this includes criteria such as connectivity and branching angle, or higher representations of branching schemes. in return, topological connectivities can be used to improve the segmentation of images in the top down-process of data processing, or it can serve as embedded analytical graphics to improve volume renditions. the mammalian central nervous system (cns) contains at least ten times more glial cells than neurons. the three major populations of cns glia are astrocytes, oligodendrocytes, and microglia. astrocytes and oligodendrocytes together are often referred to as macroglial cells and arise from primitive neuroectodermal precursor cells, while microglia are derived from the mesodermal germ layer. it is thought that common precursor cells, known as o- a progenitors, can differentiate into either astrocytes or oligodendrocytes during gliogenesis. in contrast, the origin of microglial cells remains enigmatic, although there is substantial evidence that microglial cells arise from primitive hematopoietic stem cells that gain access to the cns at a very early stage of development. much of our knowledge about glia and glial cell function has been derived from histochemical and electron microscopic studies. astrocytes can be visualized reliably using immunohistochemical methods with antibodies against the glial fibrillary acidic protein (gfap). gfap is an abundant constituent of intermediate filaments which can seen ultrastructurally in most astrocytes. in addition, astrocytes also contain enzymes, such as nadph diaphorase and glucose- -phosphatase, which are readily detected by enzyme histochemical methods. we have focused much of our attention on developing methods for detecting oligodendrocytes and microglial cells in sections of rat brain. lectin histochemistry has been a particularly useful tool in studying these types of cns glial cells. using lectins i and ii from griffonia simplicifolia seeds with carbohydrate specificities against α-d-galacatose and nacetyl-d-galactosamine (glcnac), we were able to demonstrate selective labeling of microglia and oligodendrocytes, respectively. the b -isolectin from griffonia simplicifolia coupled to horseradish peroxidase (gs i-b -hrp) can be used to detect a membrane-bound glycoprotein on the microglial cell surface at all stages of cns development ranging from the early embryonic age to adulthood. in contrast, oligodendrocytes were found to express glcnac-containing glycoproteins in the perinuclear cytoplasm using biotinylated gsl ii. the perinuclear staining was determined ultrastructurally to be associated with golgi complexes. biochemical analyses using tricine/sds-polyacrylamide gel electrophoresis and western blotting with gsl ii showed the glcnac-containing glycoproteins to be insoluble, with molecular masses ranging from to kd. having available specific markers for the three major glial cell groups, we were able to combine lectin histochemistry with immunohistochemistry to perform double-labeling studies demonstrating specificity of each stain for a given glial cell type. following the study of glia in the normal cns, we went on to investigate glial cell reactions that occur as a consequence of nervous system injury or disease. it became immediately ap-parent that microglial cells were the major glial cell type responding to neuron injury. microglia not only proliferate and change their morphology in response to cns damage, but they can also vary their membrane phenotype by expressing new molecules on their surface. we found that antigens of the major histocompatibility complex (mhc), which are largely absent from the normal cns, are expressed de novo on microglia and related perivascular cells responding to neuron injury. our studies, which have included various neuropathologic conditions including stroke and brain tumors, have shown that the expression of mhc antigens, as well as other related immunomolecules, is always restricted to cells of the microglial lineage. these findings strongly suggest a role for microglia as indigenous immunocompetent cells of the cns. jeremiah r. lowney national institute of standards and technology, gaithersburg, maryland, usa a scanning electron microscope (sem) can be used to measure the dimensions of the microlithographic features of integrated circuits. however, without a good model of the electron-beam/specimen interaction, accurate edge location cannot be obtained. a monte carlo code has been developed to model the interaction of an electron beam with lines lithographically produced on a multilayer substrate. the purpose of the code is to enable one to extract the edge position of a line from sem measurements. it is based on prior codes developed at nist but with a new formulation for the atomic scattering cross sections and the inclusion of a method to simulate edge roughness or rounding. the code is currently able to model transmitted and backscattered electrons, and the results from the code have been applied to the analysis of electron transmission through gold lines on a thin silicon substrate, such as used in an x-ray lithographic mask. there is provision for both transmitted and backscattered electron detectors. by comparing the predictions of the code with measured data, it is possible to obtain edge positions to the order of nm, which is needed for the advanced lithography projected for the year . the uncertainty of these measurements is limited by the sample geometry and surface roughness and not by the measurement process. much of the improved code is devoted to the treatment of boundary crossings by the electrons. the present code allows for a substrate of at most three layers and one or two identical lines with a trapezoidal cross section on top. there is also provision for a symmetrical jog (i.e., a discontinuous change in the width of the trapezoid) along the edges to simulate edge roughness and rounding. the three layers that form the substrate, which are typical of an x-ray mask membrane, are silicon, polyamide, and chromium. the lithographically produced lines are gold, and the chromium improves adherence of the lines to the substrate. the code is easily modified to model other media by simply changing the atomic parameters in the input subroutine. the x-ray lithography mask, which has been used as a test sample, is a very good model system for the development of accurate sem standards because it provides a measurement of both transmitted and backscattered electrons for comparison with the predictions of theoretical models. a plateau in the transmitted and backscattered electron signal occurs as the beam traverses the sloping edge of the line trapezoid. this effect can be blurred by edge roughness, and the effects on the plateau of various widths and heights for the jog can be shown as well as the effects of rounding at the bottom of the lines and the calculated noise level for various numbers of trajectories. direct comparisons with measured transmission through an x-ray mask of gold on a silicon membrane can be used to demonstrate the determination of the edge of the gold line. figure shows the transmission (measured downward) along the axis of a gold line indicating the plateaus in the data near the middle of the edges. figure shows a simulation of the data with the plateaus at nearly the same location along the edges as in the data. this work shows how high-resolution metrology of the features produced by advanced lithography can be obtained with an sem. extensions of this code to modeling secondary signals as well as the effects of charging are planned. at philips research, a monte carlo program for electron microscopy is developed, which simulates electron-solid state interactions in the energy range . - kev. it is based on the program published by l. reimer (scanning , , ) . in particular, to ensure proper operation in the low-voltage region, mott cross sections for elastic scattering are calculated by numerically solving the dirac equation. the model for inelastic scattering treats inner shell ionizations as discrete events, described by a scaled gryzinski formula, whereas the effect of valence and/or conduction electrons is incorporated as a continuous bethe loss. this model, which differs from similar models presented in the literature, provides good fits to experimental stopping power data. the program is extended to include multilayer specimens, each layer composed of multiple elements. one of our objectives is the modeling of cd linewidth measurements in a high-resolution low-voltage scanning electron microscope. our monte carlo program can generate top-view backscattered electron (bse) and secondary electron (se) video profiles of lines with variable slope and pitch. we use the se generation model of d.c. joy (j microsc, , , ) , which is extended to account for the regions near the corners of the line profile. furthermore, the recollection of se by the sample is accounted for. as a result, realistic pure se profiles are generated. the primary beam parameters include the probe size and the depth of focus. determination of the top and bottom linewidths via the commonly used heuristic algorithms reveals the systematic errors of these methods. a simple example is the probe size dependence of the peak-to-peak width, which can be related to the strong asymmetry of these peaks. we expect that improved algorithms can be developed, which use a modest database acquired by monte carlo simulation. a scanning electron microscope (sem) fitted with a helium-neon laser interferometer is used to measure the widths of features on photomasks. in this way the magnification of the sem can be known very precisely. algorithms yielding good measurement repeatability, which use the back-scattered electron (bse) signal, have been developed (nunn , nunn and turner ) but in order to be able to relate measurements made on the image to the physical dimensions of the artefact, it has been necessary to model the image formation process. the essential details of the "plural scattering" model used in this work have been described by d.c. joy ( ) . typical geometries and materials of photomasks have been modelled along with a range of accelerating voltages and beam diameters to observe how the image is affected by the different parameters. more important, the modelled image intensity profiles are studied to relate the position of the physical foot of the sloping edges of actual physical lines to the broadened image intensity profile of the edges. although the "single scattering" model, also described by joy ( ) , is more rigorous and more applicable to the type of samples used in this work, it was not used in the first instance because of the much greater computer time required. results from the modelled profiles suggest that the position of the % thresholds of the bse image intensity profiles are located a few tens of nanometres away from the physical position of the edge. the exact offset depends on the angle between the sloping sides and the horizontal. the modelled offset increases from approximately nm for a vertical edge to nm for an edge angled at ˚ to the horizontal. the effect of the bse detector size and geometry on the signal detected has also been modelled. the model shows that for best performance the detectors should collect the largest possible solid angle, but that as long as the detector is placed symmetrically, the only adverse effect of a smaller detector on the image is a loss of signal to noise. measurement comparisons between optical microscopy and the sem corrected by the "plural scattering" modelled offsets have been encouraging (nunn and turner ) ; nevertheless, if time permits, a more rigorous study using more appropriate models should be pursued. audio enthusiasts carefully read the test reports when looking for their new equipment. however, usually no one tries to find out the frequency response, distortion or signal-to-noise ratio of a very expensive, complex system such as a scanning electron microscope (sem). sems were once used mainly as image-gathering devices, and in spite of certain obvious and sometimes serious problems they served as acceptable and effective instruments for many applications. these "image grade" microscopes are now being replaced with much better quality "measurement grade" instruments. the guaranteed resolution is no longer enough to ensure good performance and other factors must be considered as well. even though these newer systems have field emission of lab electron guns, the main advantage is in the built-in, computer controlled, digital image and data-processing capabilities. there are several alternatives to upgrading an existing microscope with an external, usually desktop computer-based measuring system. soon the sem, similar to a scanner or camera, will just be a part of an imaging network, and automatic image enhancement and processing will be commonplace. this presentation deals with a short description of the electrical properties of different components of an sem; the beam scanning circuitry, the video signal chain, sampling, analog to digital (ad) and digital to analog (da) conversion. it will relate their possible influences to the detected and observed signal to improve the imaging. this will also make possible better comparisons of measured data to the computer-modeled data. essentially, the main parts of an sem essentially are the electron source, electromagnetic coils, dc power supplies, (scan) generators, detectors, amplifiers and, finally, the displays. the quality of an sem image depends on the characteristics of the primary electron beam, the correctness of the beam scanning, signal detection, and the fidelity of the video signal chain. the correctness of the beam scanning not only means good linearity and proper amplitude and direction of the deflection of the primary and displaying electron beam, but the displaying or data assigning has to be in correct synchrony also. the deflection coils are fundamentally nonlinear and inherently have hysteresis; hence, correcting circuitry is indispensable. the total distortion caused by improper magnification, nonlinearity, and hysteresis can be greater than % and it is difficult to keep it below % . the fidelity of the video signal chain depends on its transfer function, noise, and distortion figure. the main parts of the video chain of a modern sem are the detector, amplifier, ad converter, computer memory (disk, imaging network), da converter, amplifier, and the display or printer. all of these parts make their contribution and form the overall characteristics of the video chain. to produce good quality images at a certain scan rate, the bandwidth of the video chain must be high enough to show the finest possible details of the image. the transfer function that describes the frequency or bandwidth and phase characteristics of a detector or an amplifier is very useful to characterize the video chain of an sem. for good quality tv frequency imaging, - mhz bandwidth is required, but for slow-speed image collection this bandwidth can be surprisingly low, that is, - khz. noisy signal means loss or lack of information. at a certain noise level, details that otherwise would be in the video signal cannot be seen. the visual effects of noise are closely tied to the resolution, appearance, contrast, brightness, and other aspects of the image, as well as intensity distribution and other properties of the noise itself. a db ( - ), signal-to-noise ratio gives a reasonably clean image, but more noise degrades the ability to differentiate two areas of different brightness by eye. to turn an analog signal to digital data, ad conversion is needed. usually there is a sampling and hold circuitry that keeps the analog signal unchanged for the time of conversion. depending on the design, this results in smaller or larger signal loss because just a fraction of the analog signal is collected due to the relatively short sampling and long conversion time. well-designed circuitry, for example, a gated integrator, can improve the signal-to-noise ratio, but the quantization process in the ad converter itself introduces noise also. modeling data with monte carlo or other techniques has to include the shortcomings of the real, nonideal measuring tool, that is, the sem with all its associated components. by knowing the transfer function, noise, and distortion figure in digital form, it is relatively easy to obtain more accurate comparison of the measured and calculated signal (fig. the calculation of image contrast in the scanning electron microscope (sem) can be done using monte carlo techniques if the electron trajectories can be calculated through the composition profiles in the specimen. this has been done for the case of a discrete one-dimensional composition variation in the incident beam direction , but most of the programs that are available are designed to calculate signal intensities from samples of uniform atomic number and density (e.g., reimer and stelter ) . nevertheless, the calculation of electron trajectories in a three-dimensional heterogeneous microstructure has not been reported. the basic idea behind a monte carlo calculation is that an electron, in penetrating a solid, will undergo a series of predictable elastic collisions (electron-atom) which change the direction and inelastic collision (electron-electron) which change the energy. in figure , which is the example of a calculation in a lead tin eutectic with somewhat unrealistic geometry, the electron enters the sample and is scattered to a new coordinate location. since the directional change and distance scattered are sensitive to the variations of atomic number, atomic weight, and density, the electron might travel to location in the block of the lead-rich position of the pbsn eutectic, scatter into the position , and then go on to position of the tin-rich matrix. thus, depending upon the details of the microstructure, that is, the size and location of the second phase, the image will appear different. the anticipated backscattered intensities from this rather unrealistic microstructure are shown, for example, in figure as the block varies both in size and location below the sample surface. we clearly see that the backscattered signal in this case is much more sensitive to the position in the specimen of the sec- iv- scanning vol. , supplement iv ( ) fig . the geometry of the sample and a schematic of the electron trajectory for the calculation. intensity ond phase rather than the size distribution, but for more realistic microstructural geometries such a calculation would be useful in determining the presence of alternative phases in a complex assemblage. terference microscopy - is based on the observation that a simple single-arm interferometer can be constructed by allowing light from a laser to be back-reflected from a target and to reenter the laser resonant cavity to produce a modulation of the laser light-intensity. [ ] [ ] [ ] the resultant light modulation is dependent on the optical phase (optical path length traveled) and amplitude of the reentry light. if the target is moved over several wavelengths, the laser light intensity displays a cos(z) dependence; for a mirror target, the light modulation index (i-i o /i+i o ) can be as great as . . these observations were denoted as laser-feedback interferometry (lfi); this phenomenon has been characterized and analyzed several times in the past years. [ ] [ ] [ ] [ ] [ ] [ ] [ ] the theory of lfi , , provides an understanding of the harmonic content of lfi signals; they follow a bessel-function dependence, a property that is useful in designing electronic feedback circuits necessary to produce a practical laser-feedback microscope. lfi can employ either diode or gas lasers as the interactive laser source/detector. using a he-ne laser with a high-reflectivity output coupler mirror (≤ %), it is possible to measure surface profiles of highly-reflecting surfaces (e.g., metals or high index-of-refraction materials such as silicon) with axial detail as small as nm and surface vibrations up to - mhz can be measured down to picometer amplitudes. in addition, weakly backscattering materials (e.g., biological cell surfaces and cell components) give sufficient signals to provide a useful method of high-resolution imaging in biology. a versatile laser-feedback microscope (phoebe) has been constructed having the following properties. the he-ne ( . nm) laser incorporated is a milliwatt, linearly-polarized output unit with special mounting to minimize output mirror movement. access to the low-power rear laser beam allows the laser intensity to be measured directly by a silicon photodetector. the main beam is expanded to fill the back aperture of any type of microscope objective (air-, water-, or oil-immersion) suitable for the microscopic examination being undertaken. an x,y scanning stage moves the sample under the laser beam in a raster fashion; pixel × pixel image frames are obtained in < s. axial (z) motion, furnished by a tubular piezoelectric transducer, provides both a modulation signal input for the electronic feedback circuit ( khz) and repositions the sample at each point. the electronic feedback circuit maintains a fixed distance between the output coupler mirror of the laser and a point in the object being imaged. the correction (output) signal of the feedback circuit then gives a measure of the surface topography of the sample. concomitantly, the laser intensity modulation gives a measure of the surface reflectivity of each point on the sample. these two images are digitized and stored in the computer memory. all microscope control functions have been consolidated in a dedicated small computer allowing samples to be run without interruption by the resetting or realignment of the microscope. phoebe scans for biological use range from µm × µm to µm × µm. when an air objective is used, surface topography images display a quantitative measure of height; for fluid-immersion objectives, the measured heights are shortened by a factor dependent on the index-of-refraction of the fluid (water or oil). the confocal property of this scanning microscope is due to the requirement that only backscattered light reentering the cavity-resonator mode (tem oo ) of the laser is effective; the through-focus response of lfm verifies this property in comparison with a confocal pinhole placed at the focus of the laserbeam expanding lens. the lateral resolution of phoebe is nm as determined by imaging silicon-based resolution standards. an important result of measurements with the phoebe lfm is that high-contrast images can be obtained from biological samples placed on a plastic or glass substrate (e.g., a microscope slide or petri dish). in contrast to images of samples with sharply defined index-of-refraction boundaries, what is measured in this case is a point-by-point optical path length difference with the substrate furnishing the majority of the back-reflected, lfi-measured light. other imaging modes include the use of interferometric optical sectioning and the buildup of three-dimensional structures from a series of twodimensional sections. because of the coherence requirement of lfm, the use of fluorescent labels and the detection of fluorescence is precluded; however, other reflective labels may be employed to gain back some of the advantages of that method. - , - ( ) . sarid d, iams d, weissenberger v, bell ls: compact scanningforce microscope using a laser diode. opt lett , lett , - lett , ( university of california, irvine, california, usa nmr microscopy is one of the newly emerging tools for the high resolution three-dimensional ( -d) imaging of live animals and plants for biological as well as medical research. more recent applications and developments include such things as porous materials and microflow, essential for oil research. one of the main interests of nmr microscopy, however, lies in the fact that the method is truly a noninvasive -d high resolution imaging tool with which µm resolution can be achieved. however, nmr imaging, especially nmr microscopy, has a number of formidable difficulties, namely, small signal-tonoise ratio due to the inherently small object size, diffusion and bandwidth limitations, and other inhomogeneity effects such as chemical shifts and susceptibility. among others, diffusion problems due to the random brownian motion of (water) molecules appear to be one of the fundamental physical limitations to nmr imaging, especially in high-resolution microscopy where these molecular diffusion distances are close to the resolution limits. although the diffusion effect in nmr has been studied extensively, the effect on nmr microscopy has not as yet been observed experimentally due to the current limitations of the nmr microscope. theory, however, suggests substantial resolution broadening in nmr microscopy if molecular self-diffusion prevails, especially when we deal with molecules with large diffusion coefficients. resolution limits on nmr microscopy especially include diffusion limits, namely, effects of phase variation due to molecular self-diffusion during data acquisition, effects of signal attenuation and related line broadening, and some diffusion effects of molecules which, when confined in boundaries or walls, exhibit anomalies in resolution observation in nmr microscopy. the first two are based on free-water molecules, while the third is based on the model of water molecules confined by boundaries, which have significant physical consequences. diffusion effects limit resolution in microscopic imaging, as does the phase factor which is related to the finite bandwidth of the imaging instrumentation: that is, the available gradient strength and acquisition time, both of which are intimately related to the diffusion effect. an x-ray microtomographic system is being developed at our laboratories amil & arts. a generalized feldkamp cone-beam reconstruction algorithm was already developed for our system (wang et al. ) . the generalized algorithm is approximate, but quite accurate and computationally efficient. under some feasible conditions, the generalized algorithm produces exact volumetric reconstruction for longitudinal invariant specimens and exact transaxial reconstruction for a point source contained in that transaxial plane. in the generalized feldkamp cone-beam reconstruction, a transaxial slice is reconstructed using projection data collected from a scanning turn of ˚ angular range. in fan-beam reconstruction, there actually are two complete sets of projection data over a full-scan range. exact reconstruction can be achieved using only projection data corresponding to a half-scan. in our cone-beam system configuration, it can be appreciated that there are "approximate redundancies" in data acquired along geometric rays that would be identical after projection onto a transaxial plane. there are various weight functions for half-scan fan-beam reconstruction. with gullberg and zeng's weight function, a half-scan generalized feldkamp cone-beam algorithm is obtained for less longitudinal blurring: ( ) where ( ) ∆ is an additional angle for a smooth transition between essential and duplicated radon regions, and φ(z) is an offset specific to the longitudinal coordinate z. optionally, cone-beam projection values associated with appropriate pairs of opposite rays can be lineraly interpolated to synthesize needed fan-beam projections for transaxial reconstruction. at an additional computational cost, the interpolation-based cone-beam algorithm allows exact reconstruction if the longitudinal specimen variation is linear. numerical simulation results demonstrate the feasibility of our extended algorithms. wang g, lin th, cheng pc, shinozaki dm: a general cone-beam reconstruction algorithm. ieee trans med imag ( ), - ( ) biological specimens can be preserved by rapid freezing, a process which takes just a few milliseconds and is termed "cryofixation." it is an alternative to chemical fixatives which may take many minutes to penetrate a specimen and even longer to fully stabilise cellular components, thereby compromising their ultrastructural and chemical integrity. when cryofixation is performed properly, the water molecules in the specimen do not have time to form ice crystals and the specimen is preserved in a near life-like state. the time-scale of cryofixation means that short-lived phenomena can be captured and preserved. when this aspect is combined with electrical or chemical stimulation in a controlled experiment, then cellular processes can be studied by freezing the experiment at predetermined time intervals, so that a series of transient steps are preserved. besides electrical and chemical stimulation, other methods have been combined with cryofixation; these have involved electrophoresis, electroporation, temperature-jump, and flash photolysis. electrically stimulated muscle was slam-frozen by sjöström et al. ( ) and van harreveld et al. ( ) . their methods held specimens in defined physiologic states: true time-resolved freezing was introduced by heuser et al. ( ) , who showed that ultrastructural differences could be observed in synaptic events at neuromuscular junctions between and ms after stimulation. chemical stimulation is effectively performed by a chemical flow method, involving the rapid mixing of small specimens with a stimulant from different syringes and flowing them for a predetermined time along a reaction tube before spraying the reactants into a coolant, thus quenching the reaction (knoll et al. , rand et al. . electrophoresis involves the movement of particles in an electrical field and was used with freeze fracture to study the diffusion rate of intramembrane particles (sowers and hackenbrock ) . electroporation uses a radio frequency field to induce transient pores in cell membranes (chang and reese ) . optical stimulation has been used to cause a temperature jump to investigate temperature effects, for example, a ˚c jump after ms exposure to a xenon lamp, on the ultrastructure of lipid specimens (chestnut et al. ) . flash photolysis methods have been used to release caged photolabile chemicals in the study of the dynamics of the actomysin cycle (funatsu et al. , ménétret et al. . when the available stimulation methods are considered with the available freezing methods (namely, slam, plunge, jet, and microdroplet-spray freezing), then it becomes clear that there is promising scope for the study of dynamic cellular processes using electron microscopy. the combined methodology integrates the temporal resolution of rapid freezing with the spatial resolution of the electron microscope. chang dc, reese ts: changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy. biophys j , - ( - ( ) universität des saarlandes, homburg-saar, germany contrary to standard preparation at ambient temperature, there is some confusion on what is optimal in cryopreparation of biological specimens for subsequent electron microscopy (em) investigation. this results mainly from the diversity of methods as well as instruments described and the rapid development of these techniques in the preceding years. since the big advantages of cryopreparation in various fields are often hidden by the choice of unsuitable or even antiquated techniques, it seems to be justified to report about the recent developments in this field and to classify different methods for different purposes. this tutorial is based mainly on experience in transmission electron microscopy (tem). nevertheless, most of the information may also be useful for scanning electron microscopy, since a proper preparation has the same importance in both fields. without doubt, cf is the most important first step in most cryopreparations with a big variety of different techniques. the simplest procedure (plunging or immersion cryofixation = icf) is best suited for vitrification of thin suspension layers ("bare grid" or "ice embedding") for subsequent cryo-tem in the frozen hydrated state. high pressure freezing (hpf) reduces ice segregation in suitable specimens (e.g., thin plant leaves or rigid tissues such as cartilage). the main problem of hpf in soft tissues results from the indispensable dividing of the specimens into small pieces. impact freezing on a metal mirror (mmf) is well suited for suspensions such as hpf or double jet (djf), but mmf has an advantage for soft tissues, since it is suitable for larger specimens, that is, tissue slices > mm . as far as liquid cryogens are concerned (icf, djf), ln or partially frozen n -slush are not to be recommended; ethane gives the best results. propane is well suited and less expensive for routine applications. as an alternative to cryosectioning, these more time-consuming preparations gain continuously in importance for cytochemistry. even element analysis is possible in certain cases. only for simple morphology or morphometry additives (e.g., oso , uo -acetate, aldehydes) are short drying times in freeze substitution (fs) or freeze drying (fd) possible. proper drying by fs or fd without additives seems only possible for small objects (diameter < . mm) in periods between - days at − ˚ to − ˚c. otherwise, severe artifacts by thermal collapse phenomena and redistribution effects result. a suitable instrumentation is of great importance for this long drying time. the results are excellent and the advantages are striking as long as sufficiently long drying times are employed. in addition, low-temperature embedding (lte) in special acrylics improves the results. care has to be taken in handling these monomeric resins, since some of them are strong allergens. sugar protected specimens are easy to section on the dry knife at − to − (− )˚c for subsequent histochemistry of macromolecular components. overall morphologic preservation is rather poor in comparison with fs/fd/lte. larger areas are mostly not obtainable, but speedy work and results within hours instead of days or weeks are possible. sectioning can be considerably improved by the use of cryo-diamond knives together with an ionizer (useless without antistatic tool). sectioning below − ˚c down to − ˚c is possible with cryo-diamond knives on an ionizer, if the specimen is well frozen (no ice segregation) and the cutting area << . × . mm . trimming with a diamond trimming tool is advantageous. good preconditions are given after hpf or mmf. the advantage of mmf is the mirror-like, well-frozen surface, which can be easily trimmed and orientated to the knife edge. cryotransfer to the tem/stem or proper fd for fedx pose no severe problems (fd for > h at − ˚c). most of the different actual cryomethods (except fresh frozen cryosectioning < − ˚c for cryo-tem/stem) allow routine work, if the most suitable method is carefully selected and modern instrumentation is available. in all cases, the additional effort and investigation are definitely justified by better and more reliable results (literature and reprints on request). paul walther, renÉ hermann, martin mÜller laboratory of em , department of cell biology, eth zurich, zurich, switzerland the reason for using low temperatures for preparation and microscopy is the decreased mobility of atoms and molecules reducing the danger of artefact formation. for scanning electron microscopy (sem) cryotechniques have the following advantages. . cryofixation (rapid freezing) is the fastest way to immobilize a biological sample at a defined physiologic state. thereby, all processes in a cell are arrested within milliseconds. chemical fixation, in contrast, takes seconds or minutes to act, leading to unpredictable osmotic effects and redistribution of cellular compounds. . a frozen biological sample behaves like any other bulk specimen, and redistribution of substances is almost excluded. inner structures can be made amenable to the electron beam by cryofracturing or cryosectioning. . the conductive metal layers have a finer grain size when applied to cold samples, because diffusion of the metal atoms on the surface is reduced. . the frozen sample is analyzed in the sem by use of a cold stage. this prevents volume changes due to drying artifacts. in addition, hydrocarbon contamination due to irradiation by the electron beam is greatly reduced at cold temperatures. for most applications in biology, cryotechniques are only used for some but not for all preparation steps; for example, samples are cryofixed and then dehydrated by either freezedrying or freeze-substitution and afterward stored and imaged at ambient temperature. for high resolution sem, it is advantageous to coat also dry samples at cold temperatures in order to obtain a finer grain size and to observe the samples at cold temperatures in the sem to reduce hydrocarbon contamination. ( ) a major limitation of cryotechniques is distortion of the ultrastructure due to ice crystal formation during freezing. high pressure freezing allows for direct cryofixation of living samples within minimal or no ice crystal artefacts up to a thickness of several µm. ( ) the problems of water vapour contaminants that condense on the sample during preparation and microscopy were well investigated for the tem freeze-etching technique in the s and s. during the last years, this knowledge has been adapted for the construction of cryo preparation systems for the low-temperature sem. ( ) drying artefacts such as shrinkage are omitted by observing fully hydrated frozen samples. on the other hand, the ice covers many structures of interest and therefore often needs to be partially removed, either by freeze-drying or by freezesubstitution. however, removal of water bears the risk of drying artefacts. ( ) hydrated samples are extremely sensitive to the electron beam. the high surface-to-volume ratio inherent in particulate samples, and the fact that we already live in a sea of possibly contaminating particles, necessitates special care and understanding in particle preparation. selection of reagents and handling practices can be critical. for instance, in a hypothetical case, a particle analyst reports that his sample consists of approximately five major inorganic phases. the first has a rounded morphology with crystalline overgrowths, the light element content of the second is depleted, the crystalline structure of the next is damaged, the fourth occurs at a % volume level, and the fifth consists of glass shards. in this example, these results are essentially worthless. phase one was soluble in a liquid used in the preparation process and then reprecipitated upon evaporation. the light elements of the second were leached because of the use of the same solvent. the third was attacked by hf formation during ultrasonification in a fluorinated solvent. the fourth was originally present at a % volume level, but was preferentially attracted to the sides of the storage container prior to sample preparation, biasing the sampling; and the fifth is not part of the original sample, but comes from the ground glass neck of one of the reagent bottles. decisions on reagents and methods are crucial but unfortunately cannot satisfy every concern. for instance, reagents chosen for inorganic preparation should be nonpolar when easily leached elements such as boron or lithium are of interest, or if surface oxidation or ionic dissolution is a concern. but nonpolar solvents can increase particle agglomeration problems due to the lack of charge dissipation. therefore, wise choices in preparation methods are strongly tied to the objectives of the analyst, and sometimes multiple methods performed in parallel with additional analyses may be required to obtain truly representative results. contamination often is a concern, especially when performing high resolution work on a trace particle constituent. suspended urban air particulate is typically - µg/m , mostly in the . - . µm range and usually with less than particles per cubic centimeter above µm. supermicrometer-sized par-ticles can constitute µg/m . these particles may settle out gravitationally, electrostatically, or may simply happen to intercept the surface of the sample mount or sample particles. a large background can accumulate on unprotected substrates. the use of easily charged containers such as polystyrene disposable petri dishes or polyethylene centrifugation cones can further complicate dust collection or an additional problem, sample losses, due to electrostatic forces. the use of light microscopes for particle preparation makes possible a variety of preparation methods including micromanipulation. uses include ensuring even particle distributions on mounts, precise particle positioning, particle size reduction, washing unwanted films or residues off of particles, and many more. a light microscope can also be used to make some helpful observations such as specific gravity, population densities, refractive index, morphology, size, solubility, film thickness, and color. "micro world" effects often have to be provided for when performing preparation on a microscale with a microscope. for example, the polarity of solvents sometimes must be sacrificed for lower evaporation rates. reduction in evaporation rate can be aided by the use of small transparent covers, air movement shields, or by the choice of a combination of substrate and solvent that have poor affinity for one another (the resulting bead-ing-up action decreases the surface-to-volume ratio). the insertion of a "reservoir object" such as a probe tip at °"holding" a liquid droplet against the substrate, or a coverslip from the edge of which a supply of underlying liquid can be obtained, are examples of creative procedures that may be used (fig. ) .recommended polar solvents include heptane and cyclohexane. these avoid hfc environmental concerns, evaporate readily, and avoid halogen interactions. isoamyl acetate, flexible collodion, and formvar in ethylene dichloride are preferred micromanipulation and mounting media. microscale fume "hoods" protect microscope optical coatings and personnel. for containers, glass or static dissipator-coated plastics are preferred to ordinary uncoated plastics. if clean rooms are not available, static dissipators (physical or chemical), laminar flow work benches, single hepa-filter curtained areas, and modified work practices are inexpensive solutions. syringe filters help provide easy to handle point-of-use contamination control for reagents. recommended micromanipulation probes include tungsten and specific animal hairs. intermediate substrate surfaces may employ temporary teflon or paraffin coatings. other procedures include ashing, centrifugation, filtration, ultrasonification, and the exploitation of effects observed during micromanipulation. an approach for the indirect visualization of biological material in transmission electron microscopy (tem) is the freezeetch or freeze-fracture/replica method. the preparations steps ( fig. ) of this purely physical method include: (a) fast fixation and stabilization by quick freezing (in ms vs. min necessary for conventional chemical fixation); (b) creation of clean fracture faces with a fracturing cryotome (high vacuum required); (c) replication with electron beam evaporation (ebe, high vacuum required); (d) three-dimensional imaging of fracture faces and surfaces (with structural resolution of - nm); (e) high stability of the pt-c replicas in the electron beam (selection of relevant details) and possible long-term storage (weeks to months). the important drawbacks are that the replicas have to be cleaned (subsequent changing of several cleaning solutions over hours or days), picked up (disintegration of highly structured replicas happens frequently), and can be viewed only as a result of one single fracturing event. specimens providing iv- scanning vol. , supplement iv ( ) fig highly redundant structures and relatively smooth fractures, such as cell suspensions or o/w emulsions, were investigated using freeze fracture/replication and ambient temperature transmission electron microscopy (at-tem). freeze-drying is comparable to freeze-etching ( fig. ) , but with the important difference that all the water is removed from the specimen by sublimation. in tem, scanning electron microscopy (sem), or scanning tunnelling microscopy (stm), small cell components or particles, such as viruses, can be imaged together with the replication layer on top. in freeze-fracturing for low-temperature sem (lt-sem), most advantages of tem cryopreparation are kept. the preparation includes: (a) quick freezing; (b) fracturing under high vacuum conditions (c) coating with a planar magnetron sputter source (pms, ar as working gas); (d) -d imaging and observation of changed conditions (in situ etching); (e) high signal/noise ratio of pt or w coatings for secondary electron imaging and low yield from cr for backscattered electron detection of immuno-gold labelled specimens. the specimens can be imaged directly eventually after multiple fracturing (i.e., searching of distinct structures) in a different situation (i.e., fully hydrated or partly freeze-dried, coated or uncoated), in a "close to nature state." however, new problems have to be solved in order to get thin reproducible, conductive coatings, without superimposition of specimen and coating film structures and direct imaging, reducing contamination and beam damage, or long-term low temperature storage. moreover, the coating film thickness, the ice in the frozen bulk specimen, and, most important of all, the type of sem are now limiting the structural resolution. the biological relevant details of results obtained with low-temperature sem in a conventional fieldemission sem can be compared with those achieved with routine freeze fracture/replication and tem. according to honig and hook, the physics of water in high vacuum play an ultimate role in the application of these techniques (fig. ) ; therefore, only this knowledge in combination with cold stages and high vacuum technology enable the application of state of the art preparations of biological material for electron microscopy. the cryo-jet freezing technique provides a means for the in situ preservation of diffusible ions in mouse spinal cord explants after trauma. using the technique, the effects of trauma, in particular the intracellular shifts of calcium and other diffusible ions, can be analyzed and recorded by qualitative and quantitative electron probe microanalysis (epma). chemical fixation must be avoided if one intends to attempt the epma localization of diffusible substances in cells and tissues. the goal of the cryo-jet method is to confine axonal components and chemical elements to a biologically natural position. trau- matized mouse embryo cord explants were compared with the intact rat spinal cord trauma model, using epma studies of perturbations of calcium and other ions that are reported to be located in the axonal cytoskeleton. icr mouse embryo spinal cord explants were grown to - -day maturation in maximow chambers. cords that survived stringent elimination examination criteria and exhibited minimal degeneration or spontaneous necrosis were randomly divided into four groups. group i served as experimental controls; group ii was traumatized by dropping a mg weight through a . cm glass pipette positioned directly over the culture and cryojetted h after trauma. representative explants from i and ii were freeze-substituted and embedded in epoxy for comparison. by light microscopic examination of the whole mount, living culture, the site of impact could be determined within - min. the impact zone became markedly dense when compared with the surrounding, uninjured tissue. toluidine blue of the freeze-substituted sections revealed the site of impact on the surface and path of the trauma damage through the explant. control cultures with spontaneous necrosis could be distinguished from the experimental group, because the traumatic necrosis was confluent and often even wedge-shaped. within the traumatized zone, nerve fiber alterations similar to those observed in in vivo spinal cord trauma and calcium toxicity could be found, that is, granular degeneration of axoplasm, pleomorphic spheroids, tubulovesicular profiles, and myelin showing adaxonal, periaxonal, and intramyelinic vacuolization. light microscopy sections from the freeze-substituted explants were noted to be well preserved at the surface and into the culture for - µm (fig. ) ; however, some ice crystal damage was noted deep within the body of the culture. previous studies that compared metal mirror and plunge freezing results with the propane jet methodology reported here confirmed that propane jetting is the method of choice for preserving organotypic spinal cord tissue cultures. x-ray microanalysis of cryosections of the cytoskeletal tubules, cross bridge structures, mitochondria, and the myelin sheath of in vitro spinal cord explants, compared with the in vivo model of spinal cord trauma, has yielded unique data concerning the ionic changes that occur as a result of trauma or experimental manipulation. after trauma, populations of axons were found that could be distinguished by their elemental compositions. one group was similar in elemental composition to controls, although the morphology was dissimilar, while a second group had - % lower k and - -fold elevated ca (fig. ) . the response of axons and the associated myelin was paired, that is, loss of k and gain of na within the axon was always accompanied by comparable changes in the myelin and vice versa. the morphologic appearance of axons was not a predictor of the elemental composition. the cardiomyopathic (cm) hamster manifests cardiac dysfunction from an early age, with the disease ultimately progressing to congestive heart failure. on the basis of significant elevation in the ca + content of the cm hearts, as well as significantly increased ca + in the mitochondrial (mt) fractions and an increased density of voltage-sensitive ca + channels, previous studies suggested a ca + overload in the cm hearts (sen et al. ). evidence has also been presented for an increased sensitivity of cardiac muscle cells of cm hamster hearts to an external ca + "stress," that is, any manipulation which increases ca + influx into the cells (hano and lakatta , sen et al. ). on the other hand, ultrastructural studies have shown localized focal lesions from an early age, believed to be due to local cell injury. the cardiac myocytes within these areas suffer irreversible damage, becoming ca + overloaded as demonstrated by measurements of mt and a-band (ab) ca + content by electron probe microanalysis (epma) in a previous study from our laboratory (bond et al. ). this study also demonstrated that the vast majority of cardiac myocytes throughout the cm hearts not only had a normal ultrastructural appearance, but also showed no elevation of subcellular ca + content. an alternative explanation for impaired contractile function may be a decreased amount or availability of ca + stored in the sarcoplasmic reticulum (sr) for stimulated release and activation of contraction. to investigate this question, we have utilized epma to measure ca + content directly in the junctional sr (jsr), as well as in mt and ab of the cm hamster heart, either under control conditions or, alternatively, after pretreatment with the ca + channel agonist, bay k , in order to increase ca + influx into the cardiac muscle cells. isolated papillary muscles from normal and cm hearts at days of age were stimulated to contract electrically, and parameters of isometric contraction were recorded at l max . muscles were randomly assigned to one of three protocols: ( ) eleven cm and normal papillary muscles were used to construct dose/response curves to the ca + channel agonist, bay k. after stabilization at l max , cumulative doses of bay k ( - m to - m) were added to the muscle bath. all contractile parameters were recorded from the muscle at each dose. ( ) for epma, five cm muscles were frozen, after stabilization at l max , at peak +dt/dt and five muscles during relaxation. ( ) ten cm papillary muscles were incubated with single dose of - m bay k in order to elicit a maximal inotropic effect. once the contractile response to drug addition had stabilized, the muscles were frozen either at peak +dt/dt (n= ) or during relaxation (n= ). ultrathin cryosections were cut from the surface of the frozen muscles and freeze-dried overnight. subcellular ca + content (ab, mt, and jsr) of cm muscles was measured by epma in stem mode in a philips cm scanning transmission electron microscope (fig. ) . measurements of baseline contractile function revealed a significant decrease in develop tension (dt) (from . ± . g in cm muscles to . ± . g in normals), +dt/dt ( . ± . g/s to . ± . g/s) and a decrease in −dt/dt ( . ± . g/s to . ± . g/s) in cm versus normal hamster. there was no significant difference in values of resting tension (rt). the inotropic response to increasing doses of bay k was markedly blunted in the cm muscles compared with controls, suggesting that even when ca + entry into the cardiac muscle cells is increased, force development is still impaired. a comparison of elemental content (na, mg, p, s, cl, k, and ca) of ab and mt between experimental groups revealed no statistically significant differences. in addition, no differences in elemental composition of ab and mt were observed compared with our previous measurement on normal hamsters frozen during contraction and relaxation (moravec and bond ) . the amount of ca + stored in the sr of the cm muscles that were rapidly frozen during relaxation (in absence of bay k) was . ± . mmol/kg dry weight ( fig. ) (left panel, rel); however treatment with the ca + channel agonist bay k significantly increased the size of the store to . ± . mmol/kg dry weight (right panel, rel). thus we conclude that ca + uptake into the sr of cm muscles was enhanced as a result of bay k treatment. the contractile data show that the amount of ca + that can be released from the jsr of cm muscles during a cardiac twitch is very small (equivalent to the difference between the relaxed and contracted values) in "control" (untreated cm hamsters, left panel); however, treatment with bay k increases the sr ca + load in the relaxed muscle, with little demonstrable effect on the amount of ca + remaining in the sr at peak +dt/dt (right panel, cont), resulting in a significant increase in the amount of releasable ca + in the sr store. the total ca + content measured in the relaxed bay k treated muscles was, nevertheless, considerably less than previously measured in normal papillary muscles rapidly frozen during relaxation. in summary, these data suggest that ( ) a ca + deficit, as opposed to a ca + excess or ca + overload, may be an important factor contributing to the cardiac dysfunction in cm hamsters, and ( ) that, specifically, impaired ca + regulation by the sr may result in this ca + deficit. the application of automated scanning electron microscopy (sem) to the analysis of particulate populations is in many ways a unique application of microanalytical instrumentation. when applied to the analysis of large numbers of particles, the sem is used as both a microanalytical and a macroanalytical instrument. as a microanalytical instrument, the sem provides single-particle compositional and morphologic information that is not available from the conventional macroanalytical techniques used in particle analysis such as atomic absorption and instrumental neutron activation analysis. as a macroanalytical instrument, the sem, when used for automated particle analysis, provides information from which population characteristics can be inferred. for example, automated sem information often is used to determine the percent of an aerosol that originates from a given source by extrapolating the single-particle results (e.g., the number of particles containing major fe) to the entire particle population, that is, the air filter. this value is then extrapolated to the sampled air volume. while providing the analyst with a wealth of information, the dual role of the sem in automated particle analysis has several limitations that must be considered when doing an analysis. these limitations involve, among other things, the spatial dispersion of the particle sample; particle size distribution; analytical parameters such as magnification, accelerating voltage and electron dose; and the algorithms for determining particle composition and particle groupings. at nist we have been conducting a series of experiments to study the limitations associated with the quantitative elemental analysis of particles during an automated run. specifically, we have been evaluating the relationship of measured x-ray intensity to the accuracy, precision, and detection limits of automated x-ray analyses. analytical accuracy, precision, and detection will have a pronounced effect on the ability to separate particles with similar but different elemental compositions into groups. for this experiment, we developed an analytical glass series containing six glasses with varying amounts of uranium and lead ( table i) . samples of the different glasses were prepared as bulk-polished specimens and as particles. the results from the analyses of the bulk samples represented the "best case" that could be expected under a given set of analytical conditions since there were no particle effects involved. all analyses were done on an electron probe and a sem at kev and na beam current. dead times for the bulk and particle analyses were between and %. two separate counting times were selected for the experiment , s and s. these times resulted in the x-ray peak intensities for pb and u m xrays that are shown in table ii . glass k- was used as the standard for the quantitative analyses of the different runs. for the particles, both bulk and particle forms of k- were used as standards. the concentration of oxygen was determined by stiochiometry. the results of the experimental runs on the bulk and particle forms of the glasses are shown in figure a and b as plots of the u versus pb concentrations in wt.% (normalized to % total analysis) for the s data. the bulk plot shows a complete separation of the six different glasses, while the particle results show an overlap of adjacent glasses even at the s count time. the s data show a much stronger overlap between adjacent glasses for both bulk and particle morphologies. the analytical data were also processed with a clusteranalysis algorithm to determine the average silhouette width (asw) for both bulk and particle forms of the glasses at the different counting times, (fig. ) (kaufman and rousseeuw ). the asw is a robust measure of the cluster strength with a value approaching representing the maximum association among cluster members. since there are six glasses in the series, the asw for six clusters should be the highest. of all the different runs on both bulk glasses and particles, only the s data on the bulk glasses has the highest asw for six clusters. the results of this study indicate that the uncertainties associated with the shorter counting times in asem analysis may severely limit the ability to distinguish correctly between similar groups of materials. in addition, the uncertainties associated with particle analyses are considerably greater than those from bulk analyses due to absorption and mass effects. these greater uncertainties for particles underscore the need to define the strengths and limitations of asem analysis and to design automated methods that will maximize the information that can be obtained from a sample. botany department, university of georgia, athens, georgia, usa the monoblepharidales (chytridiomycetes) produce asexual motile reproductive structures known as zoospores through the cleavage of cytoplasm in the zoosporangium. although different aspects of zoosporogenesis have been studied in a number of zoosporic fungi, little is known about the events of zoospore formation in the sporangium of the monoblepharidales, or the chytridiomycetes in general. earlier studies of other zoosporic fungi proposed various spore formation events that have been challenged recently (hyde et al. ) , especially with respect to the relationship of vesicles, golgi, and cleavage furrows. hyde et al. ( ) concluded that all eukaryotic cleavage events may need reinvestigation. we are studying the various aspects of zoosporogenesis in monoblepharella using both chemical fixation and cryofixation methods to determine whether the cleavage events in this chytridiomycete are similar to those found in the study by hyde et al. ( ) . questions that remain include how the nuclei accumulate in the sporangium, how the cytoplasmic domains are established, and how each zoospore obtains its usual complement of cellular components. mycelia were induced to form sporangia by transferring the cultures to distilled water. sporangia were chemically fixed at different stages of development using a sequential aldehyde/ osmium fixation protocol. the resulting tissue was embedded in araldite/embed . freeze substitution fixation was also used for comparison of vesicle and cleavage furrow profiles. when the tissue was to be used for cytochemical localizations, osmium was omitted and lr white was used as the embedding medium for both fixation protocols. various dyes, lectins, and antibodies were used to localize organelles within the sporangium. nuclei were close to the plasma membrane as they moved from the subtending hyphal strand to the swelling tip of the developing sporangium. the centrioles were closely associated with the nuclei and oriented toward the periphery of the sporangium. nuclei then moved a short distance from the centriole toward the center of the sporangium. microtubules, originating from the centriolar region ( fig. ), were seen around the nucleus and extended into the cytoplasm, possibly delimiting the boundary of the forming zoospore. membranes forming the cleavage furrows appeared around the forming flagella near the kinetosome (fig. ) and also at specific sites along the periphery of the sporangium. the furrows continue to expand at both sites as extending sheets of membrane. dictyosomes with large numbers of vesicles were closely associated with the nuclei, suggesting a source of the membrane needed for furrow extension. er was first seen in large sheets in the central region of the sporangium. later the strands of er surrounded the nuclei prior to ribosomal aggregation. iv- scanning vol. , supplement iv ( ) fig. nucleus (n) with associated centriole (arrow) and microtubules (arrowheads) at periphery of sporangium. fig. developing cleavage furrow and flagellum (f). note coated region of furrow (arrowhead) and radiating microtubules (arrows). hyde gj, lancelle s, hepler pk, hardham ar: freeze substitution reveals a new model for sporangial cleavage in phytophthora, a result with implications for cytokinesis in other eukaryotes. j cell sci , ( ) center of ultrastructural research, barrow hall, university of georgia, athens, georgia, usa cryptocaryon irritans, a parasitic ciliate of many species of seawater fishes, has a complex life cycle consisting of feeding, resting, dividing, and infective stages. the disease, termed white spot disease, can cause extreme fish loss in marine aquaria and mariculture environments. cryptocaryon irritans attaches to the epidermis of the fish and feeds on epidermal cells. this form of the ciliate termed the "trophont" appears as large white spots on the fish. the trophonts grow in size and eventually leave the host. the free-living trophont settles down to the substrate and secretes a cyst wall. while in this resting stage known as the "tomont," the cell undergoes a series of unequal palintomic divisions to form daughter cells called "tomites." the cyst wall ruptures and free swimming ciliates known as "theronts" are released. the theronts represent the infective stage as they search for new hosts. different procedures were used to prepare tomite, theront, tomont, trophont, and cyst stages. organisms were fixed with glutaraldehyde and osmium tetroxide for transmission electron microscopy (tem). parduc's fixation (oso +saturated hgcl) was applied for the scanning electron microscopic (sem) work. the parasite's cytoskelatal framework was stained by using indirect immunofluorescence technique. for this purpose monoclonal anti-α-tubulin was used as a primary antibody, and rhodamine-labeled goat antimouse igg as a secondary antibody. fluorescently labeled cells were examined by laser scanning confocal microscopy. the trophont has an elongate body shape with a broad anterior end, a tapered posterior end, and is completely covered with somatic cilia. the cytostome is apically located and trophonts often were observed moving with their mouth part leading. kineties were arranged in a parallel fashion along the longitudinal axis of the cell and terminated in a ring around the cytostome. there is no oral membrane at the oral region. there are cirri-like structures around the oral opening as described by cheung et al. ( ) . these cilia are shorter ( - µm) and wider than somatic cilia. the cytopharynx is surrounded by ridges or oral ribs (nonciliated lining). large bundles of microtubules support the oral region. the theronts are oval to teardrop-shaped and completely covered with cilia. they have a ventral mouth with a slit-type structure in the middle. the cytostome covers almost / - / of the body. the cirri-like structures are found around the mouth and are similar to those in trophonts. the oral ribs are present but they are layered. based on sem and tem results, it was found that there were some structural differences in the cytostomes of trophonts and theronts. the mouth probably does not become functional until the theront has entered a host. the short and stiff cilia seem to be used for burrowing and the gathering of food particles. the role of the pellicle and cyto-plasm is discussed in relation to penetration into fish epidermis. we found that there are mucocysts in both trophont and theront, but not in tomont. theront mucocysts are concentrated around the slit type structure of the mouth. the secretion of mucocysts might contain enzymes that help the theront to penetrate into fish epidermis. they also might aid trophonts in feeding on fish tissue and have a function in cyst wall formation at the later stage. penetration into the fish tissue probably is started by mucocyst secretion that either enables the parasite to stick to the tissues or to help it enter the tissue by enzymatic reaction over the irritated areas. the theront may then utilize its relatively stiff oral cilia for burrowing into the these irritated areas. after burrowing into the epidermis of fish, the theront develops its oral apparatus and increases its size. trophonts eventually leave the host when they reach a certain size ( - µm). this study is supported in part by the national aquarium, baltimore, md. over the years that the fbi has practiced sem/edxa, the technology has grown in importance to be considered an essential tool for investigative forensic exams. because of the wide variety of applications, there is a corresponding vast array of preparation methods and analytical techniques. analytic techniques and sample preparation methods are inextricably linked, and although many are routinely applied, often only imaginative approaches serve to fulfill the desired analytic result. the scanning electron microscope (sem) practitioner is expected to be knowledgeable about the applications and proficient at the methods of preparation in order to utilize the sem to its greatest advantage. the main types of analysis practiced at the fbi include ( ) visualization of structure, including surface features and internal structure, ( ) inorganic elemental characterization, ( ) particle analysis, and most recently ( ) the use of a compositional data base for identification and association. sem is a powerful complement to light microscopy (lm) for low magnification morphology characterization and is unsurpassed for applications requiring greater depth of field than are available with lm. toolmark and fracture exams can be enhanced by stereo analysis. preparation can be minimal, and several electronic signals (bei, sei) are available. the preparation of cross sections permits the study and comparison of heterogeneous materials. embedment usually is necessary to support the object during cross sectioning. hard materials generally are polished by an adaption of metallurgical polishing methods, and soft materials generally are microtomed. sectioning often is possible by manual methods, without the use of a microtome. this method can reveal complex structures such as plating layers and document laminates. the most routinely applied application of edxa is the qualitative exam. it is part of the inorganic analysis scheme for material characterization. the qualitative exam frequently is combined with elemental distribution mapping to provide "compositional pictures." particle populations often are indicative of an environment and can be used to associate an item or individual with an activ-ity. too small for individual manipulation, they are most easily sampled by adhesive lift. additional methods involving separation and concentration often are effective. compositional characteristics of materials are stored in a database to permit comparison and identification of a questioned material. standard spectra are collected and a specific peak for each element is integrated above background and ratioed to the sum peaks from all elements. this value representing % x-ray counts is stored in a "periodic table" data base including standard information. in addition, the original spectrum is stored on disk and a hard copy is filed. the standard files include metal alloys, building materials, paints, tape adhesives, fingerprint powders, and cosmetics. the reference list can be queried for comparison to an unknown for alloy matching, identification of an unknown material, or manufacturer identification. the effectiveness of this method depends upon the compositional uniqueness of the material and the variation of composition within the class of materials to which it belongs. this project is in its infancy, with only several hundred entries to date. data entry currently is manual, although software currently is being developed to extract required data from spectra automatically and to export it to the database. the need for a vehicle for information exchange has been expressed within the community of sem users in crime labs. since an electronic medium such as internet was not feasible because most forensic laboratories are not electronically linked, a "newsletter" was produced to link laboratories involved with sem in forensic science and related areas, as well as individuals in industry and academia. timely and informal, it augments the professional publications and attempts to bring practical methods, reprints from obscure journals, translations from foreign publications, and questions/answers directly to the user. hamilton county coroner's laboratory, cincinnati, ohio, usa many examinations in the crime lab involve comparing questioned material from a suspect to known material from a victim. by characterizing the material it may be possible to establish a link between the suspect and victim. our trace evidence section characterizes materials by using a combination of analytical instruments. the infrared microspectrometer is used to determine the organic constituents, and the scanning electron microscope-energy dispersive x-ray spectrometer (sem/edxa) is used to analyze the inorganic composition. this approach is routinely applied to paint particles because paint formulations include both organic and inorganic constituents. analysis by sem/edxa is very valuable when more than one layer is present in the particle. the instrument can then be used in line scan mode to analyze each layer individually without separation. in addition to comparisons, the sem/edxa is used to identify materials. in bombing cases it may be difficult to identify the explosive as well as other components. large amounts of potassium and sulfur in residues from a pipe bomb indicate black powder as an explosive. if chlorine is also present then "pyrodex," a black powder substitute, may have been used. other applications, such as matching small fractures, make use of the superb imaging capabilities of the sem. the capability of maintaining excellent depth of field at high magnifications is particularly important when matching the ends of wires that have been pulled apart. this is exactly what was done in a case of tape players that were jerked out of victims' vehicles. small fractured surfaces also have been encountered in the investigation of hit-and-run accidents when pieces of chrome trim were knocked loose and left at the scene. clearly modern instrumental means of analysis, such as the sem/edxa, are critical to the work of the forensic scientist. the increased sensitivity of the instrumentation, however, may raise questions as to the relevance of the evidence found. if a single, very small paint particle is found on the jeans of a pedestrian struck in a hit-and-run accident, could the particle be from the striking vehicle, or merely from the roadway debris at the scene? such questions indicate that increases in instrument sensitivity require sensitivity on the part of the analyst to questions of contamination and weight of evidence. research division, office of laboratories and scientific services, u.s. customs service, washington, d.c., usa the u.s. customs service laboratory system provides a variety of analytic services to control the commerce and assure enforcement of numerous regulations at the border. any item which is imported into the united states may need to be analyzed to determine the answer to any number of questions. what is the item? is the item correctly described? does the item infringe upon a u.s. patent? the scanning electron microscope (sem) and eds x-ray system can be utilized to answer some of the questions which arise. several examples follow. a u.s. company holds a patent on a feature incorporated into an electronically programmable read-only memory (eprom) cell. they allege to the international trade commission (itc) that another company is incorporating this patented feature in their eproms without the patent holder's permission. they win their case and the itc issues an exclusion order. it is at this point that the u.s. customs service becomes involved. we are charged with enforcement of the exclusion order. the incorporated feature is exceedingly small on a visible scale. how will we know which shipments of eproms should be excluded from entry into the u.s.? now the ability of the sem to produce images easily at very high magnifications comes into play. with the help of the sem, the laboratories are able to determine if the infringing feature is present or not. another u.s. company holds a patent for a denim (textile) finishing process. they bring a complaint before the itc that their patent is being infringed upon. the complaint is upheld by the itc and an exclusion order is issued. the u.s. customs service laboratory system must now develop a method to differentiate among various finishing processes in use. research at the headquarters laboratory found that a combination of imaging with a stereomicroscope and an sem could make the differentiation. the sem samples were au-pd sputtercoated with a hummer vi a sputtering system (anatech ltd.). the sputtercoated denim samples clearly showed distinctive features not easily seen with an optical microscope. a sample purported to be eelskin was submitted to the headquarters laboratory for conformation of its identity. it was thought that the product might be embossed plastic or possibly leather of mammalian origin. the top surface of the sample was au-pd sputtercoated and then examined with the sem. the features displayed by this examination were convincing evidence that the sample was indeed leather. a cross section of the sample was then prepared and au-pd sputtercoated. the sem images showed cell patterns consistent with reptilian or marine origin. another sample arrived courtesy of a foreign customs service. their inspectors had seized a large statue of a roman gladiator and his horse and chariot. the original reason for suspicion was that the declared value for the statue was much higher than one would expect for an object of this type; it appeared to be a cheap plastic statue. it had been dismantled and tested for the presence of drugs. the results were negative. using our princeton gamma-tech eds x-ray system, an elemental analysis of the exterior covering of the portion of the statue we received was performed. it showed the presence of large amounts of silver. this would explain the high declared value of the statue. when the u.s. customs service ran tests on narcotics particle detection systems, it was noted that sampling for heroin was more difficult than sampling for cocaine. a brief study of samples of each narcotic using the sem showed that in general the average particle size for heroin is less than that of cocaine. this may help to account for the sampling difficulty for heroin. allan n. walters u.s. postal inspection service, forensic laboratory, dulles, virginia, usa currently, the two main applications of sem/edax are explosive residue analysis and alloy quantitation. most explosives encountered are from improvised explosive devices (ieds) and commonly are low explosives such as black powder, smokeless powder, pyrodex, and flash powder. pyrodex and smokeless powder are commercially manufactured, while black and flash powder may be either of commercial or improvised (homemade) manufacture. the following table illustrates the composition of the common low explosives: device components are placed in the sem, and edax is performed before disturbing the residue. the resulting elemental profile is then used to guide further analyses with other instrumentation such as xrd, ftir, hplc, tlc, and chemical spot tests. sputter coating of samples is not performed so as to avoid modifying the sample and interfering with subsequent analyses and examinations. quantitative analysis is performed on alloys which are of interest to the u.s. postal service engineering and development center and is required to ensure that the materials meet the re-quired specifications. samples have included lock bodies, lock springs, keys, and lock tumblers. reverse engineering using quantitative analysis of current collectors for mobile electrification systems (mail sorting machines) has been conducted. quantitations are performed using a standardless method. scanned probe microscopy has evolved significantly over the last years. beginning with the first commercial scanning tunneling microscopes (stm) and continuing through the sophisticated "multifunction" microscopes of today, the "probe" has been one of the most critical and, in some instances, the least understood component of these systems. depending on the type and geometry of the sample, the properties of a probe can be optimized to reduce imaging artifacts. examples of this will be given using two well-known techniques, scanning tunneling microscopy (stm) and atomic force microscopy (afm). also, new innovations in probes for these two techniques (and others) will be presented. scanning tunneling microscopy, as first introduced, used a probe constructed of a chemically sharpened tungsten wire. later, it was found that a suitable probe could be formed from a mechanically sharpened wire (usually pt/ir). both of these probes proved that they could produce self-consistent images under specific sample and environmental conditions. however, problems arose with the tungsten probe while imaging in air because of the formation of native oxide. in addition, the mechanically formed probe tended to produce significant imaging artifacts if used on samples with topography > nm. in many cases the distortion produced by these artifacts made the data uninterpretable. a solution to these problems was to use a chemically etched probe (to control the shape) made from an inert material with physical properties suitable for the samples involved. musselman et al. developed the techniques necessary to chemically etch pt/ir wire into tips with a controlled geometry. these probes were capable of imaging surface structures greater than µm peak-to-valley depths with significantly reduced tip-related artifacts. this controlled geometry shape also made it more advantageous for use as a coated tip for electrochemical imaging. still, the aspect ratio of this particular probe was not suitable when imaging high-aspect ratio features such as pits in optical discs, contact vias in ics, fracture surfaces, etc. to obtain images from these types of structures, it was necessary to "machine" (in a controlled way) the probe described above. by using a focused ion beam (fib) as a machining tool, it was possible to create a probe to image the above features ( fig. ) . this fib "nanomachining" technique has opened up many possibilities for specialty probes. the majority of atomic force microscopy still utilizes the basic silicon nitride (si n ) triangular cantilever and pyramidal probe combination. the base of the pyramid is on the order of µm with the sidewalls extending upward at an approximate o angle to the apex. again, for samples with features < nm, such as mica, these probes have provided very good image repeatability. in general, however, for structures much greater than nm, a convolution of the probe and the sample surface will again occur. these probes can be modified, using the fib technique, to increase their aspect ratio significantly. because of their "hollow" design, these modified probes are still limited to topographies of ≤ . µm. most of the advances in probe manufacturing (on a wafer level) have come about by utilizing silicon as the probe material. several silicon probe types are now available, which provide significantly sharper tips with aspect ratios on the order of to . for imaging structures of higher aspect ratios, such as vias or deep trenches, longer and thinner probes are needed. these are made possible by using a combination of fib and electron beam techniques to "grow" a thin probe using an existing si n pyramidal probe as the base (fig. ). these probes are available in lengths up to µm with aspect ratios of ≥ to . utilizing a combination of fib milling of existing structures and electron beam growth, probes with lengths of - µm are feasible. an obvious problem with probes of this type (and with any long, thin structure) is "flexing" during imaging. analysis of one such probes' physical characteristics has shown that the elastic modulus is relatively low (e~ . gpa), while the coefficient of friction on most surfaces is extremely low (µ< . ). thus, probes of this type should be kept as short as possible while exceeding the maximum peak-to-valley distance to be imaged. other scanned probe techniques now in the prototype phase include thermal, magnetic force, near field optical, and probes capable of imaging undercut sidewalls. probes for all of these methods have been shown to be feasible, although manufacturing techniques to provide large quantities, reliably and repeatably, have yet to be developed. when imaging with an atomic force microscope (afm), the image resolution is a complex function of the relative tip and sample geometries. when imaging or measuring high-aspect ratio features, sharp and slender tips offer the possibility of probing down into extremely small topographical features. the most commonly used contact mode afm tips are batchfabricated si n thin-film cantilevers with an integrated pyramidal structure used as the tip. it has been shown that microtips, which are fabricated by electron beam-induced growth of carbonaceous material on the apex of the pyramid, can reduce the artifacts associated with integrated pyramidal afm tips. graph of a whisker of electron beam-grown contamination, or microtip, grown on the apex of an integrated pyramid, is shown in figure . an obvious problem with the use of a long slender microtip is the lateral deflection of the microtip as it is scanned across the sample surface. the proper use and interpretation of artifacts associated with electron beam-grown microtips demands an understanding of the mechanics of microtip deflection. it has been observed that long, slender microtips scanned over flat surfaces (rms roughness of < nm) produce hysteresis in the fast-scan direction. a model has been developed to explain the observed hysteresis loop in terms of a mechanical cantilever beam deflecting because of lateral frictional forces induced by repulsive imaging forces. this model is shown schematically in figure . by applying cantilever beam mechanics to the model of microtip deflection, a method of calculating the elastic modulus of microtip material has been developed. to find the elastic modulus of microtip material, a series of experimentally determined microtip deflection distances and the respective microtip lengths are required. microtip deflection distances have been experimentally determined for different microtip lengths on two different flat surfaces; fusion deposited boro-silicate glass and polished silicon. the elastic modulus of the microtip material has been determined from the deflection data to be approximately . gpa. once the elastic modulus has been determined, the coefficient of friction between microtip material and a sample sur-face can be calculated. the coefficient of friction between a microtip and the sample surface will indicate if the sample material is suitable for afm imaging with electron beam-grown microtips. the elastic modulus of microtip material and the coefficient of friction data lead to a better understanding not only of microtips but of electron beam-induced contamination in general. the low elastic modulus rules out the possibility of the material being diamond-like and suggests a polymeric material. the use of microtips can greatly improve image resolution; however, it is important to note that since microtip deflection increases with increasing microtip length, the microtip used to image a sample surface should be as short as possible while remaining long enough to image the largest peak-valley structure on the sample surface. the magnitude of observed microtip deflection should be reduced substantially by the use of ac mode microscopes where surface friction is less of a concern. because of their ability to achieve high resolution simultaneously in all three dimensions in a wide range of ambient conditions, scanning probe microscopes are promising candidates for performing measurements of surface topography. crosssectional and perspective views can be generated, nondestructively, at any location once an image has been acquired. surface topography measurements fall into two basic classes: position (or pitch) measurement and size (or critical dimension) measurement. the ability of a microscope to perform position measurement depends more on the quality of its design and construction than on the fundamental interaction of probing beam or stylus with the sample. size measurement, on the other hand, depends strongly on the probe-sample interaction. modern manufacturing, especially semiconductor lithography, often produces high-aspect ratio, submicron structures whose size and shape must be known with tiny uncertainties. surprisingly, stylus profilometers in the guise of scanning probe microscopes can perform some of these measurements at a level unmatched by any other type of microscope. to obtain size and shape measurements in semiconductor manufacture, we have developed a scanning probe microscope with several refinements, depicted in the figure. we use capacitance-based sensors for probe force sensing iv- scanning vol. , supplement iv ( ) cantilever and microtip at rest and for probe position measurement. , many of the samples that we scan are at least partially electrically insulating. for this reason, our microscope is used primarily as a scanning force microscope, although it is capable of operating as a tunneling microscope also. our force sensor employs a small silicon beam that pivots in one dimension about a pair of magnetically constrained ball bearings. the beam forms a pair of capacitors that both sense the position of the beam and maintain its balance with a suitable servo loop. this force-balance technique allows high-force sensitivity without sacrificing the stiffness required to resist surface forces. capacitors are also used to measure the position of the probe tip. the piezoceramic tube used as a scan actuator exhibits strong hysteresis and creep, so the drive voltage is an unreliable measure of probe position. the capacitors monitor the probe position in all three dimensions during the scan, and these data are collected along with the topograph. the most important factor determining the quality of the measurements is the shape of the probe tip. geometry alone makes the probe-sample interaction strongly nonlinear. in surface roughness measurements, a blunt probe can severely limit the range of spatial frequencies that can be detected. in scans of high-aspect ratio features, the probe shape determines what parts of the features can be measured. when scanning deep trenches and holes on a patterned surface, we use either a conical probe or a cylindrical probe, depending of what part of the feature is most important. the conical probes are made using focused ion beam sputtering of iridium. a chemical etch is used to form the cylindrical probes. if the probe shape is well known, then it is possible to determine what parts of a scan were distorted by the probe tip and, in some cases, this distortion can be removed. , the probe microscope itself can be used to determine the probe tip shape if a suitable structure is available for probe characterization. since the pioneering work of binnig and rohrer in the early s on scanning tunneling microscopy (stm), the stm has evolved into a powerful tool for spectroscopy, metrology, electrochemistry, and nanolithography. many other instruments have also evolved from the stm technology under the family of scanning probe microscopes (spms) with applications in atomic force, electric potentiometry, and magnetic force imaging. in the magnetic force microscopy (mfm) mode, the technique has been applied to the imaging of magnetic bit patterns in recording media (grütter et al. , mamin et al. ) and the mapping of static and dynamic magnetic fields of recording heads (martin and wickramasinghe ) . mfm is typically performed in the noncontact atomic force microscopy (afm) mode using a silicon cantilever which is coated with a thin film magnetic material, usually co, ni-fe alloy, or co-pt-cr alloy. the force exerted on the magnetic tip by stray fields from the sample causes the deflection of the cantilever which is subsequently measured. using a novel variation of the stm technique with a flexible iron tip, rice and moreland ( ) have also performed mfm imaging on magnetic bit patterns on a hard disk in a tunneling-stabilized mfm (tsmfm) mode. standard mfm images, however, reflect both topographic and magnetic information, with the relative strengths of each signal depending on the tip-to-sample spacing. using a differential interferometric technique, schönenberger et al. ( ) have shown that the topographic and magnetic information can be reasonably separated. in this abstract, results of some applications of the spm for topographical and magnetic force imaging of magnetic materials are presented. as the critical dimensions of magnetic devices are getting smaller, the surface topography and magnetic morphology of the recording head and media are becoming increasingly important with respect to optimization for best performance. the ability of the spm to obtain submicron topographical and magnetic information makes this an invaluable metrology and failure analysis tool for the magnetic recording industry. compared with other techniques for highresolution magnetic imaging, such as lorentz microscopy, electron holography, scanning electron microscopy with polarization analysis, mfm has the advantages of ease of sample preparation and the ability to operate in air. typical resolutions of - nm are obtainable in the mfm mode, although resolutions of - nm have also been achieved with considerable effort. in our work, topographical imaging was performed in the contact mode using a v-shape silicon nitride cantilever of length l = µm, width w = µm, thickness t = µm and force constant k = . n/m. the sensing tip at the end of the cantilever is pyramidal in shape, with a × µm square base and : aspect ratio. the tip radius is typically smaller than Å. for magnetic force imaging, the instrument was operated in the noncontact amplitude resonance mode by oscillating the cantilever and measuring changes in its resonant frequency using either phase or amplitude detection. this method of detection is responsive to the force gradient. for each point of the recorded image, the cantilever was first brought to a large tipto-sample distance of nm or greater to acquire the magnetic force image and subsequently moved close to the sample surface to acquire the topography image. this allows the simultaneous acquisition of topography and magnetic force images. a - v specimen bias was also applied to provide a linearizing and stabilizing force to the servo feedback loop. the cantilever used for the mfm imaging is a diving boardshape probe made of ( ) silicon. the geometry of the cantilever are l = µm, w = µm, t = µm and k = n/m. the sensing tip is conical in shape with a height of about µm, an aspect ratio of : , and a tip radius of < Å. the cantilever, which was sputter-coated with a Å thin-film cobalt material and magnetized inside a - gauss solenoid field, has a typical resonant frequency of khz. figure shows a three-dimensional topographic profile of a defective thin-film recording head, in which the pole tips protrude slightly from the surface, using the contact afm mode. the dimensions of the two rectangular pole tips are about µm × µm and µm × µm. the gap width was measured to be about . µm. dirt particles can also be seen and these are due to the cleaning process during sample preparation whereby the head was lightly swabbed with a cotton tip soaked in methanol. figure shows the magnetic force image of the surface of a recorded computer hard disk. the bright and dark lines represent regions of different magnetization or bits on the hard disk surface. the separation between each pair of bright-dark lines was measured to be about . µm and the track width is about µm. the striations in the topographic image (not shown), representing the texture lines, are approximately perpendicular to the recorded bit patterns. the line profile of the bit patterns in figure (not shown) is very similar to the calculated force gradient contours by mamin et al. ( ) in which both the horizontal and vertical magnetization components of the tip are are being sensed. iv- scanning vol. , supplement iv ( ) fig. three-dimensional topographic profile of a defective thin-film recording head using contact afm, in which the pole tips protrude slightly from the surface. in a good recording head, the pole-tips are supposed to be slightly recessed from the surrounding region. image resolution in traditional far-field microscopy is limited by diffraction caused by the primary aperture in the optical system of the microscope being used. in practice, diffraction limits lateral resolution to approximately λ/ , or about . micron for optical microscopes. to image smaller cellular structures and biological materials which are smaller than . micron, biologists have used microscopies which "illuminate" the specimen with radiation of smaller wavelength, such as an electron beam (i.e., using an electron microscope). the nearfield scanning optical microscope (nsom) breaks the diffraction limit to lateral resolution by illuminating (or collecting light from) the specimen through a sub-wavelength aperture which is held a small fraction of a wavelength above the surface of the specimen. using this nearfield technique, resolution far better than λ/ has been demonstrated on biological specimens and resolution better than λ/ has been demonstrated on standards (betzig and trautman ) . with nsom we have recently obtained optical images of tobacco mosaic viruses ( nm diameter) and of photoreceptor rod outer segments at a sub-wavelength resolution. betzig e, trautman jk: near-field optics; microscopy spectroscopy and surface modification beyond the diffraction limit. science , in june , the initial incident involving a report of a syringe found in a canned pepsi ® product received nationwide publicity. during the following months, law enforcement authorities investigated hundreds of additional claims of tampering from nearly every state in the united states. more than cases involving foreign objects allegedly found in soft drink containers were processed by the national forensic chemistry center (nfcc). to date, the forensic and law enforcement efforts of the fda have resulted in numerous arrests and convictions for charges related to product tampering and felonious reporting of product tampering. scanning electron microscopy (sem) has been valuable, and in several cases crucial, in providing conclusive evidence of fraudulent reports of product tampering. the items reportedly found inside of suspect cans were primarily hypodermic syringes (with and without needles), but other reported objects included nails, screws, bullets, pins, sewing needles, tacks, glass and plastic shards, and rodent carcasses. since most of the cases involved syringes, primarily insulin-type syringes, the first analyses were designed to determine if the syringes were contaminated and if they contained human blood, tissue, drug, or insulin residue. syringe/needle rinses were examined by a number of techniques including sem, which was used to find any intact blood cells and/or tissue. energy dispersive x-ray analysis (edxa) was performed on residue preparations. edxa was able to detect a small k αl peak for zinc in several syringe rinses which was consistent with edxa analysis of dried residues containing some types of insulin. type identification of insulin from syringe rinsing was performed by lc/mass spectrometry in a method developed at nfcc. in another case, a broken pin was reportedly found in a soft drink can. a cross-sectional analysis of the suspect pin by edxa showed the pin was a nickel-plated, iron-core straight pin. the absence of observed iron corrosion provided evidence that the object had not been in contact with the soft drink from the time the canned product was produced until the time the pin was allegedly discovered. the most common question requested by investigators was "how long had the syringe been in the soft drink can?" to answer part of that question, a number of experiments were conducted at nfcc. one sem/edxa method investigated the corrosion of the aluminum crimp (found on some syringe needles) submerged in diet cola. new aluminum crimp needles were sealed in diet cola and removed at -h intervals. stereoscopic light microscopy was initially used to examine each needle crimp. it was discovered that the submersion produced a brown to black "corrosion flower" in less than h of soaking. of specific interest, the submersion repeatedly produced a single point of corrosion on the crimp. the single point of corrosion suggested possible electrolysis of the aluminum crimp in the soft drink. this corrosion point continued to enlarge and penetrate more deeply into the aluminum crimp over time. backscattered electron (bse) imaging and x-ray mapping were used to highlight the region of corrosion in the sem. figure shows the secondary and backscattered electron image (sei and bei) of an aluminum crimp on a syringe needle after h submersion in diet cola. the sei shows the corrosion of the aluminum crimp as pitting in the crimp surface. the bei shows the corrosion area as a darker region caused by surface oxides blocking bse generation from the aluminum below. the study also demonstrated that the corrosion of the aluminum crimp was dependent upon the amount of time the crimp was submerged in the pressurized soft drink container. figure shows a point of corrosion on an aluminum crimp after weeks of submersion. at this time interval, several points of corrosion often developed. the pitting of the aluminum mater-ial immediately beneath the corrosion was extensive. sem/edxa analyses have shown that the aluminum crimp on syringes submerged in diet cola produced a characteristic single point of corrosion after only h. the corrosion resulted in damage to the crimp surface and the production of a metal oxide. the location of the oxide was identified and plotted by x-ray mapping for oxygen (via edxa). the analysis of samples related to the pepsi "tampering" cases of are continuing to date. in many cases, the circumstances and sample condition are unique and generated specific questions which require further forensic research using additional time-related studies, multielement x-ray mapping, and/or image analysis. confocal microscopy can be used to investigate the properties of rough surfaces. based on the kirchhoff approximation (sheppard et al. a,b) , the three-dimensional ( -d) confocal image can be modelled using the -d coherent transfer function (ctf). the form of the -d ctf for confocal reflection and transmission have been derived using a high-angle scalar theory (sheppard et al. ) . in both cases the ctfs can be expressed analytically. using these allows the profile of the rough surface to be reconstructed. in many cases we need to know only the statistics of the rough surface, rather than the actual profile. ways in which the statistical properties can be extracted directly have therefore also been considered (sheppard iv- scanning vol. , supplement iv ( ) fig . a. castenholz in previous in vivo studies based on vital microscopic and fluorescence microscopic techniques it was possible to observe lymph flow in initial lymphatics and regional lymph nodes. [ ] [ ] [ ] [ ] as an expression of immunological mechanisms, the studies carried out in the rat tongue under various issues showed some remarkable phenomena such as cell traffic along lymphatic pathways and phagocytotic activity of the lymphatic endothelium. since these phenomena could not be defined with traditional fluorescence microscopy on the cytological level, confocal laser scanning microscopy (clsm) was applied to the living tissue and fixed specimens). moreover, in morphologic studies on the lymphatic and blood vascular system, clsm has proved a very suitable tool also for the representation of resin-injected tissue, which is commonly processed as corrosion casts for scanning electron microscopy. all these approaches, also including the application of special fluorescent markers, should be outlined here. in vivo studies with the clsm have been designed for the functional morphologic analysis under normal conditions and in a state of inflammation and experimental edema. after staining the lymphatic endothelium with dimethyl-carboxfluorescein, clsm revealed a distinct pattern of the so-labelled initial lymphatics consisting of bright (cytoplasm) and dark zones (nuclear portions). for labelling tissue macrophages passing the lymphatic pathways and other phagocytotic cells, fluorescent microbeads (latex standard particles and liposomes) were applied by interstitial injection. thus, clsm enabled a certain identification of moving particle-laden cells and also gave evidence of phagocytotic activity of the endothelium of initial lymphatics. in long-term experiments, phago-cytosis of cells lining the sinuses of lymph nodes could be clearly recognized. tissue (tongue, skin) and lymph nodes from different sites of the rat were examined in the clsm as unfixed or fixed (glutardialdehyde) thick sections. cells labelled with fluorescent microbeads or stained with fluorescent dyes thus could be easily detected and identified. thereby, it was possible to determine the location of single beads at the endothelial surface or within the endothelial cytoplasm (fig. ). if the tissue was conventionally stained with hematoxilin eosin or acridine orange, optimum information could be obtained from unlabelled structures in these specimens as well. by means of the two-detection system, clsm also was able to distinguish two or more different fractions of microbeads incorporated by macro-phages ( fig. ) or lying in tissue spaces as free elements. in hemal lymph nodes of rats, erythrophagocytosis related to sinus macrophages was successfully represented by the clsm after vital staining of erythrocytes with the fluorescent celllinker pkh . in this approach, mercox (acrylate) stained with rhodamin and fluorescent yellow was used to create high fluorescence in the lumen of blood or lymphatic vessels. if the resin was injected into the tissue, the interstitial spaces were filled up by resin. clsm of sections from such resin-injected specimens enabled exact distinction between casts related to the blood or lymphatic system and those of the interstitial spaces, when two differently stained resins were simultaneously injected from the arterial system and into the interstice. clsm of resin-injected specimens was also applied as a suitable method for the control of corrosion casts of sem. proceedings of scanning /seems iv- today, clsm has become an established tool in many fields of biological sciences. because of its abilities to produce images with optimum resolution and clarity, the application in experimental lymphology is striking as well. some experiments in techniques related to in vivo experiments, supra vital and fixed tissue, and resin-injected specimens have been reported here. these approaches, also comprising the application of new fluorescent markers for living cells, may demonstrate how wide the spectrum of application is spanned for clsm in experimental lymphology and immune research. this study was supported by a grant of dfg (deutsche forschungsgemeinschaft, bonn) daniel chin, ph.d agouron institute, la jolla, california, usa the regulated metabolism and distribution of nucleic acids is required for endogenous antisense or rna regulatory systems. recent interest has focused upon using exogenous agents as antisense therapeutics to treat viral infections or metastatic diseases. both endogenous and therapeutic antisense regulation of gene expression requires the formation of a hybrid between the antisense molecule and the message or gene sequence. it is not surprising that detection of such hybrids in living cells has not been reported to date. we have used fluorescence resonance energy transfer (fret) to study hybrid formation and dissociation after microinjection of oligonucleotides (odns) into living cells. two systems were examined: one system characterized the kinetics of hybrid dissociation of two synthetic odns while a second system examined the distribution of hybrid complexes formed by hybridization of injected odns with endogenous mrna. in the first system, a -mer phosphodiester odn (+pd) was synthesized and labeled with a ′ rhodamine (+pd-r). the complementary, antisense ′-fluorescein labeled phosphorothioate odn (-pt-f) was specifically quenched by addition of the +pd-r, as detected by both absorbance and fret in solution. rapid and specific hybridization between the odns occurred at µm within min and the preformed hybrid slowly dissociated (t / ≈ h) in the presence of a -fold excess of the unlabeled odn. upon microinjection into the cytoplasm of cells, preformed fluorescent hybrids dissociated with a halftime of min, which is attributed to the degradation of the phosphodiester. formation of the hybrid from sequentially injected odns was detected by fret transiently in the cytoplasm and later in the cell nucleus, where nearly all injected odns accumulate. diffuse, specific nucleoplasmic fret could be distinguished from nonspecific fret in punctate nuclear structures which may result from concentration effects similar to fret seen in late endosomes or lysosomes between endocytosed, inert fitc-dextran and +pt-r odn. a second set of experiments with two adjacent -mer pt odns complementary to mouse b-actin mrna was performed in mouse t fibroblasts or n neuroblastomas. the upstream odn was ′ labeled with fitc and the downstream odn was ′ labeled with rhodamine. when hybridized to synthetic actin mrna or a complementary -mer pd odn, quenching of fitc and fret was observed in solution. injection studies in t cells showed transient odn accumulation and fret in filopodia and extended processes. in n neuroblastomas, fitc quenching was dependent upon cytoplasmic compartmentalization. after min, both odns accumulated into the nucleus. in summary, these experiments suggest that antisense odns can hybridize to intracellular targets in both the cytoplasm and the nucleus. the goal of this work was to increase the sensitivity of the electron backscattering pattern (ebsp) technique by introducing an electron energy filter to increase contrast and improve pattern visibility. energy filtering previously has been shown to increase contrast in selected area channelling patterns (joy ) . energy filtering differs from arithmetic background subtraction in signal processing, because filtering before detection has a physical basis and eliminates the unwanted background without increasing the noise component in the signal. the electrostatic retarding filter used in this experiment was constructed as a cone with five coaxial electrodes. the primary purpose of the electrodes was to create symmetric fields that would not distort the pattern, yet allow only backscattered electrons with the least energy loss to contribute to the pattern. analysis (courtesy e. munro, micro electron beam software, ltd.) of the electron trajectories indicated that for kev electrons, filtering voltages of up to kv could be used before chromatic aberrations became unacceptable. the angular field-of-view of the filter was approximately o . another feature of the detector is the use of a microchannel plate as the initial sensing component. microchannel plates have high gain and are very sensitive to the low-energy electrons that pass through the retarding field filter. in a configuration similar to that of venables ( ) , the output of the microchannel plates was proximity-focussed to a phosphor screen that displayed the microdiffraction patterns. the gain of the assembly exceeded a simple phosphor by more than times. the light output of the assembly was focused on a kodak megaplus . ccd camera using either a macrolens alone or a hybrid fiber optic-lens combination. no vignetting was observed across the field. the digital output of the camera was displayed and analyzed on a macintosh iix using nih image. results were obtained on a variety of materials including single crystal silicon, al-sic metal matrix material, alumina, aluminum foil, and aluminum used in aluminum cans. the high voltage design limited the maximum retarding potential to − kv so that meaningful data were obtained with incident electron beam energies between and kev. filter potentials above % of the primary beam accelerating voltage increased the kikuchi band contrast relative to the background. figure contains a very low contrast pattern for alumina obtained with essentially no filtering. the effect of filtering to within kv of the beam accelerating voltage is shown in figure for the same specimen. the best patterns were obtained when the filter voltages were between and % of the accelerating potential. filtering above % degraded pattern resolution while less filtering usually produced less contrast. up to four-fold improvements in the contrast ratio were achieved on some specimens. the detector also had the ability to obtain energy-filtered backscattered electron images in sem raster mode. one advantage of this configuration is that the sem characterization of a given feature and the ebsp data obtained from it can be obtained from the same perspective. another is that filtered im-ages appeared to have improved surface detail and greater crystallographic contrast. venables ( ) showed that a detector of this kind could be used for "dark field" sem imaging. this work demonstrated the feasibility of employing an energy-filtering detector with high gain to increase the sensitivity of microdiffraction measurements in the sem. further work is planned to optimize the filter and improve pattern quality and the range of filtering voltages. the authors gratefully acknowledge the contributions of munro electron beam software, ltd. and of mark vaudin at nist, as well as the support by the national science foundation under award number iii- , and the department of commerce under contract number -dkna- - . submicron-sized elements are of great interest in physics and technology. there exists a variety of methods to produce them by using conventional microelectronics technology as well as new methods for deposition of such elements directly from the desired material onto a substrate. the current onestage maskless techniques, for example, laser-induced cvd or laser-induced etching , do not ensure resolution below nm because of their physical limitations imposed by the laser wavelength. focused ion beams, which generally allow the formation of submicron-sized elements, often are unacceptable as they can cause radiation damage in the material. moreover, the cost of the necessary equipment is high. these circumstances stimulate the development of techniques for direct formation of submicron-sized patterns with the necessary geometry from an arbitrarily chosen material directly at substrates based on electron-beam-induced cvd . the cvd experiments were performed in a temscan jem- cx electron microscope at an accelerating voltage of kev and a beam diameter ~ nm. the electron microscope was equipped with a specially designed pressure cell and an oil-free system for pumping and reagent vapor inlet. this allows to maintain a well-defined atmosphere of chemical compounds (metal carbonyls and halides, freons) around the specimen. two electronically controlled needle valves are capable of supplying two different reagents simultaneously into the specimen holder. the sample itself is placed inside a heater which provides temperatures up to ˚c required for the local electron-beam-induced etching. contacts are made to the sample, which allow in situ measurement of its electrical characteristics. the holder is supplied with two differential apertures (through which the electron beam passes) placed above and beneath the sample. these apertures keep the gas pressure around the sample up to pa without breaking the vacuum in the microscope column. w(co) and rez(co) were used as reagents. structure and composition analysis both were performed in a jem- fx electron microscope equipped with a link an- / s system for edx analysis. self-supporting rods of - nm in width, containing tungsten or rhenium, respectively, were grown up to nm long at a speed of the electron-beam movement - nm/s and at a vapor pressure near the sample of . pa. provided a constant speed of the beam is maintained, the thickness of the produced rods was found to be inversely proportional to the beam speed but decreasing towards the rod's end. this result is similar to that obtained by electron-beam-induced fabrication of self-supporting carbon-containing rods electron microscopic observation of the inner structure of these self-supporting metal-containing rods revealed many individual fibers mutually aligned in parallel. thus, the mor- iv- scanning vol. , supplement iv ( ) fig. tem image (a) and sad pattern (b) from a self-supporting tungsten containing rod section after annealing in vacuum at ˚c for min. the accelerating voltage is kv, the diffraction length on the (b) is cm. phology of these rods is similar to that of the well-known carbon rods . this observation suggests similar formation mechanisms independent from the rod material. selected area diffraction (sad) patterns show the prepared rods as being amorphous. in situ annealing of the tungsten-containing rods at ˚c for min in the heating holder inside the microscope column transforms the amorphous structure into a nanocrystalline one (fig. ) . sad patterns reveal the presence of a set of different phases: in addition to pure crystalline tungsten there are various tungsten-containing compounds. among them, in particular, two new cubic phases with lattice constants ± pm and ± pm could be identified. after annealing chemical and phase compositions of the rods grown from w(co) and rez(co) were found to differ depending on the conditions of their formation (reagent vapor pressure, electron current, beam speed). the rods of nano-sized width, grown from rez(co) are considered to be promising as tips for scanning tunneling microscopes because of their chemical stability. an important requirement for developing high-speed highpower devices is fabrication of thermally stable ohmic contacts with low resistance and smooth interfaces. metal semiconductor interface inhomogeneities, such as lateral interface phases and spiking protrusions, lead to nonuniform current flow and consumption of the gaas substrate. this type of interface morphology is not acceptable for device applications where a large electric field or shallow contact is required. in particular, devices such as a heterojunction bipolar transistor (hbt) cannot tolerate contacts with lateral and vertical interface inhomogeneities. recently, we have introduced a novel metallization scheme, pt/ti/ge/pd, which yields thermally stable and low resistance ohmic contacts to both n and p + -gaas (han et. al. ) .to insure processing reproducibility and contact reliability, one must identify and understand the mechanisms responsible for this contact's electrical per-formance. this study employed cross-sectional transmission electron microscopy (tem), auger electron spectroscopy (aes), and electrical measurements (transmission line mode) to investigate the structural, chemical, and electrical properties of this contact. the interface morphology, phase composition, and elemental diffusion were examined and correlated with the measured contact resistances at annealing temperatures which yielded the best slight degradation and the worst electrical performance. annealing at ˚c yielded the lowest specific contact resistance, ~ . × - Ω-cm . the metal-semiconductor interface was planar and structurally abrupt. the pd and ge reacted to form a pdge phase. directly beneath the pdge was a thin, discontinuous, ga-rich pd-ga-as ternary phase. the presence of this ga-rich ternary compound has important implications for contact formation on the n-gaas substrate. formation of this interface phase creates excess ga vacancies in the gaas substrate. upon heating, ge diffuses into the gaas, occupying ga vacancies at dopant levels, to form an n + surface layer, thus allowing considerable tunneling at the metal-semiconductor interface. the existence of the ga vacancies is crucial, because otherwise ge would not readily diffuse into the n-gaas to form this n + -gaas layer. for the p + -gaas, this ge indiffusion occurs also, but the ge concentration level is not such that it has a detrimental effect, that is, it does not approach that of carbon (the p-type dopant) ~ × cm - . the ti/pt layers remained stable, hence no surface degradation was present. annealing at ˚c resulted in a slightly higher specific contact resistance, ~ . × - Ω-cm . there was significant elemental diffusion within the contact metal and minor elemental diffusion into the substrate. the interface exhibited a roughness on the order of nm and possessed large areas where spiking single-phased pdgega protrusions spatially dominated the metal-semiconductor interface region. the nonplanar nature of the interface lends itself to several explanations: ( ) nonuniformity in the heating associated with the annealing process, ( ) material defects or pitting in the gaas substrate such that the contact metallization covered and filled the pitted area as it would a flat gaas surface, and/or ( ) new phases being formed as a result of the ti layer beginning to break down as a diffusion barrier. apparently, this interface nonuniformity (spiking) does not cause significant deterioration in the specific contact resistance, since the contact resistance maintains reasonable electrical integrity. it is speculated that the compositional uniformity of the protrusion phase, which is close to that of the pdge phase formed during the ˚c anneal, is responsible for this behavior. annealing at ˚c proved to have a detrimental effect on the specific contact resistance, ~ - Ω-cm . this degradation was accompanied by strong chemical intermixing between the contact and the substrate, resulting in laterally continuous and vertically deep (> nm) multiphased protrusions spiking into the gaas substrate. the surface of this contact possesses surface anomalies (~ . µm in size) with a density of ~ %. the anomalies are somewhat enriched in pt and as and depleted in ge. this nonuniform surface morphology demonstrates that pt no longer represents a smooth surface for bonding. our results demonstrate that annealing temperatures between - ˚c are of practical interest for hbt device processing. the thin base region ( Å) and narrow distance between the contact and emitter ( . - . µm) make the ˚c anneal impractical for the hbt. specifically, the composition, extent, and magnitude of the interface spiking would be totally detrimental to this device design, that is, spiking areas would penetrate through the base region, into the collector, and short the device. the present paper is a review of diagnostic availabilities as well as of physical data taken by color cathodoluminescence scanning electron microscopy (ccl-sem) used for investigation of structural, polytypic, and impurity inhomogeneities of sic crystals, epitaxial layers, and devices (saparin and obyden ) . as examined objects, the "alpha" and "beta" sic crystals, different polytypes ( h, h, c, r) sic epitaxial layers and devices have been studied. the epitaxial layers were grown by the sublimation "sandwich" method (vodakov et al. ) in the vacuum or ar-media under temperatures between and ˚c. from results of experimental studies it is possible to enumerate the following that are impossible to prove by other techniques: ( ) space distribution of impurities; ( ) static and dynamic characterization of polytype transformation. multitransformation of polytypes during the growth process was observed by way r → c → h. it was concluded that the growth conditions of epitaxial layers and surface perfection of the substrate markedly influence polytype variations. ( ) space distribution of radiation defects on dependence of annealing temperature; ( ) action of mechanical defects on the spatial distribution of luminescence centers; ( ) availability to observe the transient layers in sic devices; ( ) -d analysis of epitaxial layers with polytype transformation. saparin gv, obyden sk: colour display of video information in scanning electron microscopy: principles and applications to physics, geology, soil science, biology, and medicine. scanning , - the examination of chemical vapour deposition (cvd) diamond films by the scanning electron microscope (sem) (secondary electron mode with spatial resolution - nm) shows that the morphologies of films are strongly affected by the synthesis conditions, especially the substrate temperature, the methane concentration, total pressure in the deposition chamber, etc. studies demonstrate that the changes of ch for concentrations in the range of . - % vol. create the shape variation of crystals determined by the ratio of the apparent growth rates of the ( ) and ( ) faces r( )/r( ) in cubo-octaedrons interval from . - . . the exact cubooctahedral shape observed very frequently is determined by the value of ratio . and preferable orientation of the ( )-or ( ) faces. thus, the secondary electron mode of the sem is a very useful diagnostic technique for diamond films. less frequently researchers use the second diagnostic availability of the sem (saparin and obyden ) : cathodoluminescence (cl) mode (spatial resolution . - . mkm) with monochromatic and panchromatic (real color) images and local cl spectra. diagnostic possibilities of this mode identify the quality of diamond films in comparison with natural diamond. the cl spectra of undoped epitaxial films show the dependence on the face upon which the epitaxy was done. cl discriminates between different types of diamond. there are distinct spectral differences between natural, synthetic diamonds and cvd films. the cl emission of the a-band was associated with donor-acceptor pair recombination (blue and green regions); vacancy-rich material emits in the red spectral region ( - nm). we would like to note that the cl spectra of natural diamonds (types ia, ib, iia, and iib) lie in the range of - nm. variations in these cl spectra were attributed to differences in the defect structures formed during the growth of material. cl images and local spectra allow one to recognize, for doped and undoped films, the small amounts of impurities, such as al( ppm) and b( . ppm) and nitrogen remaining in the gas, which could be a possible source of donors. thus, the sem investigations of morphology, crystallinity, and cl emission of films led to the correlative estima-tion of diamond film quality in comparison with the natural diamond as standard. the etching process of a crystal surface during its dissolution is well-known. the process of etch pits formation, as a result of crystals dissolution, usually is explained by scientists by the presence of dislocations inside the crystal structure. however, the same process occurring near the crystal edges is studied less, although such investigations can give additional scientific knowledge for a better understanding of the dissolution mechanisms of solids. this brief communication continues the series of investigations (dorozhkin , dorozhkin et al. the results show that here can be various surface structures taking place under geometric interaction among the growing etch pits and dissolving fap crystal surfaces. as the fap has a hexagonal crystal structure, the growing etch pits mainly have a hexagonal shape also (dorozhkin , dorozhkin et al. ). on the other hand, the investigated fap crystals were obtained from natural fap-containing rock after its disintegration and concentration stages. therefore, the crystals had a very irregular shape and a lot of dislocations inside. figure shows an example of a typical hexagonal etch pit having only three faces instead of the necessary six; three lacking faces (left side) have already been dissolved by the acid. a very similar situation is presented in figure . this is another example of a former hexagonal etch pit having only three faces (center). it is easy to restore events which occurred with the pits some minutes before. the pits began to form and grow from the moment when the outputs of dislocations on the fap crystal surface began to interact with the acid solution. as the pits are always faced only by fast dissolving faces, their dissolution rate is greater than the one for the crystal surface in common. on the other hand, the growing of etch pits and the fap crystal surface dissolution are always occurring simultaneously. as a result, a layer of fap substance between the growing pit, which is situated close to any vicinal dissolving crystal face, and the vicinal crystal face itself were getting increasingly thinner. finally, this layer disappeared completely, resulting in the pits' formation having only three faces (figs. , ) . if one had dissolved the fap crystal for a few more seconds, its surface would have reached the bottom of the pits and they would have disappeared entirely. figure shows an example of interaction between etch pits with an irregular shape and a surface of the fap crystal. for this purpose a very thin and sharp spallation fragment of the fap crystal was chosen which was then etched by phosphoric acid solution. inasmuch as the spallation fragment was equal to a random and unknown crystallographic face, the irregular etch pits were obtained. it is easy to see four different states of such pits (fig. ) : they point to a pit which ( ) ( ) is only running through the fap crystal but is situated relatively far from the dissolving crystal face. finally it should be noted that all the above-mentioned cases of interaction among pits and crystal surface may only take place when directions of dislocations inside the dissolving crystals are close to lines parallel to any nearest vicinal crystal face. k. habib and p. g. caceres* materials applications department; *central analytical laboratory kisr, safat, kuwait a fundamental study of co-based metallic glasses has been conducted. the study focused on understanding the changes of the properties and structures of an fe-b-si glass as a function of co, co-ni, co-mn, and co-ni-mo additions. the separate addition of co, co-ni,co-mn, and co-ni-mo elements was successful in a way four new metallic glasses were produced. the compositions of the new glasses are fe co b si, co fe ni b si , co fe mn b si , and co fe ni mo b si . consequently, an evaluation of the physical and magnetic properties was determined. furthermore, the internal and surface structures of the glasses have been characterized by a transmission electron microscope and a scanning tunneling microscope, respectively. a comparison between the internal and surface structures of the glasses was carried out on both amorphous and crystallined forms. as a result, a correlation between the properties and structures of the glasses is established. for instance, figures a and a show surface structures of the co fe ni mo b si metallic glass in the amorphous and annealed crystalline forms, respectively. on the other hand, figures b and b show the corresponding x-ray diffraction patterns of the amorphous (fig. a) and the annealed structures (fig. a) , respectively. figures a and a are basically three dimensional line plots of the surface profile. it is clear that the amorphous structure (fig. a) represents a complete rough surface along nm × nm scanning area. in contrary, the annealed structure (fig. a) exhibits a surface profile with less surface roughness than the annealed structure. this observation is in agreement with work done by the author on other metallic glasses cited elsewhere (habib et al. ). the current status of external funding for most academic and research facilities throughout this country is meager at best. many institutions are being forced to seek financial support from sources other than the conventional governmental agencies, private funding, or contractual agreements. this facility has, for the past years, derived the bulk of its operational budget through third-party payments for services rendered as hospital charges for diagnostic pathology services. while we have been fairly successful in maintaining a relatively consistent level of service, the overall cost of this operation continues to rise. as cost containment became the buzz word and hospital admissions declined, the number of requests for diagnostic procedures also began to decline. this pattern was observed not only in the electron microscopy (em) lab but in many other specialized laboratories throughout the hospital and our affiliated clinics. as the health care debate accelerated and new concepts such as hmos, emergency care clinics, managed competition, and regional alliances all geared to assure "more health care for the dollar came into being," it became apparent that an entire new concept was needed to provide high-quality diagnostic services for these newer group practices and smaller clinics that were being created to meet these new demands. the diagnostic referral service * has been established in the department of pathology, medical college of georgia, to offer a range of state-of-the-art diagnostic services to external referral sources, including private practitioners, group practices, pathology laboratories, and hospitals. these specialized services are designed to supplement other routine analyses and assist in the diagnosis, prognosis, and clinical management of patients with a wide range of diseases. the laboratories involved are fully cap-accredited, and all diagnoses are evaluated by a pathologist certified by the american board of pathology. the following is a listing of the laboratories contributing to this endeavor and a brief summary of some of the diagnostic offerings. this laboratory provides cytogenetic analyses to identify specific chromosomal abnormalities. these are useful not only in establishing a diagnosis of malignancy, but also in classifying certain malignant disorders, monitoring remission and progression, deciding on treatment regimen, and estimating prognosis. chromosomal analysis can be conducted on bone marrow aspirates, peripheral blood, and lymph node biopsies. this laboratory offers standardized transmission electron microscopic analysis of biopsies from any organ or tumor, as well as specialized procedures for examination of cell suspensions such as bone marrow and lung aspirates and blood samples. em analysis is particularly useful in conjunction with light and/or immunohistochemical microscopy for the diagnosis of tumors which cannot be classified by conventional light microscopy. working in close collaboration with the histology laboratory, the immunohistochemical laboratory, and the renal biopsy service, the em laboratory provides standardized, reproducible ultrastructural analysis which allows a consistent comprehensive approach to the interpretation of biopsy pathology. immunophenotyping by flow cytometry details the presence or absence of surface antigen markers of cellular maturation which define cellular subsets present in specific forms of leukemia and lymphomas. this technique provides precise information to aid in the diagnosis, prognosis, and clinical management of patients thought to have varying forms of lymphoproliferative disorders. in addition, quantitative dna ploidy and cell cycle analysis in combination with standard histopathologic and cytochemical methods provides the most comprehensive assessment of clinicopathologic status, tumor aggressiveness, and the likelihood of disease progression for patients with breast, colon, and ovarian cancer. this facility performs state-of-the-art diagnostic immunohistochemical and in situ hybridization techniques for the morphologic analysis of cellular and molecular events. these techniques are adjuncts to histopathologic examination and can be applied to the study of neoplastic, infectious, and other diseases. the laboratory offers more than immunohistochemical markers as special decision-making tests to resolve differential diagnoses. special histochemical stains and molecular probes for colorimetric in situ hybridization are also provided by this laboratory. this laboratory offers a full service for examination and consultation on kidney biopsies involving a wide range of disease processes. in addition to routine histopathology, all renal biopsies are examined by immunofluorescence for the identification of immunoglobulins, albumin, and c deposits, and by transmission electron microscopy for the detection of early or submicroscopic abnormalities. this laboratory presently offers complete dau screening and gc/ms confirmation for pre-employment/employee drug testing. upon completion of certification as a forensic urine drug lab, this will be the only such certified lab in this area. department of pathology, duke university and va medical centers, durham, north carolina, usa microprobe analysis in biomedicine is usually done on an electron microscope (em) equipped with an energy-dispersive x-ray detector (edx). this type of analysis is commonly referred to as electron probe microanalysis (epma). there are also other fundamentally different new techniques for microprobe analysis which involve the use of laser or ion beams. however, these are not yet commonly employed for diagnostic studies (ingram et al. ) . whereas biomedical epma primarily used to be a research tool, that is no longer the case. epma findings now often have diagnostic, therapeutic, and/or legal significance for the patient (baker et al. , shelburne ). in addition, since much of the current work involves conditions such as the pneumoconioses, the findings frequently have public health and/or industrial medicine implications as well as implications for single patients (shelburne et al. ) . currently the most commonly studied clinical conditions include the pneumoconioses, especially asbestosis and related conditions, "hard metal" pulmonary fibrosis, and other min-eral-induced pneumoconioses (roggli and shelburne ). a second major application is the use of this technology for the analysis of stones, particularly renal stones. microprobe analysis can be more sensitive than x-ray diffraction or chemical techniques, particularly for the identification of small components of complex stones. another major application is the use of microprobe analysis to identify unexplained pigments or deposits and to study unexplained granulomas (kupke et al. , pickett et al. . as is evident from the foregoing discussion, most applications involve the study of insoluble particulates. accordingly, conventional histologic processing with chemical fixation and paraffin or plastic embedding is acceptable. an obvious limitation of this approach is that electrolytes cannot be studied. currently we are exploring the feasibility of utilizing flash freezing techniques to permit studies on electrolytes utilizing cryoultramicrotomy. one way to gauge the usefulness of this technology is to study the use of microprobe analysis in a single hospital system, that of the veterans administration medical centers. currently there are va medical centers in the united states. within these hospitals there are diagnostic electron microscopy laboratories. of these, only five currently utilize epma as a diagnostic technique. at each of these laboratories, conventional transmission electron microscopic studies are the predominant type of analysis. epma studies constitute less than % of our electron microscopy laboratory workload. nevertheless, as the chemical information available from epma is better understood by clinicians, and as cryotechniques are shown to be useful, we anticipate increased usage. in conducting these studies, it is important to be aware of several artifacts that are common problems. the major types are those caused by poor specimen fixation. not only does traditional chemical fixation remove electrolytes from the tissue, it is common in electron microscopy laboratories to add heavy metals such as osmium, uranium, and lead. these elements may produce peaks in the final spectrum that can obscure important elements of physiologic significance. for example, the m-alpha line for lead obscures the k-alpha line for sulfur. in conducting an epma study, it is important to identify all peaks obtained so that the investigator is not mislead by a contaminant. in addition, it is important to utilize several controls. the investigator should not only probe the feature of interest, but also the cytoplasm adjacent to that feature and the blank stub. only in this manner can artifacts contributed by, for example, metal in the microscope column be understood and eliminated from consideration. all living things are infected/affected by viruses. whether the subject is a tissue culture, an animal being used in research, or a human, it behaves differently when infected with a virus. in research subjects virus identification is important to prevent erroneous data due to the presence of a foreign organism. in the case of human viral illness, it is increasingly important to identify pathogens so that appropriate viral therapy can be initiated. several antiviral agents are already on the market, and many more are presently in clinical trials. advantages of using electron microscopy (em) in viral diagnosis are that it is rapid; specific standards and reagents are unnecessary; and infectious particles are not required. disadvantages include the facts that a fairly high concentration of virions must be present in liquid samples to visualize them, and that solid tissues may have focal infections that must be included in the sampling. identification of viruses by direct em is performed by two techniques: negative staining of liquid samples and thin sectioning of tissues, cells, and tissue cultures. negative stains most used in virology are phosphotungstic acid (pta) and uranyl acetate, although many others have been described. for thin sectioning, any fixation method used successfully for em of tissue will preserve viruses; this includes some form of glutaraldehyde and osmium fixation, usually followed by uranyl acetate. detailed preparatory techniques have been described. in negative stains, morphology questions used in identification are: is the virus naked (always icosahedral) or enveloped (pleomorphic); if naked, what is its size, and does it have distinguishing capsid (outer) markings; if enveloped, does it have visible spikes or fuzz around the outside; what is its size, and is the nucleocapsid (the core) visible in particles that have been penetrated by stain; what is the shape of the nucleocapsid, if visible? naked human viruses are all icosahedral; these pathogens fall into three size ranges: - nm, - nm, and - nm. the small viruses may or may not have surface substructure; those that do not are not morphologically distinguishable and have been referred to as small round viruses (srv) (fig. a) if smooth, or small round structured viruses (srsv) if rough. others may have characteristic surface markings that permit precise morphologic identification. the medium-sized (fig. b) and large (fig. c, d) viruses are identifiable. enveloped viruses (those that have a pliable covering) are harder to identify, especially if mixed together with cellular debris. if they have surface fuzz or spikes (fig. e) , they are more readily distinguished. the genetic material inside is sometimes packaged into a distinct form such as an icosahedron, similar to the naked viruses (fig. a-d) or helical filament (fig. f ). if the negative stain penetrates the membrane, this nucleocapsid may be recognizable. however, some viruses do not have a morphologically characteristic nucleocapsid. in thin sections of infected cells, dna viruses are usually seen in the nucleus ( fig. a, b) where they are constructed, and rna viruses are usually found in the cytoplasm where they are formed (fig. c, d) , but there are exceptions. enveloped viruses can be seen associated with or budding through cell membranes; the membrane type is a further clue to identity. finally, the shape of the nucleocapsid within enveloped viruses is a key. possibilities are icosahedral (round in sections, fig. a, b) , helical or filamentous (like worms in sections, fig. c ), complex (pox viruses), or morphologically nondescript. recognition of viral particles and differentiation from cellular components and debris is paramount. once the presence of a virus has been determined, one may consult an atlas to confirm identification. [ ] [ ] [ ] [ ] [ ] for specific concentration or identification of viruses, some antiviral antibodies are available. these reagents can be used to aggregate, to coat, or to gold-label viruses. use of antibodies requires an a priori hint of the identity of the potential pathogen for selection of the proper reagent. charles d. humphrey, cynthia s. goldsmith, luanne h. division of viral and rickettsial diseases, cdc, atlanta, georgia, usa an unexplained acute pulmonary illness resulting in the death of previously healthy individuals was recognized in may . the cause of the illness was quickly identified serologically, pathologically, and genetically as a close relative of prospect hill virus (a rodent-transmitted hantavirus). recently, we isolated the virus from trapped rodents near the homes of patients, cultivated it in vero e cells, and determined that it was identical genetically and morphologically to the causative infectious agent. preparations for electron microscopy (em) were made by extracting, clarifying, and concentrating the virus from unfixed and . % glutaraldehyde-fixed, supernatant fluids of infected vero e cells . uninfected supernatants were prepared similarly as controls. concentrated virus suspensions were applied to glow-discharge treated formvar-carbon grids, blotted, and stained with . % uranyl acetate (ua) or with % phosphotungstic acid (pta), ph . . infected and noninfected cells were prepared for thin section by washing with . m phosphate buffer (po ) ph . , fixing in situ with . % glutaraldehyde in po , scraping, and pelleting. cell pellets were postfixed en bloc in po buffered % osmium tetroxide, stained in . % ua, dehydrated, infiltrated, and embedded in epon-substitute araldite. thin sections were stained with ua and in lead citrate. the extracted negatively stained virus resembled a hantavirus. the nm - + nm viruses were spherical, generally nonelongated particles with typical hantavirus grid-like surface features to which surface projections were attached (fig. ) . iv- scanning vol. , supplement iv ( ) fig our previous studies have demonstrated that application of scanning electron microscopy (sem) imaging for examination of transplanted hearts gave new possibilities for evaluation and early detection of acute rejection (jakobczak et al. ) . continuation of our studies confirmed the benefits of sem for diagnostics and basic investigations in cardiac transplantations. the aim of this study was to investigate coronary vessels during acute rejection in experimentally transplanted hearts. allogeneic heterotopic heart transplants were performed in ether anaesthetized rats, using microsurgical techniques according to the ono-lindsey method. male rats of the inbred long-evans strain were used as recipients, and male inbred sprague-dawley rats served as donors. the anastomoses-the donor aorta to the recipient abdominal aorta and the donor pulmonary artery to the inferior vena cava of the recipient (end to the side)-were performed under the operating microscope (wild m ) with - microsutures (davis-geck). cardiac allograft survival was estimated daily by electrocardiogram and palpation of ventricular contractions. rejection was considered complete at the time of cessation of a palpable cardiac beat. rejection was confirmed with laparotomy and histologic examination. the animals did not receive immunosuppression. allograft survival ranged from to days. transplanted hearts were perfused with . % nacl and . % glutaraldehyde solution in . m sodium cacodylate buffer ( mosm; ph . ). hearts fixed in glutaraldehyde were cut into thin sections, mm thick slides orientated perpendicularly to interventricular and interatrial septums. slides were treated with % osmium tetroxide for h at room temperature. then the specimens were dehydrated in an ascending series of ethanol solutions, ending in rinses in % ethanol and in pure acetone. thereafter, slides were criticalpoint dried using co , mounted on aluminum stubs with conductive silver paint and coated with a thin layer of gold. for examination a jeol jsm c sem was used. microcorrosion casts of the coronary arteries and veins and myocardial microvasculature of transplanted hearts were prepared by infusion of methacrylate casting medium (mercox). following infusion of approximately ml of casting material for one heart, the preparations were left for min to allow polymerization to occur. then, the tissue was macerated, leav- empty nm particles were often seen. virus particles were seen by thin section em as shells coated with barely distinct surface projections and enclosing hair-like strands of nucleocapsid material (fig. ) . particle size ( - nm) was smaller than that seen by negative stain em. spherical particles with elongated tubules (fig. ) were often observed by both negative stain and thin-section em. we conclude that deer mice trapped near the homes of humans with unexplained acute pulmonary illness harbor hantaviruses that likely are the causative agent for the human illness. ing a microcorrosion cast. dried preparations were mounted on stubs, coated with gold, and examined with sem (murakami , potter et al. . investigations of the coronary vessels of the right and left ventricular walls, septum, and the apex region were performed. tissue fragments were taken from various heart regions each day during the first postoperative week for studies with sem, and remnant tissues were used for histologic procedure. grading of the severity of cardiac graft rejection was based upon the stanford classification. in addition, the coronary vessels of transplanted hearts (of the other experimental group) were reproduced with a casting medium and examined with sem. the observations were compared with views of nontransplanted and syngeneically transplanted control hearts. the applied vascular casting method enables one to study the three-dimensional architecture of the vascular network of the rejecting myocardium. besides the large areas with minimal damage and readily distinguishable impressions made in the cast material by endothelial nuclei, there were the regions where various signals of vascular pathology were present: changes of diameter of coronary arteries and veins; unusual size and shape of the vessels; indentations on the cast surface caused by adhesion of blood cells to the vessel wall; characteristic occlusion of the vessels; irregularity of capillary network with changes in diameter and cast surface; capillary destruction with accompanying extrusion of casting material into the interstitial tissue. the observed vasculopathies varied in mild, moderate, and severe rejection and in several areas of the hearts. studies of coronary vessels of transplanted hearts using sem imaging have made significant advances in the understanding of rejection vasculopathy possible. sem observations of sectioned transplanted hearts correlate with results obtained by application of the microcorrosion casting technique. data showed that the highest rate of acute rejection occurs in the interventricular septum, and the development of rejection in various heart regions differs significantly. observed differences in localization and dynamics of development of vasculopathies in the septum and left and right ventricular walls of transplanted hearts confirm the unequal character of rejection. sem imaging is a valuable method for investigation of vascular pathology during acute rejection in transplanted hearts. the authors are very grateful to prof. a. miodonski, director of the sem laboratory of ent dept. of the n. copernicus academy of medicine, cracow, for the enabling of sem observations. preservation of the ureteral blood supply in kidney transplant surgery and ureteral reconstruction is of paramount importance in preventing urological complications of the ureter. the incidence of complications in ureteral surgery has declined in recent years, in part due to greater understanding of the ureteral blood supply and improved surgical technique. however, the ureteral vasculature has been described primarily at the gross level and only recently received attention at the microvascular level. because of its critical role in the success of renal transplants and its potential vulnerability to surgical trauma, the ureteral vasculature merits further investigation. we studied the microvascular anatomy of the ureteral vasculature (uv) in male new zealand rabbits using transmission electron microscopy (tem) and scanning electron microscopy (sem) of vascular corrosion casts and alkalitreated tissue samples. the uv was perfuse-fixed with buffered glutaraldehyde via the abdominal aorta, washed in buffer, and treated with alkali according to the method of takahashi-iwanaga for sem. for tem, fixed tissue was embedded in epon, sectioned, and stained with uranium and lead. for vascular corrosion casts, the uv was washed free of blood with buffered saline and filled with plastic resin (mercox•/methylmethacrylate, / ) at physiologic temperature and pressure. resin-filled tissues were digested with % koh, washed in water, critical-point dried, mounted on stubs, and sputtercoated with gold palladium for sem examination. the blood supply to the male rabbit ureter originates primarily from ureteric branches of the renal artery, the testicular artery, and the vesicular artery. these intrinsic vessels run within the wall of the adventitia the full length of the ureter. perforating arterioles and venules pass through the muscle wall and divide further in the lamina propria to supply a dense plexus of continuous capillaries in the mucosa. although numerous arterioles and venules populate the lamina propria, the ureteral musculature does not possess a rich capillary bed. the capillary plexus is positioned between the transitional epithelium and the lamina propria and uniformly extends the entire circumference of the ureter. capillary orientation occasionally follows the longitudinal axis of the ureter, but is primarily plexiform and exhibits multiple "y" and "t" shaped anastomoses (fig. ) . at the points of junction with the underlying arterioles and venules of the lamina propria, the capillaries commonly exhibit "kinks" and "bends." alkali treatment revealed that the capillary bed is supported by a dense, fibrous network of collagen (fig. ) . pericytes were occasionally associated with the capillary network. intercapillary distance ranged from - µm and capillary diameters typically were - µm. venous valves were not observed in the ureter. the combination of vascular corrosion casts, alkali treat-ment, and tem provides a clear and comprehensive perspective of the microvasculature of the rabbit ureter. the limited number of intrinsic vessels supplying the ureter wall and capillary bed emphasize the importance of understanding the blood flow in this organ in related surgical procedures. rabbits were perfused with glutaraldehyde and mercox ® via the abdominal aorta and subsequently processed for preparation of vascular replicas according to a previously described method. the angioarchitecture of the tibia was studied with use of the scanning electron microscope. at low magnification the pattern of vascularisation to the tibia could be established. they are richly supplied with arteries which pass into the bone substance proper from the periosteum. the main supply is from the nutrient arteries, which divide longitudinally in a dichotomous manner after passing through a foramina in the compact bone, major branches running both proximally and distally. this is the general case in all long bones. these branches reach the epiphysis and diaphysis of the tibia. in addition, separate arterial entities supply specific regions of the tibia: i. periostal vessels, ii. epiphyseal vessels, and iii. diaphyseal vessels. arteries and veins can be readily distinguished due to their characteristic nuclear impression present on the surface of the replicas. arterial nuclear impressions are oriented longitudinally to the long axis of the vessel, whereas in veins they are situated circumferentially (miodonski et al. ) . for further details about methods, see syed ali et al. . at higher magnification it was possible to demonstrate the microangioarchitecture of the epiphyseal region, which consisted of a very fine meshwork of capillaries arising from a main epiphyseal artery. a connection between arteries of the epiphysis was observed and consisted of medium-sized arteries. the capillaries of the epiphyseal meshwork show a rich anastomosing network building open, sinus-like dilatations. capillaries are also richly present in the areas of the growth plate and stressed areas of the epiphysis of the tibia. the so-called sinuses are situated under the cartilaginous plate. the venous drainage takes place through the vessels running parallel to the corresponding arteries. the animals were premedicated with chloroform and were then anesthetized with ketanest ( . ml/kg body weight) via intraperitoneal injection. after examining the abdominal reflex the rabbits were fixed on an operating table. all preparations were carried out at room temperature. the abdomen was shaved and opened cranially at the xyphoid process down to the pubic symphysis. after gently displacing the intestines and mesenterial convolutes, the abdominal aorta and the inferior vena cava were exposed very carefully caudal to the kidney vessels. a knob cannula was inserted through a small incision in the abdominal aorta and held in place by a ligation around the cannula distal to the point of incision. these procedures should be performed very quickly, otherwise the blood pressure will fall. the hindlimb vessels were washed through the cannula with an isotonic nacl solution containing heparin ( i.e.) and sodium nitroprusside ( mg/l) in ml. the inferior vena cava was opened with an incision as exit for fixing fluid and clamped when required. soon thereafter, . % glutaraldehyde in phosphate buffer ( . m, ph . ) was passed through the same cannula. after completing the perfusion, about ml mercox (methacrylate) was applied through the cannula and observed until the mercox came out of the inferior vena cava. it was then clamped to avoid unnecessary backflow. the animals were left overnight at room temperature and then placed in a water bath for h for rapid polymerization. the bones were dissected free from other tissues and left in a % koh solution, which was changed every day. the specimens were washed with distilled water very carefully; it is very important to avoid every kind of mechanical disturbance. the solution and water can be changed with the help of a small water pump. the best results were achieved with an alternative change of % koh and % trichlor tetra acetic acid solutions. after complete maceration, the preparation was checked under a stereo microscope to eliminate all rest tissue. it was then frozen at − °c and freeze-dried. the preparation was contrasted with % os o vapour in a desiccator for about h to increase the contrast in the microscope. the specimens were fixed on scanning stubs with conducting silver and sputtered with gold ( min, - ma). they were then viewed in a psem (philips) microscope and photographed with kodak film. iv- scanning vol. , supplement iv ( ) fig. low magnification view of a rabbit tibia from the epiphysial zone with its specific end capillaries arrangement, the proliferation zone, and the connecting capillaries between the epiphysis and metaphysis ( apart from the ability to capture three-dimensional ( -d) images of microscopic structures, a confocal laser scanning microscope (clsm) equipped with multiple detectors allows one to add an extra dimension to the data acquisition process. a typical example are the ongoing studies in our laboratories. in this paper, we discuss the channel spill-over problem associated with acquiring two-color clsm images and present image processing techniques that analyze multichannel image data. for double labeling of dna replication sites, mouse t cells, exponentially grown on cover slips or synchronized at specific times in -phase, were pulsed for brief times ( - min) with cldu (chlorodeoxyuridune). the pulsed cells were then fixed and processed for fluorescence microscopy using monoclonal antibodies, appropriate extraction conditions, and fluorochrome-conjugated secondary antibodies which enabled differential recognition of sites of cldu versus idu incorporation into newly replicated dna. under the conditions used, there is no measurable cross reaction between the antibodies for cldu-versus idu-labeled replication sites. optical sections of labeled cells were collected with an olympus lsm gb- clsm with a m w ar ion laser. the microscope is operated in high resolution mode ( × pixels) with three fluorescence channels and a transmission channel. the microscope is controlled by a - mhz computer with mb ram and . gb hard drive. the digital confocal optical sections were transferred via ethernet to a dedicated sun sparc / with mb memory for further analysis. during image capture, a combination of band-pass and high-pass filters are used to mask unwanted emission at each detector. a major problem with the dyes used is their overlapping emission spectra. this seemed to contribute a significant amount of spill-over from the green channel (fitc) to the red channel (texas red or rhodamine) and only an insignificant amount in the other direction. because of the unidirectional nature of the spill-over between red and green channels, we were able to apply a correction factor to the red channel based on the green-channel intensity. to obtain the correction coefficients, a set of calibration images were obtained by using a sample which is identically labeled in both color fluorescent dyes and captured at different gain and offset parameters. for each of these images, the mean spill-over value in to the red channel (r i ) is calculated for each pixel value (i) in the green channel as follows: ( ) where i r (x,y,x) and i g (x,y,z) denotes the image intensities of the red and green channels, respectively, and n i indicates the number of pixels in the green channel having value i. a third order polynomial is fitted to the data set (<i, r i >,i= , , ... ) and the corresponding coefficients (a o a ,a ,a ) are estimated using the least square error criteria. the correction factor for each pixel of the red channel is then computed as follows and subtracted from the original intensity of the red channel to obtain the corrected image. ∆i r (x,y,z)= a o + a i g (x,y,z) + a i g (x,y,z) + a i g (x,y,z) ( ) figure shows the channel spill-over for different filter sets. the corrected images were analyzed using a set of image processing routines developed at our laboratory to obtain the boundary of replication sites in individual channels. these boundary data are then used to detect the overlapping particles between channels, and the overlap area is calculated for each overlapping particle. the results are presented in several forms including a two-dimensional histogram of particle volume and overlap percentage. the image processing routines are able to generate boundary data of a × × image with particles in approximately mins and the overlap estimation using boundary data takes approximately s. currently we are working on visualization techniques of raw and processed multichannel -d images. these fringes, these dark, granular, linear structures shed dark particles which then fused with opposite lines. a stream of particles trafficking between cells in contract, particularly at overlaps, was noticed. this pointed to a rather intense exchange of very small particles between cells of the same origin on contact. we conclude that interference reflection mode, video-rate, laser scanning confocal microscopy is a useful tool for intravital analysis of the intracellular structural dynamics in relationship to cell type, function and pathophysiological state. department of plant pathology and department of botany, university of georgia, athens, georgia, usa powdery mildew diseases of plants are caused by a group of obligately parasitic fungi belonging to the order erysiphales, class ascomycetes. the somatic hyphae of most of these fungi grow exclusively on the surfaces of infected plant organs, most typically leaves. these hyphae form specialized structures known as appressoria that attach to the host surface. each appressorium gives rise to a tiny penetration peg that penetrates the wall of the underlying epidermal cell and invaginates the host cell plasma membrane. the penetration peg then develops into a specialized structure known as a haustorium that absorbs nutrients from the host cell. in this study transmission electron microscopy was used to examine the haustoria of the powdery mildew fungus erysiphe lagerstromemiae and the relationship of these structures to epidermal cells of infected leaves of crape-myrtle (lagerstromemia indica). the haustorium of e. lagerstromemiae (fig. ) possesses a slender neck region and an expanded body that contains a single prominent nucleus. much of the neck is surrounded by a collar of host cell wall material. a single septum with a tiny central pore with which woronin bodies are associated is present in the distal portion of the neck near the haustorial body. the haustorial body is divided into numerous small, coiled branches. the entire haustorial apparatus is separated from the host cell cytoplasm by an extrahaustorial membrane (figs. , ) . though continuous with the host cell plasma membrane, the extrahaustorial membrane is much thicker than the plasma membrane and is highly convoluted in certain regions. the haustorial branches are separated from the extrahaustorial membrane by a region known as the extrahaustorial matrix. at this point it is unclear whether the finely fibrillar material comprising this matrix originates from the fungus, the host cell or both. although the haustorial apparatus of e. lagerstromemiae occupies a considerable portion of the overall volume of an invaded cell, the host cell organelles appear normal. however, numerous golgi bodies and many structures resembling microbodies are concentrated in the host cytoplasm very near the extrahaustorial membrane (fig. ) . pavel vesely and alan boyde* institute of molecular genetics, academy of sciences of the czech republic, prague, czech republic; *department of anatomy and developmental biology, university college, london, u.k. video-rate laser scanning confocal microscopy in the reflection interference mode (irm) enables the visualisation of fine intracellular structures in living cells in vitro and the observation of rapid changes of shapes, the trafficking of very small particles, and exchange of material. the dynamics of this intracellular activity can be established by optical sectioning with objectives of high magnification and numerical aperture, from within the bottom level of the cell contact to the substrate through to the top cell surface: these levels may be up to µm apart for large and/or tall cells. in the present studies, three video rate confocal microscope systems (lasertec lm , noran odyssey and biorad dvc ) were used to compare the ultramicroanatomy and dynamics of motion of intracellular structures in established cell lines (k , k , t , and a on). k cells are clonal descendents of spontaneously in vitro transformed lew/cub rat fibroblasts which give rise to low metastatic sarcomas in vivo: k cells are rous sarcoma virus transformants of the k line and both t and a on cells developed from k by neoplastic progression to a higher metastatic capacity. previously observed differences in the incidence of cells with rapidly oscillating and trafficking particles between k and the other cell lines were analysed at improved resolution. using the noran odyssey system, with zoom factor and a / . nikon lens, this phenomenon was observed in almost all the cells of all the cell lines compared. this finding provided an explanation of the previously described difference between k and the other cells which was obtained using a nikon / . wi lens (but not with an olympus / . wi using the lasertec system, zoom factor ) from the level of the cell periphery adjacent to the culture surface only. the present observations indicate that there may be a difference in the extent of this type of intracellular activity towards the periphery of the cell. more accurate mechanical targetting of the optical probe will be needed before it will be possible to measure this effect. at the free, top surface of some k cells, small pinocytic opening and closing was seen. similar images of opening and closing near the bottom sides of some cells in all cell lines were evidently produced by vertical oscillatory movements of particles in small clusters. when a . mm pinhole diameter (odyssey) was used to improve confocality, the interference fringes were observed to move, during focussing, along the steep slope of the high bodies of the k cells, optically transforming granular, linear, or randomly oriented structures inside the cell into concentric lines. above and below florida institute of technology, department of biological sciences, melbourne, florida, usa acid phosphatase (ap) is well known as one of the representative lysosomal enzymes. ap activity is recognized at the light microscopic level as intensely stained granules. although the localization and distribution of ap activity in the mammalian inner ear were researched by several investigators, we found only one report about the occurrence of ap in the avian inner ear (marmo ) . the objective of the present study is to demonstrate the occurrence of ap activity in the membranous labyrinth of the chick's inner ear, using the simultaneous coupling azo dye method (barka and anderson ) , and to discuss the significance of these findings. after newly hatched chicks were sacrificed, their ears were fixed for h in . % glutaraldehyde (ga) or . % paraformaldehyde (pfa). the membranous labyrinths were dissected out, dehydrated in either a graded series of acetone (used only for . % ga fixation specimens) or n,n-dimethylformamide (dmf), and embedded in jb- (polysciences, warrington, penna.), a methacrylate plastic. these sections were incubated for or h at °c in an incubation medium (ph . ) containing naphthol as-bi phosphate as substrate and hexazonium pararosaniline as a coupler that used the azo-coupling method. in some instances, intact, fixed whole specimens were incubated in an aliquot of the same incubation medium as that of sectioned specimens and were then dehydrated with dmf, embedded in jb- or lr white, and sectioned. for control specimens, either a substrate-deficient medium or an incubation medium containing cupric sulfate ( . m) was used. methyl green was used as a nuclear stain. intense ap activity, as represented by dense dye deposits, was detectable in the supranuclear area of almost all hair cells in the basilar papilla and vestibular sensory hair cells (fig. ). this result was in agreement with the finding of marmo ( ) . there were no differences in the distribution pattern of ap activity of the hair cells from the distal to the proximal region in the basilar papilla. in particular, hair cells of lagenar macula were most often characterized by intense ap activity in the subcuticular area (fig. ) . the functional significance of high levels of ap in the hair cell is still a matter of conjecture. for example, ishii and balogh ( ) demonstrated no morphologic evidence for phagocytic or secretory activities in hair cells, although their electron micrographs of hair cells reveal lipofuscin-like substances within lysosomes. it has long been contended that nonsensory hair cells such as stria vascularis, external spiral sulcus cells, and those around the macula and cristae ampullares help to regulate the ionic composition of endolymph (kimura et al. , kikuchi and hilding ) . in the present study, the columnar cells and the cells of the tegmentum vasculosum showed moderate to strong ap activity, and the transitional epithelia of the cristae ampullares also showed strong ap activity . the dark cells help to regulate the ionic composition of endolymph and perilymph (kimura ). in the present study, intense ap activity of dark cells was concentrated in the supranuclear area or diffusely in the cytoplasm. these results show that ap activity is highest in cells with highest levels of transport activities on the production of endolymph (marmo ) , although the metabolic linkage between ap and the transport enzymes is uncertain. the wall cells and supporting cells of the vestibular labyrinth showed no enzyme reaction. the statoacoustic and vestibular ganglion cells showed various degrees of ap activity. the sections of statoacoustic and vestibular ganglion cells of specimens that had been embedded in lr white displayed more intense ap activity than those sections from specimens embedded in jb- . ap activity was also stronger in specimens fixed in . % pfa than in . % ga. the syncytiotrophoblast layer covers the fetal villous tree which is in direct contact with maternal blood. it contains many microvilli, resembling a carpet surface, which are responsible for the absorption, excretion, and synthesis of many key hormones which are important for fetal development, secretion, and exchange of gases; of course, many earlier scanning electron microscopy (sem) studies of normal and pathologic placental villi have been described and many investigations on the histology, ultrastructure, and three-dimensional features of human placental villi have been carried out. [ ] [ ] [ ] [ ] technical handicaps causing structural deformations on the placental villi have been indicated. all of these studies depended on the normal development of placental villi but were not found to be comparable with tubal pregnancies. the purpose of this study is to describe the development of the human placental villous trees emerging from chorionic plate during the early periods of uterinal and tubal pregnancies, and to compare their three-dimensional structures using sem technique. iv- scanning vol. , supplement iv ( ) fig samples of human placentas have been studied. six early specimens obtained by curettage or by hysterectomy have been staged as described in our previous study. four additional specimens dated according to anamnestic data in comparison with the measurement of fetal weight and length at , , , and weeks p.c. were obtained from clinically normal pregnancies interrupted by legal curettage or by hysterectomy, and two samples aged and weeks p.c. from tubal ectopic pregnancies. all specimens were fixed and prepared for sem. the specimens were dehydrated in ascending concentrations of ethanol, critical-point (bio-red e ) and air dried, and coated with a layer of gold particles using a bio-red sc super coater. observations were made with a jeol-jem sem. ectopic tubal placental villi trees are not as well developed as normal uterinal villi trees. villi formation and ramification were very rare depending on the physiologic causes. some villi shoots were thinned gradually from base to its apex, and at the top these developing villi showed very interesting properties; they were curved, thinned, and crossed each other (fig. ) . the predominant villi types were immature, intermediate, and mesenchymal villi. [ ] [ ] [ ] the surface of these villi trees was completely covered by small and deformative curved microvilli and by fibrinoid particles compared with the normal uterinal villi trees. the trophoblastic shell of some villi was peeled off and degenerated. developmental retardation of placental villus trees is clearly seen. some villi were curved and crossing, and gradually thinned to the apex; on the villi surface, many wrinkles and furrows resembling very old skin were observed (fig. ) . it is well known that the placental development shows a parallel regulation due to the desidual properties. cellular and extracellular interactions take place between uterine and trophoblast during initial stages of placentation. , - khong and robertson suggested that the deficient decidua is the main reason why tubal pregnancies do not reach to term. the findings they describe may be explained as being due to absent decidua rather than being its cause. moreover, another reason for the usual failure of ectopic pregnancies to reach term is placenta accreta which becomes placenta percreta with bleeding. according to our sem observations, in tubal pregnancies the formation and development of placental villi trees were found in some patterns observed in uterinal placental samples. villi formation patterns-buds, tendrils, and shoots-are very rare on the surface of villi trees (fig. ) . placental villi shoots do not show uniform composition compared with to the uterinal villi samples. these are very thinned to the apex of villi, and are compressed in a common configuration, resembling a racket which does not appear as an alive condition. these observations are very interesting and original. the university of iowa central electron microscopy research facility (cemrf) was established in to support all faculty, staff, and students needing this technology. initially, the cemrf operated with one transmission electron microscope (tem), one scanning electron microscope (sem), and three staff members, and it supported about projects annually. during the past years, all instrumentation predating has been replaced and now includes two tems, two sems, two eds systems, a scanning probe microscope, a laser scanning confocal microscope, and all necessary supporting equipment. the facility presently consists of staff members and supports over projects yearly from departments in colleges and industrial laboratories. one of the unique strengths of the cemrf is that both biomedical and physical scientists use the facility. the development of the university of iowa cemrf was made possible due, in large part, to the central administration's support of equipment acquisition over the past decade. of the $ , , invested in equipment, more than % was provided by the graduate college and the vice president for research, with the other % obtained through equipment grants and user fees. of the operating expenses for the facility, % are recovered from a large and well-funded group of investigators who are charged on an at-need, fee-for-service, first-come, first-served basis. faculty recognize that they have a facility available to them that provides immediate access to state-of-the-art equipment and techniques, as well as one that provides training and supervision. in addition to supporting research, the cemrf offers four formal courses, as well as assisting with sections of eight other classes. the facility annually organizes at least two workshops and provides about tours for visiting scientists, faculty and student recruitment, high school students, business groups, and politicians. the facility also serves as the business office for the iowa microscopy society. concurrent with the development and success of the cemrf, several departments voluntarily divested themselves of their own electron microscopy (em) equipment ( ems on campus in compared with ems in ). this represents a savings of more than $ , annually ( dollars), as well as the release of or more rooms for other use. in addition, the interaction between the remaining six em laboratories is more positive as a result of this downsizing and centralization. the availability of user-friendly quality equipment in the cemrf assures that em is now accessible to all university faculty, staff, and students. in addition, with many investigators sharing instruments, it is easier to justify acquisition of new equipment for the cemrf. accurate dimensional metrology of the submicrometer gold absorber structures of x-ray masks can be accomplished in the scanning electron microscope (sem) with the use of electron beam modeling (postek ) . accurate metrology is possible because the x-ray masks present a unique measurement object from most other semiconductor structures viewed in the sem. this occurs because the silicon support membrane is x-ray transparent by design. this characteristic can be used as a distinct advantage in electron beam-based mask metrology since, depending upon the incident electron beam energies, substrate composition, and substrate thickness, the membrane can also be essentially electron-transparent. the areas of the mask where the absorber structures are located are essentially x-rayopaque, as well as electron-opaque. viewing the sample from a perspective below the mask by placing an electron detector beneath the mask provides excellent electron signal contrast between the absorber structure and the base membrane. the present technique utilizes a broad acceptance angle detector which is different in concept from other transmission electron (te) detectors used in the sem. in this case, the broad angle is used to detect as many of the transmitted electrons as possible (i.e., whether scattered or not) that have an energy above some predetermined threshold which is usually several kiloelectron volts. then, the electrons are physically filtered both by the signal threshold characteristics of the detector and an by an electron energy filter in front of the detector. energy filtering of the transmitted electrons excludes the highly scattered and thus lower-energy electrons from entrance into the detector. this greatly improves the contrast level over the conventional transmitted-electron detection mode for this type of application, and greatly simplifies the required electron beam/sample interaction modeling necessary for edge determination. it is, in fact, this change in electron detection philosophy that makes the present te approach so attractive for dimensional metrology and inspection of x-ray masks. monte carlo modeling of the transmitted electron signal was used extensively to support this work in order to determine the optimum electron detector position and characteristics, as well as to determine the position of the edge in the image profile. the monte carlo modeling is more accurate in this work, in contrast to the secondary electron detection mode, because in the transmitted electron detection mode the modeling of the electron beam/specimen interaction becomes far less difficult than in the modeling of typical secondary electron images of other opaque objects. the generated low-energy secondary electrons (which are complex to model) are excluded from the detector and, therefore, do not need to be included in the calculation. combining all of these factors provides a modeled signal that is extremely sensitive to wall slope. wall slope variation can result in large differences in the modeled profiles. the comparison between the data from the theoretically modeled electron beam interaction and actual fitted experimental data is shown in figure for a wall angle of ˚. the theoretical profiles were shown to agree extremely well with experiment, particularly with regard to the wall slope characteristics of the structure obtained from the sem images and video profiles. a plateau in the transmission is seen in the modeled profile as the beam traverses the edge, which can be used to identify the loca-tion of the edge of the absorber line and thus allows accurate measurements to be made. this work provides an approach to improved x-ray mask linewidth metrology and a more precise edge location algorithm for measurement of feature sizes on x-ray masks in commercial instrumentation. the transmitted electron detection mode is also useful in both mask inspection and mask repair, because the high contrast of the image allows for rapid determination of mask defects and high-density contamination particle detection because the transmitted electrons simulate transmitted x-rays. this work represents the first time that electron beam modeling has been used to determine the accurate edge location in an sem image. this also represents an initial step toward the first sem-based accurate linewidth measurement standard from nist, as well as providing a viable metrology for linewidth measurement instruments of x-ray masks for the x-ray lithography community. postek using monte carlo models that take into account the gaussian beam width of the incident electron beam, a noticeable broadening of the linewidth measurements is simulated for aluminium linewidths under glass. this simulated effect correlates closely with measurements of linewidth using energy dispersive x-ray analysis of an aluminium ka line under glassivation. detailed simulation of a full linewidth measurement has been performed using a point source and gaussian incident electron beam for the multiple scattering and single scattering models. the modeling effort was undertaken to better understand the effect that incident electron beam width would have on the accuracy of scanning electron microscope measurements. the multiple scattering (ms) model was adapted from pas-cal code for a single material to simulate layered materials, specifically, sio over aluminium. further, application-specific code was added to simulate an electron beam scanning across a linewidth on a fabricated microelectronic circuit. the linewidth selected for these simulations was one micrometer and one micrometer thick with a selectable sio thickness for the overcoat. in this case, the sio thickness was . micrometers. the single scattering (ss) model was adapted also from available pascal code for layered materials with the same application-specific code used in the multiple scattering model. the differences in the results from the two models used for the simulation are shown in figures and . the results for the ms and ss models at k trajectories are shown in table i . the gaussian beam width was selected to be . micrometers. the gaussian beam noticeably broadens linewidth measurement over point source, based on the results in table i . the linewidth measurement was taken at fwhm by extrapolation between points. the large differences between the ss and ms models is under further study. the calculation of the image contrast from samples with surface topography can be done using monte carlo techniques as long as the electron trajectories can be calculated through a surface profile. the image simulations that are described here were done using the same methodology that has been applied to the calculation of the electron backscattered signal from samples where the composition variation is taken into account (ly and howitt and these proceedings) . the design of the program is such that the scattering cross sections are reassessed as the electron passes from one region of the specimen into another, which in this case includes free space. the program takes longer to run than that for a homogeneous specimen with a flat surface because the parameters, such as the atomic number and atomic weight and density, need to be constantly updated at each point in the calculation. depending upon the image resolution that is required, we divide the three-dimensional specimen into blocks of either specimen or vacuum. the modifications to the conventional monte carlo approach to such calculations include not only the specimen geometry but also the determination of the energy of the electron that is backscattered and the direction in which it travels. in this way the signals at the backscattered and secondary detectors can be distinguished because almost all the low-energy electrons find their way to the secondary electron detector, whereas only those in the line of sight to the backscattered detector contribute to this signal. in most practical situations, where the geometry of the specimen is difficult to predict, it is useful to have specimens of standard shape, such as a sphere cone or box, to compare directly with the images. the calculation of the image at the secondary electron detector for spheres of the same size but of different atomic weight is shown as an example in figure . the spheres are assumed to be on a graphite substrate and the electron-beam energy is kev. the signals are displayed as they would appear in a micrograph, that is, in an intensity scan across the image and as a two-dimensional profile of the image intensity. we have also found that pseudo color images, where the various gray levels are replaced with different colors, are very useful when it comes to comparing calculated and experimental images. calculated secondary electron images from nm spheres of silicon, titanium, nickel, and molybdenum on a carbon substrate at an electron beam energy of kev. the signals are displayed as they would appear in a micrograph, that is, in an intensity scan across the image and as a two-dimensional profile of the image intensity. pierre hovington, dominique drouin, raynald gauvin, david c. joy * department of mechanical engineering, university de sherbrooke, sherbrooke, québec, canada; * em facility, university of tennessee, knoxville, tennessee, usa quantitative analysis of resolution < nm can be achieved at low accelerating voltages. however, to exploit all the emitted signals (x-rays, secondary, and backscattered images) fully, specialized programs have to be used. in this paper we present some results that can be achieved with such programs. it is shown that our single scattering monte carlo program can effectively predict the effect of surface oxidation on aluminum (al), model the backscattered coefficient even at very low energy (< kv) and accurately predict the backscattered profile around a spherical inclusion. at low voltages, mott elastic cross section has to be used. the tabulated mott scattering cross section of czyzewski et al. ( ) , combined with the energy loss of joy and luo ( ) , is used as the physical basis of the program. hence, the program can be used accurately for simulation even at very low energy (e < kv) (fig. ) . numerical experiments can be made with one or several regions of different composition and shapes. the regions can be defined as horizontal (i.e., layered samples) or vertical (i.e., grain boundary). in addition, based on the results of gauvin et al. ( ) , spherical inclusions can also be modeled. the program is written in c language and makes use of a fully graphic environment both for input and output of data. in figure , we present a typical output of the program of a simulation on a multilayer electronic component [a nm ga ( % at ) al ( % at) as ( % at) on a gaas substrat] covered with nm of contaminated carbon. the effect of the backscattered electrons on the resolution can be clearly determined since their paths are marked with a different color. in addition, at the end of the simulation, the interaction volume ranges at , , , and % are plotted. to increase the range of applicability of our program, backscattered, secondary, and x-ray images (k, l, and m lines) can be generated. generated and experimental images can then be used for metrology and microanalysis. we present in figure a backscattered linescan of a nm diameter beam (eo = kv) with a hypothetical nm diameter hemispherical mns inclusion in a fe matrix. it is important to note in figure that the difference between the "screen dimension" and the "real dimension" is over % . in figure , we present an experimental backscattered profile taken across an mns inclusion in steel at kv. we note the similarity of the theoretical and the simulated backscattered profile (slight decrease between the center and the end of the inclusion and the presence of peaks at the interface between the matrix and the inclusion). iv- scanning vol. , supplement iv ( ) fig . when low voltages are used, the geometry and composition of the surfaces greatly influence the resulting signals. in figure , we present an experimental spectrum from a pure aluminum (al) sample taken at kv with a windowless eds detector and a theoretical generated spectrum (hovington et al. ) . the difference between the two spectra is mainly due to the presence of oxide (al o ) at the surface of the sample. in figure , we compute the j(rz) curves for an oxide thickness of and nm and for a nonoxidized sample. the decrease of the al k intensity for the and nm compared with the nonoxidized sample is and %, respectively. the experimental to theoretical ratio found on the spectra (fig. ) is approximately %, indicating that the oxide layer is < nm thick. it is important to note that because the standard and the unknown may not have the same thickness of al o , the use of theoretical stan-dards may become critical in quantitative x-ray microanalysis at low voltages. material investigation methods based on the interaction of the electron beam with a solid requires detailed information about the electron distribution. among the various approaches which allow one to calculate the electron distribution, the monte carlo simulation seems to be most suitable. this is due to the fact that the monte carlo method generally can be applied to any target. the main problem of this approach is the accuracy of the electron distribution approximation. one important criterion of the quality of the approximation is the coincidence of the calculated and experimentally observed x-rays in depth distribution. note that the correct calculation of this function plays the essential role in epma data interpretation. as have been shown in the monte carlo process developed by murata et al., which is based on the mott cross section for elastic and on a knock-on model for inelastic interactions, it provides a rather good agreement between the calculated and experimental φ(ρz) function for cdlα line in al and au targets at the electron beam energy e = kev. but a more careful comparison of φ(ρz), obtained according to this model with the experimental data for sikα in al, ni, ag, and au at e = , , kev, respectively, and cukα at e = , kev, reveals some discrepancy, especially in the tail part. this discrepancy increases when the atomic number grows and the electron beam energy decreases. the origin of this discrepancy can be connected with the errors of the approximation of the differential cross sections for elastic and inelastic interactions, as well as with the ionization cross section. as the mott formula for elastic cross section uses the model of the atomic potential, then the approximation of this potential can influence the results of the scattering. to avoid the errors connected with the choice of the atomic potential approximation, we have used the elastic differential cross section obtained by riley et al. with the help of the static approximation theory (sat). the main features of the developed monte carlo program are as follows: . the elastic interaction is calculated according to the sat cross-section . the energy dissipation process is described by the knockon model. the bethe formula for de/ds is used in the high-energy region (e > . j i ). in the low-energy region (e < . j i ), the equation used in [ ] is exploited instead of the bethe law . the fast secondary electrons produced by electron-electron interactions in an inelastic collision are taken into account . the mean ionization potential is chosen according to the berger and seltzer formula . to calculate the ionization cross section, the formula of gryzinski is used. we have calculated the energy and angular spectra of the electrons transmitted through the films of the various compositions and thicknesses. comparison of these results with the data obtained with the help of the mott cross section shows that the use of the sat approximation for the elastic interaction does not influence essentially the electron distribution and cannot improve the accuracy of its determination. the comparison of φ(ρz) functions which are found in accordance with these two models confirms this conclusion. therefore, one can state that the details of the mott cross section together with the errors in atomic potential approximation cannot influence the electron distribution in targets. the factor which can affect the φ(ρz) in targets independently of electron distribution is the ionization cross section. to take into account this factor, we have used the experimental results obtained by long et al. during differentiation of the heart there appears to be a sequential pattern to the formation of individual muscle fibers. phenotypic changes result in the expression, synthesis, and organization of complex proteins to form the terminally differentiated myocytes. the formation of the basic pattern of myofibers ultimately determines the physiologic performance of the final form of the heart. pattern formation involves both intracellular events associated with myofibrillogenesis and extracellular events associated with cell:cell and cell:matrix interactions. this basic pattern is repeated to form complex layers which results in the final form of the heart. it is essential to understand the sequence of expression associated with these patterns of fibers in order to understand errors that may result in congenital defects in heart formation. in this study time pregnant ( . - . days of gestation, ed) sprague dawley rats were obtained from harlan sprague dawley laboratories (indianapolis, ind.). animals were anesthetized with % chloral hydrate in normal saline solution and the individual embryos, including uterine tube, were removed and placed in . m phosphate buffered saline solution containing . % azide (pbs-a) at °c. the embryos were removed from the uterine tube under a dissecting microscope and fixed for - h in % paraformaldehyde in . m hepes (n- -hydroxyethylpiperazine-n'- -ethanesulfonic acid) at ph . . the embryos were scored and gestational ages were assigned. the embryos ( . - . days of gestation) were stained using a modification of the procedure described by tokuyasu and maher ( ) . after fixation, the embryos were placed in pbs-a for h and immersed in . % triton x- in pbs-a for h. after washing for an additional hour in pbs-a, the embryos were incubated in units/ml bovine testicular hyaluronidase for min, washed in . % triton x- with . m glycine in pbs-a for h and immersed in % bovine serum albumin (bsa) in pbs-a for h. the individual embryos were incubated overnight with rhodamine-labelled phalloidin from molecular probe, eugene, ore. ( µl of stock solution/embryo), or control buffer. following incubation, the embryos were washed in pbs-a at °c for h and immersed in % bsa for h. hearts from . - . day gestation embryos were removed and fixed as above. after fixation, the embryos were encased in % polyacrylamide gel using a modification of the procedure described by hansen and dryer ( ) . after fixa-tion, the hearts were washed in pbs-a for h and immersed in . % triton x- in pbs-a for / h. the hearts were embedded individually in blocks containing % polyacrylamide with . % bis-diamine which was polymerized with % ammonium persulfate. the polyacrylamide blocks containing the hearts were sectioned at µ with an oxford vibratome model g before being stained as described above. the tissue was examined using a bio-rad mrc- confocal laser scanning microscope (biorad, cambridge, mass.) using × (na= . ) and × (na= . ) objectives. at ed . to . , the cells of the myocardium are round and tightly packed with no discernable pattern. the first indication of cell orientation into fiber patterns occurred in the outflow tract and ventricle. the outflow tract myofibers developed circumferentially and maintained this pattern throughout the study time. the ventricular myofibers also developed circumferentially; however, they gradually changed into a net-like pattern ( fig. ) followed by a thickening of the ventricular wall and protrusion of trabeculae. with continued maturation, the ventricular trabeculae appeared to coalesce especially in the re-gion of the muscular interventricular septum (fig. ) . the myofiber pattern developed later in the atria. the atria expressed the same early pattern seen in the ventricles; however, they retained the net-like appearance throughout development. the results indicate that there are region-specific differential changes in the orientation of myocytes that result in the unique myofiber patterns of the heart. these regional pattern changes may be correlated with mechanical forces and hemodynamic alterations during development. the preparation of thick, optically clear sections of fragile tissue structures greatly aids the power of confocal laser scanning microscopy in imaging three-dimensional structures. hale and matsumoto ( ) have presented confocal images of agarose-embedded, vibratome-sectioned tissues; earlier workers have embedded soft tissues in polyacrylamide gels for frozen sectioning and lectin or immunohistochemical staining of structures in - micron sections (bronson et al. , hausen and dreyer , johnson and blanks . we report here conditions for embedding, sectioning, and staining embryos in polyacrylamide gels for a variety of confocal imaging techniques. in general, infiltration of - mm specimens for - h in a mixture of - % acrylamide monomer ( part bisacrylamide cross linker to parts monomer) yields, upon polymerization (with . volume of % ammonium persulfate), blocks that cut easily by vibratome between - microns. these conditions work well for tissues previously stained (with fluorescent probes or dii tattoos) or for staining gel sections water-soluble fluorochromes with low molecular weight (e.g., propidium iodide, phalloidin). for immunostaining after sectioning, the acrylamide concentration must be reduced to - % acrylamide to facilitate access of immunoglobulins to antigenic sites, and the gel must be supplemented with % agarose to aid sectioning and handling. illustrated below are confocal images from acrylamide sections of a stage chick embryo, fluorescence-immunostained in whole mount for cadherins. this specimen was fixed in % dmso/ % methanol (dent and klymkowsky ) and incubated overnight with a : dilution of commercial antiserum against a synthetic peptide common to all known cadherins (sigma, pan cadherin, #c ). rinsing preceded overnight reaction with tritc-conjugated secondary antibody, brief formaldehyde fixation, and embedment in % acrylamide. a survey ventral view was taken before transverse vibratome sectioning across the entire embryo at micron intervals, yielding eight sections that were spanning the thoracic region and were mounted in % glycerol for closer inspection. figure illustrates a stereo pair of projections from a series of confocal images across the atrioventricular junction. this gel-embedding and vibratome-sectioning method yields abundant, optically clear, and easily handled sections for confocal examination of fluorescent structures in water-miscible media. greater detail concerning procedures and technical problems with this technique are provided elsewhere (germroth et al. in press) . department of anatomy and cell biology, suny health science center, syracuse, new york, usa endothelial cells arise in the early embryo from precurser cells called angioblasts. in the quail embryo, the emergence of these cells can be observed as an epithelial to mesenchymal conversion of cells from the mesoderm which may be observed by scanning electron microscopy (sem) after removal of the endoderm or by immunolabelling the whole embryo or sections with a monoclonal antibody (qh- ) which labels angioblasts. the process of angioblast migration and assembly into the solid cords, which are the rudiments of the earliest blood vessels, is called vasculogenesis. simple embryological manipulations have been used to distinguish the role of cell migration in the early vessel rudiments. the dorsal aortae arise ventrolateral to the forming somites, and inserting blockages revealed that little cell migration is involved in their formation. the endocardial cells which form the endothelial cell lining of the heart, in contrast, undergo an extensive migration from more lateral regions of the embryo which has been studied by blockages and the construction of quail/chick chimeras. iv- scanning vol. , supplement iv ( ) the first rudiments of veins in the embryo are the cardinal veins. these form beneath the ectoderm of the embryo in an area with few angioblasts. the early stages of cardinal vein formation have been studied by using immunogold-labelled secondary antibodies after qh- labelling and imaging in the sem backscatter mode after silver enhancement. these studies revealed that small groups of angioblasts appear over the lateral mesoderm, possibly by migration from the angioblastrich mesoderm adjacent to the endoderm, and then migrate medially to assemble into a solid cord of cells in association with the developing wolffian duct. the ability of scanning probe technology to image atomic topography of a surface, to manipulate individual atoms, and even to probe the internal structure of a molecule confirms the significance of these super-resolution microscopies used at the nanometer-scale analysis of molecular systems. so far, a number of interactions between the sample and the probe tip gives access to a variety of local properties (wickramasinghe ) . the latest applications of the local probe microscopies on organic molecules include photoemission, tunneling potentiometry, electrostatic, elastic and tribo logic properties, or near-field thermal measurements and as many other phenomena on which various contrast formation mechanisms are based. topics include imaging of individual biomolecules, highly ordered molecular assemblies, and polymeric materials under various environments. the development of additional imaging techniques,based on near-field electromagnetic interaction between the probe tip and the surface (betzig and trautman ) , or even by means of magnetic resonance (rugar et al. ) , indicates that new technologies with subnanometer spatial resolution could be achieved in principle. recent advances in near-field optical microscopy (nsom) confirmed spatial resolution in the range of - nm but still limited by the diameter and optical penetration depth of the aperture. here, we demonstrated a new concept for an aperture near-field scanning optical microscope which combines force microscopy and optical scattering for imaging the sample at subwavelength resolution without the use of an aperture. the end of a silicon tip is illuminated in transmission mode by a laser beam through a transparent substrate. both the tip and the sample undergo perpendicular motion, each at a different frequency, with amplitudes chosen comparable to the desired measurement resolution. the scattered light from the tip and the surface is detected at the difference frequency for imaging and sample at sub-wavelength resolution without the use of an aperture. we describe the novel experimental scheme and present the most recent results obtained from our system. the resolution demonstrated to date is nm using helium-neon wavelength and optical line scans are shown in figure . finally, a consideration of the basic theory demonstrates that much higher resolution can be easily anticipated. these preliminary results firmly establish the great potential of this new near-field optical microscopy for biological research. high-resolution scanning electron microscopy (sem) studies of enamel crystals from remineralized enamel have provided clues as to the changes in crystal diameters within specific zones of artificially created carious-like lesions. , these data have supported the evidence obtained through polarized light microscopy of zones of demineralized and remineralized enamel. the dark zone, viewed in polarized light, has been identified as the zone of remineralization. these initial studies were conducted using sputtered coatings of gold/palladium (au/pd). high-resolution images of the crystals within the zone of remineralization revealed large crystallites in excess of µm, often with what appeared to be remineralized "nodules." however, it was difficult to determine whether these were one crystal, or two crystals which had fused during the remineralization phase. a study was conducted using an ultrathin coating of sputtered chromium (cr) film and se-i imaging to provide topographic contrasts of crystal surfaces. since the majority of the se-i information should be produced close to the surface, the resultant image is specimen-surface specific. , artificial caries-like lesions were prepared in noncarious human teeth (second and third molars) and sectioned into µm sections which were then viewed in polarized light to identify the dark zone of remineralization. the sections were then fractured through the dark zone producing a spicule of enamel with an edge of fractured enamel free from sectioning artifacts. each specimen was then mounted onto supports with silver paste with its fractured edge facing up. measurements were then made to determine the location of the dark zone so that it would be easily located within the sem. specimens were degassed and then sputtercoated with nm au/pd and nm cr. , specimens were imaged at high magnification in the se-i mode by placement on the condenser/objective (c/o) lens stage of an isi ds- /lab sem at kv and a hitachi s- cold cathode field emission (fe) sem operated at kv. high magnification se-i images (s- fe sem) of au/pd coated enamel crystal surfaces within the dark zone, revealed particulate features decorated by metal giving it a continuous granular appearance (fig. ) . analysis of a specimen coated with a . nm cr film revealed crystal surfaces with well defined particulate features that were clearly delineated and separate from one another. this study documents the effect au/pd coating has upon the examination and evaluation of enamel crystals at high magnifications. results obtained document that an ultrathin coating of sputtered chromium film of - nm does not produce the coating artifacts found with conventional sputtered au/pd. an ultrathin coating of cr, together with the use of se-i imaging, allows surface features to be more accurately imaged and measured. iv- scanning vol. , supplement iv ( ) fig . previous results have shown that the ability of intercalative dyes to modulate the antiviral activity of poly r(a-u) was related to the groove through which the dyes intercalated into the poly r(a-u). when poly r(a-u) was combined with the minor groove intercalating dyes such as ethidium bromide (eb) or the minor/major groove intercalating dyes, optimum enhancement of antiviral activity was observed at a dye/ribonucleotide ratio of / . no enhancement of antiviral activity was observed when poly r(a-u) was combined with the major groove intercalating dyes. when eb was combined with poly r(a-u) and then added to human foreskin fibroblast (hsf), the % effective dose of the poly r(a-u) was -fold lower. the results of additional studies demonstrated that the enhanced antiviral activity was not due to superinduction of interferon, direct viral inactivation, or host cell cytotoxicity. phase contrast, fluorescence, and confocal micrographs of hsf cells following a -h exposure to µm eb alone or a µm eb/ µm poly r(a-u) combination stained the nucleolus, but not the chromatin. negatively stained transmission electron microscopy (tem) preparations (fig. a) and replicas (fig. b) (fig. a,b) which may be associated with the enhanced antiviral activity of this combination. under the conditions employed in this study, poly r(a-u) exhibits an elongated conformation ( - nm in length) that possesses a number of hairpin loops as well as single-stranded and double-stranded domains (fig. a,b) . the double-stranded domains are found predominantly at the base of nm hairpin loops. in contrast to the poly r(a-u) alone, micrographs of the eb/poly r(a-u) combination illustrate the presence of condensed structures with diameters ranging from to nm. results from scanning force microscopy corroborate the results of both tem preparations. tem of unstained and uranyl-stained eb/poly r(a-u)-treated hsf cells illustrate the endocytosis of electrondense material into acidic compartments of the hsf cells. subsequently, the electron-dense material escaped from the acidic compartment and formed electron-dense bodies with dimensions that closely approximate the dimensions of the eb/poly r(a-u) combination visualized in the negative staining preparations. these electron-dense bodies are detected near the nuclear membrane and in the nucleolus. the nucleolus of an unstained, eb/poly r(a-u)-treated hsf cell demonstrates the segregation of the nucleolar components so that the fine fibrillar component, which comprises the nucleolar organizer region, is being peripheral to a dense granular material occupying the major central area of the nucleolus. the results of the current study and our previous work suggest that the elevated antiviral activity of the eb/poly r(a-u) combination may be related to the ability of the eb to complex with poly r(a-u) and condense it into a conformation with dimensions that can be accommodated by endocytotic vesicles. exit from the acidic compartment is promoted by unbound eb that induces endosomal or lysosomal swelling. subsequently, changes in the topology and surface charge of the poly r(a-u) induced by the eb allow increased access to the nucleolus which results in the modulation of additional cellular processes, especially rrna synthesis and processing. aromatic constituents in plant cell walls are associated with other wall components and affect strength and other characteristics of plant cell walls. ultraviolet (uv) absorption microspectrophotometry has been useful in characterizing aromatics within specific cell types in plant organs. , , this technique was applied to walls of bran and endosperm cells in a series of hard and soft wheats. kernels from hard and soft cultivars were fixed in glutaraldehyde ( % in . m cacodylate buffer at ph . ), and sections were cut with a microtome at µm thickness. the sections were mounted on quartz slides in glycerin and covered with a quartz cover slip. cell types in the sections were analyzed for uv absorption using a computer-controlled zeiss umsp microspectrophotometry system. transmitted illumination was provided by a xenon lamp (xbo w) with a connecting grating monochromator using a bandwidth of nm. a -× quartz objective lens with a final aperture of . µm, which was delimited within about onethird of the area of a field-limiting diaphragm to reduce stray light, was positioned over walls of selected cell types. absorbance of transmitted uv illumination was recorded over a range from - nm at -nm increments. the system was standardized at µm. spectra were collected, displayed, and evaluated using the zeiss lambda scan software. uv absorption maxima or distinct shoulders occurred near , , and nm. while the absorption near nm is not defined, absorption near nm is believed to be due to lignin (i.e., polymerized phenolic constituents) and that near likely is due to ester-linked ferulic acid. synthetic lignin, from polymerization of coniferyl alcohol with horseradish peroxidase, has a strong absorption at nm and no absorption at higher nm. further, ferulic acid linked to arabinoxylans, which has been isolated from bermudagrass cell walls, gave a shoulder near nm and a strong max at nm. other work, using the addition of cinnamic acids to milled lignins or removal of phenolic esters by alkali treatment, supports the above spectral interpretations. the kernel of wheat consists of several distinct cell types (fig. ) . spectra were obtained of walls of these various cell types (fig. ) . epidermal and parenchymal walls gave the highest absorption and both had spectra indicative of lignin (near nm) and also of ester-linked ferulic acid (near nm). nucellar epidermis walls have spectral patterns similar to the cell types above, but absorption was much less. aleurone walls gave spectral patterns indicative of mostly ester-linked ferulic acid and less lignin than previous cell types; the side walls gave a considerably higher absorbance than upper or lower walls. endosperm cell walls, which enclose the starch/protein matrix, were totally lacking in uv absorption maxima, indicating no aromatic constituents in these walls. variations occurred in uv absorption and spectral patterns for walls of various cell types in wheat kernels. however, no consistent differences occurred among the various kernels that might relate to hard-or soft-rated wheats. iv- scanning vol. , supplement iv ( ) fig . food microscopists are continually searching for noninvasive methods of examining the internal structure of foods, especially those with delicate labile structures. x-radiation is of considerable potential because of the great penetrative power and the minimal need for sample preparation. examination can be carried out at atmospheric pressure, and there is no intrinsic reason why temperature-controlled or temperature-ramped studies are not possible. the term x-ray microscopy covers several techniques and can be defined as the use of x-rays to produce a magnified image, or to give information about specific microscopically identifiable parts of a specimen. our current equipment consist of an electron gun, two electromagnetic lenses (condenser and objective), and x-ray target. x-rays are generated from a µm spot on a tungsten target. the target usually is positioned so that the microfocused electron beam glances the side of the target. x-rays from the microfocal spot form a cone which diverges through a thin window. focusing is achieved by placing a grid with bar widths of and µm into the beam and adjusting the current in the condenser and objective lenses. the image is captured on a tv monitor. finally, test radiographs are recorded on x-ray sensitive film. the x-ray radiograph is a shadowgraph and, where the sample is thick, the overlaying shadows will be complicated to interpret. two methods can be used to help interpret the image. one method is to take stereo pairs of the sample and view these images with a suitable stereo viewer which will show a threedimensional image. the other method is tomography. in the projection x-ray microscope, the entire specimen is in focus at once with the same resolution, determined by the spot size, although different depths within the specimen are magnified by different amounts. three-dimensional images can be made by moving the specimen either by tilting or translating it between exposures in the divergent x-ray beam. these images are then viewed with a suitable stereo viewer which will show a three-dimensional image of the structure removing some of the confusing overlay of detail to be found. we have also obtained images from pieces of tissue subject to tensile elongation. in the design of our x-ray microscope there is considerable distance separating the x-ray film from the specimen, and this space allows the apparatus for tomography to be set up. tomography is a form of imaging whereby a section or "tome" can be taken through a specimen by using x-rays, and details above and below the sectional area of interest will be blurred out, giving a fairly clear image of this area. this technique is nondestructive to the sample. the method is similar to that de- scribed by lindegaard-andersen and thuesen, originally proposed by watson. in their method, the x-ray source is stationary and the subject and recording film are rotated synchronously. the x-ray beam strikes the film at near glancing incidence producing transaxial summation images. however, the associated point spread function has a /r radial dependency, which means that smearing of detail will have occurred. the results are sufficiently encouraging, nevertheless, to stimulate the search for improved contrast and resolution, with provision for three-dimensional mapping of internal structures. the images shown here are of food products containing large air cells, giving maximal contrast in the x-ray beam. we have obtained comparable success with water-filled cellular structures such as vegetable and fruit tissue. in the future, we anticipate further enhancement of contrast and resolution by the use of improved x-ray flux, image capture, and image processing. centre for food and animal research, agriculture and agri-food canada, ottawa, ontario, canada over the past years, electron microscopy (em) has proven to be a useful tool in assessing the effects of various physical and chemical treatments on the microstructure of egg components (related to the production of egg products, baking, behaviour in various food systems, nutrient bioavailability, etc.) much of this information has been acquired using transmission electron microscopy (tem) to view yolk samples which have been biochemically extracted after removal from the egg. the author studied microstructural changes in yolk during its in vivo digestion by the epidermal cells of the chick embryo yolk sac. yolk is digested within these cells, as signified by its presence in varying degrees of microstructural alteration, compartmentalized within cell organelles. a temporal sequence can be inferred with the degree of microstructural change observed. it was possible to follow the fate of yolk granules and low density lipoprotein (ldl) particles during digestion, because yolk granules retain their integrity during preparation for em. ldl particles were made visible by crosslinking action of the fixatives and by the use of a specific enhancement technique. initial work focused on the microstructure of native, undiluted yolk which had been fixed using conventional and novel fixatives. thin sections were examined by tem. these experiments allowed us to optimize fixation conditions and confirmed earlier results by other workers that yolk is composed of granules which have a subgranular structure and lack a boundary membrane. the granules sit in a suspension of closely packed ldl particles. yolk granules from incubated eggs were identical to those from unincubated eggs and showed no microstructural changes to indicate that digestion had taken place outside of the cell. after clarifying that yolk is digested intracellularly, the next phase of the study was the microstructural description of intracellular yolk and yolk sac epithelial cells during incubation, prepared using half-strength karnovsky's fixative and the imidazole-buffered osmium tetroxide protocol (ibo) of angermuller and fahimi ( ) which enhanced lipid staining. epithelial cells were examined systematically using em, from apex to base, to study the three processes associated with digestion: uptake, breakdown, and transport. using tem, granules and ldl particles were observed to enter the cells in uptake structures, the microplicae and coated pits, respectively. when samples were viewed using sem (fig. ) , these uptake structures appeared as fossae of different sizes, which have not previously been described in the literature. yolk granules and ldl particles were observed by tem, within the cells, in phagosomes, endosomes, and acid phosphatase-containing vacuoles (secondary lysosomes), components of an active intracellular digestion system as it is presently known. the secondary lysosomes contained yolk granules exhibiting various degrees of microstructural alteration (fig. ) . the microstructure of these intermediates in yolk digestion appears to be very similar to that appearing in the literature describing known biochemical manipulations of yolk ex ovo, and indicates that a re- iv- scanning vol. , supplement iv ( ) fig. sem of apices of yolk sac epithelial cells from an embryo incubated for days. yolk granules have entered the cell, and a fossa (f) is observed. mp = microplicae. lationship may exist among structural, functional, and biochemical information. the results of these individual experiments were used to propose a scheme of yolk digestion based on progressive microstructural changes of intracellular yolk. in addition, a transport system for yolk lipid and its digestion products to the embryo was microstructurally demonstrated using ibo. this transport system shared ultrastructural characteristics with that reported for lipid transport in intestinal cells, especially with respect to the formation and transport of chylomicronlike particles. laser scanning confocal microscopy and digital image processing were used to visualize the pattern of de novo blood vessel formation in the quail embryo. stage quail embryos were fixed on the yolk, excised, then immunolabeled with qh , an antibody marker for angioblasts and vascular endothelial cells . after incubation in fluorescein-conjugated secondary antibodies, the embryos were mounted in pbs/glycerol ( : ratio) under a # glass coverslip. specimens were examined on a biorad mrc confocal laser scanning device equipped with zeiss optics. a × objective lens was used to image the entire caudal half of each embryo during the laser scanning step. ten µm optical sections were acquired in a plane parallel to the embryonic axis (en face). the ten optical sections were then collapsed into one composite image using biorad's proprietary software (fig. ) . the image was imported into photoshop ™ software on a macintosh quadra . using this software, the single composite fluorescence image was processed with the "emboss" tool (fig. ) . other image processing routines such as edge tracing, sharpening, and contrast enhancement can also be applied with ease (not shown). in contrast to conventional microscopy, this procedure yields a map of the entire endothelial network of the quail embryo in sharp focus, and with a highly favorable signal-to-noise ratio (fig. ) . while some geometric distortion occurs when ten sections are collapsed into one imaginary plane, the flat- ( ) and ldl particles ( ) are found in separate organelles, and are also found together within the same organelle ( ) . myelin bodies (m) and lipid drops (l), products of intracellular digestion of yolk, are also observed. fig . this immunofluorescence image depicts a map of all the vasculature elements in the caudal half of a developing quail embryo (stage ). the wide vessels on each side of the embryonic axis are the dorsal aortae; lateral to the aortae are two fields of rapidly forming vascular networks. tened nature of the early avian embryo minimizes this problem. also, since the qh epitope (a carbohydrate) is present on multiple cell surface molecules, the image gives a reasonable approximation of vessel morphology and the protrusions of the vascular endothelial cells. the rendering of the data into a pseudo three-dimensional relief provides the observer with a more familiar visual format (fig. ). the type of image shown in figure facilitates comparison of endothelial sprouts and anastomotic foci during formation of the first vasculature in the quail embryo. based on images from embryos at progressively older developmental stages, we suggest that morphogenesis of the lateral anastomotic network occurs by mechanisms that involve angioblastic tractional structuring of the extracellular matrix (madri et al. , montesano et al. , stopack and harris . this hypothetical mechanism more recently has been elaborated upon by vernon and colleagues ( ) . according to the latter workers, cellular responses to morphogenic cues within the highly planar extracellular matrix underlie early vascular patterning. in addition, the digital imaging approach shown here offers an improved method of comparing normal vasculogenesis with experimentally produced malformations (drake et al. ) . ( . - . ). chemical composition and ph did not change during extended heating. the shmp sample was the firmest and the sc sample was initially the softest. the firmness of the sc and tsp samples increased with extended heating whereas the firmness of the dsp sample did not change. meltability decreased with extended heating in all samples, except the shmp sample which had the lowest meltability of all samples throughout the experiment. there were marked differences in the microstructure of the process cheese protein matrices after heating. osmiophilic areas gradually developed during heating and their incidence was related to the melting salt used. after h of heating, they were numerous but considerably smaller in the dsp sample ( fig. ) than in the tsp sample (fig. ) . the highest and the lowest incidence was in the shmp and the sc samples, respectively, which were the samples with the lowest and highest meltability. new york, usa mozzarella cheese contains parallel protein fibres created by stretching the curd in hot water during manufacture. the protein fibres are oriented parallel to the direction of extrusion and are separated by milkfat or whey (kaláb , taneya et al. . by varying manufacturing parameters and storage time, cheeses with different sensory/functional properties, as well as with different microstructural characteristics, can be produced (kiely et al. ). in the present study, the impact of varying the stretching temperature ( °c, °c, and °c) and storage time ( days, weeks, and weeks) at °c on the microstructure of mozzarella cheese was investigated using light microscopy (lm) and scanning electron microscopy (sem). for both lm and sem, samples were fixed in % glutaraldehyde in mm pipes containing mm cacl , before cryosectioning ( µm sections). sections were cut both parallel to the protein fibres (longitudinal) and across the fibres. for lm, sections were stained with ice-cold oil red o and methyl- ene blue. because the fat is not well fixed by aldehyde fixation, slides were held on a bed of ice during staining to prevent fat migration. for sem, the sections were stained with uranyl acetate in methanol, washed in methanol, and stained with lead citrate, dried, mounted on aluminum stubs, and gold-coated. this procedure removed most of the free fat and water/whey, allowing the association of the protein fibres to be clearly visualized. increased stretching temperature increased the size of the fat globules, which was particularly noticeable in cross sections. with °c stretching, the fat globules were relatively small and uniformly distributed across the section, while with °c and °c stretching there were almost two populations of globules: some small and some very large. in longitudinal sections, the protein strands formed during stretching at °c and °c were slender and smooth, while those formed during stretching at °c were thicker and less regular. little difference was seen with storage time in either longitudinal or cross sections for the three different stretching temperatures using lm. with sem, however, after removal of the fat, the ability of the fibres to hold together decreased with storage time. this is best seen in longitudinal sections, where the protein fibres are closely associated in cheese stored for days (fig. ) , and are able to withstand the stress of drying, while in cheese stored for weeks (fig. ) , the fibres have been weakened (presumably by proteolytic activity from coagulant and culture enzymes) and pulled apart as the section dried. indian dairy association, sector-iv, r.k. puram, new delhi, india milk is a canvas of seemingly silent molecular structure. there is a large variety of "resident molecules" with molecular harmony in milk which are of mammary gland origin. in general, the "structural matrix" of milk comprises three classes of biomolecules: molecules in suspension (casein micelles), molecules in solution (lactose, proteins, vitamins, and salts) , and molecules in emulsion (fat globules). because of its excellent nutritive composition, milk is ubiquitous and the most popular "ready-to-consume" food from time immemorial. today, in iv- scanning vol. , supplement iv ( ) fig . different parts of the world, milk from various species of animals is used for food. in the u.s., however, the cow furnishes virtually all of the available market milk, whereas in my country more than % of the total milk produced is of buffalo origin. hence, structural studies particularly on milk and milkfood products made from various animal species are more fascinating and challenging from a global angle. it is the local consumer habits that determine the final structural orientation of the product in the country of origin, especially in a country such as india where to % of milk produced is converted into a variety ( plus) of traditional milk products, using processes such as coagulation (heat and/or acid), desiccation, and formulation. hence, a presentation on the "structural style" of milkfoods which are developed on the basis of "environment-friendly green technology" in the tropical countries might generate a new dimensional need for future studies on milkfood products. a comprehensive literature scan of published papers in the areas of "food" and "food structure" during the last years reveals the following publication status: during the period to : . global: of the total number of , food articles published, , are related to "food structure." the number on "food microstructure" is . when i think of their contributions, i claim no merit for my presentation in this conference today. these data provide a "food-for-thought" why food structural studies do not receive the desired attention. we may ponder this issue. while preparing this presentation, the excellent review article by kalab on food structure and milk products was extensively consulted. keeping in view the existing and reported studies on the subject, the situation in the tropical countries, particularly regarding indian milk such as buffalo milk, deserves special attention. according to kalab the structure of milk and milkfoods determines their properties such as firmness, spreadability, elasticity, viscosity, and susceptibility to syneresis, which are globally recognized as texture. the understanding of the processes both conventional and traditional and their relationship between "structural style" and "textural mood" is important in product development. in the tropical countries, the preprocessing exposure of milk to the environment, that is, temperature, microbes, and humidity has to receive extra attention. the dynamic status and kinetics of molecular interaction in different species of milk should be a vital area of structural studies in asian countries. the structural studies on milk and milkfood are generally conducted by use of microscopy. the methods used are optical microscopy such as fluorescence microscopy and confocal scanning laser microscopy, and electron microscopy consisting of two major types: scanning electron microscopy (sem) and transmission electron microscopy (tem). these techniques meet the particular requirements of the study. the application and findings derived using sem and tem in various milk food systems have been extensively covered recently by kalab . repeating and reproducing the published data may not be necessary for the participants in this conference. however, a brief microcosm might be refreshing, and is hence documented below: . milk casein micelles ( - nm), being small, cannot be seen using a light microscope, but tem shows them as globules that are apparently composed of submicelles. . the casein micelles, if deprived of their stabilizing factor, aggregate and form a gel of a regular structural "crowd." . in the case of acid-gel, the micelles start disintegration as a result of the solubilisation of their structural partners such as calcium phosphate. . there is an interstructural and intrastructural relationship between the suspended casein micelles and soluble blactoglobulins. heat disturbs their stability equilibrium and their molecular relationship. . among the milkfoods, cultural milk products like yoghurt are highly influenced by heat in relation to their "set-style" and "stirred-style." the indian counterpart, "dahi," offers its advantage when made from buffalo milk because of its high curd tension due to high calcium level. this area deserves a close look using sem technique. . as far as cheese is concerned, because of its inherent multinatural processing steps using chymosin on one hand and starter culture on the other, it is an evergreen exploratory platform using either sem or tem. . dried milkfoods as spray-dried milk particles are globular of larger diameter fat-globules, however, they undergo some changes as revealed by sem studies. lactose plays a commanding role in a so-called instantisation process. while making a presentation before a "highly-structured" qualified scientist using sem and tem, it is tempting for me to present our own work of limited nature. in our laboratory, during the last decade, we carried out a sem study on buffalo milk and its casein. the micelles are of larger size and have higher-bound calcium unlike cow casein micelles. regarding the casein fractions, an elaborate study on the b-casein in relation to structure and function as affected by heat and enzymic cleavage were carried out. work relating to microstructure development on another indian milk product, namely "paneer," from buffalo and cow milk has been reported. more recently, while working on casein micelles in csiro dairy research laboratory in australia, the author made an original contribution when ascertaining the micellar integrity of casein micelles in milk. he used a very simple technology not dependent on sem or tem. it was on the "colour communication" property of molecules as an integrated system or in isolation. using this new technique, it is possible to declare the miscellar integrity and to identify milk from different animal species, based on the different miscellar structure-dependent light-reflecting phenomena. the time has come to develop a "global strategy" on milk structural studies with a view first to understand the local product and then to ensure marketing of "rightly-structured milkfoods" of the right type to meet the countries' marketing needs. starch is a major component of many foods and is commonly used to modify the texture of a system. starch gels are composites of swollen gelatinized granules embedded in a continuous amylose network. the rheological characteristics of the starch pastes or gels depend on the shape and swelling power of the granules, amount of amylose and amylopectin leached outside the granule, network entanglement, and interaction between the paste components. many food systems also include in their formulations a lipid phase, thus, starch-lipid interaction becomes a major concern in starch paste rheology. the interactions of triglycerides might be different from those of monoglycerides due to their amphiphilic character, dispersion states, and steric hindrances. triglycerides owe their characteristics to their fatty acid composition (chain length and insaturation) and distribution (davis et al. ) . the objectives of this work were to analyze the effect of different lipid phases on the swelling power of the starch granules, as well as to analyze the rheological behavior of the starch pastes (apparent viscosity and viscoelasticity). commercial corn starch (cs) (refinerías de maíz, argentina) was used as thickener. the lipid phases used were: ( ) shortening (sh) (molinos río de la plata s.a., argentina) containing . % of mono-and . % of polyunsaturated and . % of saturated fatty acids; ( ) sunflower oil (so) (molinos río de la plata s.a., argentina) containing . % of monoand % of polyunsaturated and % of saturated fatty acids, and ( ) commercial glycerol monostearate (gms) mivaplex (eastman kodak co., u.s.a.), which contained % of monoglycerides and % of diglycerides. seven percent starch suspensions with and without lipid phase ( % of sh or so or % of gms) were prepared using a modification of the method of eliasson ( ) . swelling power was calculated as the weight of sedimented gel divided by the original dry weight of starch. samples were placed on slides and micrographed in a leitz ortholux ii microscope with a photographic camera leitz vario orthomat (leitz, germany). the suspensions were gelatinized in a thermostatic bath at ± . °c under standardized conditions. a rotational viscometer haake rotovisko rv (germany) with a sensor mvip of concentric cylinders was used. the measurements were performed at °c and the transient shear stress curves (τ vs. time) were obtained at different constant shear rates (d) from to s − . apparent viscosities were calculated as τ/d ratio at d= s − . iv- scanning vol. , supplement iv ( ) table i shows the swelling power obtained from the starch suspensions with and without lipid phase. when either of both triglycerides, sh or so, was added, the swelling power values were higher than the value of the control (cs). the so, with the highest insaturation content, presented the higher swelling power. the addition of the monoglyceride (gms) led to the lowest value. the relative sizes of the swollen granules shown on the micrographs of figure confirm these observations. gms forms a helical inclusion complex with amylose, which might delay the transport of water into the granule and consequently decreases the swelling power (eliasson ) . the formation of inclusion complexes between the triglycerides and the amylose seems to be difficult because of the steric characteristics of these lipids. apparent viscosities correlated well with the swelling power (table i) ; the larger the swelling power the higher the viscosity obtained from the rheological curves at long shear times. bird-leider model (dickie and kokini ) was used to analyze the viscoelastic behavior and the shear time dependence of the different pastes: where n and m are the power law parameters and b and c are adjustable parameters related to the viscoelasticity and to the structure breakdown of the samples. a satisfactory goodness of fit was obtained (r min= . ) (fig. ) . when the swelling power increases, b parameter decreases (table i) . since viscoelasticity (b) depends mainly on the network entanglement (leached amylose) and the volume of the swollen granules, both factors should be considered to explain this behavior. because of the health risk and other environmental factors that airborne grain dust presents to the working population, our laboratory has initiated studies on the isolation, identification, and characterization of bacteria that possess multiple resistance to a series of antibiotics and other compounds (e.g., insecticides and pesticides) at the molecular level. samples of grain dust were collected from various grain elevators in the duluth-superior regions of the u.s. during the diverse growing seasons. each sample collected consisted of a heterogenous population of constituents that vary with encountered geographic, climatic, and handling differences. in addition, the geographical growth regions and the mechanism of storage of grains appear to be directly associated with the microbial flora and occurrence of toxic substances. scanning electron micrographs of the concentrated grain dusts were morphologically consistent with the observation of previous investigators. the dust from various plants consisted of a distinct assortment of particles; small husk fragments or pericarp (seed coat in case of flax) and "trichrome-like particles" were also present. numerous bacteria spores were seen at high magnification, particularly durum wheat and barley. light photomicrographs showed a heterogenous population of both gram negative and gram positive bacteria. the bacteria consisted of different shapes such as short rods, long rods, and cocci. transmission electron photomicrographs revealed the isolated strains to consist of one or more flagella attached to the membrane surfaces. at least three of the bacterial strains isolated were encapsulated. we exposed, selectively, twelve of the isolated bacterial strains to a variety of antibodies, pesticides, and insecticides. as a result, of the bacterial strains tested showed resistance to both ampicillin and bacitracin ( µl/mg). of the strains, showed resistance to insecticides (sevin) and the pesticides (enforcer) as high as µl/mg. three of the five strains that were resistant to both ampicillin and bacitracin were also resistant to the insecticide sevin at high concentrations ( µl/mg). these data suggest that bacteria found in grain dust may be directly or indirectly related to occupational health disorders. chlordiazepoxide (librium) is a commercial antianxiety agent, but its use has not been associated with any striking health improvements. nevertheless, millions of chlordiazepoxide (cdz) prescriptions have been written and the drug is widely employed clinically as a muscle relaxant, anticonvulsant, anxiolytic, and hypnotic. adverse effects vary from skin rash, nausea, headache, and impaired sexual functions to vertigo and lightheadedness, as well as complications from the drug's administration during pregnancy. the ciliated protozoan, tetrahymena pyriformis, has been of significant importance to research since its successful axenic culture years ago. the drug's tranquilizing effects on humans may possibly be elucidated by investigating cdz-induced impairment of the protozoan's growth and motility through modifying microtubular-directed ciliary function (bell et al. ) . here, uptake, recovery, and attempted localization of cdz, as well as its alterations of cellular ultrastructure are reported. thin layer chromatography of cdz (hoffman laroche, nutley, n.j.) in three different mobile phases revealed optimal cdz stability when dissolved and stored in isoamyl alcohol; benzene. after days of liquid culture, t. pyriformis removed % of the radioactivity from [ c] -cdz in the growth medium. liquid scintillation counting of cell washes during the -day time course suggested surface, nonspecifically-bound radioactivity. following days of culture, [ c] -cdz together with its metabolites and/or breakdown products were recovered from homogenates of tetrahymena. to detect the intracellular sites of cdz localization and possible action, both transmission electron microscopy and immunoelectron microscopy were performed. the former revealed that cdz disrupted cytoplas-buffer or cacodylate-buffered dopa revealed dopa/ppo reaction product but failed to reveal a definitive, substrate localization of the enzyme. instead, cytoplasmic morphologic distortions of buffered dopa-treated hyphae were apparent, mandating modification of the employed higher-plant, cytochemical procedure for localizing fungal ppo. thus, attempts to establish the route of ppo secretion to the growth medium utilizing liquid cultured hyphae may be of limited value as the sheath appears to be sloughed during shake culture. in this connection, c. versicolor grows upon a solid substratum when decaying timber in situ. support: doe-bctr program. the evaluation of surface topography is conventionally done with the scanning electron microscope (sem) by visually reconstructing stereo images in a stereo viewer. the monocular clues in an sem image, shown for example in the image in figure , can also be used to map the topography of a sample surface. the first attempt to identify the surface gradient from a secondary electron detector in this way was reported by suganuma ( ) who found that ( ) and described the inclination of the surface to the specimen plane, where a and b are the signal outputs from the two detectors and a n and b n are the signal outputs from the same material when it is perfectly horizontal. although it is possible to add additional detectors and to modify their signal outputs in a conventional electron microscope, the technique can also be implemented in a straightforward manner by using the differences in signal intensity from the two halves of a split backscattered detector. thus if a and b are the signal outputs from the two different halves of the detector, the backscattered signal, which is usually collected as topographic information (a − b) or composition information (a + b), can be multiplied together (a − b ) to produce the basis of the empirical relationship described by suganuma iv- scanning vol. , supplement iv ( ) fig . ( ) to fit the contrast variations. indeed, since the denominator in this equation is also a constant and there is invariably a flat spot somewhere in the image, it is easier to simply use the expression to try and describe the surface gradient. this function can then be scaled to an absolute magnitude of about to take maximum advantage of the contrast variations that exist. the simplest way to evaluate the surface topography without actually modifying the microscope is to process a digital image in a computer. such an image can be collected directly or a polaroid micrograph can simply be scanned into the machine. in either event the processing is fairly straightforward to accomplish the use of software such as the nih image program. the surface profile is obtained by integrating the surface gradient over the scan distance, and in figure we show, by way of an example, a faceted surface displayed as a conventional secondary image, as a topographic profile, and as a grey scale image indicating the faceted surfaces with the same orientation to the electron beam direction. suganuma t: measurement of surface topography using sem with two secondary electron detectors. j electron microsc ( ), - ( ) corn kernels of an unknown italian cultivar were surfacesterilized in full strength bleach ( . % sodium hypochlorite), rinsed in sterile distilled water, and germinated on difco potato dextrose agar (pda) under fluorescent light at room temperature. isolation of bacteria was made either directly from the rhizosphere of seedlings or from colonies produced on agar. bacteria were identified by their fatty acid profiles (microbial identification system, newark, del.) and diagnostic biochemical test (micro-id, durham, n.c.) . fusarium moniliforme used to test for bacterial antagonism were isolated from corn. experiments designed to infect seedling roots with the isolated bacterium were performed on corn kernels that were subjected to a double sterilization process in which the kernels were surface-sterilized with bleach and then subjected to a mild heat treatment to remove internal bacteria and fungi. this process produced sterile seedlings which remained so at least up to the to leaf stages of growth. the corn cultivars used were trucker's favorite, silver queen, reid's yellow dent, and an inbred cultivar, pr. samples were fixed in % glutaraldehyde in sodium cacodylate buffer and postfixed in % osmium tetroxide in buffer. the tissues were dehydrated in an ethanol series, critical-point dried and coated with gold-palladium or embedded in spurr's medium and sectioned. samples were examined using a philips scanning or jeol cx transmission electron microscope. the bacteria isolated from roots of the italian corn cultivar were gram-negative rods and were further characterized as being positive for voges-proskauer reaction, nitrate reductase, and ornithine decarboxylase and b-galactosidase. the isolate fermented arabinose and malonate. it was negative for phenylalanine deaminase, lysine decarboxylase, urease, h s, and indole production. it lacked the ability to utilize adonitol, inositol, and sorbitol, and was negative for esculin hydrolysis. these biochemical characteristics served to place this bacterium in the family enterobacteriaceae, the tribe klebsielleae, and it was identified as e. cloacae according to ewing ( ) . microscopic studies established that the bacterium was endophytically associated with the roots of the corn cultivar. scanning and transmission electron microscopy demonstrated that the bacterium was distributed uniformly over the corn root epidermis but was randomly distributed intercellularly in the root cortex and outer margin of the pericycle, usually adjacent to phloem cells. although there was a proliferation of bacterial cells in the intercellular spaces of roots (figs. , ) , there was no evidence of damage to host cells, decline in seed germination, nor seedling growth from e. cloacae infection during the week observation period. the presence of the bacterium on kernels of several corn cultivars enhanced the growth of corn seedlings and inhibited growth of f. moniliforme when the bacteria-infected kernels were germinated in fungal-amended soil. this indicated that e. cloacae is biologically associated with corn plants. the bacterium exhibited strong antagonism to several f. moniliforme isolates when grown on nutrient agar and pda plates. the fungus growth was inhibited and restricted to aerial growth on agar plates. the endophytic nature of e. cloacae in corn roots and its antagonism against isolates of f. moniliforme indicate that this bacterium has potential for the biocontrol of f. moniliforme, a corn pathogen. tracheal organ cultures have been widely used for the demonstration of the cell-and organ-destroying capacities of bacteria and viruses colonizing the respiratory tract. however, the assessment of the cell-damaging abilities of fish parasites, the gill organ culture appeared to be a suitable tool (stadtländer and kirchhoff ) . piscine gill epithelium represents the relevant target tissue for cytotoxicity studies of fish parasites, but this tissue is diffi- iv- scanning vol. , supplement iv ( ) fig cult to cultivate in vitro. excessive mucus production complicates the investigation with the light and electron microscope. this is even more difficult when infected specimens are investigated. here, cell debris due to released cells and cell fragments require special attention of the investigator to obtain interpretable electron micrographs. a fine balance is required between preservation of the status quo of the in vitro situation and the necessary procedures (multi-step preparation) for scanning electron microscopy (sem). in this abstract, we describe the detailed and improved preparation of piscine gill epithelium for sem. this will allow other investigators using piscine tissue for research to obtain scanning electron micrographs with high resolution at high magnification. gill filaments were obtained from rainbow trouts (salmo gairdneri, richardson) and were infected in vitro with mycoplasma mobile k, a wall-less prokaryote causing severe damage in gill organ culture (stadtländer and kirchhoff ) . each gill filament (noninfected and infected) was rinsed in several changes of . m cacodylate-trihydrate [cacodylic acid buffer (cb)] at ph . to free the tissue surface from mucilage, medium, and unattached mycoplasmas. the washing step turned out to be critical for obtaining satisfactory results. excessive washing led to complete removal of cell debris and did not reveal the same status quo of infection as seen with the light microscope during the in vitro cultivation of gill filaments. on the other hand, insufficient washing complicated the interpretation of electron micrographs, especially the identification of m. mobile on gill epithelium, due to excessive mucus and cell debris released from infected tissue. the pre-fixation was performed with . % glutaraldehyde (v/v) (in cb) for h at ˚c. the aldehyde was removed by careful rinsing in cb (three times for min). samples were not postfixed, but dehydrated in acetone covering a graded series of solutions (acetone/water) from through % (v/v). all specimens were critical-point dried (cpd) using carbon dioxide as the transitional fluid. cpd was performed in a critical point drying apparatus e (polaron equipment ltd., england) by filling the chamber with / of liquid carbon dioxide. the heating process was conducted in ˚c per min steps until the critical point was reached ( o c and bar). samples were immediately placed in an exsiccator containing anhydrous calcium chloride to avoid rehydration of samples by air contact. after being attached to aluminum mounting stubs using double-sided sticky tape, each specimen was coated with a layer of to nm of gold in a hummer v sputter coater (technics inc., alexandria, va., usa). samples were examined in an etec auto scan b (etec, calif., usa) operating at an accelerating voltage of . - kv. results were documented on a polaroid × land film (type , positive-negative). the method described above allows the detailed investigation of noninfected and infected piscine gill epithelium at high magnification with an sem (fig. , ) . good structural preservation of noninfected, apparently intact secondary lamellae (fig. ) and destroyed lamellae after infection with m. mobile (fig. ) document the usefulness of the described method for the study of the infectious process with this mycoplasma on fish tissue and will certainly also give convincing results in investigations with other fish pathogens. the arrows in figure indicate the presence of the flask-shaped mycoplasma on damaged piscine gill epithelium. bars represent figure : µm, figure : µm. for this study, the sem was superior to light microscopy (lm) and transmission electron microscopy (tem). lm does not demonstrate details in the damaged tissue and tem is much more laborious and time consuming. the mammalian bladder functions in urine storage and expulsion and is thus subject to alternating distension and contraction. blood flow in the bladder wall is compromised by distension (dunn , levin et al. , and acute and chronic overdistension result in ischemia and necrosis, respectively. the latter often leads to loss of mucosal integrity. yet, the vasculature of the bladder wall has rarely been studied (inoue and gabella ) except at the gross level. in this study we describe several unique features of the bladder microvascular anatomy using light microscopy (lm), transmission and scanning electron microscopy (tem and sem), vascular corrosion casting, and alkali digestion (takahashi-iwanaga ). twenty four male new zealand white rabbits were anticoagulated, anesthetized, and cannulated via the abdominal aorta. for routine lm, tem, and sem, the bladder vasculature was flushed with buffered saline at physiologic temperature and pressure, then perfuse-fixed with buffered glutaraldehyde; for corrosion casting, the vasculature was filled with resin (mercox/methylmethacrylate monomer, / ); and for alkali digestion, tissue was treated with n naoh at ˚c for min. thin sections were stained with lead and uranium, and casts and digested tissues were mounted on sem stubs with silver paste or colloidal carbon and viewed at - kv. the bladder is supplied by left and right vesicular arteries, branches of the internal ( %) or external ( %) iliac arteries. within the adventitia, the vesicular arteries send coiled branches dorsally and ventrally over the bladder surface. secondary arteries penetrate the muscularis to supply a rich capillary plexus (fig. ) closely apposed to and located in grooves within the base of the transitional epithelium (fig. ) . capillaries measure - µm in diameter and are often fenestrated and invested with pericytes. veins exhibit abundant valves primarily in the basal half of the bladder. the lamina propria of the bladder consists of very loose, flexible, collagenous connective tissue. unusual capillary "glomeruli," associated with accessory vessels paralleling the primary bladder vessels, are present in the adventitia on either side of the bladder wall. these glomeruli consist of one to four contiguous capillary spheres. these various methods provide a clear, three-dimensional view of the microvasculature of the rabbit bladder, reveal the very close association between the urothelium and the underlying capillary plexus, and describe the fine structure of the mucosal capillaries. several unique features of the bladder vasculature including capillary glomeruli require further char- iv- scanning vol. , supplement iv ( ) acterization. the latter may be associated with sensory ganglia related to pressure sensation, but their function has not been determined. these results form the basis for comparison of normal bladder vasculature with that of experimentally compromised vasculature. the epithelial cell layer lining the nasal turbinates of humans is functionally and histologically consistent with that of the lower airways. this anatomic site is easily sampled using an inexpensive, disposable curette, and the epithelium obtained can be evaluated for both clinical and experimental objectives. the original rationale for ultrastructural evaluation of clinical specimens of nasal epithelium in this facility was to document index ciliary lesions consistent with the diagnosis of primary ciliary dyskinesia (pcd) (figs. , ) among individuals considered at risk. patients referred for study encompassed both adult and pediatric populations and had lifelong histories of chronic sinusitis, bronchiectasis, and/or otitis media, but with normal immunoglobulin levels. several patients presenting with situs inversus, polysplenia, or infertility problems-clinical findings considered risk factors for pcd-also were evaluated. approximately nasal biopsies are submitted annually for evaluation, of which approximately % demonstrate impaired ciliary motion and ultrastructural abnormalities that could be the basis for altered ciliary motion and a diagnosis of pcd. this finding is consistent with the suspected prevalence of pcd in the general population although the rate appears higher among individuals of scandinavian descent, pacific islanders, and inbred populations. the diagnosis of pcd is confirmed by the electron microscopic documentation of any of three ultrastructural level lesions of airway cilia. these lesions are: ( ) dysmorphology of dynein arms, ( ) absent radial spokes, and ( ) microtubular transpositions. in our experience, dysmorphology of the dynein arms represents the major form of ciliary abnormality associated with pcd. missing radial spokes and microtubular transpositions have not been documented among any of our patients. these clinical studies also have provided a unique opportunity for defining a spectrum of ciliary abnormalities which distinguish the heritable primary ciliary abnormalities associated with pcd from acquired cil- iary abnormalities associated with chronic disease or acute injury due to infectious processes or exposure to irritant gases in the ambient air. in addition, other histopathologic features such as mucus cell hyperplasia have been encountered occasionally among children presenting with chronic respiratory disease and referred for evaluation. in summary, our experience shows that ultrastructural evaluation of nasal epithelium can provide a clinically significant perspective on respiratory health. this work was supported by scor grants hl , hl , and hl from the national heart, lung, and blood institute and by u. s. environmental protection agency cooperative agreement cr to philip a. bromberg. this is an abstract of a proposed presentation and does not necessarily reflect epa policy. using conventional light microscopy, silicone has been described as a translucent, clear, mucoid, refractile material which is often difficult to visualize. it is not birefringent, as is silica, by polarized light microscopy. silicone gel tends to form homogenous, rounded "globules," "vacuoles," or "droplets" unlike the angulated, sharp spicules observed with silica. in paraffin-embedded tissue sections, silicone gel often appears to be slightly out of the plane of focus. in addition, silicone gel "globules" occasionally drop out of standard µm histologic sections during tissue processing, leaving partially or totally vacant holes or "cysts." because silicone extravasation and deposition into surrounding fibrous breast capsules is difficult to visualize by standard light microscopy techniques, the electron microscopist must often "blindly" examine multiple fields/grids in a labor-intensive fashion. to decrease the time generally required for the positive identification of silicon by electron probe microanalysis (epma), alternative light microscopy techniques for preliminary screening purposes were investigated. six periprosthetic capsulectomy specimens, synovium from a previously reported silicone breast augmentation patient with arthritic pain and a silicone granuloma from another patient with a ruptured prosthesis were utilized in this study. sections were cut at , , , and µm and mounted on glass slides. in addition to standard permount mounting media, aqua-mount was stained with ink preparations: black stamp pad ink, india ink and aniline artist dyes-black, blue, brown in a : mixture for coverslip application. each series was stained with a battery of common histochemical stains. the sections were then viewed with a zeiss axioplan microscope utilizing conventional incidental light, polarized, non-koehler, phase contrast, and darkfield microscopy. a commercial silicone gel and silicone gel extracted from a previously implanted silicone breast prosthesis were smeared and examined unstained or stained with papanicolaou and "diff quik." silicone was noted to be refractile, nonpolarizable, and nonstainable. the results confirm the refractile, nonpolarizable, and nonstainable properties of silicone in histologic and cytologic preparations (fig. , scale bar = µm). decolorizing techniques (toluidine blue o, etc.) that were not completely differentiated, especially with thicker sections, occasionally demonstrated nonspecific dye "trapping" on the larger silicone globules. the relative ease of silicone localization was greatly increased in histologic specimens with non-koehler, phase contrast, and darkfield microscopy when compared with conventional light microscopy. although standard h&e staining was adequate for silicone localization in tissue sections, uniform dark staining with toluidine blue o increased the contrast between the stained tissue and the unstained refractile silicone. the contrast was also enhanced by aqua mount black stamp pad ink mounting media, with the silicone appearing milky white. in thicker sections, negative staining was slightly accentuated because of the increased concentration of stain as well as thicker, more refractile silicone globules, especially with darkfield microscopy. by utilizing these sensitive screening techniques, we were then able to sample paraffin-embedded tissue selectively, which dramatically decreased the time and effort for correlating confirmatory identification of silicon by epma (fig. ) iv- scanning vol. , supplement iv ( ) the majority of biological samples are high in water content. preparing tissues for scanning electron microscopy (sem) requires removal of this water and often produces severe structural distortions due to surface tension forces during phase transitions. this can result in bulk shrinkage artifact, surface cracking, curled cell borders, clumping or flattening of cilia, and collapse of surface vesicles. failure to postfix biological material with oso can lead to extraction of surface membrane lipids during solvent dehydration. it is likely that many shrinkage artifacts are related to incomplete or improper fixation and drying. critical-point drying (cpd) has become the standard procedure for most biological materials. although it produces a relatively intact end product, wollweber et al. have reported shrinkage of as much as % after cpd of glutaraldehyde and osmium-fixed macrophages and lymphocytes. the use of mordant techniques to complement or enhance oso and uranyl binding may aid in preserving fine structural details regardless of the drying method. numerous substitution/transition fluids have been introduced for both critical-point and direct-evaporative drying, some with more success than others. an advantage in using solvent drying techniques as alternatives to cpd is the ability to process large numbers of samples simultaneously. early attempts at direct drying of nonosmicated samples from ethanol and other solvents produced a generalized cell shrinkage and collapse. freon evaporative drying was shown to be a useful rapid drying technique by liepins and de harven. gamliel further refined the freon direct-drying technique to include the use of guanidine hcl as a bifunctional mordant, prior to osmification, to minimize shrinkage of leukocytes. direct drying from hexamethyldisilazane(hmds) was introduced by nation to dry insect tissue and has subsequently been used for drying various other tissues (adams et al. ) . peldri ii has since been shown to be an effective solvent drying medium. it is a solid at room temperature. a comparative study by bray et al. of peldri ii, hmds, and cpd, using both plant and animal tissues, has produced identical results with animal tissues but not plant tissues. the purpose of this study is to introduce acetonitrile as a potential solvent for direct evaporative drying. acetonitrile has been used as a less toxic propylene oxide replacement transition fluid for transmission electron microscopy (tem) dehydration and infiltration (edwards et al. ) . to date, no studies have been published indicating the of use of acetonitrile as both a dehydration and intermediate transition fluid for direct solvent-drying of tissue-cultured cells for sem. this study compares solvent drying of kb (hela) cells from freon , peldri ii, hmds, acetonitrile, and ethanol to critical pointdried cells grown on thermanox coverslips. the mordant (gtgo) technique of gamliel is included to illustrate its usefulness in shrinkage control. solvent-plasticware compatibility should always be tested before preparing cultured cells for sem; whenever possible, the use of glass is best. cells grown on thermanox coverslips were fixed in . % glutaraldehyde, . m cacodylate, . m sucrose, . mm cacl , ph . . the gtgo fixation protocol of gamliel was found to be the most useful one in preserving cell architecture (figs. , ) . in figure , cells were evaporative-dried under a mild aspirator vacuum after gtgo fixation and dehydration in acetonitrile ( %, %, %, %- ×, min each). as a comparison, cells in figure were identically fixed and criticalpoint dried after ethanol dehydration. the overall appearance of the cells from both treatments is similar at low magnification (figs. a, a) , with some shrinkage evident in either case. under these conditions, it appears that the cpd cells retain more fine structural details than those evaporative dried from an (figs. b, b) . the results of this study are encouraging and may lead to future studies to develop a simple fixation protocol which enables evaporative drying from solvents directly miscible with water such as acetonitrile. snow, which may occasionally cover up to % of the earth's land, supplies about one-third of the water that is used for irrigation and the growth of crops (gray ) . for this reason, estimating the amount of water in winter snow pack is an extremely important forecast activity that attempts to predict the amount of water that may be available for the following growing season. unfortunately these estimates can be easily iv- scanning vol. , supplement iv ( ) fig . confounded by the sizes and shapes of the snow crystals that comprise the snowpack. a snow crystal is a single frozen ice grain that generally results from a process known as nucleation in which atmospheric water vapor condenses on a solid particle or nucleus at temperatures below °c. when nucleation occurs, the water molecules form a hexagonal crystal lattice resulting from the specific orientation and binding that occurs between the oxygen and hydrogen atoms. depending on the temperature and moisture that prevails during formation and descent of snow crystals, nucleation may result in plates, stellar crystals, columns, needles, or dendrites-all of which are based on the hexagonal lattice structure. an individual snow crystal may range in size from µm to mm (gray ); aggregations of two or more of these crystals form a snowflake. the shapes of snow crystals have been extensively studied and photographed with the light microscope (bentley and humphreys , nakaya ) . although these studies have resulted in a classification system that currently recognizes distinct classes and over subclasses of snow crystals (hobbs ), detailed examinations have been hampered by the difficulty of working with a frozen specimen, which is susceptible to sublimation and melting, and by the limiting resolution of the light microscope. for these reasons, an attempt was made to determine whether snow crystals could be collected/stored and prepared for observation and recording in the low-temperature sem. attempts to allow snowflakes merely to settle on a precooled specimen holder were unsuccessful; the snowflakes tended to "bounce" off the holder and those that did alight did not remain attached during subsequent handling. a successful procedure consisted of placing a thin layer of methyl cellulose solution on a holder and precooling it to the prevailing outside temperature during a snow fall. next, snowflakes were allowed to settle on the surface of the methyl cellulose solution. after a few minutes, the holder was plunged into liquid nitrogen and transferred to the laboratory where it was retrieved from liquid nitrogen, mounted on the transfer rod of an oxford ct- hr cryosystem, moved into the prechamber for sputter coating with au/pd, and then inserted into a hitachi s- field emission scanning electron microscope (sem) equipped with a cold stage that was maintained at − °c. these procedures allowed us to observe several forms of the individual snow crystals as well as their nucleation centers. at low magnification, the specimens, which did not appear to be altered by the sputter coating, resembled those that had been previously photographed with the light microscope. the snow crystals were stable in the beam, did not sublime, and could be observed at magnifications of , × or more to reveal microcrystalline water deposits or rime on the surface of some the snow crystals. this procedure, which was used to collect specimens during several snow falls in beltsville, maryland during the - winter season, was also capable of preserving sleet, graupel, and hail. furthermore, storage holders were devised that allowed capture of the snowflakes and their storage in liquid nitrogen until the specimens could be processed for examination in the sem. finally, the specimen stage of the sem allowed specimen tilt so that stereo images of the snow crystals could be recorded (fig. ) . in conclusion, low-temperature sem is a viable technique for examining snow crystals at magnifications that far exceed the resolution of the light microscope. furthermore, the ability to collect and store samples enables investigators to accumulate samples from numerous locations or different time intervals so that detailed observations and comparisons can be done in a convenient and orderly manner. these were identified and returned into the corresponding reconstruction sections in an automatic tracing procedure, programmed and executed on vidas . (zeiss/kontron germany). in addition it is possible to obtain a plastic, -d-like impression by applying the rcm with oblique illumination. this is achieved by decentralization of stach's slide (central diaphragm). . two main problems in -d reconstruction of histologic specimens are the horizontal distortion during the preparation of serial thin-tissue slides and the following vertical readjustment (alignment). we have recently shown that optical sections can be obtained by rcm within thick tissue-slides. its confocal-like principle provides an alternative to mechanical slices. the preserved integrity of the examined object allows the precise movement within the optical axis in one (vertical) dimension, thus avoiding manual alignment. applying the rcm on histochemical and immunohistochemical stains, reflections can be observed within the tissue. the depth of penetration of the light beam amounts to approx. µm, equivalent to about optical tomolevels (fig. ) . using the example of neurons of the supraoptic nucleus of the rat, we demonstrate this new technique on chrome-alum haematoxylin stained neurosecretion. the reflections of the dye particles associated with neurosecretory granules allow the precise localization of these subcellular structures. the visualization of the neurosecretion and its distribution is more distinct and of sharper contrast than in bright-field microscopy. reflected light is like a binary signal and therefore generates a suitable prerequisite for automatic discrimination in greyscale image analysis. thus identified black and white negatives were reconstructed with the module rec d on vidas . . this paper introduces two extended applications of the rcm for -d reconstruction. the generated optical slices and isohypses allow qualitative and quantitative investigations of intracellular structures and surfaces. quantitative chemotaxis is of great interest in a broad field of cell research (e.g., receptor-ligand interaction, ionic channels, cytoskeletal and metabolic processes, embryogenesis) as well as in clinical studies (e.g., immune reaction, wound healing, infection, tumor invasion). the most accepted assay to measure chemotactic behaviour of cells is the boyden filter assay, in which cells move against a gradient of chemical or biological substances. the chemotactic response is analyzed by measuring either the distance travelled by the leading front of cells or by quantifying the number of cells on the lower surface of the filter. this method is laborious and limited by the observer's subjective errors. we have developed a computer-based image analysis system to estimate the three-dimensional distribution of the migrated cells inside the filter. using one-micron optical sectioning, we can determine the position, size, and shape of many individual cells. within min, the position of thousands of cells can be recorded and the migration profile in the filter can be determined. it is possible to distinguish between different cell populations by selecting particular cell parameters. the system consists of a nikon labophot- a microscope with video microscopy and programmable focus control, used in connection with a hasotec-image processor with fast data acquisition and user-friendly software (mswindows). the high throughput, the consistent accuracy, and the simple operation of the system optimize all aspects of routine chemotaxis analysis in basic research and clinical studies. the atomic force microscope (afm) is being used increasingly in the life-sciences field. with this increase in usage, a concomitant increase in the need for both better developed specimen preparation techniques and better defined operational parameters for the afm instrument has occurred. lifesciences afm methodology can be divided into three main areas: ( ) choosing the appropriate support substrate for the specimen, ( ) choosing the most appropriate immobilization techniques for attaching the specimen to the support substrate, and ( ) selection of the optimum instrument scan parameters to ensure reliable transfer of data from the specimen to the image. of central importance to life-science afm is the nature of the substrate to be used. the most reliable substrates are those with a well-documented surface, whose features are at least an order of magnitude smaller than the specimens of interest. furthermore, the material should be transparent so that other forms of microscopy can be used (i.e., light, fluorescence, near-field scanning optical, etc.) and the surface chemistry should be both well documented and susceptible to chemical derivitization. the two most common substrates to fit the above descriptions are glass and freshly cleaved mica. both of these surfaces can easily be manipulated to produce a wealth of reactive primary amines, either by coating the substrate with poly-l-lysine or by treatment with -aminopropyltriethoxy silane (aptes). an analysis of the modified and unmodified substrates show that treatment with either aptes or poly-l-lysine resulted in an increase in surface roughness (ra). based on the roughness information, we recommend modified mica as being suitable for biological material ranging from whole cells to dna, whereas modified glass is unsuitable for samples with heights < . nm. the red blood cell (rbc) cytoskeleton was examined by atomic force microscopy (afm). samples were placed on either glass or mica and imaged in air. no fixative or stain was used; this allowed modification of the samples between images so that molecular components could be identified. the meshlike structure, which is observed when the rbcs are lysed, is identified as a complex of the cytoskeletal integral proteinsspectrin, actin, and band . . the identification was accomplished by imaging the intact cytoskeleton, then treating the sample to selectively remove these proteins and re-imaging the sample. to support the identification of the cytoskeletal proteins further, images were obtained of samples which had been treated with detergent to remove the lipid membrane and leave the cytoskeleton behind. this is the first study of the intact rbc cytoskeleton which identifies specific proteins. more generally, it shows that afm is a useful tool for examining biological systems in their native state since sample preparation is simple and, once attached to the substrate, the sample can be treated in a variety of ways. we have reported some features about the structural and morphologic changes in nylon transformed from β-alanine single crystal and nylon polymerized from ε-amino-ncaproic acid one, which are members of the ω-amino acid family, through the solid-state polycondensation procedure. it was also found that p-aminohippuric acid [n-( -aminobenzoyl)glycine] single crystal could be converted into an aromatic polyamide crystal by heat treatment below its melting point. in this report, crystalline and morphologic structures of polyglycine produced from glycine (the simplest amino acid) single crystal were examined mainly by means of scanning electron microscopy (sem) and x-ray diffraction technique. the monomer solution was prepared by dissolving g of commercial glycine powder into ml of distilled water at °c. the monomer single crystal was precipitated at °c from the solution, which was transparent and prism-shaped. definitive cleavage planes were observed in the monomer crystal, which is found to be parallel to the a-c plane in the αform crystal of glycine. it has been reported that such cleavage is caused by the characteristic structure of α-form glycine crystal. the monomer single crystal was used as the original specimen for the polycondensation reaction, where the original specimen was annealed in decaline at and °c up to h. morphology of the polymerized material was observed by using a hitachi s- sem with accelerating voltage of kv after being coated with gold. x-ray photographs were taken by a flat camera mounted on an x-ray generator with ni-filtered cu-kα radiation, where the crystal took three orientational positions so that a-c (cleavage plane), a-b, and b-c planes made a right angle with the incident beam, respectively. figure shows two types of x-ray diffraction patterns from a specimen polymerized at °c for h with the a-c plane perpendicular (fig. la) and parallel (fig. lb) to the incident beam, where patterns reflected from the polymerized material were observed as well to have spots from the original monomer single crystal. in figure la , two strong arcs are observed, which are indexed as (inner reflection) and (outer reflection) from type-i modification of polyglycine crystal. a more complicated diffraction pattern is shown in figure lb , which seems to be a fiber diagram. almost all molecular chains in polyglycine-i crystal are considered to be normal to the a-c plane and parallel to the hydrogen bond in the original monomer single crystal. figure a shows a sem photograph for the surface of the original monomer crystal. lamination layers of lamellae are observed on the cleavage plane, edges of which are parallel to the b-axis of the monomer crystal. voids were observed in the grain boundary or crack region. a sem picture of the specimen polymerized at °c for h is shown in figure b , where the laminal materials are seen to overlap each other crosswise. such intersecting laminal structure seems to be responsible for the biaxial crystalline orientation observed in the x-ray diffraction pattern shown in figure la. in the case of the specimen polymerized at higher temperatures, the fibrillar structure was observed over the surface. figure c shows the results for the specimen annealed at °c for h. hiroshi toyoda, takashi itoh, hiroshi sakabe, takashi konishi department of polymer science, kyoto institute of technology, kyoto, japan it is well known that polytetrafluoroethylene (ptfe) is produced by emulsification polymerization in the form of powder or aqueous dispersion to be processed industrially. the dispersion-type material is mainly composed of fibrillar and/or lamellar crystallites, where the spherulite structure generally is unseen because of stiffness of the molecular chain. such morphologic feature is considered to be responsible for the remarkable repellent property of the polymer to liquid. the authors prepared thin films of ptfe from the aqueous dispersion to observe change in the fibrillar structure through heat treatment, considering that from such a viewpoint the fibrillar morphology in ptfe is the aspect that is most different from other polymeric materials. the polymerized ptfe used in this study was produced by dupont ltd. a glass plate was dipped into the solution and then withdrawn vertically. the thin film was prepared by drying the solution on the plate at room temperature. the heat treatment was performed at , , , , and °c for h in an air-drying oven. the samples were immediately quenched in ice water (cooling rate: , °c/min) or cooled in air at controlled rates ( , , l, . , and . °c/min). any serious oxidation and/or degradation effects were not observed even during annealing at °c. a scanning electron microscope (sem)(jeol, jsm- lv) was mainly used to observe the au-coated surface structure of the sample, when an accelerating voltage was set at kv to make the resolution of the sem high. composite materials were produced by coating ptfe on textile of glass-fiber and that of carbon fiber. after the coating and subsequent annealing at °c for min, the material was cooled to room temperature. the surface was found to be covered with balls of a diameter of about nm, composed of several hundred nodules of a diameter of about nm as shown in figure . in this sample, the degree of crystallinity of ptfe was about % and no fibrillar structure was observed. by increasing the annealing temperature to > °c, the fiber bun- iv- scanning vol. , supplement iv ( ) dles grew up. such phenomena may be explained by stiffness of the molecular chain including f instead of h, that is, the chain does not fold even if the molecular motion is stimulated at elevated temperatures. any sheaf-like or microspherulitic structure (a precursor of the spherulite), which is often observed in thermoplastic polymers such as polyethylene, did not appear in ptfe. when decreasing the cooling rate, the fibrils grow laterally and interfibrillar space tends to become larger. it is unknown how such space affects the physical properties of the material. thinner fibrils appeared, which connected the original thick ones, in the case of . °c/min cooling as shown in figure . such morphologic structure characteristic of ptfe seems to be responsible for the processing efficiency. schott glaswerke, department of instrumental analysis and mineralogy, mainz, germany fused-cast refractories of the zac-type (zirconia-alumina cast) are of great importance in the manufacturing of glass. especially glass-tanks for the melting of specialty glasses are built with these materials. zac refractories are produced by melting the raw materials at very high temperatures and subsequently casting them into suitable molds. on cooling, part of the material crystallizes, forming al o (corundum) and zro (zirconia, baddeleyite), where the zirconia crystals are also growing within or into the corundum crystals. besides the crystalline phases there also exists a glassy phase, composed mainly of sio , al o , and zro , and small amounts of alkali (na o, k o), tio , and fe o . this microstructure of the material must have an influence on the corrosion behavior when subjected to the aggressive melt in a glass tank. the aim of this work was to establish a method to characterize quantitatively the microstructure of these refractories. the inhomogeneous nature of the material can already be discerned in the light microscope; however, the resolution is not sufficient to measure the sometimes very small (< µm) crystal sizes of zro , not to mention the determination of a form factor! therefore, the electron microprobe (epma) was used to perform this work. the instrumentation used was a jeol jsm- scanning electron microscope, equipped with an optical microscope for reflected and transmitted light, motorized stage (x, y, z), and coupled with a combined eds/image analyzing system voy-ager from noran, which also controls the stage movement. the following parameters of the microstructure, for example, the geometric and chemical properties of the crystalline and glassy phases, were to be determined: maximum and minimum diameters, area of the individual phases and fractional area within the material, form factors and orientation to a reference plane, and the chemical composition of the glass phase. furthermore, it was necessary to distinguish between the zirconia within the corundum and the more isolated crystals. since the material is inhomogeneous, a rather large number of image fields had to be analyzed (at magnifications of × and higher the individual image field is very small!); as a consequence, the whole procedure had to be automated as much as possible. sample preparation: a flat and polished surface of a section of the material had to be produced. although the grinding and polishing was done with diamond wheels and paste, a certain amount of relief between the hard corundum and the "soft" glass is unavoidable. the first step in analyzing the microstructure is to recognize/discriminate the three phases in the material. the signal used for image formation and subsequent phase discrimination is the backscattered electron (bse) image; however, the abovementioned relief due to differences in hardness made the distinction between glass and corundum impossible, since (a) brightness differences between glass and corundum are not very strong in the first place, and (b) edge effects in the corundum crystals showed brightness values of the glass, and vice versa (discrimination, i.e., tranformation of a particular phase into a binary image, is based on such brightness differences). contrast enhancement or image filters were not sufficient for clear separation of these two phases. therefore, for the recognition of the glass phase, an element distribution for si (x-ray map) was used; combined with the image of zro , which is easily discriminated from other phases because of its high average atomic number/high backscatter signal, it yields the inverse of the corundum image. this sounds quite simple; however, since the resolution of the x-ray signal is considerably less than the bse signal, quite some effort had to be put into the treatment of the si-distribution to give correct areas and outlines of the glass phase. another rather complicated step was the recognition of the zirconia crystals grown within or into the corundum. crystals fully enclosed by the corundum presented no problem, but those growing from the edge into the al o were difficult to discriminate as to belonging to this particular structure. the problem here is to tell the machine what the eye and judgement of the operator consider to be part of this feature. an additional treatment of the glass phase (binary image filters) was necessary to solve this task. the binary image of the glass phase is used as a template for the determination of the chemical data of the glass; it provides the control for stepping the electron beam only over the desired areas while acquiring data with the energy dispersive spectrometer (eds). all image analysis procedures, including the storage of images and eds spectra together with the evaluation set-ups (selection of properties to be determined), were combined within a schedule. the stage control and automation software then connects the stage movement to predetermined points (or a number of points along a line) with the execution of the schedule at each of these points on the sample. after completion of the analysis run, which needs about hours for image fields and is therefore done unattended overnight, the large amount of stored data are statistically evaluated (for the zirconia particles alone, there are easily more than , data sets, consisting of seven measured or derived properties each!). this task is performed using the lotus - - calculation program. the chemical data are extracted from the stored spectra with the zaf-correction procedure to yield wt-% oxide data, and also statistically evaluated with the lotus program. although the aim of this work was only to set up an analysis procedure, the test runs on various samples have already shown differences in the microstructure as determined by the geometric and chemical properties of the individual phases. the application of this procedure to "real" samples will then establish correlations between the microstructure and the properties of the material with respect to their use in glass melting. approximately , tons of electric furnace flue dust accumulated in an industrial area in tifton, georgia. vehicles transporting the flue dust, classified as k hazardous waste, initially dumped the material in a warehouse. once the warehouse was full, the flue dust was dumped in an uncovered pile. run-off from the pile and wind-driven particles contaminated nearby industries, residential buildings, and soils over a period of many years. scanning electron microscopy-energy dispersive x-ray spectrometry (sem-eds) was used to compare the morphology and chemical composition of fly ash dust from the suspect pile ( fig. ) with samples collected from the surrounding buildings and soil. post-it notes (millette et al. ) , modified with a strip of conductive carbon tape, were used to collect dust that had accumulated in buildings surrounding the fly ash dump site. suspect dust particles were analyzed by sem-eds to compare with known dust particles from the fly ash pile. soil samples were sieved, with "fines" from the dry soil analyzed by sem-eds and compared with samples from the fly ash pile. particles similar in chemical composition and morphology were identified in most of the buildings sampled that surround the fly ash dump site. soil samples from areas surrounding the dump site were also found to contain fly ash iv- scanning vol. , supplement iv ( ) fig. backscattered electron image of fly ash spheres from the flue dust pile. scale bar = µm. particles similar in morphology and chemical composition to fly ash from the suspect pile. in conclusion, soil and dust samples taken from homes and outdoor areas surrounding the fly ash pile were found to contain particles similar in morphology and chemical composition to particles from the fly ash pile. scanning electron microscopes (sems) are widely used in detection and quantification of material microstructure and imperfections. results obtain with sems need a high expertise to be fully exploited. specialized programs such as a single-scattering monte carlo simulation can effectively predict the electron beam interaction with solids and thus help the quantification. one of the most powerful advantages of monte carlo simulation to help microscopists is to generate images. with high-speed computers we can now simulate images in a reasonable amount of time. in this paper we present images of spherical inclusion of mns in a fe matrix. it is shown that there exists a difference between the image dimension and the real dimension. also, it is shown that geometric effects can alter the resulting image. the monte carlo program used for low-energy simulation is described elsewhere . figures - show simulated image of mns inclusions (the dark center of the figures) in a fe matrix (lighter part). to build this image we need to simulate , primary electron trajectories and then calculate the associate backscattering coefficient for each pixel of the screen. the image will then need , simulations for a total time of approximately , cpu min on a risc workstation. because of the symmetry of the image we can use an better method. starting from the center of the inclusion we simulate points moving on a radius to the border. then we rotate this line on °and add random noise for the monte carlo simulation to obtain the full image. this image is coded in windows bitmap format and can then be converted to tiff or another format. for our simulation, we used a diameter of nm for the electron beam, at which value the incident beam current would be × − amps in our jeol sem. figure shows a spherical inclusion with a radius and at a depth of nm simulated with a beam energy of kev. figure shows the same inclusion but simulation at kev. figures and show the inclusion with a ratio depth/radius of . , simulated at and kev, respectively. in figure we can observe the discrepancy between the image dimension and the real dimension as a function of the ratio depth/radius. as was expected, the error increased with the depth/radius ratio. it is also interesting to note that increasing the energy of the incident electrons increases the image error. the geometry of the solids affect the backscattering coefficient. we can obseve a white border in figures - a similar problem occurs in the middle of the inclusion which appears lighter. it is interesting to see the same phenomena in figure for an image of a real inclusion. the photograph of the embedded particulates in a matrix taken by sem in backscattering mode should be analysed carefully because this method usually overestimates the dimension of such particulates. the measurement of strain, using lattice parameter changes, can be determined from channeling patterns in the scanning electron microscope (sem) using holz lines. the method used by kuzubowski, for example, in the [ ] orientation of silicon, utilizes the change of the height of the triangle from the intersections of the ( ) and two { } holz lines. more recently we have been investigating the channeling patterns for silicon to calibrate accurately the voltage in an sem and we have utilized a pin wheel pattern formed from the { } holz lines in [ ] pattern at . kev. at this voltage, the ( - - - ) and ( ) are very close to each other, within . degrees, and these two lines split as the voltage increases or decreases. the width of the splitting is quite sensitive to any voltage changes as are the { } intersections with these lines. an experimental electron channeling pattern (ecp) of these lines, along with a simulation are shown in figure . the voltage calibrated from the simulation is . kev as shown in figure c . these same holz lines are, of course, also appropriate for strain measurement since the variation in the voltage is equivalent to an isotropic strain field that would uniformly change the lattice spacings. the shifting of the holz lines in the electron channeling patterns due to strain are from both the change in the magnitude of bragg angle and the change in the orientation of the diffracting planes. the former corresponds to a change in the distance of reciprocal lattice point from the origin(i.e., d spacing) and the latter is due to the rotation of reciprocal lattice point about the origin. thus the total angular change can be written as where θ b is the bragg angle, ∆θ p is the rotation of the plane, g is the diffraction vector, λ is the electron wavelength, and | | represents the length of a vector. e is a matrix equal to ε + i where ε is the strain tensor and i a unit matrix. the approximation ~ is due to the small angle approximation for sin(∆θ b )~∆θ b and − ∆θ b ~ . the holz lines toward the center of the channeling patterns are more sensitive to lattice parameter variations because they have a larger value of g and the change of the bragg angle is in proportion to this magnitude. the angular iv- scanning vol. , supplement iv ( ) ∆θ = ∆θ b + ∆θ p~e g − g widths of these channeling lines are also smaller and so they are well defined. when the strain is not isotropic, the sensitivity is also determined by the orientation relationship between the strain ellipsoid and the g vector. thus the appropriate choice of the g vector can be used to maximize the second term in the equation. in figure a , for example, we show the simulation of a channeling pattern for a strain along only one direction (e.g., [ ] ) which breaks the four-fold symmetry of the [ ] pattern. it can be seen that the strain effects on the { } are all the same since all the planes in this family are symmetrical to the strain direction. however, the sensitivities to the strain in the { } are not all equal, ( ) being more sensitive than ( ). the comparison of the sensitivity can be seen from the simulation and from figure b , in which the angles of splitting are plotted as a function of the strain. for years lab has been the industry standard for thermionic emission cathode material. in , fei introduced ceb as an improved alternative to lab . ceb directly replaces lab and has certain distinct benefits. ceb has a lower volatility than lab , increasing the lifetime of the cathode. this reduces the frequency of cathode purchases and replacements. in addition, greater beam stability and faster startup are achievable from ceb 's greater resistance to contamination. however, to gain the benefits of ceb , it must be operated correctly. studies have shown that the operating characteristics of ceb , such as total emission current, are different from lab . proper operation of ceb comes from understanding the operating characteristics of ceb and how they interact with the system in which it is operating. to compare the operating characteristics of ceb and lab , we performed parametric studies in a jeol scanning electron microscope. the jeol is a self-biased system where the bias voltage (vb) is dependent upon the total emission current as vb = ie*rb, where rb is the bias resistor and ie is the total emission current. the results show that ceb operates at a lower total emission current than that of lab (fig. ) . therefore, for the same bias resistor settings, the bias voltage on ceb is less than that on lab (fig. ) . please note that the lower emission current of ceb does not imply a lower probe current. ceb has high transmission and has been shown to provide probe currents similar to lab . to optimize the operation of ceb in a self-biased system, the operating conditions must be optimized for a low emission current source. these operating conditions include the bias resistor value and wehnelt-to-tip spacing. we have found that increasing the bias resistor improves the performance and lifetime of ceb . other experiments show that adjustments to the wehnelt-to-crystal tip distance further improve the performance of ceb . the effects of adjusting the wehnelt-to-tip distance vary for different electron microscope systems. for independently biased systems, we recommend setting the filament temperature to k (the corresponding filament current is provided with the cathode) and subsequently adjusting the bias until the desired emission image is obtained. this is possible because the emission image of ceb is the same as the lab emission image. the wehnelt-to-tip distance should be set to the distance recommended for lab . in transmission electron microscopes (tems), the operating point is set by viewing the source image. because the emission image of ceb is similar to lab , the procedures for obtaining the lab operating point in tems still apply to ceb . however, the total emission current at the operating point will be lower than with lab . by following these guidelines, the operation of ceb can be optimized and with this optimized operation, the benefits from additional lifetime and increased stability over lab can be achieved. takeshi hatsuzawa, yoshihisa tanimura, kouji toyoda, makoto nara*, syuuji toyonaga*, shin-ya hara*, hirotaka iwasaki*, kazuhiko kondou* national research laboratory of metrology, miti tsukuba; *nikon corporation, tokyo, japan a compact laser interferometer with a piezo-driven scanner has been developed for metrological micro-linewidth measurement in regular scanning electron microscopes (sems). so far, special sems combined with various scanners and interferometers (postek , hatsuzawa et al. ) are necessary to perform absolute and precise measurements; however, this device solved the problem by miniaturizing a one-dimensional mechanical scanner and a multi-optical path interferometer. the arrangements of optical components are illustrated in figure . a mechanical scanner is constructed in the center of a mm diameter base disk. the square part, slit by using a electric discharging machine, is suspended by thin elastic suspensions at each corner. the table is driven by a piezo-electric actuator of a traveling length of micrometers. a he-ne laser beam is introduced on the disk by a single mode fiber through a collimator lens, and it is split and deflected by beam splitters and lenses so that two beams are facing each other and form a differential interferometer. at both ends of the table, right-angle prisms are facing each other so that the laser beam goes back and forth five times. by using the differential arrangement and optical-path multiplication technique, the resolution of the interferometer is improved ten times from the original michelson's arrangement. the reflected beams are surperimposed by a half-mirror to generate an interferometric signal, and it is detected by four photo diodes (pd -pd ) after changing its phase by ˚ through a half-and a quarter-wave plate arrangement. this detection method and an electrically operational processing enhance the resolution iv- scanning vol. , supplement iv ( ) times from its original. eventually, by using physical and electrical methods, the resolution of the compact interferometer is . nm (âλ/ ). to evaluate the performance of the compact interferometer, it was installed in the vacuum chamber of a regular sem (joel jsm- a) as shown in figure . the fiber and electric wires are introduced through two flanges next to the chamber. a software servosystem is constructed by using the interferometer and a piezo driver. according to the positional information read through the interferometer counter, a d/a converter commands the piezo driver to change table position precisely, allowing simultaneous sampling of the secondary electron intensity distribution. in the system, the resolution is improved to . nm (âλ/ ) by using the counter function. thus, a precise measurement system is realized for the absolute measurement of micro-linewidth in a regular sem. a maximum drift of the interferometer counter of nm/h was observed by fluctuations in room temperature, however, the influence of the drift can be neglected in actual measurements since a line-scan is finished within a dozen seconds. a comparison of measurements between the metrological sem (hatsuzawa et al. ) and the compact system was made by using silicon micro-line artifacts, ranging from . to . micrometer. the measured linewidths agree within a couple of nm in both measurements, although the measurement conditions are different in acceleration voltage, etc. the results show that the measurement system using the compact interferometer has the same performance as the metrological sem. this means that absolute and accurate measurements can be obtained everywhere by using the compact laser interferometer and a regular sem. this device can be applied to various types of scanning probing microscopes as well as to optical microscopes by improving the table mechanism and the arrangement of optics. institute of ecology and department of botany, university of georgia, athens, georgia recent studies of lake lanier, georgia, revealed a fungal epidemic on the planktonic alga, synedra acus. clonal isolates of the fungus were identified as zygorhizidium planktonicum (chytridiomycetes), an obligate parasite of freshwater diatoms. although frequently present in lakes and reservoirs of western europe, the occurrence of z. planktonicum in north america has not been previously confirmed. earlier studies have described the morphology of z. planktonicum on asterionella formosa; however, little is known of the fine structure and infection process on s. acus. as a basis for further investigations, morphology and mechanism of infection were characterized by scanning and transmission electron microscopy. these observations provided exceptional accounts of germinating spores and developing thalli. moreover, conjugation was characterized as the fusion of heterogametangia by means of an extended smooth-walled tube, emanated from the smaller "male" gametangium. upon attachment, the club-shaped conjugation tube adhered to a small region of the adjoining thallus. this point of contact became continuous with the maturing "female" gametangium which appeared smooth-walled and often highly vacuolate. ultrastructural examinations also illustrated a shared cytoplasm between conjugating gametangia and apparent migration of organelles; however, fusion of nuclei was not observed. the mechanism of infection on s. acus appeared identical to earlier descriptions of z. planktonicum on a. formosa (beakes et al. ) . following encystment, spores typically produced a single germ tube which grew over the frustule valve in an unwavering, linear fashion. penetration occurred between an overlapping region of the outer and inner frustule. at the point of intrusion, the rhizoid appeared slightly swollen, often further displacing the outer diatom wall. a program for monte carlo simulation of electron energy loss in nanostructures a monte carlo calculation of the backscattering coefficient for a multilayer sample monte carlo program for minicomputers using mott cross sections an improved method of measuring biological submicron motion and displacement using laser amplified motion detection and analysis friedlander sk: smoke, dust and haze, fundamentals of aerosol behavior subcommittee on airborne particles: airborne particles micromanipulators and micromanipulation moor h: recent progress in the freeze-etching technique optimization and application of jet-freezing platinum-iridium/carbon: a high-resolution shadowing material for tem, stm, and sem of biological macromolecular structures freeze-fracturing for conventional and field emission low-temperature scanning electron microscopy: the scanning cryo unit scu cryo-preparation and planar magnetron sputtering for low temperature scanning electron microscopy imaging of intramembranous particles in frozen hydrated cells (saccharomyces cerevisiae) vapor pressure data for some common gases. r.c.a. review an improved cryo-jet freezing method in vitro spinal cord trauma early post trauma changes in rat spinal cord: electron probe microanalysis surface studies by stm fabrication technique for tips with controlled geometry for scanning tunnelling microscopy scanning probe metrology low temperature thermal oxidation sharpening of microcast tips microfabrication of afm tips using focused ion and electron beam techniques dimensional metrology with scanning probe microscopes a rocking beam electrostatic balance for the measurement of small forces a scanning tunneling microscope with a capacitance-based position monitor scanning probe tips formed by focused ion beams envelope reconstruction of probe microscope images surface recovery in scanning probe microscopy probe characterization for scanning probe metrology comparison of diffraction techniques for the sem. scan electr microsc electron channelling in the sem a review of excimer laser projection lithography m: direct electron-beam patterning for nanolithography direct stem fabrication and characterization of selfsupporting carbon structures for nanoelectronics die entstehung einer vielzahl von kontaminationsfäden unter der electronen-mikrosonde transition from chemical etching to chemical polishing studied by the sem chemical preparation of dielectrics for studying their microtopography by the sem microprobe analysis in human pathology shelburne jd: preparation of biological tissue sections for correlative ion, electron and light microscopy negative staining: applications and methods detection and identification of viruses by electron microscopy diagnosis of viral infection by electron microscopy electron microscopy in diagnostic virology electron microscopy in viral diagnosis genetic identification of a hantavirus associated with an outbreak of acute respiratory illness isolation of muerto canyon virus, causative agent of hantavirus pulmonary syndrome distinction between bunyaviridae genera by surface structure and comparison with hantaan virus using negative stain electron microscopy anatomic complications of abdominal surgery with special reference to the ureter urological complications of renal transplantation can be prevented or controlled the microvasculature of the guinea pig ureter. a scanning electron microscopic investigation application of an naoh maceration method to a scanning electron microscopic observation of ito cells in the rat liver sem blood vessel cast-analysis microangioarchitecture of the islets of langerhans in the snakes, naja naja, vipera russelli and echis carinatus histochemical methods for acid phosphatase using hexazonium pararosanilin as coupler acid phosphatase activity in the inner ear the development of the stria vascularis in the mouse kimura rs: distribution, structure and function of dark cells in the vestibular labyrinth secretory epithelial linings in the ampullae of the guinea pig labyrinth la fosfatasi acida del labirinto membranoso dell'embrione di pollo durante lo sviluppo the development of human placental villous tree scanning electron microscopic observations on the surfaces of chorionic villi of young and mature placentas ultrastructure of the epithelium of the chorionic villi of the human placenta some new findings about hofbauer cells in the chorionic villi of the human placenta the fine structure of human placental villus as revealed by scanning electron microscopy monte carlo simulation with ebic a monte carlo calculation backscattering coefficients calculations of mott scattering cross section simulation of sem screen image by a monte carlo method quantitative x-ray microanalysis of spherical inclusions embedded in a matrix using a sem and monte carlo simulations a standard procedure for the modeling of the decrease in detection efficiency with time for low-energy eds spectra an empirical stopping power relationship for low-energy electrons measuring the backscattering coefficient and secondary electron yield inside a scanning electron microscope applications of a knock-on process monte carlo simulation based on the mott cross section to quantitative electron microprobe analysis x-ray production as a function of depth for low electron energies theoretical electron-atom elastic scattering cross section cross section for k-shell ionization by electron impact the use of polyacrylamide as an embedding medium for immunohistochemical studies or embryonic tissue immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos i: presence of immunofluorescent titin spots in premyofibril stages novel applications of acrylamide for cryosectioning of isolated cells, tissues, and arthropods whole-mount analyses of cytoskeletal reorganization and function during oogenesis and early embryogenesis in xenopus confocal microscopy of thick sections from acrylamide gel embedded embryos resolution of subcellular detail in thick tissue sections: immunohistochemical preparation and fluorescence confocal microscopy the use of polyacrylamide as an embedding medium for immunohistochemical studies of embryonic tissues application of acrylamide as an embedding medium in studies of lectin and antibody binding in the vertebrate retina developmental angiogenesis: quail embryonic vasculature embryonic vascular development: immunohistochemical identification of the origin and subsequent morphogenesis of the major vessel primordia vasculogenesis and angiogenesis: two distinct morphogenetic mechanisms establish embryonic vascular pattern endothelial cell origin and migration in embryonic heart and cranial blood vessel development morphogenetic mechanisms in avian vascular development near-field optics: microscopy, spectroscopy and surface modification beyond the diffraction limit mechanical detection of magnetic resonance related scanning techniques morphology and diameters of crystallites in remineralized enamel the shape of enamel crystal within human enamel densitometric study of polarized light images from carious lesions conditions required for detection of specimen specific se- secondary electrons in an analytical sem a high resolution se- sem study of enamel crystal morphology high resolution topographic imaging of enamel crystal surfaces analysis of metal films suitable for high resolution se- microscopy antiviral activity of rnadye combinations microspectrophotometry and digestibility of alkali-treated walls in bermudagrass cell types simplified highly efficient apparatus for photographic transaxial x-ray tomography embryonic vascular development: immunohistochemical identification of the origin and subsequent morphogenesis of the major vessel primordia in quail embryos antibodies to b -integrins cause alterations of aortic vasculogenesis, in vivo capillary endothelial cell cultures: phenotypic modulation by matrix components in vitro rapid organization of endothelial cells into capillary-like networks is promoted by collagen matrices connective tissue morphogenesis by fibroblast traction. i. tissue culture observations reorganization of basement membrane matrices by cellular traction promotes the formation of cellular networks in vitro consumption of various process cheese products has been steadily increasing in the u.s.a. and worldwide. these products are manufactured in various styles depending on composition and physical properties sodium citrate (sc), trisodium phosphate (tsp), disodium phosphate (dsp), and sodium hexametaphosphate (shmp) were used at . % levels as melting salts. after cooking, the samples were held at °c for up to h. they were removed after , , . , and h, cooled at °c, and stored for days, after which they were analyzed for moisture, fat, protein, ph, firmness, and meltability. for transmission electron microscopy, small samples ( mm ) were fixed in . % glutaraldehyde and % osmium tetroxide, embedded in spurr's low-viscosity medium, sectioned ( nm thick sections), stained with uranyl acetate and lead citrate, and examined at kv accelerating voltage the data in figure were converted to a pseudo three-dimensional relief map of the embryonic vessels using digital image processing software. it can be argued that this digital rendering, which contains apparent overhead illumination (and shadows below), provides a more comfortable format for assessing visual information. references caric m, kaláb m: processed cheese products textural properties and microstructure of process cheese food rework microstructure of processed cheese products milk gel structure. vi. cheese texture and microstructure effect of draw ph on the development of curd structure during the manufacture of mozzarella cheese structure and rheology of string cheese mozzarella cheese: impact of coagulant type on functional properties encyclopedia of food science & technology food texture and microstructure electron microscopic observations on the casein micelles of buffalo milk: a preliminary study development of microstructure in raw, fried, and fried cooked paneer made from buffalo, cow and mixed milks the role of casein micelles in changes in the colour of milk microstructural evaluation of model starch systems containing different types of oils use of the bird-leider equation in food rheology starch gelatinization in the presence of emulsifiers. a morphological study of wheat starch fixation analysis of tetrahymena pyriformis ultrastructure distribution of polyphenol oxidase in organelles of hyphae of the wood-deteriorating fungus, coriolus versicolor. biodeterioration research a study of the bladder blood flow during distention in rabbits a vascular network closely linked to the epithelium of the urinary bladder of the rat the effects of acute overdistention of the rabbit bladder applications of an naoh maceration method to a scanning electron microscopic observation of ito cells in the rat liver light microscopy techniques for the demonstration of silicone synovial metaplasia of a periprosthetic breast capsule demonstration of silicon in sites of connective tissue disease in patients with silicone-gel breast implants biological specimen preparation for sem by a method other than critical point drying comparison of hexamethyldisilazane (hmds), peldri ii, and critical point drying methods for scanning electron microscopy of biological specimens acetonitrile as a substitute for ethanol/propylene oxide in tissue processing for transmission electron microscopy: comparison of fine structure and lipid solubility in mouse liver, kidney, and intestine optimum conditions may allow air drying of soft biological specimens with minimum cell shrinkage and maximum preservation of surface features a rapid method for cell drying for scanning electron microscopy nation jl: a new method using hexamethyldisilazane for preparation of soft insect tissue for scanning electron microscopy the use of a simple method to avoid cell shrinkage during sem preparation handbook of snow: principles, processes, management and use hobbs pv: ice physics snow crystals: natural and artificial interference reflection microscopy in cell biology: methodology and applications rekonstruktion des oberflächenreliefs von erythrozyten mit hilfe der leitz-reflexionskontrast-einrichtung. leitz-mitt wiss tech vii/ reflection contrast microscopy within chrome-alum haematoxylin stained thick tissueslides reflexionskontrastmikroskopie in der immunhistochemie lichtmikroskopische untersuchungen zum einfluss von atrialem natriuretischem peptid (anp) am nucleus supraopticus scanning electron microscopy of post-it ™ notes used for environmental sampling development of a monte carlo program for low energy work measurement of small elastic strains in silicon using electron channeling patterns electron channeling patterns in the scanning electron microscope a metrological electron microscope system for microfeatures of very large scale integrated circuits scanning electron microscope-based metrological electron microscope system and new prototype of scanning electron microscope magnification standards comparative ultrastructural ontogeny of zoosporangia of zygorhizidium affluens and z. planktonicum, chytrid parasites of the diatom asterionella formosa iv- scanning heterogametangia attached by a fully developed conjugation tube (arrowhead) infection sites of diatom host showing intruding germ tubes (arrowheads) and developing thalli. scale bar = µm the author is grateful to dr. david howell for critically reviewing this manuscript and to ms. lara muffley for technical assistance.this research is supported in part by nih grant nddk r dk - . the authors gratefully acknowledge dr. steven armstrong for his assistance in animal preparation. mic integrity and induced mitochondrial hypertrophy without altering microtubular ultrastructure (fig. a, b) . to localize exogenously administered cdz, a polyclonal antibody to the drug conjugated to keyhole limpet hemocyanin (pierce, rockford, ill.) was prepared by immunizing new zealand rabbits and subsequent bleeds. the antibody titer was both detected and quantified by an indirect elisa assay (pierce elisa starter kit). the detected antibody was separated from other serum proteins by immunoaffinity chromatography utilizing pharmacia's mab trap g and then tagged with nm immunogold particles. transmission immunoelectron microscopy employing tagged antibody and glutaraldehyde/ paraformaldehyde-fixed, dmf dehydrated, and lowicryl-embedded cdz-treated and nontreated tetrahymena revealed no immunogold association with either microtubules or any other cytoplasmic organelle. this suggested that intracellular cdz was cytosolic and leached out during em processing. thus, cdz appears to impair growth and motility through an effect on general metabolism, for example, protein synthesis and/or respiration, rather than a direct action upon microtubules. however, isolation, purification, and characterization of microtubular proteins from tetrahymena cultured with and without cdz are required to substantiate this tentative conclusion. support: nsf-rimi grant no. rii- . coriolus versicolor, a white-rot, wood-decay basidiomycetous fungus, elaborates extracellular ligno-cellulolytic enzymes which possess marked industrial and agricultural applications. thus, we have been attempting to overproduce and enhance/regulate the secretion of these enzymes employing polyphenol oxidase (ppo) as a model enzyme. it catalyzes the conversion of o-diphenols (tree-generated resistance factors) to o-diquinones and oligomerizes syringic acid, alignin derivative. previously, we (moore et al. ) employed biochemistry and immunoelectron microscopy to map the route of ppo secretion through intracellular endomembrane and possible wall-associated components for hyphae cultured in defined liquid (biochemistry) and solid (microscopy) media. here, the ultrastructures of c. versicolor hyphae cultured in kirk and kelman's defined liquid or solid media are compared. in addition, ultrastructural cytochemistry to define further the intracellular route of ppo secretion to the growth medium in liquid cultured hyphae is described. hyphae of various culture ages ( - days) were prefixed min in . - . % glutaraldehyde buffered with . m cacodylate/cacodylic acid, ph . and after washing with buffer postfixed for h in buffered % s . for cytochemistry, prefixed hyphae were washed and treated with either cacodylate buffer or buffered mg ml - tlc pure, dihydroxyphenylalanine (dopa) on ice, followed by h at °c and then s postfixed. the hyphae were dehydrated through a graded acetone series and embedded in spurr's low epoxy resin. the ultrastructures of hyphae cultured in defined medium containing or lacking agar were similar (fig. a , b) except that hyphae grown upon agar possessed a sheath (hs) external to the cell wall. comparisons of numerous micrographs of aldehyde-fixed hyphae treated with cacodylate christian h. rickert and timm j. filler institute of anatomy, westfälische wilhelms-universität, münster, germany until today, quantification in cytochemistry has mainly been performed on supracellular level and by subjective estimation which makes it difficult to compare results of different investigations, even for standardized cytochemical procedures. reasons for this are the lack either or of calibrations or of relative reference points. greyscale image analysis principally allows the quantification of dye-density, but in practice stain intensities depend on many technical circumstances, that is, slide thickness, density of materials, light conditions, or costaining effects, and do not allow automatic identification because of weak grey-contrast. thus, greyscale image analysis for cytochemical quantification on subcellular level necessitates four prerequisites: ( ) applicability within tissue-slides combined with high resolving power, ( ) thin optical tomolevels to avoid superimposition of stained structures, ( ) clear distinction of structures (specificity), and ( ) high contrast. the reflection contrast microscope (rcm; leica germany) meets the above mentioned requirements. it combines effective suppression of aspecific reflected light with epi-illumination. because of its confocal-like principle, the rcm can be applied on thick tissue slides to obtain distinct optical sections. , at a magnification of ×, the depth of these sections amounts to approximately - µm, circumventing an accumulative effect as seen in transillumination. some cytochemical stains show specific reflections in the rcm, increasing the detection sensitivity to a level of objects nm in size. , reflections are like a binary signal (all-or-nothing principle) and deliver an intense contrast against a dark background, thus facilitating image processing by substituting the common density measurements with field measurement corresponding to areas of reflections.we designed an analysis program on vidas . (zeiss/kontron germany) for quantification of rcm images. one of the main parts of this application handles the greyvalue manipulation. apart from standard routines for image optimization and automatic region selection, the mean greyvalue of the whole image was determined. this was used as a reference parameter for the identification threshold in order to minimize the deviation of the identified area from the real area of reflection caused by inconsistencies of the light intensity.applying the rcm with consecutive image analysis on gomori-stained neurons of the supraoptic nucleus (son), we verified the validity of our measuring routine by employing it on a recognized system. a linear correlation between the process of neurosecretion and nuclear volume of the son is well known. switching from rcm to bright field, it is possible to obtain topographically identical images of the chosen tissue region. thus, we photographed reflecting neurosecretory granules in the former while karyometry was performed on the latter. the nuclei were classified by area and matched with the corresponding nucleus area of the same cell. a linear regression of these parameters was computed within a % confidence interval. our results in general confirm former findings about the measurable relationship between nuclear size and specific cell activity, surpassing earlier methods by increasing the sensitivity and decreasing the duration.this paper introduces the rcm combined with digital processing as a useful tool for quantification in the investigative gap between light and electron microscopy. the reflection contrast microscope (rcm; leica germany) is a light microscopic instrument, making reflections along interfaces visible by means of centrally polarized epi-illumination. these reflections cause interference patterns that are suitable for the analysis of superficies or detection of contact zones . we present two properties of this widely unknown technique allowing three-dimensional ( -d) reconstructions in the following manners: . applying the rcm on the surface of air-dried unstained erythrocytes, we made use of the phenomenon that neighbouring zones of equal altitude are joined by closed interference fringes; these can be interpreted as isohypses. to quantify the angle of declivity within a period of the resulting dark-light pattern, it is necessary to know the mean wavelength. the difference of layer thickness between two interference lines of equal tone is about nm for monochromatic light of λ= nm. the advantage of this technique is the simultaneous presentation of the profile of all the isohypses in just one picture (fig. ) . key: cord- - gh tg authors: uversky, vladimir n title: the alphabet of intrinsic disorder: ii. various roles of glutamic acid in ordered and intrinsically disordered proteins date: - - journal: intrinsically disord proteins doi: . /idp. sha: doc_id: cord_uid: gh tg the ability of a protein to fold into unique functional state or to stay intrinsically disordered is encoded in its amino acid sequence. both ordered and intrinsically disordered proteins (idps) are natural polypeptides that use the same arsenal of proteinogenic amino acid residues as their major building blocks. the exceptional structural plasticity of idps, their capability to exist as heterogeneous structural ensembles and their wide array of important disorder-based biological functions that complements functional repertoire of ordered proteins are all rooted within the peculiar differential usage of these building blocks by ordered proteins and idps. in fact, some residues (so-called disorder-promoting residues) are noticeably more common in idps than in sequences of ordered proteins, which, in their turn, are enriched in several order-promoting residues. furthermore, residues can be arranged according to their “disorder promoting potencies,” which are evaluated based on the relative abundances of various amino acids in ordered and disordered proteins. this review continues a series of publications on the roles of different amino acids in defining the phenomenon of protein intrinsic disorder and concerns glutamic acid, which is the second most disorder-promoting residue. intrinsically disordered proteins (idps) and intrinsically disordered protein regions (idprs) are new exciting members of the protein kingdom. , they are highly abundant in nature, [ ] [ ] [ ] [ ] [ ] possess numerous intriguing properties, are intimately involved in various cellular processes [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and are commonly found to be related to the pathogenesis of various diseases. , [ ] [ ] [ ] [ ] [ ] [ ] the common theme of protein disorder-based functionality is recognition, and idps/idprs are frequently involved in complex protein-protein, protein-nucleic acid and protein-small molecule interactions. some of these interactions can induce a disorder-order transition in the entire idp or in its part. , [ ] [ ] [ ] [ ] , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] furthermore, intrinsic disorder opens a unique capability for one protein to be involved in interaction with several unrelated binding partners and to gain different bound structures. , some idps can form highly stable complexes; others are involved in signaling interactions where they undergo constant "bound-unbound" transitions, thus acting as dynamic and sensitive "on-off" switches. these proteins typically return to their intrinsically disordered state after the completion of a particular function. many of the idps/idprs can gain different conformations depending on the environmental peculiarities. , all this constitutes an important arsenal of the unique physiological properties of idps/idprs that determines their ability to exert different functions in different cellular contests according to a specific conformational state. the folding-at-binding principle is believed to help idps or idprs to obtain maximal specificity in a protein-protein interaction without very high affinity. this combination of high specificity with low affinity defines the broad utilization of intrinsic disorder in regulatory interactions where turning a signal off is as important as turning it on. although some partial folding during the idp/ idpr-based interactions is a widespread phenomenon, with significant fraction (~ / ) of the interacting residues in idps/idprs adopting α-helix, β-strand and irregular structures, , there are still many other idps/idprs that are involved in the formation of "fuzzy complexes," where an idp/idpr keeps a certain amount of disorder in its bound conformation. , [ ] [ ] [ ] often, the interacting regions in idps are observed as loosely structured fragments in their unbound forms. these disorderbased binding sites are known as molecular recognition elements or features (mores or morfs), , preformed structural elements or pre-structured motifs (presmos). although the existence of such loosely structured regions suggests that idps can adopt their bound structure(s) at a free-energy cost that is not too high, it is important to remember that increasing the stability of the bound conformation does not necessarily enhance the binding affinity. another important feature of the disorder-based interactions is their increased speed due to the greater capture radius and the ability to spatially search through interaction space (the so-called "fly-casting" mechanism) and to the fact that fewer encounter events are required for the binding because of lack of orientational restrains. linking all these form at phs greater than its pk a . (and thus glu is negatively charged at the physiological ph ranging from . - . ) . therefore, glutamic acid is one of two acidic amino acids found in proteins that play important roles as general acids in enzyme active centers, as well as in maintaining the solubility and ionic character of proteins. in fact, glutamic acid residue has a nonpolar surface of Å , and the estimated hydrophobic effect associated with the burial of this residue is . kcal/mol. in ordered proteins, glutamic acids are predominantly located on protein surface so that they have access to the solvent. in fact, % of glutamic acids in known structures of folded proteins are classified as exposed since they have solvent exposed areas of > Å , and only % of glutamic acids in folded proteins possess solvent exposed areas of < Å and therefore are buried. the carboxylate anions and salts of glutamic acid are known as glutamates. glutamic acid is one of the most common natural amino acids and the most abundant amino acid in the diet. besides being an important component of proteins and polypeptides (see below), being a substrate for the production of the krebscycle-related α-ketoglutarate intermediate, glutamine and proline, and being the precursor for the synthesis of the inhibitory γ-aminobutyric acid (gaba) in gaba-ergic neurons, glutamate is the principal excitatory neurotransmitter within the vertebrate nervous system. in fact, glutamate is known to act on several different types of receptors and has excitatory effects at ionotropic receptors [such as n-methyl-d-aspartate (nmda), α-amino- -hydroxy- -methyl- -isoxazolepropionic acid (ampa), and kainite, which all incorporate ion channels that are permeable to cations] and modulatory effects at metabotropic receptors [which are g protein-coupled glutamate receptors (mglur) that modify neuronal and glial excitability through g protein subunits acting on membrane ion channels and second messengers such as diacylglycerol and camp]. at chemical synapses of the glutamatergic neurons, glutamate is stored in vesicles and is released from the pre-synaptic cell by nerve impulses. in the opposing post-synaptic cell, binding of glutamate lead to activation of specific glutamate receptors such as nmda or ampa. glutamate plays an important role in synaptic plasticity in the brain and is involved in various cognitive functions, such as learning and memory. in fact, long-term potentiation (one of the plasticity forms) takes place at glutamatergic synapses in the neocortex, hippocampus and other parts of the brain. another important role of glutamate is its ability to generate volume transmission, where extrasynaptic signaling is created via the summation of glutamate released from a neighboring synapse. in addition to glutamate receptors, neuronal and glial membranes contain glutamate transporters that are responsible for rapid remove of glutamate from extracellular space. under stress conditions (such as brain injury or disease), glutamate transporters work in reverse leading to the accumulation of considerations with the recent report showing that idp affinities are tuned mostly by association rates suggests that the degree of pre-adoption of binding conformations in idps has to be limited, but not unfavorable. all the functional and structural peculiarities of idps/idprs are encoded in their amino acid sequences. it was recognized long ago that there are significant differences between ordered proteins/domains and idps/idprs at the level of their amino acid sequences. , , in fact, in comparison with ordered proteins, idps/idprs are characterized by noticeable biases in their amino acid compositions, , , , [ ] [ ] [ ] containing less of so-called "order-promoting" residues (cysteine, tryptophan, isoleucine, tyrosine, phenylalanine, leucine, histidine, valine, asparagines and methionine, which are mostly hydrophobic residues which are commonly found within the hydrophobic cores of foldable proteins) and more of "disorder-promoting" residues (lysine, glutamine, serine, glutamic acid and proline, which are mostly polar and charged residues, which are typically located at the surface of foldable proteins) (fig. a) . glutamic acid is second of the most common disorder-promoting residues. figure b and table represent the result of a statistical analysis of the amino acid compositions of proteins in four standard data sets (disprot, uniprot, pdb select and surface residues ) and shows that the glutamic acid content in these data sets is . ± . %, . ± . %, . ± . % and . ± . %, respectively (cprofiler.org/help. html). in other words, idps/idprs contain . -and . times more glutamic acid residues than the average natural proteins from uniprot or ordered proteins from pdb, respectively. furthermore, the glutamic acid content in idps/idprs is . times higher than that on the surfaces of ordered proteins. this article continues a series of publications on the intrinsic disorder alphabet dedicated to the exploration of the amino acid determinants of protein intrinsic disorder. i overview below some functions of glutamic acid in idps/idprs (as well as in ordered proteins and domains) and show that there is a variety of glutamic acid-specific functions in disordered proteins and regions. chemical structure of glutamic acid. glutamic acid (glutamate, glu, e, see fig. a ) is one of the proteinogenic amino acids encoded by the standard genetic code and its codons are gaa and gag. glutamic acid is a dibasic nonessential amino acid that has a molecular mass of . da (molecular mass of glu residue is . da), surface of Å , volume of . Å , pk a of side chain of . and pi . at °c. intriguingly, free glutamic acid is not very soluble, possessing solubility of . g/ g at °c, which is significantly lower than the solubility of free prolines ( . g/ g at °c), and the solubility of the vast majority of free amino acids (www.fli-leibniz.de/ image_aa.html). the side chain of glutamic acid contains two methylene group and the carboxylic acid functional group (see fig. a ) that exists in a negatively charged deprotonated carboxylate with stroke, autism, amyotrophic lateral sclerosis, lathyrism, some forms of mental retardation and alzheimer's disease. the decreased glutamate release is associated with phenylketonuria leading to the developmental disruption of glutamate receptor expression. , the excess glutamate in the extracellular space and promoting entrance of calcium to the cell via the nmda receptor channels. this process is known as excitotoxicity, and it results in neuronal damage and eventual cell death. the excitotoxicity might occur as part of the ischemic cascade that is associated figure . amino acid determinants defining structural and functional differences between the ordered and intrinsically disordered proteins. (a) fractional difference in the amino acid composition (compositional profile) between the typical idps from the disprot database and a set of completely ordered proteins calculated for each amino acid residue. the fractional difference was evaluated as (c disprot − c pdb )/c pdb , where c disprot is the content of a given amino acid in a disprot databse, and c pdb is the corresponding content in the data set of fully ordered proteins. positive bars correspond to residues found more abundantly in idps, whereas negative bars show residues, in which idps are depleted. amino acid types were ranked according to their decreasing disorder-promoting potential. (b) amino acid compositions of several data sets discussed in the text (disprot, uniprot, pdb select and surface residues ). linkages, or ion pairs. an electrostatic interaction is a non-covalent bond that is based on the attraction of two oppositely charged groups. it can easily be broken and reformed and is characterized by the optimal distance of . Å between the interacting groups. the strength of these interactions depends on the distance of the two charges and the properties of the medium between them. in proteins, electrostatic interactions typically occur between coo − in the side chain of glutamic and aspartic acids and nh + in the side chains of lysines and arginines. hydrogen bond (h-bond) is another non-covalent bond. this interaction depends on the sharing of one hydrogen atom (h-atom) between two other atoms, where the h-atom has a covalent bond to one of them (which therefore serves as the h-bond donor), and where the other atom, to which the h-atom has a weaker bond, serves as the acceptor, a. hydrogen bond is weaker than a covalent bond but stronger than a van der waals bond. similar to electrostatic interactions, h-bonds can easily be broken and reformed. among established geometrical criteria for h-bond are a set of optimal distances between the non-h atom of donor and acceptor (dono-acceptor < . Å) and between the h atom of donor and acceptor (h-acceptor < . Å). being negatively charged at physiological ph, glutamic acid can serve as a hydrogen bond acceptor, whereas at acidic ph, it also can be a hydrogen bond donor. glutamic acid in the ramachandran plot. the structure of a protein can be described using torsion angles-φ and ψ-of its backbone that provides a simple view of the conformation of a protein. in sequence order, φ is the n i- -c i -cα i -n i torsion angle, and ψ is the c i -cα i -n i -c i+ torsion angle. since most combinations of φ and ψ are sterically forbidden, the d plot of the torsion angles of the protein backbone, known as the ramachandran plot, provides a simple view of the conformation of a protein, since the φ-ψ angles cluster into distinct regions in the ramachandran plot, where each region corresponds to a particular secondary structure. in the generic ramachandran plot (see fig. b ) that refers to the non-glycine and non-proline amino acids, there are four distinct regions of density (the α (right-handed α-helix region), α l (mirror image of α), β s (region largely involved in β-sheet formation) and β p (region associated with extended polyproline-like helices but also observed in β-sheet). the shape of the generic ramachandran plot is determined mainly by the presence of specific steric clashes and backbone dipole-dipole interactions. [ ] [ ] [ ] glutamic acid in electrostatic interactions and hydrogen bonds. glutamic acid participates in electrostatic interactions, which are also known as ionic bonds, or salt bridges, or salt gcn leucine zipper dimer revealed that the free energy of helix stabilization associated with the hydrogen-bonding and hydrophobic interactions in this capping structure is − . kcal/ mol, illustrating that helix capping might play a significant role in protein folding. based on the analysis of α-helices the normalized frequencies for finding particular residues at the c cap position, the average fraction of buried surface area and the hydrogen bonding patterns of the c cap residue side-chain were calculated. this analysis revealed that the residue found in the c cap position is on average % buried and that there is a noticeable correlation between the relative burial of this residue and its hydrophobicity. furthermore, c cap residues with polar sidechains were shown to be involved in hydrogen bonding, where the longer side-chains of glutamic acid, glutamin, arginine, lysine and histidine form hydrogen bonds with residues located more than four residues apart, whereas the shorter side-chains glutamic acid and protein secondary structure. although protein secondary structure is determined by hydrogen bonds between donor and acceptor groups in the protein backbone, different amino acids are known to favor the formation of different secondary structure elements, such as α-helices, β-pleated sheets or loops. the α-helix-formers include alanine, cysteine, leucine, methionine, glutamic acid, glutamine, histidine and lysine, whereas valine, isoleucine, phenylalanine, tyrosine, tryptophan and threonine favor β-structure formation, and serine, glycine, uncharged aspartic acid, asparagine and proline are found most often in β-turns. it was pointed out that there is no apparent relationship between the chemical nature of the amino acid side chain and its secondary structure preferences. for example, although glutamic and aspartic acids are closely related chemically, glutamic acid is more likely to be found in helices and aspartic acid is predominantly located in β-turns. in fact, the helical propensity of glutamic acid is . , whereas aspartic acid has an helical propensity of . , the third largest value after proline and glycine. note that the helical propensity is defined as the difference in free energy Δ(Δg) estimated in kcal/mol per residue in an α-helical configuration relative to alanine, which has been set to zero because it is usually the amino acid with the most favorable helix propensity. here, the higher helical propensity values correspond to more positive free energies and therefore are related to residues which are less favored in α-helix. glutamic acid in α-helix caps. since α-helices in peptides and proteins have an overall dipole moments caused by the cumulative effects of all the individual dipoles from the carbonyl groups of the peptide bond pointing along the helix axis, the overall helical structure is destabilized due to the noticeable entropic effects. the effect of this helical dipole moment can be approximated by placing . - . positive unit charge near the n-terminus and . - . negative unit charge near the c-terminus of the helix. , one of the nature's strategies to neutralize this helix dipole is the specific capping of the n-terminal ends of α-helices by negatively charged residues, such as glutamic acids. , furthermore, careful analysis of α-helices revealed that their first and last four residues differ from the remaining residues by being unable to make intrα-helical hydrogen bonds. instead, these first four (> n-h) groups and last four (> c = o) groups in an α-helix are often capped by alternative hydrogen bond partners. [ ] [ ] [ ] physico-chemical and statistical analysis suggested that certain residues are more preferable at the c-and n-termini of an α-helix (the helical c-and n-caps). for example, based on the analysis of series of mutations in the two n-caps of barnase, it was concluded that a single n-cap can stabilize the protein by up to ~ . kcal/mol. importantly, the presence of a negative charge of the n-cap was shown to add ~ . kcal/mol of stabilization energy mostly due to the compensation effects for the macroscopic electrostatic dipole of the helix. from a global survey among proteins of known structure, seven distinct capping motifs are identified-three at the helix n-terminus and four at the c-terminus. one of these motifs is the helix-capping motif ser-x-x-glu, a sequence that occurs frequently at the n-termini of α-helices in proteins. [ ] [ ] [ ] thermodynamic analysis of this ser-x-x-glu motif from the ramachandran plots for backbone conformations of the non-glycine and non-proline amino acids. marked regions of density correspond to the right-handed α-helix region (α), mirror image of α (α l ), region largely involved in β-sheet formation (β s ), and region associated with extended polyproline-like helices, but also observed in β-sheet (β p ). by the four glutamic acid residues located at homologous positions within each of the four pore-forming segments and which form a single or multiple ca + -binding site(s) that entrap calcium ions, thus giving them a possibility to be electrostatically repulsed through the intracellular opening of the pore. in the bacterial kcsa and inwardly rectifying k + (kir) channels, glutamic acid is also involved in the action of the selectivity filter. here, the network of residues stabilizing the pore of kcsa involves a glu -asp carboxyl-carboxylate interaction behind the selectivity filter, whereas the structure of the pore in kir channels is stabilized by a glu-arg salt bridge. therefore, although glu is quite conserved among both types of channels, the network of interactions is not translatable from one channel to the other. this clearly shows that different potassium channels are characterized by diverse gating patterns. the presence of a highly conserved glutamic acid residue in the middle of a transmembrane domain is a characteristic feature of a family of transmembrane glycoproteins with two immunoglobulin-like domains, such as basigin (bsg, also known as cd or emmprin), embigin and neuroplastin. finally, a critical glutamic acid residue was recently identified in clc proteins, which constitute a large structurally defined family of cl − ion channels and h + /cl − antiporters which are found in prokaryotes and eukaryotes, and which perform their functions in the plasma membrane or in various intracellular organelles such as vesicles of the endosomal/lysosomal pathway or in synaptic vesicles. mutations in human clc channels are known to cause a set of very diverse diseases such as myotonia (muscle stiffness), bartter syndrome (renal salt loss) with or without deafness, dent's disease (proteinuria and kidney stones), osteopetrosis and neurodegeneration, and possibly epilepsy. the side chain of the aforementioned critical glutamic acid occupies a third cl − ion binding site in the closed state of the channel and moves away to allow cl − binding. glutamic acid valve. glutamic acid is known to play a unique role in regulation of the cytochrome-c oxidase (cco) activity. cco is the last enzyme of the respiratory electron transport chain in mitochondria (or bacteria) located in the inner mitochondrial (or bacterial) membrane, and it is responsible for reducing ~ % of the oxygen taken up in aerobic life. this protein powers the production of atp by generating an electrochemical proton gradient across the membrane via the catalysis of the oxygen reduction to water that takes place in the binuclear center (bnc) of the enzyme. cco uses four electrons taken up from the cytochrome c located at the positively charged p-side (outside) of the membrane and four "chemical" protons taken from the negatively charged n-side (inside) to reduce the dioxygen to two water molecules. in addition to this oxygen reduction reaction, four "pump" protons are translocated from the n-side to the p-side across the membrane against the opposing membrane potential, doubling the total amount of charge separated by the enzyme. [ ] [ ] [ ] [ ] therefore, the main role of cco is to serve as a proton pump and a generator of the electrochemical proton gradient or charge separation across the membrane, which is achieved via two separate processes. first, the reduction of oxygen to water by electrons and protons taken up from opposite sides of the membrane leads of aspartic acid, asparagine, serine and threonine form hydrogen bonds with residues located close in sequence. finally, based on the analysis of α-helical propensity of a series of dodecapeptides containing alanine, asparagine, aspartate, glutamine, glutamate and serine at the n-terminus and arginine, lysine and alanine at the c-terminus, it was concluded that the α-helix-stabilizing abilities of these residues can be ranged as follows: aspartate > asparagine > serine > glutamate > glutamine > alanine at the n-terminus and arginine > lysine > alanine at the c-terminus. glutamic acid and protein solubility. based on the analysis of solubility-changing substitutions in proteins it has been pointed out that together with two other hydrophilic residues (aspartic acid and serine) glutamic acid contributes significantly more favorably to protein solubility than other hydrophilic residues (asparagine, glutamine, threonine, lysine and arginine). based on this observation, an important strategy for solubility enhancement was proposed, were the hydrophilic residues that do not contribute favorably to protein solubility can be replaced with the hydrophilic residues that contribute more favorably. glutamic acids inside the pores of ion channels. being negatively charged at physiological ph, glutamic acid is perfectly suited for binding metal ions. this property is used in specific regulation of a variety of ion channels. for example, in cyclic nucleotide-gated (cng) channels (which are found in vertebrate photoreceptors and olfactory epithelium, elsewhere in the nervous system [ ] [ ] [ ] and in a variety of other cell types including kidney, testis and heart, and whose activation represents the final step in the transduction pathways in both vision and olfaction [ ] [ ] [ ] ), a single glutamic acid strategically located in the pore represents the binding site for multiple monovalent cations, the blocking site for external divalent cations and the site for the effect of protons on permeation. this is not too surprising since the pore region of the channel controls both the singlechannel conductance and the pore diameter of the channel. importantly, cng channels are permeable to ca + , which is an important element in the activation of intracellular targets, and which in addition to permeating cng channels can profoundly block the current flow carried by monovalent cations through the cng channels. this capability of ca + to block the monovalent cation flow is determined by the high-affinity binding of ca + to a single acidic amino acid residue located in the pore of the channel, which is glu for the rod cng channel and glu for the catfish olfactory cng channel. this same glutamic acid residue is also responsible for the external rapid proton block of cng channels, another characteristic that the cng channels share with ca + channels. glutamic acid also plays an important regulatory role in the voltage-dependent calcium channels that are located in the plasma membrane and form a highly selective conduit by which ca + ions enter all excitable cells and some nonexcitable cells. for these channels to operate, ca + ions must enter selectively through the pore, bypassing competition with other extracellular ions. the high selectivity of a unique ca + filter is determined pathway for protons utilized in the catalytic no reduction; the carboxylate group of glu , which is located at the backside of glu , contributes to the electro-negative environment of the binuclear center of cnor, and to the low redox potential of heme b iron; finally glu and glu are positioned in the loop connecting the transmembrane helices iii and iv, with glu serving as one of the ca + ligands (which is crucial for maintaining the configuration of heme b and b ) and assisting in the water-mediated proton transfer through interactions with a number of water molecules, and with glu serving as a key residue for maintaining the unique conformation of the long loop through interactions with the residues in transmembrane helix ii, which would stabilize the coordination of glu to ca + . mono-adp-ribosyltransferase, which is responsible for the mono-adp-ribosylation of proteins, possesses a critical glutamic acid at the catalytic cleft which functions to position nad for nucleophilic attack at the n-glycosidic linkage for either adpribose transfer or nad hydrolysis. the pronounced na + /k + selectivity of na,k-atpase relies on the strategic positioning of glutamic acid residues. here, intramembrane glu in transmembrane segment m , glu in m , asp and asp in m are essential for tight binding of k + and na + , whereas asn and glu in m , together with thr , asn and glu in the -ytltsnipeitp motif of m contribute to the na + / k + selectivity. in the family of thiamin diphosphate enzymes, a highly conserved glutamate is known to promote the c -h ionization and the thiamin diphosphate activation. the direct catalytic role of glutamic acid can be seen in matrix metalloproteinases, which are ubiquitous endopeptidases characterized by an active site where a zn + atom, coordinated by three histidines, plays the catalytic role, assisted by a glutamic acid that acts as a general base. for example, one of the wellknown zinc-binding metalloproteases that uses a glutamic acid residue as the fourth ligand to coordinate the zinc ion is thermolysin. in thermolysin, glutamic acid is amino acids downstream from the second histidine in the first motif and present in a small conserved motif (nexxsd). in the zincin and pdf groups of metalloproteases, the catalytic zinc-binding site contains the hexxhxxg motif. also, a glutamic acid residue may be catalytically active in the substrate-binding cleft of plant lysozymes. each enzyme in the α-amylase family of multidomain hydrolases and transferases has one glutamic acid and two aspartic acid residues necessary for activity. the irreversible dealkylation reaction catalyzed by the o -alkylguanine-dna alkyltransferase (agt) that directly repairs alkylation damage at the o -position of guanine is accomplished by an active-site cysteine that participates in a hydrogen bond network with invariant histidine and glutamic acid residues, reminiscent of the serine protease catalytic triad. the spore germination protease (gpr) that degrades small, acid soluble proteins (sasp) protecting spore's dna against damage, is a structurally and functionally unique protease that utilizes glutamic acid residue to catalyze sasp degradation. in the hydrolytic aldehyde dehydrogenases (aldhs), catalytic but flexible glutamic acid residues located within the active site serve as the general base that activates the hydrolytic water molecule in the deacylation step. to the net translocation of one electrical charge across the membrane per electron consumed. second, an additional proton is translocated vectorially across the membrane for each electron consumed, resulting in a net transport of two electrical charges per electron. the protons for the chemical reaction are extracted from the n-side of the membrane via two proton pathways, the d-and k-channels. the d-channel starts at a highly conserved residue, asp (bovine numbering; subunit i) near the n side, and continues to another highly conserved residue glu that donates protons to the bnc, whereas the key residue in the k-channel is a highly conserved lysine (k ). the d-channel is responsible for the delivery of four "pump" protons that are first transferred from glu to a "loading" site above the bnc and then delivered to the p side via a proton-exit channel. the mystery of this mechanism is in the ability of glu located at the end of the d-channel to somehow sort "pump" protons from "chemical" protons. to explain this behavior, the glutamate valve model has been proposed according to which the side chain of glu shuttles between a state protonically connected to the d channel, and a state connected to the bnc and the pump site. in this proton valve model, the glu motion depends on its protonation state, where the unprotonated residue remains predominantly in a "down" conformation, pointing toward the n side, and therefore facilitating the uptake of a proton, whereas protonation shifts the glu to the "up" conformation, where the side chain of this important residue is swung toward the p side by ~ Å. glutamic acid in the active sites of enzymes. in addition to serve multiple structural roles and being involved in regulation of various channels, glutamic acid residues, being positioned within or in the close proximity to the active sites, might have roles in the catalytic activities of various enzymes. one of the illustrative examples of the functional roles of glutamic acid can be found in bacterial nitric oxide reductase (nor), which is a membraneintegrated enzyme that catalyzes the reduction of nitric oxide no to nitrous oxide n o using a type of anaerobic respiration where cytotoxic no is immediately decomposed after its production from nitrite no − via the nitrite reductase-catalyzed reaction. [ ] [ ] [ ] three different nor types are found in bacteria, with the cytochrome c dependent nor (cnor) that consists of two subunits, norb and norc, being the most extensively studied enzyme. precise description of the complex catalytic mechanism of this important enzyme is outside the scopes of this review, and therefore only a small piece of the entire picture, where the roles of glutamic acid are emphasized, is briefly described below. the characteristic feature of cnors is the presence of five conserved glutamic acid residues (glu , glu , glu , glu and glu in p. aeruginosa cnor) within the norb subunit consisting of trans-membrane helices and containing the heme b and the binuclear center (heme b /fe b ) buried in the hydrophobic interior of its trans-membrane region. here, glu is involved in the coordination of fe b and its carboxylate functions as the shuttle for catalytic protons from glu to the bound-no; glu , which interacts with glu but is not involved in direct interaction with fe b , is an important player of the thr -ser -glu -glu network that acts as a delivery matrix communication) and their ligands, it has been concluded that divalent cations are critical for integrin interactions with almost all ligands. importantly, although divalent cations are bound to integrins, their coordination sphere is not completed and the interactions between integrin and its ligands typically involve completing the metal ion coordination with an acidic ligand residue. for example, complexes between the human intercellular adhesion molecule- (icam- ) and the i domain of its integrin receptor αlβ are stabilized by a critical glutamate residue that completes the magnesium coordination in integrin. similarly, in the crystal structure of a complex between the i domain of a b integrin and a triple-helical collagen peptide containing a critical gfoger motif, glutamate residue from the collagen peptide completes the coordination sphere of the i domain metal ion. based on these observations it has been concluded that a metalglutamate handshake represents a basic mechanism of integrin i domain interaction with its binding partners. furthermore, it is believed now that the general mechanism by which integrins, these αβ-heterodimeric cell-surface receptors that are vital to the survival and function of nucleated cells, recognize their structurally diverse ligands relies on specific glutamic-acid-or aspartic-acid-based sequence motifs that function in a divalent cation-dependent and conformationally sensitive manner. the levels of intracellular zinc in living cells are crucial for managing various cellular processes, such as growth, development and differentiation. zinc is involved in protein, nucleic acid, carbohydrate and lipid metabolism and also plays a role in the control of gene transcription and the coordination of other biological processes controlled by proteins containing dna-binding zinc finger motifs, ring fingers and lim domains. the physiologically relevant intracellular levels of zinc are controlled by specific zinc transporters which mostly transport zinc into cells from outside. members of one of the subfamilies of these transporters, liv- subfamily of zip zinc transporters (lzt), being similar to other zip transporters in secondary structure and ability to transport metal ions across the plasma membrane or intracellular membranes, possess a unique hexphexgd motif containing conserved proline and glutamic acid residues, that fits the consensus sequence for the catalytic zinc-biding site of matrix metalloproteinases (hexxhxxgxxh), and which is unprecedented in other zinc transporters. in addition to this set of specific examples, one should keep in mind that all structures of the ca + -binding domains have in common a high negative surface potential usually associated with asp or glu residues. therefore, important glutamic acid residues responsible for calcium coordination can be found in various members of the major ca + -binding proteins, such as ef-hand domains, egf-like domains, γ-carboxyl glutamic acid (gla)-rich domains, cadherin domains, ca + -dependent (c)-type lectin-like domains and ca + -binding pockets of family c g-protein-coupled receptors. a particularly intriguing role was described for the n-terminal glutamic acid residues in the canonical ca + -protein, α-lactabumin, which is frequently used as a model protein in folding studies and in studies on the effect of calcium binding on protein structure, stability and folding. for example, in nudix hydrolases (which is a family of mg + -requiring enzymes that catalyze the hydrolysis of nucleoside diphosphates linked to other moieties) there is a specific motif, nudix box (gx ex reuxeexgu, where u is a bulky hydrophobic residue), that forms a loop-α helix-loop structural motif that functions as a common mg + -binding and catalytic site. it was emphasized that the overall catalytic powers of nudix hydrolases consists in accelerating the reaction rate by to times. the reactions are accelerated - -times by general base catalysis by a glutamate residue within, or beyond the nudix box, or by a histidine beyond the nudix box. the additional - -fold rate acceleration is due to the lewis acid catalysis provided by one, two, or three divalent cations. one divalent cation is coordinated by two or three conserved residues of the nudix box, the initial glycine and one or two glutamate residues, together with a remote glutamate or glutamine ligand located outside the nudix box. glutamic acids at various binding sites. hemopexin is an important multifunctional plasma protein involved in the sequestering of heme released into the plasma from hemoglobin and myoglobin as the result of intravascular or extravascular hemolysis and due to skeletal muscle trauma or neuromuscular disease. it also possesses hyaluronidase activity, serine protease activity, pro-inflammatory and anti-inflammatory activity and is involved in the suppression of lymphocyte necrosis, inhibition of cellular adhesion, and binding of divalent metal ions. finally, hemopexin possesses two highly exposed arg-gly-glu sequences that may promote interaction with cell surfaces. glutamic acid plays an important role in defining the retinal binding site geometry of rhodopsin, which is the photoreceptor in vertebrate rod cells responsible for vision at low light intensities. -cis-retinal is the photoreactive chromophore located in the interior of the protein where it is covalently attached to a lysine side chain through a protonated schiff base (psb) linkage. based on the c-nmr chemical shift data, it was concluded that glu of rhodopsin is involved in charge interactions with the retinal psb, which are crucial for maintaining rhodopsin in the inactive state in the dark and whose breaking leads to the protein activation. a centrally located glutamic acid residue in position of transmembrane segment vii of the main ligand-binding crevice of the chemokine tm receptors (gluvii: ) is crucial for recognition and binding of small molecule non-peptide ligands that contain one or two centrally located, positively charged nitrogen atoms and are characterized by relatively similar elongated overall structure with terminal aromatic moieties. furthermore, since this gluvii: is crucial for the binding and hence the function of a number of non-peptide ligands in several chemokine receptors, such as the ccr , ccr and ccr receptors, it serves as a selective anchor point for the centrally located, positively charged nitrogen of the small molecule ligands. glutamic acid and metal binding. the role of glutamic acid residues in coordination of various metal ions was already emphasized in sections discussing ion channels. a few other illustrative examples are listed below. based on the analysis of the complexes formed between integrins (which are central molecules in the adhesion processes that mediate cell-cell and cell-extracellular group giving rise to the pyrrolidone carboxylic acid (pyro-glu). however, it was emphasized that pyro-glu is exclusively found at the n-terminal end of the thermal polymers when glutamic acid is a predominant amino acid in a mixture of amino acids subjected to thermal polymerization. another important glutamic acid-based ptm is gammacarboxylation catalyzed by the vitamin k-dependent carboxylase that transforms specific glutamate residues in proteins to gammacarboxy glutamic acid (gla) in the presence of reduced vitamin k, molecular oxygen and carbon dioxide. this modification is widely distributed in the animal kingdom and has a wide range of physiological implications, such as hemostasis, bone calcification and signal transduction. in addition to be a target for various ptms, glutamic acid itself can be used as an important protein modifier, giving raise to polyglutamylation, which is a specific ptm where polyglutamate chains of variable lengths are added to the modified protein. polyglutamylation is evolutionarily conserved and is commonly found in the microtubule (mt) building block, tubulin. this ptm, being primarily found within the tubulin c-terminal tail that participates in binding of many structural and motor mt-associated proteins, is believed to be crucial for the functional adaptation of mts. polyglutamylation is catalyzed by a family of specific enzymes and in addition to tubulin can be found in some other proteins. high content of charged residues is one of the tricks used by nature to make stable proteins in thermophilic and hyperthermophilic organisms. in fact, based on the correspondence analysis of the completely sequenced genomes available from the three domains of life (seven eukaryotes, archaeal and bacterial species) it has been concluded and the amino acid composition permits discrimination between the three known lifestyles (mesophily, thermophily or hyperthermophily). the most specific amino acid compositional biases that represent specific signatures of thermophilic and hyperthermophilic proteomes are a relative abundance in glutamic acid, concomitantly with a depletion in glutamine and a significant correlation between the relative abundance in glutamic acid (negative charge) and the increase in the lumped "pool" lysine + arginine (positive charges). being absent in mesophiles, these correlations could represent a physico-chemical basis of protein thermostability. curiously, the distribution of the remaining charged amino acid, i.e., aspartic acid, appears to be quite homogeneous throughout all the species suggesting that this residue does not participate significantly in the aforementioned compensatory negative/positive (charged) correlation in thermophiles and hyperthermophiles. on average, thermophilic and hyperthermophilic proteomes were shown to contain . %, . %, . % and . % of glutamine, glutamic acid, aspartic acid and lysine + arginine residues, respectively. importantly, some of these numbers are rather different from those found in idps/idprs, as shown in table . α-lactabumin was shown to possess significantly different thermal and structural stability in its calcium-bound and calciumfree apo-forms, with the apo-protein possessing molten globule-like properties at slightly elevated temperatures. , this strong dependence of the α-lactabumin structural properties on metal-binding is determined by the simple fact that in the apo-form, many acidic side chains have unfavorable chargecharge interactions, with residues (glu , glu , glu , asp , asp , asp , asp , asp , asp , asp and asp ) possessing significantly unfavorable charge-charge repultion. although calcium binding has the most pronounced effect on residues directly involved in cation coordination (asp , asp and asp ) and strongly affects the other two residues in the ca + -binding loop, asp and asp , ca + binding has relatively minor effects on residues more distant from the ca + -binding site (glu , glu , glu , asp and asp ), which mostly preserve unfavorable electrostatic interactions seen in the apo-form. it was also shown that the mutation-induced neutralization of unfavorable charge-charge interactions in the n-terminus (residues - of which are characterized by a high proportion of negatively charged residues that cluster on the surface of the native protein) results in stabilization of both the apo-and ca +bound protein. unexpectedly, the Δglu mutant, where the glu residue was removed, leaving an n-terminal methionine in its place, possessed almost one order of magnitude higher affinity for calcium and higher thermostability (both in the absence and presence of calcium) than the native protein isolated from milk. this unique tuning of the α-lactabumin structure and calcium binding suggested that the n-terminal region of this protein might have a direct effect on the calcium-binding loop (and perhaps other regions of the structure). the side chains of glutamic acid residues are subjected to several ptms. some cytoplasmic and nuclear proteins are known to be methylated, i.e., enzymatically modified by the addition of methyl groups from s-adenosylmethionine. methylation reactions typically occur on carboxyl groups (such as the side chain of glutamic acid) and modulate the activity of the target protein. glutamate methyl ester formation plays a major role in chemotactic signal transduction in prokaryotes. for example, methyl-accepting chemotaxis proteins are a family of chemotactic-signal transducers that respond to changes in the concentration of attractants and repellents in the environment, transduce a signal from the outside to the inside of the cell, and facilitate sensory adaptation through the variation of the level of methylation. , in some proteins and peptides, glutamic acids can be amidated. also, some glutamine residues in proteins undergo spontaneous (nonenzymatic) deamidation to glutamate with rates that depend upon the sequence and higher-order structure of the protein. functional groups within the protein can catalyze this reaction, acting as general acids, bases, or stabilizers of the transition state. in rare cases, glutamate residues can be modified by cyclization via condensation of the α-amino group with the side-chain carboxyl neutral ph was shown to be accompanied by the instantaneous formation of a gel-like precipitate with intermolecular antiparallel β-structure. in bacteria, pga may be composed of only d-, only l-or both d-and l-glutamate enantiomers, and pga filaments may be poly-γ-l-glutamate filaments (plga), pdga filaments or poly-γ-l-d-glutamate (pldga) filaments. the production and maintenance of sufficient d-glutamate pool levels required for the normal bacterial growth is controlled by the glutamate racemase, which is a member of the cofactor-independent, twothiol-based family of amino acid racemases. this enzyme is conserved and essential for growth across the bacterial kingdom and has a conserved overall topology and active site architecture. therefore, it represents an attractive target for the development of specific inhibitors that could act as possible therapeutic agents. in gram-negative bacteria, the complex responsible for the polyglutamate synthesis is encoded in specific loci. if the pga is associated with the bacterial surface and forms a capsule, then the corresponding genes are named cap (for "capsule"); however, if the pga is released, then the corresponding genes are named pgs (for polyglutamate synthase). the minimal gene sets contain four genes termed cap or pgs b, c, a and e, with all cap genes and the four pgs genes (pgsb, pgsc, pgsaa, pgse) being organized into operons. since pga is an idp, whose biochemical and biophysical properties are environment-dependent, and since pga can be found in an anchored to the bacterial surface form or in a released form, this biopolymer can play different roles in different organisms and in different environments. for example, when anchored to the bacterial surface, pga forms a capsule and act as a virulence factor. , in fact, the virulence of bacillus anthracis (a gram-positive sporulating bacterium, which is the causal agent of anthrax) was found to be determined by its capsule composed solely of pga. similarly, the virulence of staphylococcus epidermidis (another gram-positive bacterium that causes severe infection after penetrating the protective epidermal barriers of the human body) is dependent on the pga-based capsule. furthermore, pga in capsules of these bacteria consists of either a mixture of l-and d-enantiomers (s. epidermidis) or solely d-enantiomer (b. anthracis), which makes them particularly non-immunogenic. the released form of pga is used by the producing organism for rather different purposes, starting from the sequestration of toxic metal ions that increases the resistance of some soil bacteria to harsh conditions, to serving as a source of glutamate for bacteria in a starvation state during late stationary phase, to playing a role in decrease of the high local salt concentrations that helps extremophilic bacteria and archaea to survive in a hostile environment, , and in hydra, to control explosion of the special stringing cells, nematocysts, that are used to capture prey, for locomotion and for defense. in addition to have multiple functional roles, bacterially produced pga has found its way to serve as an important biodegradable component with multifarious potential applications in foods, pharmaceuticals, healthcare, water treatment and other fields. , a large commercial advantage of pga is that this although some amount of glutamic acid residues is crucial for the structure and function of ordered proteins/domains, when a protein or a peptide contains a large number of glutamic acid residues and, as a consequence, possesses a small number of hydrophobic residues, it is likely to be disordered at physiological ph due to strong charge-charge repulsion and weak hydrophobic attraction. an illustrative example of such charge-infused proteins is glurich human prothymosin α, in which out of residues are charged (there are asp, glu, arg and lys residues), the overall content of hydrophobic residues (leu, ile and val) is very low, and aromatic residues (trp, tyr, phe and his) and cystein are absent. based on this amino acid composition, it was not a big surprise to find that prothymosin α behaved as a highly disordered coil-like chain, since one cannot expect that a highly charged polypeptide (that contains % of glu+asp residues) will have a strong tendency to fold under physiological conditions. , the lack of stable structure also explains the extreme thermal and acid stability of prothymosin α, since one cannot break what is non-existent. the peculiar amino acid composition of prothymosin α, this biologically active random coil, was one of the defining factors behind the charge-hydropathy plot (ch-plot) development. in fact, based on the analysis of prothymosin α and of other non-globular proteins that lacked almost any ordered secondary structure under physiological conditions in vitro, it was concluded that a combination of high net charge and low hydropathy represents the necessary and sufficient factor for a polypeptide to behave as a natively unfolded protein. strategically positioned glutamic acid residues can modulate conformational stability and function of ordered proteins too. in fact, the role of a glutamic/aspartic acid cluster located outside the ca + -binding site, and of the n-terminal glu residue in destabilizing the structure and weakening the calcium-binding capabilities of α-lactabumin has been already discussed (see above). , therefore based on these observations, protein regions and whole proteins enriched in glutamic acids are expected to be substantially disordered. poly-γ-glutamate (pga) is a natural homopolymer synthesized by several bacteria, one archaea (natrialba aegyptiaca) and one eukaryote (cnidaria). one of the most known sources of pga is the japanese specialty natto, a fermentation product made by bacillus subtilis grown on soybean. pga is a highly soluble polyanionic polymer that sequesters water molecules and can be found in surface-bound and released forms. in structural studies, polyglutamic acid is traditionally used as a biopolymer with a well-characterized secondary structure response to changes in the environmental ph, where pga is in a random coil-like conformation at neutral ph, but gains monomeric α-helical structure at acidic ph and is transformed into a β-sheet structure at alkaline ph. [ ] [ ] [ ] curiously, the addition of polylysine to an aqueous solution of polyglutamic acid homopolypeptide at ebd is not a structurally stable entity in the conventional sense, since for this protein region there are no folded states that exist for any appreciable amount of time. instead, the ebd represents a time-average d region of a protein derived from the thermally driven motion of certain polypeptide chains, including those that are part of an otherwise stable folded protein. therefore, the ebd which is defined by the time-averaged occupancy of space by a polypeptide chain, can exclude lager molecules while allowing small molecules and water to move freely through it. it was proposed that since functions of ebd depend on the intrinsically rapid thermal motion of the polypeptide, and the free energy changes that result when that motion is confined, this domain can be used to control binding events, confer mechanical properties, and sterically control molecular interactions. obviously, to be able to serve as an ebd, a given fragment of a protein has to possess specific amino acid composition that would preclude it from folding. therefore, ebds are expected to possess low hydropathy and high net charge; i.e., in the ch-plot, they can be found well above the boundary separating compact and extended disordered proteins. one of the illustrative examples of biologically active ebds (which are not tightly folded, but expected to have a very extended conformation) is given by side-arms of neurofilament (nf) proteins. the side-arms of the nf heavy polypeptide, nf-h (which are ~ amino acids long), were shown by rotary shadow electron microscopy to be ~ nm long. since there was not enough mass to form a stiff folded structure to occupy such a volume, it was proposed that the side-arms were not folded but were in constant thermal motion. analysis of the amino acid sequence of the porcine nf medium polypeptide (nf-m, which has an apparent molecular mass of kda and is one of the two high molecular mass components of mammalian neurofilaments) revealed that this protein has several peculiar features. the n-terminal residues contain a non-α-helical arginine-rich headpiece (residues - ) with multiple β-turns followed by a highly α-helical rod domain that forms double-stranded coiled-coils (residues - ), followed by a c-terminal tailpiece extension (approximately residues) that represents an autonomous domain of unique amino acid composition, being characterized by a high content of lysines and particularly glutamic acids. in human nf-m, there are glutamic acids ( . %), most of which are concentrated within the c-terminal tail, where glutamate accounts for . % ( out of residues). similarly, human nf-h (a polypeptide comprising , residues) has glutamic acids, of which are found in the residues-long c-terminal tail of this protein, whereas in the human nf-l (nf light polypeptide which has residues), there are glutamic acids, with almost half of which ( ) being located within the acidic c-terminal subdomain (the last residues of the protein). in addition to neurofilament polypeptides, ebds were found in microtubuleassociated protein (map ) and numa. analysis of the amino acid compositions of these proteins revealed that they follow the trend established by nfs and contain significant amount of glutamic acid residues ( out of , residues in human map are glutamates and there are glutamic acids in the , residues-long human numa). natural biopolymer is nontoxic, biocompatible and nonimmunogenic. it can be produced by various bacterial strains in a controllable way. as a result, pga is commonly used in cosmetics/ skin care, bone care, nanoparticle for drug delivery system, hydrogel, etc. for example, the pga-based medusa system has been recently developed for slow release of therapeutic proteins and peptides. here, a poly l-glutamate backbone is grafted with hydrophobic α-tocopherol molecules, creating a colloidal suspension of nanoparticles in water that contain hydrophobic nanodomains suitable for the reversible binding of various drug molecules. the potential multifarious applications of pga in the areas of biomedical materials, drug delivery carriers, and biological adhesives have been studied extensively. in general, γ-pga is recognized now as an important biomaterial in drug delivery applications, with γ-pga-based nanoparticles being considered as promising delivery carriers for anticancer therapeutics. recently, a high molecular weight γ-pga was shown to be used as an immune-stimulating agent. finally, conjugation of paclitaxel, a widely used chemotherapeutic agent whose therapeutic index is limited by low tumor exposure and high systemic exposure, with biodegradable poly-lglutamic acid generates paclitaxel poliglumex (ppx, ct- ). this macromolecular drug conjugate enhances tumor exposure to the drug, since the release of paclitaxel from the polymeric backbone was shown to be dependent on the ppx degradation by the lysosomal protease cathepsin b, which is upregulated in many tumor types. glutamic acid as a part of the protein degradation targeting signals, pest motifs. pest sequences (i.e., sequences enriched in proline (p), glutamic acid (e), serine (s) and threonine (t)) are known to serve as specific degradation signals. [ ] [ ] [ ] [ ] these degradation signals define cellular instability of many proteins and direct them either to the ubiquitin-proteasome degradation or to the calpain cleavage. , this controlled protein degradation is important for activation and deactivation of regulatory proteins involved in signaling pathways that control cell growth, differentiation, stress responses and physiological cell death. [ ] [ ] [ ] [ ] pest-containing sequences were shown to be solvent exposed and conformationally flexible, which preclude them from been resolved in x-ray structures. based on the comprehensive bioinformatics analysis of experimentally characterized disordered and globular regions and of pdb chains containing pest regions, it has been concluded that the pest motif is most frequently located within idprs. furthermore, analysis of the prolinerich motif pro-x-pro-x-pro in pest sequences revealed that these sequences contain glutamic acids much more often than aspartic acids. in addition to this pro-x-pro-x-pro motif, many pest sequences are highly enriched in negatively charged residues and are characterized by a very specific distribution of negative charged patterns. glutamic acids in entropic bristle domains. the entropic bristle domain (ebd) concept was proposed to describe a characteristic behavior of some highly mobile protein regions. the several metals of the transition and main groups (ib-va, z = − ) of the periodic table of elements. phytochelatins are synthesized by a constitutive enzyme, γ-glutamylcysteine dipeptidyl transpeptidase, that uses glutathione (gsh) as a substrate and catalyzes the following reaction: γ-glu-cys-gly + (γ-glu-cys) n − gly→(γ-glu-cys) n+ − gly + gly. fertilization promoting peptide. another important glutamaterich peptide is fertilization promoting peptide (fpp; pglu-glu-pronh ), which is produced by the prostate gland and secreted into seminal plasma. fpp was shown to stimulate capacitation, which is the penultimate step in the maturation of mammalian spermatozoa required to render them competent to fertilize an oocyte. furthermore, although fpp inhibits spontaneous loss of acrosome (an organelle that develops over the anterior half of the head in the spermatozoa), cells retain high fertility in vitro. gala peptide. recently, a synthetic amino acid-long gala peptide with a glutamic acid-alanine-leucine-alanine (eala) repeat was designed to analyze how viral fusion protein sequences interact with membranes. this gala peptide was long enough to span a bilayer when in the α-helical state, and the eala repeat was adjusted so that the peptide would have a hydrophobic face of sufficient hydrophobicity to interact with the bilayer when the peptide was in an α-helix. glu residues were used in gala as a ph-responsive elements. when the ph is reduced from . to . , gala converts from a water soluble random coil conformation to an amphipathic α-helix that binds to bilayer membranes. functional analysis revealed that gala promoted fusion between small unilamellar vesicles and was able to form a transmembrane pore comprised of ~ gala α-helical monomers that were oriented perpendicularly to the plane of the membrane. based on these observations, it has been proposed that ph-controlled membrane permealization induced by gala can serve as a model for the design of environmentally responsive peptidic vehicles for drugs and genes delivery. other type of pests: ptp-pests. protein tyrosine phosphatases (ptp) with proline-, glutamate-, serine-and threoninerich sequence, ptps-pest, are a ubiquitously expressed critical regulators of cell adhesion and migration. , this family of ptps includes three intracellular phosphatases known as prolineenriched phosphatase (pep) in mice or lymphoid tyrosine phosphatase (lyp) in humans (also known as ptpn and ptpn ), ptp-pest (also referred to as ptpn ) and ptp-hematopoietic stem cell fraction (ptp-hscf, which is also known by several other names, such as also termed brain-derived phosphatase (bdp ), ptp , ptp-k , fetal liver phosphatase (flp ) and ptpn . all these phosphatases possess a common structural organization that includes an n-terminally located phosphatase domain, followed by a highly divergent central region that contains various motifs for interactions with other proteins, and a conserved c-terminal domain known as carboxyl-terminal homology (cth) domain. human ptp-lyp (ptpn / ptpn ) is a residues-long protein that contains and glutamic and aspartic acids and , and prolines, serines and threonines, respectively. human ptp-pest (ptpn ) consists of residues and has , , , and glutamates, aspartates, prolines, serines and threonines, respectively, most of recently, we proposed that ebds can be used as protein solubility enhancers. in fact, we showed that highly charged protein sequences (both natural and artificial) can act as ebds, and that translational fusion of such sequences to target proteins can serve as an effective solubilizing means by creating both large favorable surface area for water interactions and large excluded volumes around the partner. this suggests that intrinsically disordered ebds (which extend away from the partner and sweep out large molecules) can enable the target protein to fold free from interference. all artificial fusions used in our study had low sequence complexity and high net charge, but were diversified using distinctive amino acid compositions and lengths. among successful solubilizers were artificial ebds containing the most disorder-promoting residues (glu, pro, gln and ser) in the proportion glu:pro:gln:ser = : : : ; i.e., sequences containing > % glutamic acids. therefore, it seems that glutamic acid is crucial for the successful function of ebd-containing proteins. glutamic acids in intrinsically disordered chaperones. the high content of glutamic acids in artificial ebds designed as solubilization means was chosen because of the earlier observation that proteins with high net charge densities can function as effective intra-and intermolecular chaperones. - for example, polyglutamate among other polyanions was shown to act as a chaperone and to accelerate the in vitro refolding of the arc repressor protein. small heat shock proteins (hsps) have flexible c-terminal extensions that, although variable in length and sequence, are rich in acidic amino acids. the shsp α-crystallin can act as a chaperone on the fibroblast growth factor (fgf- ), and this chaperone action is mediated by electrostatic interactions between the basic regions of the growth factor and acidic regions of α-crystallin. nucleolar chaperone b ( residues, of which are glutamic acids) has two acidic regions (residues - and - ) that contain glutamic residues each and that are necessary for the b chaperone-like activity. tubulin has chaperone-like activity being able to suppress the aggregation of soluble lens proteins, equine liver alcohol dehydrogenase, malic dehydrogenase and insulin, but only if its acidic c-terminus (that contains % and . % of glutamic acid residuess in the porcine αand β-tubulins, respectively) was intact. [ ] [ ] [ ] many polyanionic propeptides were shown to serve as intramolecular chaperones to aid folding of the respective proteins. [ ] [ ] [ ] [ ] for example, propeptides of human neutrophil defensins contain up to . % glutamic acids. also, the c-terminal solubilizing domain of human α-synuclein (residues - ) contains . % glutamates, whereas erd ( residues) and erd dehydrins ( residues) from arabidopsis thaliana contain . % and . % glutamic acids respectively. some functions of glutamate-rich peptides. this section presents several illustrative examples of important biological functions attributed to glutamate-rich peptides. phytochelatins. heavy metal detoxification in higher plants is dependent on a set of heavy-metal-complexing peptides, phytochelatins, with structure of (γ-glutamic acid-cysteine) n -glycine (n = - ) [(γ-glu-cys) n -gly]. the longest of these peptides possesses a molecular mass of . kda, a pi . and a net charge of − . these peptides are induced by the exposure of plants to arglu . transcriptional activators and rna polymerase ii are bridged via the central transcriptional coactivator complex, the mediator complex. it has been recently shown that the arginine and glutamate rich protein (arglu ) colocalizes with the mediator subunit (med ) in the nucleus, being in contact with the far c-terminal region of med . this arglu -med interaction is crucial for the estrogen-dependent gene transcription and breast cancer cell growth. human arglu is a residues-long protein that contains arginines and glutamates. there are two regions with significant composition biases in this protein, an arginine-rich region (residues - ) that contains arginines and a glutamic acid-rich region (residues - ) containing glutamic acids. pelp . proline-, glutamic acid-and leucine-rich protein- (pelp ) plays an important role in mediation of genomic and nongenomic signaling of β-estradiol. this potential protooncogene functions as a co-regulator of estrogen receptor, and expression of pelp is deregulated during breast cancer progression. pelp contains ten nuclear receptor-interacting boxes (lxxll motifs), which allow it to interact with estrogen receptor and other nuclear hormone receptors, a zinc finger, a glutamic acid-rich domain and two proline-rich domains. there are several consensus pxxp motifs within the proline-rich regions, via which pelp couples the estrogen receptor (er) with sh domain-containing kinase signaling proteins, such as src and pi k p regulatory subunit. there are glutamic acids in pelp (which is , residues long), and the majority of them ( ) are concentrated within the glutamic acid-rich domain (residues - ). eif . eukaryotic translation initiation factor (eif ) is a monomeric protein of about kda that functions as a gtpaseactivating protein (gap) in translation initiation. eif is involved in initiation of protein synthesis in eukaryotic cells, where, after binding to the s initiation complex ( s-eif -mrna-met-trna f -eif -gtp) at the aug codon of an mrna, it promotes gtp hydrolysis. this initiates a cascade of events that starts from the release of bound initiation factors from the s subunit and ends with the joining of the s ribosomal subunit to the s complex to form the functional s initiation complex ( s-mrna-met-trna f ). although eif binds gtp and is able to promote gtp hydrolysis reaction, it does not hydrolyze gtp by itself acting as a typical gtpase-activating protein (gap). in fact, eif forms a complex with eif via its glutamic acidrich c-terminal region that binds to the lysine-rich n-terminal region of the β-subunit of eif thus activating the gtpase activity of eif . in human eif , the d structure is known for the n-terminal nucleotide binding domain (residues - , pdb id: e h) and for the w domain (residues - , pdb id: iu ). the linker region connecting these two domains is highly disordered and contains one of the functionally important glutamic acid-rich regions (residues [ ] [ ] [ ] [ ] [ ] [ ] [ ] . overall, there are . % glutamic acid residues in the residues-long amino acid sequence of human eif . histone-interacting proteins. since histones are polycations, they are known to be involved in interactions with several polyanionic proteins, particularly with proteins containing glutamic which are located outside the catalytic domain, with respectively , , , and glutamates, aspartates, prolines, serines and threonines being found in the non-catalytic region (residues - ). finally, among the residues of the human ptp-hscf (bdp /ptp /ptp-k /flp /ptpn ), there are glutamic acids, aspartic acids, prolines, serines and threonines. importantly, glutamate-rich, non-catalytic regions of all these ptps are known to be involved in interactions with multiple binding partners. for example, ptp-lyp is involved in interaction with grb , c-cbl, and the c-terminal src kinase (csk), which is the inhibitory protein tyrosine kinase (ptk). the interaction between the ptp-lyp and csk is mediated by the proline-rich motif in pep and by the src homology (sh ) domain of csk. ptp-pest promiscuously associates with various proteins involved in the organization of the cytoskeleton, such as cas (and cas-related proteins sin and casl), paxillin (and paxillin-related polypeptides hic- and leupaxin) and the ptks fak and pyk . this protein also associates with shc, grb and csk. finally, ptp-hscf is involved in association with csk and tec. multifarious functions of glutamic acid-rich proteins. delta factor. in addition to γ-pga, bacillus subtilis produces another important polyanion, delta factor, which is an important component of the bacterial rna polymerase. this delta factor is a . kda highly acidic (pi = . ) protein that contains two distinct regions, a kda n-terminal domain with uniform charge distribution and a glu-asp-rich c-terminal region. the overall contents of glutamic and aspartic acids in delta factor are . % and . % respectively, whereas these numbers increase to . % and . % in the glu-asp-rich c-terminal domain. the ordered n-terminal domain contains % α-helix and % β-sheet, whereas the c-terminal . kda domain is highly charged (net charge of − ) and therefore is largely unstructured. importantly, the c-terminal intrinsically disordered domain has an important biological function, since the ability of delta factor to displace rna from rna polymerase requires the activities of both the n-terminal core-binding domain and the polyanionic c-terminal region. marcks. myristoylated alanine-rich c kinase substrate (marcks) is an abundant kda protein which is unusually rich in alanine and glutamic acid, with glutamic acid and alanine in this proteins accounting for . % and . % residues, respectively. marcks is a very prominent cellular substrate for protein kinase c (pkc), and its serine residues and threonines are phosphorylated. human marcks is an acidic protein with a pi of . which in addition to ala-glu enriched n-and c-terminal domains possesses a compact "effector domain" (ed), which is responsible for interaction with calmodulin, is located near the middle of the sequence and is enriched in lysines, serines and phenylalanines. marcks is a typical idp with a labile conformation and little ordered structure. in addition to calmodulin this protein can interact with synapsin and actin, and can serve as filamentous actin (f-actin) cross-linking protein. furthermore, being myristoylated, marcks is able to interact with membrane and serves as a cytoskeleton-membrane linkage crucial for controlling cell shape changes. since although the molecular mass of the phosphoprotein was shown to be about kda by sedimentation equilibrium analysis, it runs on - % sds-page (sds-page) as a protein with a molecular mass of kda. later studies revealed that bsp is capable of nucleating the bone mineral hydroxyapatite and that this nucleation involves one or both of the glutamic acid-rich sequences suggesting that polycarboxylate sequences might represent a specific site for growth-modulating interactions between proteins and biological hydroxyapatite crystals. similarly, the ability of another acidic, non-collagenous protein of bone and dentin, osteonectin (also known as secreted protein, acidic, rich in cysteine), to bind to hydroxyapatite crystals is determined by its n-terminal region containing glutamic acid-rich sequences. sparc is a highly conserved acidic calcium-binding extracellular-matrix protein. this matricellular glycoprotein is composed of three functional domains that are evolutionarily conserved in organisms ranging from nematodes to mammals. starting from the n-terminus, these functional domains are: a ca + -binding glutamic acid-rich acidic domain (domain i), a follistatin-like module (domain ii), and an extracellular ca + -binding (ec) module that contains two ef-hands and two collagen-binding epitopes (domain iii). since domain i was not found in sparc isolated from the starlet anemone nematostella vectensis, it has been proposed that sparc first evolved as a collagen-binding matricellular glycoprotein. human sparc is a residueslong protein that contains glutamic acids, of which are located within the n-terminal calcium binding region (residues - ). although xenopus laevis sparc has a molecular mass of . kda, based on sds-page analysis this protein has a molecular mass of kda. nbp- . in nuclei of mice cells, there is a nuclear protein nbp- related to the nuclear proteins hmg- /- . nbp- can function as a transcriptional activator, binds specifically to nucleosome core particles, preferentially binds to euchromatin and modulates cellular transcription by counteracting linker histone-mediated chromatin compaction. nbp- is composed of amino acids and has several functional regions and domains: the n-terminal region (residues - ) contains three segments that are highly homologous to functionally important domains in the hmg- /- protein family, namely a nuclear localization signal, a nucleosome binding domain and a chromatin unfolding domain, whereas the c-terminal region (residues - ) has . % of negatively charged residues. in fact, of the glutamic acids and aspartic acids found in nbp- , glutamic and aspartic acids are located in this highly acidic region. garps in rod photoreceptors. glutamic acid-rich proteins (garps) are common in different organisms and have numerous biological functions. for example, rod photoreceptors contain three different glutamic acid-rich proteins (garps), two soluble forms, garp and garp , and the n-terminal cytoplasmic domain (garp part) of the b subunit of the cyclic gmp-gated channel (also known as cyclic nucleotide-gated cation channel β- , cngb ), that are involved in the control of the ca + propagation from the site of its entry at the cyclic nucleotidegated channel to the cytosol of the outer segment. the cyclic acid-rich domains or regions. for example, the non-epithelial intermediate filament (if) subunit protein (e.g., human vimentin, which is attached to the nucleus, endoplasmic reticulum and mitochondria, either laterally or terminally and that contains . % glutamic acids) can specifically bind core histones with a stoichiometry of core histones per a nonneuronal if protein dimer. glutamic acids clearly play a crucial role in this interaction since the kd neurofilament protein, which was already discussed in the ebd section and contains a glutamic acid-rich c-terminal extension, can bind more core histones per dimer ( molecules of core histones) than the dimer of the non-neuronal if proteins. in the nuclei of physarum polycephalum, there is an alanine, lysine and glutamic acid-rich nuclear protein (p ) with a molecular mass of ~ . kda that can specifically interact with histones and therefore is co-extracted with histones. based on amino acid sequence analysis, it has been concluded that p is a hmg-like protein, which, according to cd measurements, contains only % secondary structure and is, therefore, essentially unstructured under in vivo conditions. titin. the gigantic protein titin (there are , residues in the human protein) is a key component in the assembly and functioning of vertebrate striated muscles. among numerous cellular functions of titin (also known as connectin) are contribution to the fine balance of forces between the two halves of the sarcomere which is crucial for the elasticity of muscle cells, as well as participation in chromosome condensation and chromosome segregation during mitosis of non-muscle cells. the ability of titin to reversibly extend relies on a set of pevk segments, rich in proline (p), glutamate (e), valine (v) and lysine (k) residues. the single molecule analysis of the recombinant titin fragment, containing approximately -residue pevk repeats and glutamic acid-rich motifs, revealed that the bending rigidity of the pevk fragments can be reduced due to calcium-induced conformational changes. furthermore, the glutamic acid-rich motif was shown to be critical for this process. based on these observations, it has been concluded that the glutamic acid-rich motifs embedded into the pevk segments make titin a calcium-dependent molecular spring that can adapt to the physiological state of the cell. curiously, titin has , glutamic acids, of which are found in the glutamic acid-rich region (residues - ) that contains pevk motifs. glutamates are not evenly distributed within the glutamic acid-rich region; e.g., glutamic acids are concentrated within the first residues of this region (residues - , ). in other words, although the glutamic acid-rich region comprises just . % of the whole titin, it has . % of all the titin's glutamates. bone phosphoproteins. bone sialoprotein ii (bsp ii) is an important component of the bone mineralized matrix. this bone-specific glycoprotein contains phosphoserine and sulphotyrosine residues and two regions of contiguous glutamic acid residues (residues - and - ) . in one of the first studies dedicated to the analysis of bone phosphoprotein it has been shown that this glycoprotein can be purified from the mixture of proteins extracted by demineralization of rat bone with . m edta in m guanidinium chloride. it was also emphasized that this protein possessed an abnormal electrophoretic mobility this protein was shown to be involved in sensing infertile nutrient conditions in infected cells to promote a transfer from saprophytic to dormant microsclerotia for long-term survival. vdgarp is a short ( residues) extremely acidic protein with a pi of . that contains . % negatively charged residues ( glutamic acids and aspartic acids). there are also several other garps in various organisms, the functions of which are not known as of yet. small garp (a -amino acid protein, with a molecular mass of . kda and an isoelectric point of . , residues of which are glutamic acids) was found in euplotes octocarinatus. plasmodium falciparum garp consists of residues, of which are glutamic acids. rhox /tox. reproductive homeobox protein (rhox or tox) is a homeodomain protein which is distantly related to the members of the paired/pax family belonging to the pepp subfamily of paired-like homeobox proteins. in mice, tox is predominately transcribed in the testis and ovary and potentially plays an important role during gametogenesis. this residues-long protein contains glutamic acids organized in two poly-glutamic acid stretches (residues - and - ) and several glu-rich regions, which together with aspartic acids makes rhox highly acidic (pi . ). kibra. kidney and brain protein (kibra) is a large ( , residues) protein that serves as a potential regulator of the hippo/ swh (sav/wts/hpo or salvador/warts/hippo) signaling pathway that restricts proliferation and promotes apoptosis therefore being crucial for tumor suppression. , kibra has glutamic acids and possesses two n-terminal ww domains, an internal c -like domain and a c-terminal glu-rich stretch (residues - ). cellular functions of kibra are modulated via phosphorylation by protein kinase c zeta (prkcz). some cellular activities of kibra may be associated with memory performance. , furthermore, in mammalian cells, this protein co-activates functions of the dynein light chain , is involved in regulation of the collagen-stimulated activation of the erk/ mapk cascade and modulates directional migration of podocytes. kibra interacts with histone h via its glu-rich region, and this interaction might play an important role in conferring an optimal transactivation function to the estrogen receptor-α (er) and also may be involved in the proliferation of ligand-stimulated breast cancer cells. sh bgr. sh domain-binding glutamic acid-rich protein (sh bgr) is a highly acidic (pi . ) residues-long protein that possesses glutamates and aspartates and that is expressed in heart and skeletal muscles. the majority of glutamates are located within the c-terminal glu-rich region (residues - ), ~ % of which are glutamic acid residues. in addition to sh bgr, several other members of the sh bgr family were found in humans. these are the so-called sh bgr-like proteins, such as sh bgrl ( residues, glutamic acids), sh bgrl ( residues, glutamic acids) and sh bgrl ( residues, glutamic acids) encoded by chromosomes xq . q - , and p . - , respectively. it was shown that the sh domainbinding glutamic acid-rich-like protein is upregulated in glioblastoma. also, this protein was noticeably downregulated in nucleotide-gated (cng) cation channel of rod photoreceptors is a heterotetramer consisting of homologous subunits, α and β (also known as cnga and cngb a). cnga is known to be indispensable for channel activation, whereas cngb a plays mostly regulatory structural roles. in fact, the n-terminal glutamic acid-rich protein (garp) domain of cngb a and the soluble garp were shown to decrease the opening probability of the cng channel and therefore these garps serve as important autoinhibitors or molecular gate keepers that control the activation of heteromeric rod cng channels. furthermore, cngb and garp , in concert with a retinal tetraspanin (peripherin- or peripherin rds ), were shown to contribute to the organization of the specific organelle, outer segment (os), which possesses a characteristic membranous "stacked pancake" architecture that has to be partially renewed daily to maintain cell function and viability. in fact, a mouse knockout of cngb and garp attenuated rod function and caused structural alterations and slowly progressive retinal degeneration. bovine garp (or cngb ) is a , residues-long transmembrane protein which plays important roles in both visual and olfactory signal transduction. cngb has glutamic acids. garp is a -residues-long cngb splice variant that possesses glutamic acids. garp is another cngb splice variant that has residues of which are glutamic acids. native garp and garp purified from bovine rod photoreceptors were shown to be typical idps. mgarp. mitochondria-localized glutamic acid-rich protein [mgarp, which is also known as ovary-specific acidic protein (osap), corneal endothelium-specific protein (cesp- ) and hypoxia upregulated mitochondrial movement regulator protein (hummr)] is one of the highly expressed proteins in retina. mgarp is highly enriched in steroidogenic tissues and the visual system, and early in development, this protein is mainly detected in the retina and adrenal gland. during the estrous cycle, mgarp levels correlate with estrogen levels in the ovaries. furthermore, the expression of mgarp is regulated by estrogen in a tissue-specific manner and through a feedback regulatory mechanism. as it follows from a long list of names, this protein has numerous important functions. in fact, among functions listed for this protein in the uniprot are ( ) plays a role in the trafficking of mitochondria along microtubules, ( ) regulates the kinesin-mediated axonal transport of mitochondria to nerve terminals along microtubules during hypoxia, ( ) participates in the translocation of trak /grif from the cytoplasm to the mitochondrion and ( ) plays a role in steroidogenesis through maintenance of mitochondrial abundance and morphology. , there are residues in mouse mgarp, of which are glutamic acids exclusively located in the glu-rich region (residues - ). based on the spectroscopic analysis of this protein, it has been concluded that mouse garp is an idp. some other garps. the life cycle of the phytopathogenic fungus verticillium dahliae kleb causing wilt disease in a wide range of crops, including cotton, includes three vegetative phases: parasitic, saprophytic and dormant. one of the genes tagged in a pathogenicity encoded a glutamic acid-rich protein (vdgarp ), which shared no significant similarity to any known proteins. linked to a glutamic acid and alanine repeat (eg-ea repeat). there are , and glutamic acids, glycines and alanines in this latency associated nuclear antigen. although there is low sequence identity between lana , ebna and orf , all three proteins determine the poor recognition of viruses by cd + cytotoxic t lymphocytes (ctl). however, the mechanisms of their action are rather different. in the epstein-barr virus and kaposi's sarcoma-associated herpesvirus the repeat domains were shown to enhance the stability of ebna and lana and decrease their translation rates, whereas the eg-ea repeat has no effect on the stability of hvs orf or its rate of translation, but results in decreased steady-state levels of orf mrna. intriguingly, the motif eeaeeaeee of hvs orf was sufficient to cause a reduction in recognition of orf by cd + ctl, suggesting that the eg-ea repeat of hvs orf is crucial for the immune evasion. nsp a. the n-terminal domain of the severe acute respiratory syndrome coronavirus (sars-cov) nonstructural protein (nsp a) is a typical idp of residues characterized by the presence of an ubiquitin-like globular domain (residues - ) and a flexible, highly extended glu-rich domain (residues . nsp a is a highly acidic protein (ph . ) that contains glutamic acids, of which are located within the c-terminal glu-rich domain. ppe antigens. proline and glutamic acid rich proteins (or pperepeat containing proteins, or ppe proteins) are important t-cell antigens produced by mycobacterium avium subsp paratuberculosis (map). one of the ppes is a . kda protein ( residues, pi . ) which following recombinant expression in e. coli was shown to elicit significant delayed type hypersensitivity skin reaction in mice sensitized with map, suggesting that this recombinant ppe protein of map was definitely associated with cellular immune response. curiously, this ppe contains alanines, glycines, prolines, aspartic acids but just glutamic acids. pt l . cassava storage roots differentially produce an interesting pt l protein with low sequence complexity characterized by a reduced amino acid alphabet (just amino acids). this residue-long protein contains glutamic acids, alanines, valines, prolines, serines and lysines, but does not have any arginines, asparagines, cysteins, histidines, phenylalanines, tyrosines and tryptophanes. glutamic acid-rich protein from cassava roots. based on the analysis of changes in the cassava root proteome during physiological deterioration of cassava root after harvesting, it has been concluded that the glutamic acid-rich protein was one of the proteins that were upregulated after harvesting. cp . eukaryotic genomes contain a set of specific functional elements, chromatin insulators or boundary elements that regulate gene transcription by interfering with promoterenhancer communication. in drosophila melanogaster, the centrosome-associated zinc finger protein cp protein (cp ) is a component of the gypsy chromatin insulator complex, which is composed of cp , mod(mdg ) and su(hw) and is required for the function of the gypsy chromatin insulator and other endogenous chromatin insulators organized by su(hw), ctcf the hippocampus and cerebral cortex of app(e Δ)-transgenic mice that are used as a model to study the pathological effects of aβ oligomers in alzheimer's disease. abra. the acidic-basic repeat antigen (abra) is a -residues-long protein found in the vacuolar space surrounding merozoites in plasmodium falciparum-infected erythrocytes, being localized in the parasitophorous vacuole and associated with the merozoite surface at the time of schizont rupture. due to its surface location, abra is one of the potential vaccine candidates against erythrocytic stages of malaria. this protein is one of the antigens enriched in the clusters of merozoites formed with growth inhibitory immune serum and possesses chymotrypsinlike activity, which can be inhibited with serine protease inhibitors such as chymostatin and phenyl methyl sulfonyl fluoride (pmsf). it was shown that the n-terminal half of the protein is responsible for the protease activity, whereas the highly charged c-terminal part of the protein was not required for this activity. furthermore, the n-terminus contains an erythrocyte-binding domain located within the cysteine-rich n-proximal region of abra. there are glutamic acids and lysines in abra, and in agreement with its name, the amino acid sequence of this protein is characterized by the presence of eight tandem repeats of [vt]-n-d-[ed]-[ed]-d (residues - ) and by a lysinerich c-terminal region (residues - ). kerp . the parasite entamoeba histolytica that colonizes the large bowel and provokes an asymptomatic luminal gut infection contains a peculiar lysine and glutamic acid-rich protein (kerp ), which is associated to parasite surface, involved in the parasite adherence to host cells and plays a role in the entamoeba histolytica liver abscess pathogenesis. an interesting feature of kerp ( residues) is a very high content of lysines ( %) and glutamic acids ( %). proteins with long simple repeat elements from herpesviruses. one of the mechanisms employed by herpesviruses to evade the immune response, allowing them to persist life-long in their hosts, relies on the use of specific proteins that function as cis-acting inhibitors of antigen presentation. among these inhibitors are the nuclear antigen (ebna ) and pgzr in the epstein-barr virus (ebv) and the latency-associated nuclear antigen (lana ) of the kaposi sarcoma herpesvirus. the common feature of all these proteins is the presence of long simple repeat elements in their amino acid sequences. for example, pgzr is a amino-acids long glycine, glutamine, and glutamic acid-rich repeat ("gz" repeat) protein that which is encoded by a large nested open reading frame located in the ebna mrna and is highly similar ( % amino-acid identity) to the acidic repeat of lana . latent nuclear antigen of human herpesvirus (hhv- ) (kaposi's sarcoma-associated herpesvirus) is a large ( , residues) highly acidic protein (pi . ) that contains glutamic acids, glutamines, prolines and aspartic acids. in herpesvirus saimiri (hvs) that infects squirrel monkeys, the functional homolog of epstein-barr virus ebna and kaposi's sarcoma-associated herpesvirus lana proteins is the residues-long product of the open reading frame known as orf or latency associated nuclear antigen. orf contains a repeat domain composed of a glutamic acid and glycine repeat mutation affects the ability of hla-dpb to present beryllium to pathogenic cd + t cells. sickle cell anemia and glu val mutation in hemoglobin. sicklecell (sca) or drepanocytosis is an autosomal recessive genetic blood disease with over-dominance, characterized by red blood cells that assume an abnormal, rigid, sickle shape. the disease is caused by a single point mutation in the β-globin chain of hemoglobin where the hydrophilic and negatively charged amino acid glutamic acid is replaced by the hydrophobic amino acid valine at the sixth position. as a result of this substitution, sickle hemoglobin polymerizes inside the affected erythrocytes. it was pointed out that such sickle hemoglobin polymerization occurs by homogeneous and heterogeneous nucleation mechanisms, which are both highly sensitive to macromolecular crowding. in fact, the rates of homogeneous nucleation were shown to be enhanced by when the initial concentration was augmented by % nonpolymerizing hemoglobin. retinitis pigmentosa and mutations in a glu-rich domain of rpgr. retinitis pigmentosa (rp) is an inherited, degenerative eye disease associated with the progressive loss of photoreceptor genes that causes severe vision impairment and often blindness. among other factors, rp is caused by mutations in the retinitis pigmentosa gtpase regulator (rpgr) gene which accounts for - % of rp cases in caucasians. genetic analysis revealed that of rpgr mutations % are associated with x-linked retinitis pigmentosa (xlrp), % are found in cone, cone-rod dystrophy or atrophic macular atrophy, and % are related to syndromal retinal dystrophies with ciliary dyskinesia and hearing loss. importantly, all disease-causing mutations occur in one or more rpgr isoforms containing the c-terminal exon open reading frame (orf ), and % occur in a glu-rich domain within exon orf , which accounts for only % of the protein. rpgr ( , residues) contains glutamic acids, more than half of which ( ) are located within the c-terminal glu-rich domain (residues - ). pyoderma gangrenosum and glu gln mutation in pstpip . pyoderma gangrenosum is a condition that causes tissue to become necrotic, causing deep ulcers that usually occur on legs. pyoderma gangrenosum is one of the most common extra-intestinal manifestations of chronic inflammatory bowel disease. the disease is caused by the alterations in the pathway that links the members of the proline-rich, glutamic acid-rich, serine-rich and threonine-rich (pest) family of protein tyrosine phosphatases (which are critical regulators of adhesion and migration) to their substrates. a major player in this pathway is a cytoskeleton-associated adaptor protein, namely proline-serine-threonine phosphatase-interacting protein (pstpip , also known as cd -binding protein , cd bp ). defects in pstpip are the cause of papa syndrome (papas), also known as pyogenic sterile arthritis, pyoderma gangrenosum and acne or familial recurrent arthritis (fra). papas is characterized by an autosomal dominant inheritance of early onset, primarily affecting skin and joint tissues. missense mutations glu -gln and ala -thr in pstpip /cd bp were identified in two families. these mutations were shown to affect the ability of pstpip to interact with its natural partners. , and beaf . although cp is a large protein ( , residues) that possesses a complex multidomain structure, only three domains were shown to be essential for the insulator function and for the viability of flies: the btb/poz domain, an aspartic acid-rich (d-rich) region and a c-terminal glutamic acid-rich (e-rich) region. here, the n-terminal cp fragment containing the btb/poz domain and the d-rich region was shown to be involved in regulation of the cp interaction with insulator complexes, whereas the c-terminally located e-rich region was necessary for the cp dissociation from chromosomes during heat-shock. importantly, the glutamic acids are not equally distributed within the protein, with the n-terminal half containing just glutamic acids and with the remaining glutamates being concentrated within the c-terminal half of cp . therefore, although the overall glutamic acid content of this protein is %, its c-terminal half is especially enriched in these residues ( . %). also, this uneven distribution is seen not only for glu, but for all the charged residues. in fact, the n-terminal fragment (residues - ) has a net charge of + (asp + glu = + = ; arg + lys = + = ), whereas the c-terminal half of cp (residues - ) has a net charge of − (asp + glu = + = ; arg + lys = + = ). pcp l . purkinje cell produces two closely related proteins containing iq motifs, purkinje cell protein -like (pcp l ) and pcp /pep- . although pcp /pep- is able to interact with calmodulin and inhibit calmodulin-dependent enzymes, and although the synthetic peptide constituting only the iq motif of pcp l binds calmodulin and inhibits calmodulin-dependent kinase ii, the full-length pcp l does not interact with calmodulin. the lack of ability of the full length pcp l to interact with calmodulin was ascribed to its nine-residue glutamic acid-rich sequence that lies outside the iq motif in pcp l . mutational analysis showed that calmodulin binding can be restored not only by the deletion of this inhibitory motif, but also by exchanging it with the homologous region of pep- and by simple point mutation converting a single isoleucine (ile ) within this motif to phenylalanine or to other aromatic residues. therefore, although pep- and pcp l possess noticeable sequence similarities, their functional properties are very different due to the presence of the glu-rich element in pcp l that can functionally suppress an iq motif. glutamic acid mutations and human diseases. chronic beryllium disease and lys glu mutation in hla-dpb . chronic beryllium disease (cbd) is a hypersensitivity disorder that affects - % of workers professionally exposed to berillium in the workplace. cbd is characterized by a granulomatous inflammation and accumulation of beryllium-specific cd + t cells in the lung. the susceptibility to this disease depends on both genetic factors (genetic susceptibility) and the nature of the exposure. genetic analysis revealed that a single point mutation at the th position of the human leukocyte antigen (hla) class ii histocompatibility antigen dp β chain (hla-dpb ), where lysine is substituted by a glutamic acid, makes the carriers more susceptible to cbd. it has been proposed that the k→e point of enzymes, or be related to metal binding. in idps/idprs, overabundance of glutamic acids defines the extended conformation of native coils and native pre-molten globules. glutamic acid is an important part of the pest motif related to protein degradation. it is crucial for function of entropic bristle domains and several chaperones. stretches of glutamic acid residues have a lot of specific functions that range from unique metal binding properties of phytochelatins and bone phosphoproteins, to regulation of cell adhesion and migration, to defining specific immunochemical reactivity of several antigens. no potential conflicts of interest were disclosed. this review illustrates that glutamic acid is differently used in ordered proteins/domains and in idps/idprs. in ordered proteins, glutamic acid residues are crucial for protein solubility and, being strategically placed within protein structure, play several structure-forming and structure-stabilizing roles. here, glutamic acid is involved in electrostatic interactions and hydrogen bond formation, serves as an important α-helix former, and participates in the α-helix cap formation. glutamic acid is an important functional residue of ordered proteins, where it can be involved in the 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selectivity within calcium channels diverse gating in k+ channels: differential role of the pore-helix glutamate in stabilizing the channel pore basigin (cd ): a multifunctional transmembrane protein involved in reproduction, neural function, inflammation and tumor invasion unique structure and function of chloride transporting clc proteins physiological functions of clc cl − channels gleaned from human genetic disease and mouse models proton pump coupled to cytochrome c oxidase in mitochondria oxygen activation and the conservation of energy in cell respiration cytochrome c oxidase: exciting progress and remaining mysteries proton-pumping mechanism of cytochrome c oxidase: a kinetic master-equation approach coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism glutamic acid is a valve in the proton pump of cytochrome c oxidase cell biology and molecular basis of denitrification inorganic nitrogen metabolism in bacteria molecular structure and function of bacterial nitric oxide reductase mono-adp-ribosylation: a reversible posttranslational modification of proteins structure-function relationships of na(+), k(+), atp, or mg( +) binding and energy transduction in na,k-atpase narberhaus f. alpha-crystallin-type heat shock proteins: socializing minichaperones in the context of a multichaperone network distinct roles of the n-terminal-binding domain and the c-terminal-solubilizing domain of alpha-synuclein, a molecular chaperone polyanions and the proteome physical stabilization of acidic fibroblast growth factor by polyanions acceleration of the refolding of arc repressor by nucleic acids and other polyanions complex effects of molecular chaperones on the aggregation and refolding of fibroblast growth factor- mapping the functional domains of nucleolar protein b chaperone-like activity of tubulin. binding and reactivation of unfolded substrate enzymes role of the carboxy-termini of tubulin on its chaperone-like activity chaperone-like activity of tubulin productive folding of human neutrophil alpha-defensins in vitro without the pro-peptide pro-sequence of subtilisin can guide the refolding of denatured subtilisin in an intermolecular process the alpha-lytic protease proregion does not require a physical linkage to activate the protease domain in vivo analysis of prepro-alpha-lytic protease expression in escherichia coli reveals that the pro region is required for activity heavy metal detoxification in higher plants--a review modulation of mammalian sperm function by fertilization promoting peptide (fpp) microbial biosynthesis of polyglutamic acid biopolymer and applications in the biopharmaceutical, biomedical and food industries new biological functions and applications of high-molecular-mass poly-gamma-glutamic acid review on medusa: a polymer-based sustained release technology for protein and peptide drugs biomedical applications of chemically and microbiologically synthesized poly(glutamic acid) and poly(lysine) production and characterization of gamma-polyglutamic acid nanoparticles for controlled anticancer drug release biological and clinical characterization of paclitaxel poliglumex (ppx, ct- ), a macromolecular polymer-drug conjugate pest sequences and regulation by proteolysis amino acid sequences common to rapidly degraded proteins: the pest hypothesis intrinsic unstructuredness and abundance of pest motifs in eukaryotic proteomes coupling caspase cleavage and proteasomal degradation of proteins carrying pest motif functional protein domains from the thermally driven motion of polypeptide chains: a proposal entropic exclusion by neurofilament sidearms: a mechanism for maintaining interfilament spacing hybrid character of a large neurofilament protein (nf-m): intermediate filament type sequence followed by a long and acidic carboxy-terminal extension electron microscopy of map (microtubule-associated protein ) a complex of numa and cytoplasmic dynein is essential for mitotic spindle assembly sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression conformational studies on poly-l-glutamic acid and copolymers of l-glutamic acid and l-phenylalanine study of the "molten globule" intermediate state in protein folding by a hydrophobic fluorescent probe salt bridge induced changes in the secondary structure of ionic polypeptides glutamate racemase as a target for drug discovery cape, a -amino-acid peptide, is necessary for bacillus anthracis polyglutamate capsule synthesis fusobacterium nucleatum, the first gram-negative bacterium demonstrated to produce polyglutamate ueber ein spezifisches protein der kapsel des milzbrandbazillus key role of poly-gamma-dl-glutamic acid in immune evasion and virulence of staphylococcus epidermidis polyglutamic acid from bacillus anthracis grown in vivo; structure and aggressin activity metal-binding characteristics of the gamma-glutamyl capsular polymer of bacillus licheniformis atcc characterization of bacillus subtilis gamma-glutamyltransferase and its involvement in the degradation of capsule polygamma-glutamate occurrence of poly-γ-d-glutamic acid and poly-α-l-glutamine in the genera xanthobacter, flexithrix, sporosarcina and planococcus transfer of natrialba asiatica b t to natrialba taiwanensis sp. nov. and description of natrialba aegyptiaca sp. nov., a novel extremely halophilic, aerobic, nonpigmented member of the archaea from egypt that produces extracellular poly(glutamic acid) poly(gamma-glutamic acid)s are the major constituents of nematocysts in hydra (hydrozoa, cnidaria) microbial degradation of poly(amino acid)s poly (glutamic acid)--an emerging biopolymer of commercial interest cloning and characterization of a novel trinucleotide repeat-containing gene garp from euplotes octocarinatus structure of a plasmodium falciparum gene that encodes a glutamic acid-rich protein (garp) a novel pepp homeobox gene, tox, is highly glutamic acid rich and specifically expressed in murine testis and ovary kibra functions as a tumor suppressor protein that regulates hippo signaling in conjunction with merlin and expanded kibra is a regulator of the salvador/warts/ hippo signaling network characterization of kibra, a novel ww domain-containing protein kibra is a novel substrate for protein kinase czeta temporal-spatial expression and novel biochemical properties of the memory-related protein kibra essential role of kibra in co-activator function of dynein light chain in mammalian cells kibra interacts with discoidin domain receptor to modulate collagen-induced signalling kibra modulates directional migration of podocytes cloning a new human gene from chromosome q . encoding a glutamic acid-rich protein expressed in heart and skeletal muscle the identification of a novel human homologue of the sh binding glutamic acid-rich (sh bgr) gene establishes a new family of highly conserved small proteins related to thioredoxin superfamily biomarker discovery: a proteomic approach for brain cancer profiling molecular evolution of sparc: absence of the acidic module and expression in the endoderm of the starlet sea anemone nbp- , a novel nucleosomal binding protein with a tissue-specific and developmentally regulated expression the interaction of nsbp /hmgn with nucleosomes in euchromatin counteracts linker histone-mediated chromatin compaction and modulates transcription binding of ca + to glutamic acid-rich polypeptides from the rod outer segment the glutamic acidrich protein is a gating inhibitor of cyclic nucleotide-gated channels in situ visualization of protein interactions in sensory neurons: glutamic acid-rich proteins (garps) play differential roles for photoreceptor outer segment scaffolding age-related changes in cngb -x knockout mice: prolonged cone survival glutamic acid-rich proteins of rod photoreceptors are natively unfolded a mitochondria-localized glutamic acid-rich protein (mgarp/osap) is highly expressed in retina that exhibits a large area of intrinsic disorder the expression of a mitochondria-localized glutamic acid-rich protein (mgarp/osap) is under the regulation of the hpg axis expression of ovary-specific acidic protein in steroidogenic tissues: a possible role in steroidogenesis hummr, a hypoxia-and hif- alpha-inducible protein, alters mitochondrial distribution and transport a glutamic acid-rich protein identified in verticillium dahliae from an insertional mutagenesis affects microsclerotial formation and pathogenicity gala: a designed synthetic ph-responsive amphipathic peptide with applications in drug and gene delivery pest family phosphatases in immunity, autoimmunity, and autoinflammatory disorders regulation of tumor cell migration by protein tyrosine phosphatase (ptp)-proline-, glutamate-, serine-, and threonine-rich sequence (pest) structural analysis of the bacillus subtilis delta factor: a protein polyanion which displaces rna from rna polymerase marcks: a case of molecular exaptation? arginine and glutamate-rich (arglu ) interacts with mediator subunit (med ) and is required for estrogen receptor-mediated gene transcription and breast cancer cell growth pelp --a novel estrogen receptorinteracting protein growth factor regulation of estrogen receptor coregulator pelp functions via protein kinase a pathway functional significance and mechanism of eif -promoted gtp hydrolysis in eukaryotic translation initiation interaction in vitro of non-epithelial intermediate filament proteins with histones isolation, characterisation and growth-related changes of an hmg-like protein from microplasmodia of physarum polycephalum calcium-dependent molecular spring elements in the giant protein titin isolation, characterization, and biosynthesis of a phosphorylated glycoprotein from rat bone modulation of crystal formation by bone phosphoproteins: role of glutamic acid-rich sequences in the nucleation of hydroxyapatite by bone sialoprotein acidic amino acid-rich sequences as binding sites of osteonectin to hydroxyapatite crystals molecular analysis of xenopus laevis sparc (secreted protein, acidic, rich in cysteine). a highly conserved acidic calciumbinding extracellular-matrix protein pcp l contains an autoinhibitory element that prevents its iq motif from binding to calmodulin genetic susceptibility and immune-mediated destruction in berylliuminduced disease crowding and the polymerization of sickle hemoglobin retinitis pigmentosa rpgr mutation analysis and disease: an update mutations in cd bp disrupt binding to ptp pest and are responsible for papa syndrome, an autoinflammatory disorder pyrin binds the pstpip / cd bp protein, defining familial mediterranean fever and papa syndrome as disorders in the same pathway reduction in rna levels rather than retardation of translation is responsible for the inhibition of major histocompatibility complex class i antigen presentation by the glutamic acid-rich repeat of herpesvirus saimiri open reading frame nuclear magnetic resonance structure of the n-terminal domain of nonstructural protein from the severe acute respiratory syndrome coronavirus expression of a gene encoding . ?kda ppe antigen of mycobacterium avium subsp isolation and characterization of the promoter sequence of a cassava gene coding for pt l , a glutamic acid-rich protein differentially expressed in storage roots itraq-based analysis of changes in the cassava root proteome reveals pathways associated with post-harvest physiological deterioration the chromosomal association/dissociation of the chromatin insulator protein cp of drosophila melanogaster is mediated by the btb/poz domain and two acidic regions proteomic analysis of the brain tissues from a transgenic mouse model of amyloid β oligomers amino terminus of plasmodium falciparum acidic basic repeat antigen interacts with the erythrocyte membrane through band protein immunogenicity of recombinant fragments of plasmodium falciparum acidic basic repeat antigen produced in escherichia coli plasmodium falciparum: chymotryptic-like proteolysis associated with a -kda acidic-basic repeat antigen expression and characterisation of plasmodium falciparum acidic basic repeat antigen expressed in escherichia coli the lysine-and glutamic acidrich protein kerp plays a role in entamoeba histolytica liver abscess pathogenesis the nested open reading frame in the epstein-barr virus nuclear antigen- mrna encodes a protein capable of inhibiting antigen presentation in cis key: cord- - quigar authors: nan title: posters date: - - journal: j pept sci doi: . /psc. sha: doc_id: cord_uid: quigar no abstract is available for this article. laboratory of molecular biology and immunology, department of pharmacy, university of patras, patras, greece antimicrobial peptides (amps) are an important component of innate immune system of most living organisms. they have recently gained much attention as new anti-infective drugs with new modes of actions and few or no side effects. their antimicrobial spectrum covers gram-positive and -negative bacteria as well as fungi and certain viruses . fish have proven to be a rich source of antimicrobial peptides. three chrysophsin peptides (chrysophsin- , - , - ) have been identified in the gills of the red sea bream, chrysophrys major, which are all bactericidal to pathogenic bacteria at low concentrations . they are cationic α-helical peptides, rich in histidine residues and all end in an unusual rrrh motif. however, in addition to its high antimicrobial potency, chrysophsins have considerable hemolytic activity. the development of new analogues which would preserve high antimicrobial potency, but would lack the undesired hemolytic activity, could be a useful tool with possible commercial and clinical applications. in the present study, we synthesized a series of analogues of chrysophsin- with different ratios of lys and leu residues, utilizing the fmoc/but solid phase methodology . the synthesized analogues were purified and isolated by rp-hplc. the antimicrobial properties of the above peptide analogues are currently testing in gram positive (s. aureus, s. epidermidis, e. faecium) and gram negative (e. coli, p. aeruginosa) bacteria. the goal is to identify the minimum bacteriostatic and bactericidal concentrations of the analogues, under conditions that simulate the best possible that of the human organism. hemolytic or cytotoxic activity of the peptides will also be determined. the rise of antibiotic resistance demands the development of new antimicrobial agents. these should exhibit a novel mechanism of action so as to overcome the resistance and be invulnerable to 'not yet acquired resistance mechanisms'. such criteria are difficult to meet. however, cationic host defence peptides (hdps) have emerged as promising candidates. hdps target and disrupt bacterial membranes. in order to evade such a threat a bacterium would need to make substantial changes to its membrane composition disfavouring the development of resistance ( ) . however, exact role and mechanism of hpds in the regulation and monitoring of microbial invasions remain to be established. herein we will present new potential mechanisms of antimicrobial regulation by helical hdps using de novo ( ) and native systems ( ) . biophysical and microbiology aspects of the experimental designs will be discussed. the low number of the newly discovered antibiotics, emergence of multiple-drug resistance, and the alarming death rate due to the infection disease led to development the alternative means to combat the infections. the researchers accumulate information about antimicrobial drugs that could be result of the innate immunity mechanisms. armed only with the innate immunity, the insect has developed into the most widespread class in living kingdom. they produce several antimicrobial peptides with complementary and rapid mode of action. so far there are hundreds of antimicrobial peptides isolated from insect and lot of them are waiting to be discovered. the fleshly neobellieria bullata was chosen for isolation of these active compounds. its larvae in the third instar were squeezed to collect the haemolymph, which was gradually centrifuged and precipitated by acidified methanol. supernatant was subsequently separated by chromatographic methods (spe column, rp-hplc) to obtain fractions of short peptides. identification and characterization of these fractions were performed by tricine electrophoresis, mass spectrometry maldi-tof analysis and n-terminal sequencing. several fractions showed antimicrobial activity against institute of chemical kinetics and combustion, novosibirsk, russian federation in this work, we extracted d-structural information on newly synthesized, medium-length, double spin-labeled peptaibiotics using peldor spectroscopy. we investigated the magnetic dipole-dipole interactions between spin labels and the orientation selectivity effects. in particular, the medium-length peptaibiotics tylopeptin b , and heptaibin , double spin-labeled with the nitroxyl probe toac ( -amino- -oxyl- , , , -tetramethylpiperidine- -carboxylic acid), were studied by means of x-band peldor spectroscopy. this study was conducted on tylopeptin labeled at positions and (t ) and heptaibin labeled at positions and (h ) in frozen glassy methanol solutions at Κ. peldor data analysis was carried out using the theory developed for short interspin distances. the distance distribution functions between spin labels for Τ (maximum at . nm, halfwidth of . nm) and Η (maximum at . nm, half-width of . nm) were determined. the intramolecular distances observed between the labels allowed us to assign an essentially α-helical conformation to Τ and a largely prevailing -helical structure to Η under the aforementioned experimental conditions. are amidated at the c-terminus, as a result of a posttranslational enzymatic reaction. temporins are particularly active against gram-positive bacteria and are not toxic to eukaryotic cells. in this study we designed a series of analogues of tb with the aim to improve the peptide antimicrobial activity against both gram negative and gram positive strains and then to structurally elucidate the mechanism of interaction of active peptides with lps. the peptides have been synthesized substituting one or two amino acids with an alanine and lengthening the sequence with positively charged amino acids. among the designed peptides, one of the analogues, tb_kkg a, showed highly increased activity against gram negative bacteria and also a slightly increased activity against gram positive bacteria with a total lack of hemolytic activity. to develop ll- -derived short amps with prokaryotic selectivity and lipolysaccharide (lps)neutralizing activity, a series of amino acid-substituted analogs based on ig- (residues - of ll- ) were synthesized. analog a showed the highest prokaryotic selectivity, but much lower lps-neutralizing activity compared to ll- . the analogs, a , a , a and a with higher hydrophobicity displayed lps-neutralizing activity comparable to that of ll- , but much lesser prokaryotic selectivity. these results indicated that the proper hydrophobicity of the peptides is crucial to exert the amalgamated property of lps-neutralizing activity and prokaryotic selectivity. to increase lps-neutralizing activity of the analog a , we synthesized trp-substituted analogs (a -w and a -w ), in which phe or phe of a is replaced by trp. despite their same prokaryotic selectivity, a -w displayed much higher lps-neutralizing activity compared to a -w . this result suggested that the effective site for trp-substitution when designing novel amps with higher lps-neutralizing activity, without a remarkable reduction in prokaryotic selectivity, is the amphipathic interface between the end of the hydrophilic side and the start of the hydrophobic side rather than the central position of the hydrophobic side in their α-helical wheel projection. furthermore, d-enantiomeric peptides (a -w -e and a -w -e) of a -w and a -w possessed not only more improved prokaryotic selectivity and retained lpsneutralizing activity compared to a -w but also protease stability. taken together, a -w -e and a -w -e can serve as promising templates for the development of therapeutic agents for the treatment of endotoxic shock and bacterial infection. department of zoology, faculty of science, charles university, prague, czech republic antimicrobial peptides (amps) are among the most promising lead compounds for developing medicines in the fight against resistant pathogenic bacteria. we have already shown that the venom of wild bee is a rich source of pharmacologically interesting antimicrobial peptides [ ] [ ] [ ] [ ] . from the venom of solitary bee macropis fulvipes, we isolated and characterized the novel antimicrobial peptide named macropin (mac- ). by edman degradation and mass spectrometry, its primary sequence was established as gfgmalkllkkvl-nh . mac- possesses potent antimicrobial activity against both gram-positive andnegative bacteria and moderate hemolytic activity against human red blood cells. cd spectra confirmed that mac- can form an amphipathic α-helical secondary structure in the presence of membrane-mimicking substances as sodium dodecyl sulfate or organic solvents like trifluoroethanol. we prepared a series of mac- analogs to study the effect of incorporating d-amino acid residues into the sequence in various positions on antimicrobial and hemolytic activity, α-helicity and serum stability. the substitution of l-amino acid residues at n-terminal part of sequence by d-amino acid residues led to the improving hemolytic activity with maintaining or increasing antimicrobial activity. these modifications increased peptide stability in human serum. effect of the incorporation of d-amino acid residues into the mac- sequence on its α-helical structure will be discussed. the neutralization of endotoxins (lipopolysaccharide, lps) by suitable compounds has been shown to be a key step in the treatment of infectious diseases, in particular in the case of gram-negative bacteria. the active endotoxic center of lps is lipid a, its lipophilic part. an effective antimicrobial peptide against gram-negative bacteria is magainin , which was originally found in the skin of an african frog. here, we studied the interaction of hexa-acyl bisphosphoryl lipid a prepared from erwinia carotovora lps with magainin with some minor substitutions in the amino acid pattern. by using fourier-transform infrared spectroscopy, the gel to liquid crystalline phase transition of the acyl chains of lipid a, the conformation of their phosphate groups due to peptide binding, and the profile of the secondary structure of the peptides was investigated. the zeta potential of lipid a aggregates in the presence of the peptides was determined by measuring the electrophoretic mobility. small-angle x-ray scattering was performed for the elucidation of the aggregate structures in the absence and presence of the peptides, and isothermal titration calorimetry was applied for evaluating the thermodynamics of binding between peptides and lipid a. the data show that asp-or glusubstituted peptides improved the binding activity to lipid a correlated with characteristic changes in the physical parameters, which were stronger expressed for the aspsubstituted peptide. the new hydrogen bond connection between glu and asp by carboxylic acids apparently leads to a more pronounced -structure of the peptide. the conformation change of the peptide enhanced the activity of incorporation into the lipid a aggregates, along with changes in biochemical and biophysical parameters. royal college of surgeons, dublin, ireland cationic antimicrobial peptides (caps) have been reported to exhibit anticancer activity . one such peptide, p , has been shown to inhibit the growth of several cancer cell lines, with inhibiting concentration (ic ) in the range of to μm . however the concentration at which p and other caps act is too high to be clinically relevant. the enhancement of their activity can be achieved through the modification of their amino acid composition or the addition of other molecules. conjugation of naturally produced hydroxylated fatty acids to p showed a -fold improvement in its anticancer activity on a variety of human-derived cell lines. in addition to the enhancement of activity we wished to understand the mechanism of action of the peptide and conjugates. we investigated the uptake of conjugated and unconjugated peptides into hela (cervical) and miapaca (pancreatic) human cancer cells and the localisation of the peptide in the cell once taken up. we investigated the effect of altering the carbon number of the hydroxylated fatty acids ranging from hydroxyhexanoic acid (r ) to hydroxydodecanoic acid (r ) conjugated to p peptide and tested on hela and miapaca cell lines. circular dichroism studies were performed to investigate the effect on α-helical content due to amino acid composition alteration and hydroxyalkanoic acid conjugation. the effect of the position of the hydroxyl moiety on enhancement of activity was also investigated. in the current study p and its derivatives also lacked haemolytic activity with concentrations up to fold higher than ic values needed to observe any haemolysis. when current antibiotics become less efficient, there is a promise that some antibiotics can be replaced by other nature's substances, e.g. peptides. halictines are novel antimicrobial peptides isolated from the venom of the eusocial bee halictus sexcinctus. we obtained four analogues of the native peptide hal from iocb av cr. they already characterized structural properties of these peptides and their antimicrobial activity against selected bacteria . the analogues were prepared by point mutations of native peptide, which could increase antimicrobial activity and decrease undesirable hemolytic activity. our aim was to characterized membrane permeation activity of halictines through the use of a basic model of biological cells -large unilamellar phospholipid vesicles luvs. we prepared two basic types of leakage assays based on luvs with free dyes entrapped inside and one assay with laurdan content. we used classical steady state fluorescence spectroscopy and advanced fluorescence methods for study of dyes escape from luvs and we also used laurdan generalized polarization technique gp for better understanding peptide insertion into membrane. in this way we received complementary information and we can conclude that the most active peptides are the native hal and analogue hal / . however hal / requires presence of negatively charged phospholipids in membrane which may explain its higher selectivity against bacteria. furthermore, fcs results have shown that the leakage happens via pore formation. results from gp revealed that peptide insertion in the membrane do not lead directly to formation of pores. against a wide range of microorganisms, mainly by perturbing the permeability of bacterial membranes through the formation of pores. however, amps effects on membrane properties probably extend beyond poreformation. we performed a systematic spectroscopic analysis of the effects on membrane structure and dynamics of two very different amps: the cationic pmap- , which creates pores according to the "carpet" model , and alamethicin, which forms "barrel-stave" channels . by using fluorescence anisotropy measurements on liposomes comprising probes localized at different depths in the bilayer, we measured peptide effects on membrane fluidity and order. laurdan spectral shifts provided indications about water penetration in the bilayer. in the case of pmap- , it was possible to focus specifically on the lipids surrounding the peptide by following the membrane-probe fluorescence due to fret from the peptide trp residues. finally, peptide-induced perturbation of lateral mobility and domain formation were determined by several methods. all experiments were compared with liposome-leakage measurements: while for pmap- all membrane-perturbing effects are correlated with the vesicle leakage process, alamethicin does not significantly influence membrane dynamics at the concentrations in which it forms pores. surprisingly, in all cases the most significant peptide-induced effect is a reduction in membrane fluidity. we have reinvestigated -residue peptaibols named metanicins from an ascomycetous fungus originally described as metarhizium anisopliae strain cbs . (cbs = centraalbureau voor schimmelcultures, utrecht, the netherlands). however, due to unusually shaped conidia and based on rna-sequencing of its internal transcribed spacer (its) region, the identification of cbs . as metarhizium has been withdrawn and this particular strain is currently under taxonomic reinvestigation . sequencing of four isolated peptides by fab-ms, esi-ms and edman degradation of partial hydrolysates revealed structural relationship to -residue peptaibol antibiotics paracelsins from trichoderma reesei (=hypocrea jecorina). sequences determined are: ac-u-a-u-a-u-a(u)-q-u-v-u-g-l-u-p-v-u-u(j)-q-q-fol (exchange positions in parenthesis; ac, acetyl; u, aib, α-aminoisobutyric acid; j, d-isovaline; fol, l-upmc univ paris laboratoire des biomolécules; cnrs umr ; ens lbm; address: laboratoire des biomolécules, ens dpt de chimie, , rue lhomond f- , paris, france current data suggest that the cellular uptake of cellpenetrating peptides (cpps) occur by two processes: direct translocation across the plasma membrane and endocytosis . the large diversity of cpp sequences described in the literature (derived either from fragments of proteins, structurally constrained synthetic peptides, peptide libraries or dendrimers) has hampered the identification of general rules for their efficacy of internalisation. we have used a reductionist approach, restricting the cpp functional groups (amide and guanidinium) and tailoring cpp amphiphilic properties. two families of cpps have been designed: ) primary amphiphilic cpps corresponding to tetra-arginines functionalised with fatty acid chains of different lengths and ) secondary amphiphilic cpps containing arginine and alanine or tryptophan residues . these cpps were linked by a disulfide bridge to a peptide inhibitor of protein kinase c (pkci). the efficiencies of internalisation of the conjugates were quantified by a method based on maldi-tof mass spectrometry previously developed in our group . the mechanism of internalisation was studied by comparing the amounts of cell-surface bound and internalized pkci cargo on cho-k cells and glycosaminoglycan-deficient cho cells at o c and o c. conjugates were found to enter by both direct translocation and glycosaminoglycandependent endocytosis. in addition, the primary amphipathic cpps were found to be more efficient than the secondary amphipathic ones. furthermore, structural or mechanistic novelty does not guarantee immunity from resistance, with strains resistant to linezolid identified prior to fda approval. therefore, modifying existing antibiotics to overcome resistance mechanisms presents an opportunity to rationally develop effective new drugs more rapidly than screening for new structures. vancomycin is a glycopeptide commonly used as a front line treatment for infections caused by methicillinresistant staphylococcus aureus (mrsa). the emergence of vancomycin-resistant enterococci (vre), vancomycinintermediate s. aureus (visa) and vancomycin-resistant s. aureus (vrsa) has prompted the development of semisynthetic glycopeptides . we have generated a variety of glycopeptide derivatives that show superior antibacterial activity against mrsa and vre compared to vancomycin and second generation lipoglycopeptides. this was undertaken by employing a combination of solid phase and solution phase chemistry to attach a membraneassociative element that selectively binds to bacterial membranes in preference to eukaryotic membranes, thus increasing the local concentration at the lipid ii d-ala-d-ala peptidoglycan cell wall precursor target site. three novel antimicrobial peptides, named panurgines (png), were isolated from the venom of wild bee panurgus calcaratus. one of them is dodecapeptide with sequence lnwgailkhiik-nh (png- ). the next two peptides are almost identical. these are cyclic peptides containing amino acid residues and two intramolecular disulfide bridges ldvkkiicvackixpnpackkicpk-oh (x=k png-k and x=r png-r). all peptides exhibited antimicrobial activity against gram-positive bacteria and gram-negative bacteria, antifungal activity and low haemolytic activity against human erythrocytes. we prepared analogues of α-helical amphipathic png- with the aim to improve its biological properties and a linear analogue of png-r to elucidate the importance of disulfide bridges for its activity. in the second part of the study, we followed the effect of panurgines on the degree of membrane disruption by observing the leakage of fluorescence dye (calcein) entrapped in artificial phospholipids vesicles [ ] . specifically, we investigated membrane interactions of pngs with the vesicles made from negatively charged : dopc/dppg and : dopc/dopg vesicles as a general model of bacteria membrane and : : dopc/dopg/cl as a possible model for a membrane of bacillus subtilis. the membrane interaction of pngs was also investigated on uncharged dopc vesicles as potential model membrane for erythrocytes. pngs exhibited weak dye-leakage activity for neutral vesicles, while they effectively induced dye leakage in the presence of negatively charged vesicles. these results indicate that pngs have stronger potency to disrupt bacteria-mimicking anionic membranes than those which mimic eukaryotic cell membrane. department of biochemistry and toxicology, university "lucian blaga", sibiu, romania a common tool to bias the conformation of linear peptides is the insertion of side-chain modified amino acids or sidechain/main-chain conformationally restricted building blocks. an alternative approach is a simple backbone modification. in this connection, backbone amide replacements with (almost) isosteric surrogates were extensively used. these modifications may impart resistance to enzymatic degradation and better bioavailability to the peptides, but also influence the secondary structure. a thioamide (ψ[cs-nh]) is perhaps the closest structural mimic of an amide. however, it possesses different and attractive features: (i) its nh group forms stronger hydrogen bonds, being more acidic than that of the amide. (ii) its c-n bond undergoes cis/trans isomerization by irradiation at nm (π→π* transition). (iii) it may act as a "minimalist" fluorescence quencher. for all these reasons, we started a programme aimed at exploring how the endothioamide bond affects peptide folding and bioactivity. in this communication, we describe the synthesis and conformational results of the three analogs of the membrane-active peptaibiotic trichogin ga iv listed below: n-octanoyl-aib-gly-ψ[cs-nh]-leu-aib-gly-gly-leu-aib-gly-ile-leu-ome ( / ) n-octanoyl-aib-gly-leu-aib-gly-ψ[cs-nh]-gly-leu-aib-gly-ile-leu-ome ( / ) n-octanoyl-aib-gly-leu-aib-gly-gly-leu-aib-gly-ψ[cs-nh]-ile-leu-ome ( / ) the syntheses of the three peptides were accomplished in solution according to a fragment condensation approach. appropriate thioamide-containing tri-or tetrapeptides were prepared by treating the corresponding all-amide precursors with the lawesson reagent. ft-ir absorption, d-nmr and cd conformational investigations on the three analogs were conducted in comparison with the naturally occurring peptaibiotic. all three analogs maintain the capability to interact with the dope/dopg model phospholipid membranes and exhibit a comparable bioactivity against s. aureus. peptide-peptide interaction of lactococcin g class iib two peptide bacteriocin h. etayash, w.soliman and k. kaur* faculty of pharmacy and pharmaceutical sciences, university of alberta, edmonton, alberta, t g e lactococcin g, a class iib two-peptide bacteriocin, consists of two complementary peptides lcng-α and lcng-β that act as one functional unit with optimal antimicrobial activity achieved by the presence of both peptides in approximately equal amounts. in this study we have investigated the mechanism of pairing of the two complementary peptides as well as explored any specific interaction that could take place between the peptides. molecular dynamics (md) simulation was employed to study the interactions at the atomistic level. four different md simulations with the peptides in a lipid bilayer system were conducted. md results from these simulations confirmed and pointed out that (i) the two putative gxxxg motif, g xxxg in lcng-α and g xxxg in lcng-β, were attracted and came closer to each other, showing the role of these motifs in attracting the two peptides to each other. closer views, however, showed no clear interactions between these two motifs. most likely, nonspecific interactions play a role in bringing the two peptides together; (ii) variations and loss in the secondary structure in both the peptide fragments were confirmed among the four simulations. on the contrary, stability of helical regions was identified between residues w -g and d -q in lcng-α and v -e in lcng-β; and (iii) role of tryptophan at the n-terminal regions in positioning and setting the peptide orientations were confirmed which matched the previous reported results. faculty of pharmaceutical sciences, unesp -univ. estadual paulista, araraquara, sa͂ o paulo, brazil antibiotic resistant bacterial strains represent a global health problem. antimicrobial peptides (amps) are promising novel antibiotics because they have displayed little or no resistance effects. it is well known that the charge, amphipathicity, hydrophobicity and helicity of the peptide are fundamental for the biological activity. in addition, covalent dimerization appears as a new parameter to be studied. in this way, several bioactive sequences were dimerized obtaining pharmacotechnics advantages like enhanced antimicrobial activity, solubility and proteases resistant. however, the effect of this modification is unclear since dimeric versions of some amps are toxic . to evaluate the effects of dimerization on the structure and biological activity of the amp aurein . , the monomeric version (au) and the c-and n-terminal dimers, (au) k and e(au) , respectively, were synthesized. circular dichroism results indicated that dimeric versions showed more defined structures in aqueous solution. e(au) showed "coiled coil" structure while (au) k an αhelix structure. in contrast, au displayed typical spectra for disordered structures. in tfe and lpc, all the peptides acquired a high amount of α-helix structure. the antimicrobial activity against bacteria and yeast decreased with dimerization. however, dimeric peptides promoted the aggregation of c. albicans. hemolytic and vesicle permeabilization assays showed that au has a concentration dependence activity, an effect that can be assigned to a "carpet" like mechanism peptide, whereas this effect was less pronounced for dimeric versions, suggesting that dimerization may change the mechanism of action. in conclusion, our studies showed that the effects of amp dimerization are complex and still unclear. , the first antimicrobial peptide generated in vivo and isolated from the gut contents of the cattle tick boophilus microplus . we have shown that these peptides are equally lethal to candida albicans mdm and practically not active on human erythrocytes . to examine the properties and mode of action of hb - a, we synthesized it and its fluorescently labeled analogue (fam-hb - a) by the solid-phase method at o c, purified them by rp-hplc and characterized their purified forms by lc-esims. at low salt concentration, both peptides were found to inhibit the growth of candida albicans atcc , candida parapsilosis atcc and candida krusei atcc , but hb - a was two-fold more active (mics of . ; . and . μm, respectively). at those concentrations, both peptides also kill the fungi. assays with human erythrocytes showed that, likewise hb - a, fam-hb - a present activity lower than % at μm. apparently, hb - a targets the membrane cell because confocal microscopy analysis revealed that, at the half of mic value, fam-hb - accumulates on the fungal cell membrane. in contrast, fluorescence activated cell sorting (facs) analysis revealed that, at the mic, more than % of the fam-hb - a penetrates into the cell. membrane permeability assay using hb - a, c. albicans atcc and the kit live/dead funga light confirmed progressive membrane damage associated with an increase in peptide concentrations. the use dibac ( ) and facs analysis showed that hb - a alters the plasma membrane potential, leading to cell death. supported by fapesp, cnpq and capes. lasso peptides form a growing class of to residue ribosomally-synthesized and post-translationally modified peptides produced by bacteria. they share a rigid and compact interlocked structure consisting of a macrolactam ring at the n-terminus and a c-terminal tail that is looped back and threaded through the ring, forming a typical [ ] rotaxane , . the macrolactam is formed by condensation of an asp or glu side-chain with the free amino group of a gly or cys . the lasso fold is stabilized either by steric hindrance assumed by bulky amino acid side-chains and/or by disulfide bonds between cysteines from the tail and the ring. given this structure, lasso peptides display a high stability against proteolytic and chemical degradation. they are biologically active on various enzymatic targets, which confer them in some cases an interesting antimicrobial activity. given its characteristics, the lasso scaffold thus represents a promising tool for biotechnological application in the development of bioactive peptides. until now, nine peptides had been structurally characterized as lasso peptides. based on a genomics-based approach, we identified a novel lasso peptide from streptomyces sviceus that we termed sviceucin. it was produced in high yield by heterologous expression in s. coelicolor and submitted to structural analysis by mass spectrometry and nmr. sviceucin is residue long and stabilized by two disulphide bonds. their connectivities were identified mainly from the typical noes between the beta-protons of the cysteines. the lasso structure of sviceucin was obtained by nmr-based molecular modelling. sviceucin was shown to exhibit antibacterial activity directed against gram positive bacteria, while gram-negative bacteria and fungi showed resistant. the penicillium chrysogerum antifungal protein (paf) is a cysteine-rich, cationic protein that inhibits the growth of a variety of filamentous fungi without toxic effect on mammalian cells . although paf is used to be produced in p. chrysogerum or a similar microorganism, preparation of analogues of the protein for structural and functional investigations requires an efficient chemical method. the unsuccessful continuous synthesis of the -mer small protein prompted us to use native chemical ligation . the syntheses of the fragments were performed by solid-phase method applying tboc chemistry. using the acid-labile tboc protecting group, the thioester end of the n-terminal fragment remains intact during the course of the synthesis. the first attempt was the synthesis of peptides with pmethylbenzyl groups on the side chains of all of the six cysteine residues. under no circumstances oxidative folding provided the natural disulphide bridge pattern. the failed attempts led us to orthogonal protection of the sulphydryl groups. different sets of protecting groups were tried and evaluated. our experiments showed that basic treatment triggered rearrangement of the previously formed disulphide pattern. thus, base-labile protecting groups (such as -fluorenylmethyl, fm) have to be avoided in the synthesis of paf. the alarming increase and spread of antibiotic resistance among bacterial pathogens has stimulated the development of new antibacterial agents with innovative mode of action. antimicrobial peptides with broad spectrum activity are widely distributed in nature and play an important role in innate immunity in several species, including humans. tigerinins are a unique family of -to -residue antimicrobial peptides found in skin secretion of the indian frog rana tigerina , . characterized by a disulfide-bridged loop composed of nine amino acids, tigerinins do not show primary structural homology to any known antimicrobial peptides from amphibians. tigerinins could provide novel lead compounds for the design of effective antimicrobial peptides with a new mode of action. the peptide murdp has been identified after the screening of phage display libraries against pseudomonas aeruginosa cell wall biosynthesis murd amide ligase enzyme . murdp is a low micromolar range inhibitor of murd enzyme and showed good antimicrobial activity. composed of nine amino acids, it is also characterized by a nine residues disulfide-bridged loop containing two prolines. this great similarity with tigerinins, led us to investigate if murd enzyme could be a potential target for these peptides. in silico analyses using modelling, molecular dynamics and docking with p. aeruginosa murd showed that murdp and tigerinin- and - make similar interactions in the binding site. these results suggest that murd may be an intracellular target for tigerinin- and tigerinin- . synthesis, murd enzymatic inhibition assay, antibacterial activity evaluation and structure-activity relationships of murdp and tigerinins analogs will be presented. h. etayash, l. norman, t. thundat*, k. kaur* faculty of pharmacy and pharmaceutical sciences, department of chemical and materials engineering, university of alberta, edmonton, alberta t g e , canada listeria monocytogenes is a gram positive bacterium that accounts for about % of the deaths resulting from food borne illnesses in north america. moreover, l. monocytogenes is considered one of the most difficult bacteria to detect in contaminated food products. while standard microbiological and biochemical assays currently used are accurate and sensitive, they are time consuming and often require specialized instruments operated by a trained user making on-site testing difficult. to this end, we propose the development of an antimicrobial peptide (amp) or peptide fragment sensor for the on-site detection of l. monocytogenes. leucocin a, which is a naturally occurring amp consisting of a amino acid sequence, is known to exhibit specific activity against l. monocytogenes at pico to nanomolar concentrations. for this reason, we have synthesized a shorter peptide fragment of leucocin a consisting of amino acids using solid phase peptide synthesis. the peptide was purified by reversed phase hplc and maldi-tof mass spectrometry indicates the desired biological entity was achieved. by including an n-terminal cysteine group, the tailored amp was readily immobilized at a gold interface. the resulting thickness and molecular orientation, determined by ellipsometry and grazing angle infrared spectroscopy, respectively, indicate that the helical peptides were adsorbed on the interface with a preferred orientation parallel to the surface. the bacterial specificity of the anchored leucocin a fragment was tested against three gram positive bacteria and results reveal that the adsorbed amp exhibits a limit of detection of approximately one bacterium/μl which is a clinically useful detection range. faculty of science, university of south bohemia, České budějovice, czech republic during the last few years we have identified three novel defensins from arthropods. two of them, lucifensin and lucifensin ii were purified from various tissues of lucilia sericata and l. cuprina larvae, respectively. larvae of these flies are routinely used in the hospitals around the world for the treatment of non-healing infected wounds in the procedure known as maggot therapy. these amino acid residues and three disulfide bridges peptides differ from each other only in one amino acid residue in position (val-ile). linear precursor of lucifensin was prepared by fmoc-spps chemistry which was then subjected to the oxidative folding yielding a peptide with a pattern of disulfide bridges identical to that of native lucifensin and other insect defensins. this was examined by the identification of the fragments resulting from the thermolysin digestion of lucifensin by means of mass spectrometry. however, this cyclization reaction proceeded via an intermediate having incorrect pairing of disulfide bridges. from the hemolymph of blood sucking tick dermacentor marginatus (d.m.) we purified defensin containing amino acids and three disulfide bridges. its sequence determined by edman degradation and mass spectrometry was identical to that previously determined by molecular biology methods . sequence of d.m.defensin shows no homology to insect defensins. by spps prepared linear precursor of d.m.-defensin was subjected to oxidative folding under the open air. the linear peptide was straightforwardly folded into cyclic one which was identical to the native peptide. in antimicrobial assay using a set of different bacteria all three studied defensins show activity preferentially against gram-positive bacteria including staphylococcus aureus but are inactive against gram-negative ones. the importance of disulfide bridges on tertiary structure of defensins and their antimicrobial activity will be presented. recently, the chemical structure and conformation of pseudodesmin a has been determined through x-ray diffraction and nmr spectroscopic analysis . in this way pseudodesmin a was identified as a new member of the viscosin group of antimicrobial peptides (amps). in addition, it was demonstrated that individual molecules self-assembly in apolar environment into a supramolecular pore-like structure, providing structural support for its biological activity , . to further explore the structure-function relationship, a viable synthesis strategy for pseudodesmin a analogues was developed, based on side-chain attachment of the first amino acid to the solid support, followed by stepwise fmoc solid-phase synthesis of the linear peptide precursor and on-resin head-to-tail cyclization. nmr study confirmed the molecular structure and thus the development of an efficient and successful synthesis of this type of amp's. these results and the synthesis route will be presented. trichogin ga iv, isolated from the fungus trichoderma longibrachiatum , is the prototype of lipopeptaibols, a subclass of short-length peptaibiotics exhibiting membranemodifying properties. its primary structure is as follows: n-oct-aib -gly-leu-aib-gly-gly-leu-aib-gly-ile -lol, where n-oct is n-octanoyl, aib is α-aminoisobutyric acid, and lol is the , -amino alcohol leucinol. this peptaibol is predominantly folded in a mixed -/αhelical conformation with a clear, albeit modest, amphiphilic character . in this work, we synthesized by solution and solid-phase methodologies a set of trichogin ga iv analogs in which the four gly residues, lying on the poorly hydrophilic face of the helical structure, are substituted by one (or more) strongly hydrophilic lys residues. moreover, we synthesized another set of analogs where one (or more) aib residues are replaced by leu. the conformational preferences of these analogs were assessed by x-ray diffraction, cd, and d-nmr techniques . we tested the role played by the substitutions on the peptide bioactivity, e.g. protease resistance, cytotoxicity, and hemolysis. cytotoxicity was tested using three in vitro cell-based assays: (i) human red-blood cells lysis; (ii) cell mortality in total human blood leukocytes and in separate subpopulations; (iii) cell mortality in three tumor-derived stable cell lines (hela, a , and a ). our data show that some of our trichogin analogs are active against tumor cells, leaving the leukocytes unaffected. a convenient post-screening ring opening approach for the decoding of one-bead one-compound cyclic peptide libraries a. girard, e. biron* faculty of pharmacy, université laval and chuq research center, quebec, canada combinatorial chemistry has been widely used as an effective method for the generation and screening of synthetic peptide libraries. amongst the different combinatorial methodologies to discover new bioactive peptide-based compounds, we were particularly interested in the one-bead one-compound (oboc) approach . this powerful approach fully exploit the great molecular diversity accessible with peptides and has been used to identify a great number of ligands and modulators for a wide variety of biological targets. however, its use with cyclic peptides is limited by difficulties in sequencing hit compounds by edman degradation or tandem mass spectroscopy due to the lack of free n-terminal amine and complicated fragmentation patterns, respectively. this problem has been overcome by pei and coworkers by using a bead segregation strategy in which the outer layer exposes the cyclic peptides and the inner layer the linear counterpart for sequencing . more recently, lim et al. reported an elegant method to prepare and sequence oboc cyclic peptoid libraries without encoding by using a ring opening approach with triazine-based cyclic derivatives . unfortunately this method is incompatible with amino acids bearing some functionalized side chains. based on this strategy, we have developed an efficient method to prepare oboc cyclic peptide libraries that does not require encoding by using a simultaneous ring opening/cleavage approach. the procedure is compatible with commonly used amino acids and allows rapid and efficient sequencing of selected hits after on-bead screening. the synthesis of an oboc cyclic peptide library, ring opening methodology and sequencing by mass spectrometry will be presented. cyclotides are a very abundant class of plant peptides that display immense sequence variability around a conserved cystine knot motif and a head-to-tail cyclized backbone conferring them with remarkable stability . their intrinsic bioactivities combined with tools of peptide engineering make cyclotides an interesting template for the design of novel agrochemicals and pharmaceuticals . however, laborious isolation and purification prior de novo sequencing limits their discovery and hence their use as scaffolds for peptide-based drug development . here we extend the knowledge about their sequence diversity by analyzing the cyclotide content of a violet species native to western asia and the caucasus region . using an experimental approach, which we named 'sequence fragment assembly' by maldi-tof/tof-based peptidomics, we were able to characterize novel cyclotides from viola ignobilis. amino acid sequencing of various enzymatic digests of cyclotides allowed the accurate assembly and alignment of smaller fragments to elucidate their primary structure, even when analyzing mixtures containing multiple peptides. using in-source decay and high energy collision induced dissociation of digested cyclotides allowed to distinguish isobaric residues ile and leu. overall this work underlines the immense structural diversity and plasticity of the unique cyclotide framework. the presented approach for the sequence analysis of peptide mixtures facilitates and accelerates the discovery of novel plant cyclotides. glycation is a nonenzymatic reaction occurring between reducing sugars and reactive amino groups of biomolecules. the reaction leads to a formation of a heterogeneous mixture of compounds which are classified as early, intermediate or advanced glycation end products (age). these compounds, especially advanced glycation end products, are involved in many pathological processes, mainly diabetic complications, and could be markers of certain diseases. detection of early products of glycation (amadori products) is a relatively easy task and can be performed by various methods including e.g. ms/ms techniques, isotopic labeling and affinity chromatography on immobilized boronic acid , . however, the diverse structures of ages make detection of these compounds more challenging. the aim of the study was testing a new method of ages identification based on isotopic c labeling. a model protein (hen egg lysozyme) was modified with an equimolar mixture of [ c ]glc and [ c ]glc. then the glycated protein was subjected to reduction of the disulfide bridges followed by enzymatic hydrolysis. the obtained digest was analyzed by lc-ms methods. the glycation products were identified on the basis of characteristic isotopic patterns resulting from the use of isotopically labeled glucose. this method allowed for identification of early maillard reaction products and different structures of the glycation end products. isotopic labeling technique combined with lc-ms is a new and very sensitive method for identification of the advanced glycation end products even if their structures are unknown. this method could be also used as an alternative method of detection of amadori products. in the course of a project aimed to assess the significance of antibiotics for the producing organism(s) in the natural habitat, we screened a specimen of the fungicolous fungus hypocrea phellinicola growing on its natural host phellinus ferruginosus . using a peptaibiomics approach , , we detected -and -residue peptide sequences by (u)hplc/hr-esi-qqtof-ms. structures of peptaibiotics found were independently confirmed by analyzing the peptaibiome of an agar plate culture of h. phellinicola cbs (ex-type) grown under laboratory conditions. notably, h. phellinicola could be identified as a potent producer of -, -, (culture) and -residue (specimen) peptaibiotics of the suzukacillin-type . minor components of the -residue peptaibols, herein named suzukacillins c, are assumed to carry a c-terminal residue tentatively assigned as tyrosinol (tyrol). in addition, the previously isolated suzukacillin b was sequenced and shown to be a microheterogeneous mixture of -residue peptaibols. in order to further investigate the significance of antibiotics for the producing organism(s) in the natural habitat, we screened specimens of the fungicolous fungus hypocrea pulvinata growing on its natural hosts piptoporus betulinus and fomitopsis pinicola . using a peptaibiomics approach , we detected -, -, -(major sequences), and -residue peptide sequences in the five specimens analyzed by (u)hplc/hr-esi-qqtof-ms. structures of peptaibiotics found were independently confirmed by analyzing the peptaibiome of pure agar cultures obtained by single-ascospore isolation from the specimens . major, -residue peptaibols were assigned as deletion sequences of the trichosporins b lacking the ala/aib residue in position . our results corroborate that: i) peptaibiotics are, indeed, biosynthesized in the natural habitat, thus, ii) their membrane-perturbing formation of ion channels may support the parasitic life style of a fungicolous fungus. based on methodology that we have developed in our lab , we identified specific and selective substrates for these serine proteases. we used a , membered pnaencoded peptide library to screen , possible peptide substrates in a single experiment. the library was incubated with the protease of interest and then hybridized on a custom designed dna microarray. microarray scanning and data analysis allowed the measurement of the changes in fam/tamra ratios resulting from the protease activity and the determination of the protease specificity. to verify the predicted activity and specificity, fret peptides were synthesized, incubated with the enzymes and the hydrolysis reaction was followed by monitoring fluorescence emission. specificity constants kcat/km were calculated and the cleavage sites of the peptides were identified. dubs were, moreover, found to be associated with several diseases and as such are emerging as potential therapeutic targets . several directions have been pursued in the search for lead anti-dub compounds. however, none of these strategies have delivered inhibitors reaching advanced clinical stages due to several challenges in the discovery process, such as the absence of a highly sensitive and practically available high-throughput screening assay . in this study, we report on the design and preparation of a fret-based assay for dubs based on the application of our recent chemical method for the synthesis of ub bioconjugates . in the assay, the ubiquitinated peptide was specifically labeled with a pair of fret labels and used to screen a library comprising compounds against uch-l . such analysis identified a novel and potent inhibitor able to inhibit this dub in time-dependent manner with kinact = . μm and ki = . μm. our assay, which was also found suitable for the uch-l enzyme, should assist in the ongoing efforts targeting the various components of the ubiquitin system and studying the role of dubs in health and disease. . more recent work based on rna interference experiments on a mouse model suggested that isoform-specific inhibitors against nmt might be effective anti-cancer agents as a knockdown of nmt inhibits the tumour growth, whereas knockdown of nmt has no effect . if residual nmt activity can compensate for loss of nmt function in healthy cells, potential toxicity may also be minimised. we developed a method to identify peptide or protein substrates of nmt and/or nmt . peptides/ proteins are exposed to nmt and/or nmt and an alkyne-tagged analogue of myristoyl coa. subsequent azide-alkyne "click" cycloaddition allows visualisation of the myristoylated substrates in fluorescence or chemiluminescence, using a fluorescent or a biotin moiety on the capture reagent. this labelling technology was applied to peptide libraries prepared on microarrays to investigate nmt / isozyme substrate specificity using recombinant nmt and nmt . peptides made of the first or amino acids at the nterminus of known myristoylated proteins were functionalised with a biotin moiety at the c-terminus and immobilised on an avidin-functionalised glass plate before being screened for activity. selective peptide substrates will be developed as isozyme-specific inhibitors and applied in cancer cell lines. using chemical proteomics and the labelling technology, a selective nmt or nmt inhibitor could also be used to identify protein substrates of one isozyme. for this purpose computer programs are created which can generate fragments of one compared structure and to reveal homology by their scanning along the amino acid sequence of another. our analysis was performed by comparing the primary structures of all possible protein fragments with the amino acid sequences of all presently known natural regulatory oligopeptides. the oligopeptides were extracted from the erop-moscow database which at the time of analysis contained data on the structures and functions of more than , natural oligopeptide regulators. the structure-function analysis was performed using a specialized software package. the input data were the complete amino acid sequences of the proteins used as a source of fragments with a specified length. then the initial sequence was fragmented in a stepwise manner. for example, in the case of dipeptide fragments, this procedure produced fragments with the following numbers of amino acids from the n-terminus - - , - , and so on until the fragment that started at the second residue from the c-terminus. the cases when the amino acid sequence of a fragment coincided with part of the primary structure of a natural oligopeptide were recorded in the total protein chemical synthesis requires a case by case design and optimization which is governed by factors such as the solubility of the individual peptide segments, their primary sequence and in particular the presence of "difficult" amino acid residues at ligation junctions such as proline or the location of cysteines. usually, a subset of chemical tools are selected among a vast array of methodologies to match the specificities of the target protein. in this context, methods enabling the assembly of three peptide segments in the n-to-c and c-to-n direction play a central role and must be considered as complementary as they can be selected for building subdomains of the target protein. to date, most of the proteins were assembled in the c-to-n direction. only few methods are available for the n-to-c sequential assembly of proteins, whose design is highly challenging. we have recently reported that sea ligation, that is the reaction of a bis( -sulfanylethyl)amido group (called sea) with a cysteinyl peptide, allows the formation of a native peptide bond in water and at neutral ph . in this communication we will show that native chemical ligation and the unique chemical properties of sea group , can be combined in order to design a highly efficient one-pot three segments protein assembly procedure, working in the n-to-c direction amylin is one of the most amyloidogenic peptides, its fibrils are responsible for causing type ii diabetes. amyloid formation mechanism is investigated both to find amyloid inhibitors as potential medical drugs, and to use amyloids as potential self-assembling biomaterials [ ] . amyloid formation of amylin - , its reverse and designed analogue beta-sheets and beta-sheet stacks was studied by molecular dynamics (md), amber . , f force field. md revealed that for amylin - and its reverse analogue both the parallel and antiparallel beta-sheet and beta-sheet stack structures are stable suggesting that this could explain the high tendency of amylin to form amyloid fibrils. parallel amylin - beta-sheet stacks are kept together by two hydrophobic cores, while for the antiparallel system the dominating is the backbone hydrogen bonding between neighbor strands. also the bent form of the amylin - beta-sheet is stable. this is in concordance with transmission electron microscopy (tem) experiments stating that all three peptides, amylin - , its reverse and designed analogues, exhibited significant fibrillar polymorphism [ ] university of gdansk, poland molecular dynamics (md) of two peptides dlsfmkge (mk) and dlsfkkge (kk) not related to any known disease was run to investigate the mechanism of the amyloid formation. the parallel and antiparallel [ ] betasheets of mk and kk peptides were simulated by molecular dynamics (md), amber . , f force field, ntp protocol. it was found that antiparallel beta-sheets both of mk-and kk-peptides show much higher stability than the corresponding parallel beta-sheets. this md result was supported by atr-ftir spectroscopy [ ] . the betasheet stacks built from six ten stranded antiparallel beta-sheets of mk-and kk-peptides: x xmk and x xkk, were subjected to md. it was found that the mk-system, x xmk, is strongly kept together due to hydrophobic core built from two metionines, two phenylalanines and two leucines, but the kk-system, x xkk, which differs only by one mutation m k dissolves already at ns of md run, because the separate beta-sheets don't hold togather in the betasheet stack due to lost hydrophobic core. the hydrophobic core of the mk-system consists of hydrophobic units centered on the two phenylalaninetwo metionine hydrophobic interactions, and two leucines from the both sides stabilize the unit. this mechanism could be used in amyloid based biomaterials. urokinase plasminogen activator (upa) is a serine protease involved in the metastasis of several tumor types. upa is therefore an interesting target in cancer therapy. upain- is a new analogue of a highly specific peptidic inhibitor (upain- ) of upa. the peptide contains twelve amino acids and is cyclized through the cysteines at its termini (s -s cyclo-ac-cswrglenhaac-nh ). upain- inhibits upa with a ki of approximately μm. one method to improve binding affinity is multivalent exposure of the inhibitor, where the local concentration at the binding site is increased. fusion of upain- to the trimeric tetranectin showed improved binding affinity compared to the single peptide. here, we report efforts towards novel chemically linked upain- peptides to allow multivalent display. the ki value of an upain- dimer, linked by a short peg chain through the n-termini, was almost halved compared to that of the single peptide ( μm). this motivated us to explore the role of the site (n-or cterminal) and the size of the linking segment on the binding affinity. additionally, the influence of the number of upain- peptides in the molecule (two vs. four) was investigated by synthesizing a carboprotein that displayed four upain- peptides. we present two novel nmr spectroscopic approaches to study reversible self-assemblies in solution. both methods were applied on the self-assembling pseudodesmin a, a pseudomonas produced cyclic lipodepsipeptide that has the capacity to form pores in cellular membranes. , the first method is based on the dependence of the c α relaxation rate constants on the anisotropy of the assembly. when the monomer conformation is known and the multiple ch bonds in the monomer sufficiently sample all orientations, the rotational diffusion coefficients can be assessed, revealing assembly shape information. in addition, the orientation of the monomer within the assembly is obtained. the second method is based on fitting translational diffusion coefficient data as a function of concentration in a model-free way, i.e. without assuming an oligomer shape beforehand. here, it is assumed that the diffusion coefficient's dependence on the oligomer size behaves as a power law, which dramatically simplifies the expression for the average diffusion coefficient (measured by pfg-nmr) as a function of concentration. the fitted value of the exponent of the power law fully embeds all shape information of the assembly, and may be related to the socalled fractal dimension of the oligomer. moreover, this approach reveals mechanistic information concerning the assembly formation. both methods thus allow structural information of the assembly to be obtained, even when there is little or no prior knowledge available on the mechanism of the selfassembly. nucleotides and α-amino acids are crucial building blocks for living organisms. these chiral molecules are the biosynthetically precursors of two of the most important classes of biopolymers, dna and proteins, respectively. the d-structures of biomolecules are currently studied using a variety of techniques, while helical handedness is routinely detected by means of light pulses of opposite circular polarization. the difference in the uv absorption of these two circularly polarized pulses is called electronic circular dichroism (ecd). in nature, biomolecules explore a wide range of conformations with intrinsically strong ecd signals in the - nm region, but these signals are essentially absent in the visible. nanomaterials such as metallic nanoparticles (depending on their sizes) display absorptions in the visible region but are achiral. as a result, when biomolecules are co-assembled with nanomaterials their chirality is transferred to create a plasmon-induced ecd signal in the visible region. in this work, we present our results which underscore the occurrence of moderately strong ecd bands in the range - nm resulting from a series of appropriately thiolfunctionalized peptide oligomers (based on alternating l-ala and aib residues) covalently anchored to - . nm sized gold nanoparticles. we related the (positive or negative) signs of the ecd plasmonic signal with the oligopeptide length, that in turn is strictly associated with their secondary structure. this latter property was simultaneously monitored via ecd in the - nm range. we believe that in our systems a peptide-tometallic surface chirality transfer would take place. light can be controlled with high temporal and spatial precision. if a specific molecule is made light-sensitive, then a precise spatiotemporal control of some of its properties can be achieved. azobenzene is the most widely used photochromic group due to its propensity to pass reversibly from the cis to the trans state under irradiation with light of the appropriate wavelength. the cisand transazobenzene isomers exhibit different spatial arrangement of the aromatic moieties that give rise to significantly distinct physical and chemical properties. the design of novel azobenzene-based molecules with precisely placed photochromic groups able to induce photomodulation of macroscopic properties is currently attracting much interest. in this work, we explored the behaviour of the conjugate formed by linking each of the four hydroxyl groups of pentaerythritol to the carboxylic function of bis[p-(phenylazo)benzyl]glycine. this c α -tetrasubstituted α-amino acid bears two side-chain azobenzene groups. the resulting system exhibits tetragonal symmetry, with a total of eight azobenzene moieties, and can be viewed as a central core surrounded by a shell of azobenzene groups at the periphery. up to eleven (out of the possible fifteen) discrete states produced by sequential trans-to-cis isomerization of the individual azobenzene units have been observed depending on the time of exposure to uv-light. this process is fully reversible (cis-to-trans) under vis-light irradiation for several cycles. in addition, this compound has been shown to exhibit photomodulated physical properties, such as polarity and hydrodynamic volume. moreover, it shows a high propensity to self-assemble in aqueous solution, giving rise to supramolecular vesicles. light-scattering and electron microscopy experiments confirmed that a conformational reorganization of the vesicles can be triggered under exposure to uv or vis light. the total chemical synthesis of native or modified proteins is gaining increase importance in the study of protein function, but also in the development of protein therapeutics. it is usually achieved by assembling in water unprotected peptide blocks using so-called native peptide ligation methods. recently, our group has developed a novel native peptide ligation method based on a peptide featuring a bis( sulfanylethyl)amido (sea) group on its c-terminus in reaction with a cysteinyl peptide in water at ph . we will discuss in this communication the scope and limitations of sea native peptide ligation. for this, model sea peptides featuring all the possible proteinogenic amino acids were synthesized. their rate of sea native peptide ligation with a model cys peptide were determined in the absence of presence of guanidinium hydrochloride or other additives frequently used in ncl. we will present also experiments intended to clarify the mechanism of sea ligation such as the effect of ph on the rate of ligation, or the ability of the transient thioester sea form produced by in situ n,s-acyl shift to participate in thiol-thioester exchange , . overall, the data show that sea ligation is an interesting method for native peptide ligation at various x-cys junctions, and thus an interesting alternative to ncl. plga copolymers were used as the support for inducing controlled biomarkers releasing system. visualization of the penetration in the hippocampus of mice with confocal microscope was carried out by testing both peptide-free and peptide-bearing nanoparticles, previously labeled with the phthalocyanine fluorescent probe. the encapsulation degree of the larger ( - ) segment was less effective than the others thus stressing the importance of the peptide length to this internalization process. the results showed that all peptide-containing nanoparticles were able to cross the blood-brain-barrier thus indicating improved bioavailability and uptake for peptide delivery into the brain. in regard to the radiolabeling approach, the m tc radioisotope was used to label the peptide sequences at his residues, as previously described . stable metal-peptide complexes were obtained in - - - m peptide concentration range. noteworthy, higher metal labeling yield was achieved with peptide segments bearing his residues at peptide c-terminal position, thus pointing to a positiondependent effect for the m tc coupling reaction. in conclusion, the findings indicate potentials for the proposed encapsulation and radiolabeling strategies applicable for in vitro and in vivo diagnostic assays with these peptides for the study of amyloid plaques. we have used bifunctional short peptides (ac-cg n c-nh , n= , , ) to selectively link gold nanorods in an end-to-end manner. additionally, we have manipulated the gap distance between the rods by changing the length of the peptide linker. the presence of the peptide in the gaps was shown by incorporating a propargylglycine residue in the sequence, which was detected with surface-enhanced raman spectroscopy (sers). the acetylene moiety will allow further chemical modification of the linker in the gaps, opening a wealth of interesting molecular systems to be placed and studies inside self-assembled nanogaps. in this work, the fragmentation pathways of alitame, neotame and andvantame in comparison to those of aspartame and aspartame-d , were studied by negative ion electrospray ionization (esi) high resolution mass spectrometry (thermo orbitrap mass analyzer). accurate mass spectra of the dipeptides allowed proposing specific fragment ions. neotame and advantame, which are the n-( , dimethylbutyl) and n-[ -( -hydroxy- -methoxyphenyl)propyl] derivatives of aspartame, presented similar fragmentation to that of aspartame. for neotame and advantame, the "diketopiperazine'' pathway seemed to be the major one, while a pathway resulting to the formation of a pyrrolidine- , -dione derivative, through the involvement of the side chain carboxyl group of aspartate, was also observed. for alitame, the "pyrrolidine- , -dione" pathway was recorded. similarities in the fragmentation using either orbitrap or triple-quadrupole mass spectrometry have been observed. elucidation of the fragmentation is very useful for the trace-level determination of the artificial dipeptide sweeteners in complex matrices. generation of silver nanoparticles in the presence of oligoproline derivatives p. feinäugle, h. wennemers* eth zürich, switzerland in the last years, the generation of silver nanoparticles (agnps) attracts, due to its unusual physical and chemical properties, more and more attention. agnps offer great opportunities for applications in molecular electronics, catalysis, imaging and for antimicrobial coatings. the characteristics depend on their shape and size. many efforts have been made to optimise the generation process by, for example, varying the reducing agents, which usually are used for the synthesis or using manifold additives which should guide the nucleation and also stabilize the resulting particles. nevertheless, the generation of agnps in defined sizes and shapes still remains a challenge. we address this goal by utilizing functionalized oligoprolines that form a conformationally well-defined and rigid helical secondary structure (ppii) as additives. recently, we showed that by decorating this template with aldehydes which allow for in situ reduction of the silver, they act as scaffolds in the generation process and allow the formation of defined nanoparticles. we will report the results of the generation of agnps with various oligoprolines as additives, which differ in the attached functional groups as well as in the length of the peptides. laser desorption/ionization mass spectrometry (ldi-ms) using specific inert surfaces to promote ion formation has been widely investigated the last decade [ ] . in addition to porous silicon through the original dios technique, different materials were tested as potent ldi-promoting agents. we explored a variety of inert silicon-based uvabsorbing materials that were presenting different physico-chemical properties for the analysis of peptides [ ] [ ] [ ] . both material architecture (amorphous powders, structured particles, structured surfaces) and material hydrophilic/hydrophobic character tuned by specific chemical derivatization (oxidation, silanization) were probed as crucial parameters for achieving efficient and robust detection of an home-made array of model peptides covering a wide structural and mass diversity. through this set of experiments, we were able to compare the performances of all investigated silicon-based supports, especially taking into account peptide detection sensitivity (down to femtomolar concentrations) and reproducibility/repeatability (intra-spot/inter-spot signal variations) as well as the method robustness using conventional maldi-tof/tof instrument. having illustrated the capability to achieve both peptide detection and sequencing on these ionizing surfaces in the same run, high-throughput identification of protein tryptic digests by a rapid ms profiling and subsequent ms/ms analyses was achieved. comparison of the ms and ms/ms data with those obtained with sample conditioned in organic matrix [ , ] showed a great behavior for low mass responses demonstrating the capability of ldi on nanostructured silicon supports to be a complementary method to maldi in proteomic workflow. the dipeptides aspartame, alitame, neotame and advantame are low caloric artificial sweeteners. advantame , which is the n-[ -( -hydroxy- -methoxyphenyl)propyl] derivative of aspartame, is the most recent among them. an application for its approval has been applied in usa, australia and new zealand. such sweeteners are used in food products and beverages and they can help in managing body weight and disorders like obesity and diabetes. in this work, the simultaneous determination of aspartame, alitame, neotame and advantame by negative and positive electrospray ionization (esi), under hydrophilic interaction chromatography (hilic), is presented. advantame, neotame and intermediates were synthesized in our laboratories for the present application. the key-step for the synthesis of advantame and neotame was the reductive amination of h-asp(obu t )-phe-ome with -( -hydroxy- methoxyphenyl)propanal and , -dimethylbutanal, respectively. the chromatographic behavior of the artificial sweetener dipeptides was studied on two hilic columns: kinetex hilic (a fused core silica column) and zic-hilic column (a sulfoalkylbetaine column). the separation of dipeptides was achieved on kinetex hilic using mm ammonium formate buffer ph . / methanol / acetonitrile ( / / ), with a flow rate of μl/min at o c column oven temperature. at this ph, silica is neutral and the dipeptides are in positively charged form. the retention mechanism of all analytes seems to be partition to the water layer as well as hydrogen bonding. département de pharmacologie, université de sherbrooke, sherbrooke, qc, canada plasma and in vivo stability are essential requirements for the successful development of potential drug candidates or diagnostic imaging probes. rapid degradation of compounds in plasma may result in insufficient concentration to produce the desired pharmacological activity or to be used as a diagnostic agent. there are several strategies to improve plasma half life of peptides including pegylation, modification of nand cterminal fragments of peptide, replacement of labile amino acids, and cyclization . we have previously reported on probes which specifically detect matrix metalloproteinase- (mmp- ) activity with magnetic resonance and optical imaging , . mmps are zincdependent endopeptidases degrading the extracellular matrix (ecm) and involved in cancer progression. the main goal of this work was to find more stable probes without sacrificing enzyme specificity. we have selected specific mmp- substrates and their stability was evaluated in three different conditions: in plasma, in plasma with a mmp inhibitor and in a mmp- solution. the samples were analyzed by hplc to detect the degradation pattern of our compounds and by lc-ms to determine the molecular mass of peptide fragments. based on these studies, the most stable peptide was selected and incorporated in a solubility switchable probe with radiolabelled ( )ga-dota. its in vivo stability was estimated up to minutes, making it a suitable candidate for further investigations. cancer of thyroid gland is the most common malignancy of the endocrine system. the treatment improvement could be achieved by early diagnosis. the aim of the study was to identify cancer specific markers using the libraries of artificial receptors immobilized on the cellulose. an array of supramolecular structures formed from n-lipidated peptides attached to cellulose via aminophenylamino- , , -triazine was prone to formation of monolayer of "holes" and "pockets" in dynamic equilibrium. this selforganized structures were found capable of binding small guest molecules very efficiently recognizing the shape, size, and polarity of ligands, thus resembling arti cial receptors . recognition and binding properties of guest molecules by artificial receptors depends mainly on the character of peptidic pockets and structure of the fatty acid. proper construction of the binding pocket allows selective binding components of mixtures of compounds from a living organism . the preliminary data indicates that it is possible to construct an array of artificial receptors with diversified structures of peptidic pockets which are able to distinguish between components of homogenates from tumor and normal tissue. century. most of opioid alkaloids and their derivatives have μ-opioid affinity, while endogenous enkephalins are rather δ-than μ-selective. morphine is still the drug of choice for treating severe pain caused by cancer or surgical operation, but its side effects are the reason for the searching and development of new, selective mor agonists. the aim of our study is to choose within recently published crystallographic structures templates for homology modeling of the human μ-opioid receptor. we generated several models using different templates and all of them were evaluated by docking procedure (gold . ) ligands used in this investigation were synthesized and evaluated for their biological activity in our previous studies. they are enekphalin analogues with substitutions in second position. the best model of the human mu-opioid receptor was chosen according to data obtained from docking and in vitro biological activity of analogues and endogenous enkephalins. acknowledgments: this work was supported by nfsr of bulgaria project dvu / and cost action cm project do - / . . . pneumoniae, h. pylori, proteus sp. are considered as important factor contributory to development of rheumatoid arthritis (ra). the aim of this study was to investigate the level and specificity of antibodies binding to the synthetic peptides corresponding to the bacterial ureases "flap" region sequences in the rheumatoid arthritis patient's sera. for these investigations, peptides with amino acid sequences derived from "flap" regions of different ureases were synthesized using -( , -dimethoxy- , , -triazin- -yl)- -methylmorpholinium tetrafluoroborate (dmt/nmm/bf ) as coupling reagent. peptides were immobilized on a cellulose membrane. the level of antibody binding as well as specificity of them was analyzed by quantitative dot blot method using sera sera from rheumatoid arthritis patients (rap) and sera from volunteer blood donors (vbd). the results of studies suggest that "flap" region may be involved in arising antibodies participating in autoimmunological processes but not to fight infection. this effect indicates that the peptides analyzed by us could be useful for investigation of ra pathogenesis. this suggestion was confirmed by the antibodies absorption experiment which indicates that specificity of antibodies present in rap serum is slightly lower in comparison with vbd serum. it has been found that antibodies present in rap serum recognize not only a specific peptide but also peptides containing fragments with different amino acid sequences. it means that immune system of rap is unstable and may produce a wide spectrum of antibodies recognizing not only a specific epitope but also a set of similar structures. autoantigen-specific t-cells also play a crucial role in the initiation and perpetuation of dsg /dsg -specific t-cell responses. t-cells recognize epitopes from dsg protein and produce different cytokines, e.g. interferon-γ (ifnγ). functional t-cell epitopes of dsg protein have outstanding importance in immunopathological research, development and the design of novel diagnostic tools. our previous studies have shown that certain t-cell epitope peptides are able to stimulate the peripheral blood monomorphonuclear cells (pbmc) of pv patients more effectively than those of healthy donors. our aim was to select a set of t-cell epitope peptides as potential synthetic antigens which are reliably able to distinguish between donors based on the in vitro t-cell stimulating activity. we have prepared synthetic dsg oligopeptides by fmoc/tbu solid phase methodology. after cleaving from the resin with tfa the peptides were purified by rp-hplc, and they were characterized by rp-hplc, mass spectrometry and amino acid analysis. pbmc of pv patients and healthy donors were isolated; and the cultures were stimulated by dsg peptides in a concentration of . mm for hours, and the rate of ifnγ production was determined from the supernatants in sandwich elisa. synthetic dsg oligopeptides induced different in vitro ifnγ production rate on pbmc obtained from pv patients and healthy controls determined by elisa. our approach identified a synthetic antigen set as a promising biomarker for pemphigus vulgaris. [ ] . in particular, cap b (pelyafprvamide) has been shown to elicit antidiuretic activity in the green stink bug acrosternum hilare [ ] , an important pest of cotton and soybean in the southern united states. analogs of cap b containing either an (e)-alkene, cispro or a transpro isosteric component [ ] were synthesized and evaluated in an in vitro stink bug diuretic assay, which involved measurement of fluid secretions of malpighian tubules isolated from a. hilare [ ] . at a concentration of μm, the conformationally constrained transpro analog demonstrated significant antidiuretic activity, whereas the cispro analog failed to elicit any activity. the results provide strong evidence for adoption of a trans orientation for the pro in cap b neuropeptides during interaction with the receptor associated with the antidiuretic process in the stink bug. the work further identifies a scaffold with which to design biostable mimetic cap b analogs as potential leads in the development of environmentally favorable pest management agents capable of disrupting cap bregulated diuretic systems. the enkephalins are pentapeptides (tyr-gly-gly-phe-met/leu) with a proven antinociceptive action. it is believed that the interaction between them and the lipids composing the membranes is important for converting the peptides into a "bioactive" conformation , . using langmuir's monolayer technique the interaction of a synthetic methionine-enkephalin (met-enk) and its amidated derivative (met-enk-nh ) with mixed lipid monolayers composed of palmitoleoylphosphatidylcholine (popc), sphingomyelin and cholesterol was studied. the surface pressure-area (π-a) isotherms with regard to πmin, π max and the hysteresis curve shape of the pure lipid monolayers and after the addition of the respective enkephalins were detected. in addition, by using brewster angle microscopy (bam), the surface morphology of the mixed lipids-enkephalins monolayers were determined. our results suggest that there is a strong penetration effect of the enkephalins studied into the mixed monolayers. moreover, our results demonstrate the potential of lipid monolayers formed in langmuir's through in combination with bam to be successfully used as an elegant and simple membrane models to study lipid-peptide interactions at the air/water interface. acknowledgments: this work was supported by bulgarian ministry of education, youth and science, projects n do - / , drg / and my-fs- / . dept pharmacolgy, temple univ, philadelpha, pa , usa bioinformatic algorithms has predicted the existence of several potential hormone-like peptides transcribed from the ecrg gene . previous publications indicated a highly level of gene expression of ecrg products has been found in the pancreas , choroids plexus, epithelial cells, leukocytes, and macrophages . however, the presence in the hypothalamus and the major form of derived peptides in each tissue haves not been clearly identified. knowingledge of the precise peptide generatesd within a given tissue is essential to understanding its functions. we have generated the peptide specific antibodies to against ecrg -derived pprepro-augurin( - ) and developed a specific ria kit for the quantification of the such peptide in question. a method for the purification of endogenous ecrg -derived peptides from bovine hypothalamus also has been established. using ria to monitor the immunoreactive fractions and maldi-tof to identify the endogenous peptides, we foundhave detected the presence of ecrg derived molecular of bovine preproaugurin( - ) from the homogenates of bovine hypothalamus. immunohistochemistrycal staining by antibody aalso confirmed the presence of thee peptide in some of the hypothalamic cells. of hypothalamus. the amount of prepro-augurin( - ) in the hypothalamus although not soas high as pancreas, but is one third of the augurin level of the pituitary. conclusions: the native peptide derived from augurin preproteinecrg has been discovered.identified. we have confirmed the property of purified peptide,s prepro-augurin( - ), along with the synthetic peptide standards. the present study provides the necessary procedures such as the elution from ( ) c column, ( ) p sizing column, and ( ) a further purification conditions for hplc in order to enhance the immunoreactivity from tissue fractions and yield enough amount for identification. this dsip-related peptide (kn-dsip or knd) differs from dsip by only amino acid residues in positions and . we do not consider the homology between dsip and knd as accidental, bearing in mind functional significance of histone demethylases of the jmjc-group. methylation-demethylation of histones is known as an important mechanism of posttranslational modification playing a prominent role in epigenetic regulation of chromatin structure and gene transcription. dsip is also known as an effective "normalizer" and protector from homeostatic disorders induced by stress related disturbances. we suggest that histone demethylase of the jmjc-group containing dsip-related region can be considered as a possible protein precursor of endogenous peptides with dsip-like activity. in order to test our hypothesis we synthesized knd and studied its biological effects. in a preliminary assay cited below [ ] knd showed similar and probably more pronounced effects than dsip as an agent that stimulates endurance and stress-resistance of animals in the forced swimming test. also knd provided a more active detoxifying action after administration of a semi-lethal dose of the cytostatic agent. in the present work we assessed neuroprotective and antioxidative potency of both peptides in vivo and confirmed the higher efficiency of knd. this study is supported by the moscow government. is a tridecapeptide (pglu -leu -tyr -glu -asn -lys -pro -arg -arg -pro -tyr -ile -leu ) highly expressed in the central nervous system. this peptide elicits an analgesic response following peripheral or central administration. importantly, nt exerts a more potent analgesia than morphine at an equimolar dose, without having the associated side effects of opioid drugs. structure-activity studies have identified the c-terminal fragment nt( - ) as the biologically active minimal sequence. however, nor the full or truncated peptides cross the blood-brain barrier (bbb), thus hampering its clinical development. the substitution of pro by an unnatural amino acid silaproline (sip) increased bioavailability and plasma stability. structural properties conferred by the pro were also retained as determined by nmr and ir. aiming at delineating the mode of action of cl, three new cl derivatives bearing suitable labeling moieties, i.e the fluorescent molecule fitc, the streptavidin-counterpart biotinyl-group and the m tc-radiometal chelating unit dimethylgly-ser-cys, were designed, synthesized, purified, and characterized to be applied in in vitro and in vivo evaluation studies. the structure of the cl derivatives in aqueous solutions was studied with nmr, in parallel and in comparison with the parent molecule cl, in order to examine whether the presence of the labeling moieties has induced changes to the structure of the biologically active part of cl. cell survival assays with cl and the cl derivative bearing the fitc moiety were conducted in the pc cell line in order to explore their rescue effect. in parallel, the cl derivative bearing the dimethylgly-ser-cys moiety was successfully radiolabeled with m tc and its stability was assessed over time in its synthesis reaction mixture and in plasma. this m tc-radiolabeled derivative was subsequently administered to swiss albino mice in order to determine the biodistribution of cl in the living organism and its route of excretion, a study that has not been carried out so far for any peptide of the humanin family. furthermore, the potential interaction of cl with β-amyloid peptide, the hallmark of ad pathogenesis, was explored with circular dischroism. the results of this multifaceted approach to the biological action of cl will be presented. institute of biochemistry and biotechnology, martin-luter university, halle-wittenberg, germany kinins, such as the nonapeptide bradykinin, are important mediators of various physiological and pathophysiological responses including inflammatory disease, asthma, rhinitis, cell division, pain, vascular permeability, allergic reactions, pathogenesis of septic and endotoxic shock. there are two types of receptors for kinins, known as b and b . b receptors are constitutively expressed in wide variety of cells and required entire bk sequence for recognition, while b receptors have normally very limited expression and respond to [desarg ]bk. b receptors gene is turned on following either tissue damage or inflammation. accumulated evidence indicates that most of the clinically relevant effects of bk are functions of b receptors this being the reason why research on their antagonists is a topic of great interest. in our previous study we described the synthesis and some pharmacological properties of four new analogues of bradykinin (bk), designed by substitution of position or of the known [d-arg ,hyp ,thi , ,d-phe ]bk antagonist with l-pipecolic acid (l-pip) (both analogues were also prepared in n-acylated form with -adamantaneacetic acid (aaa)). our results showed that presence of l-pip in position slightly increased antagonistic potency in the blood pressure test, but it turned the analogue into an agonist in the rat uterus test. replacement of thi by l-pip in position also enhanced antagonism in the rat pressure test but preserved the antagonism in the rat uterus test. in the present study we continue our previous investigations to find structural requirements which in the case of bk analogues result in high b antagonistic activity. several new bradykinin analogues modified in their cterminus with d-pipecolic acid were synthesized using spps method. the biological properties of the analogues were assessed by their ability to inhibit vasodepressor response of exogenous bk in conscious rats and by their ability to inhibit the contractions of isolated rat uterus evoked by bk. acknowledgements this work was supported by the university of gdansk (ds/ - - - ). peptides with beta-turn structure in peptide/mhc complexes a. stavrakoudis department of economics, university of ioannina, greece major histocombatibility complex (mhc) molecules interact with small peptides and form complexes. in most of the cases, peptide's structure in these complexes is found in extended conformation. however, notable exceptions exist where the peptide forms a beta-turn structure. this happens mainly in the central part of the peptide in class i complexes [ ] , or at the c-terminal of class ii complexes [ ] . several peptide/mhc complexes were, derived with xray studies, were extensively subjected to molecular dynamics simulations [ ] in order to investigate the stability of this turn-like structural feature and to explore the factors that possibly contribute to this stability. it was found that both intra-peptide and peptide/mhc interactions might be responsible for peptide's conformation. the peptides were found to undergo several structural transitions indicating conformational plasticity and not a completely rigid structure inside the mhc groove. the results might be of special importance in designing defective peptide vaccines and beta-turn pharmaceuticals. the heptapeptide met-enkephalin-arg -phe (merf) with the sequence of yggfmrf is a potent endogenous opioid located at the c-terminus of proenkephalin-a (penk), the common polypeptide precursor of met-and leuenkephalin. our systematic bioinformatic survey revealed considerable sequence polymorphism at the heptapeptide region of different penk prepropeptides among vertebrate animals. four orthologous heptapeptides with single or double amino acid replacements were identi ed among animals, such as yggfmgy (zebra sh), yggfmry (newt), yggfmkf (hedgehog tenrek) and yggfmri (mudpuppy). each novel hepta-peptide, together with the mammalian consensus merf and metenkephalin, were chemically synthesized and subjected to functionality studies, using radioligand binding competition and g-protein activation assays in rat brain membranes [ ] . equilibrium binding af nities changed from good to modest as measured by receptor type selective [ h]opioid radioligands. the relative af nities of the heptapeptides reveal slight mu-receptor (mop) preference over the delta-receptors (dop). [ s]gtpγs assay, which measures the agonist-mediated g-protein activation, has demonstrated that all the novel heptapeptides were also potent in stimulating the regulatory g-proteins. all peptides were effective in promoting the agonist induced internalization of the green uorescence protein-tagged human mu-opioid receptor (hmop-egfp) stably expressed in hek cells. thus, the c-terminally processed penk heptapeptide orthologs exhibited satisfactory bioactivities, moreover they represent further members of the so-called "natural combinatorial neuropeptide library" emerged by evolution. corticotropin releasing factor (crf) exerts most of its physiological and pathophysiological actions by interacting with its type receptor (crf ) and activating different intracellular signalling pathways. the crf is a plasmamembrane protein, which belongs to the family b of g-protein coupled receptors (gpcrs) and like the other gpcrs consists of an amino-terminal extracellular region, a carboxyl-terminal intracellular tail and seven, mostly hydrophobic, membrane-spanning segments (tm -tm ), connected by alternating intracellular (il) and extracellular loops (el). binding of crf and its related peptides, such as sauvagine, to the extracellular regions of crf is associated with receptor activation and subsequent activation of different g-proteins and regulation of diverse signalling pathways. using a mutagenesis approach in combination with a radioligand binding study we found that trp and phe in the second extracellular loop of crf interacted with the amino-terminal portion of crf and sauvagine. interestingly only the interaction of sauvagine with trp and phe is important for crf -mediated stimulation of camp accumulation. in marked contrast the interaction between crf and the residues trp and phe was unimportant for the activation of adenylate cyclase. thus it is possible for trp and phe of crf to regulate distinct signalling pathways, or different sets of them, after their interaction with different peptides. we are now performing experiments to fully elucidate the signalling pathways that are regulated by the interaction of crf and sauvagine with trp and phe . these studies will advance the development of crf -selective selective signalling-specific peptides that would be extremely useful for the elucidation of the role of crf in many physiological and pathophysiological situations, and possibly for the treatment of several crf -related diseases. thymus humoral factor gamma- (thf-γ ), an octapeptide, purified from crude thf, retains essentially all the biological properties of thf [ ] [ ] . it regulates clonal expansion, differentiation and maturation of t-cell precursors, stimulates the production of lymphokine, maitains the normalization of impaired ratios between helper(cd +) and suppressor / cytotoxic (cd +) subsets and augments il- production in spleen cells. thf-γ has a calculated molecular weight of and has the following amino acid sequence: leu-glu-asp-gly-pro-lys-phe-leu. its poor stability towards protein enzyme limits its extensive application. with the inte ntion to promote its bioavailability, bioactivity and develop ideal immunoregulatory drug candidat, four series of derivatives of thf were designed and synthesized: . n-and cterminal acylation. .restitution the flexible segment gly-pro by unnatural amino acids -aminohexanoic acid (aca) in order to shorten the synthetic steps and simultaneity improve the bioavailability and biostability of peptide; . reserve protected group of some amino acid residus as spot mutation. . mannich-based cyclization was carried out on resin [ ] , phe was replaced by tyr serving as the active hydrogen component, a proline was introduced at the n terminal as the amine component and formaldehyde was used as the only component in solution. the bioactivity of synthesized products were detected. the leukocytopenia model in mice was induced by cyclophosphamide intraperitioneal injection. white blood cell count, thymus index and spleen index were detected to evaluate the immune function of compounds in mice. the results show that those compounds play a significant role in improving immune function in mice. the activity of compound lhl and lhl are also better than authentic compound tp- and tα . marine organisms have been recognized as a promising source for the development of new pharmaceuticals. in the course of screening for antitumor substances from marine organisms, we found cyclic peptides containing many nonribosomal amino acids such as hydroxyasparagine, hydroxyleucine, or other supporting a hydrophobic side chain that were shown to be a key element for their biological activity. the laxaphycine b, a cyclic lipopeptide isolated from marine cyanobacteria anabaena torulosa harvested in french polynesia constitutes an example of this peptide class. this compound has attracted our attention because of its micromolar cytotoxic activities on different cancer cell lines as well as its antiangiogenic properties which seems to be due to an interaction with the vegf receptor- - . the synthesis of the non-natural amino acids - and of laxaphycine b analogues will be presented along with their preliminary biological activities. immune response suppressors are used in the medical praxis to prevent graft rejection after organ transplantation and in the therapy of some autoimmune diseases including dermatology. cyclolinopeptide a (cla) c(pro -pro -phe -phe -leu -ile -ile -leu -val -), a cyclic, hydrophobic nonapeptide isolated from linseed, possesses strong immunosuppressive and antimalarial activity. it has been suggested that both the pro-pro cis-amide bond and an 'edge-to-face' interaction between the two aromatic rings of adjacent phe residues in tetrapeptide unit are important for biological activity. this edge-to-face interaction can be influenced when phenyl rings are replaced by naphtyl substituent. in this communicate new analogues of cla modified by naphtylalanine ( -nal) in positions or or both and ( - linear analogues, - cyclic analogues) will be presented. the synthetic strategy and biological activity as well as conformational analysis will be evaluated. the onset of type ii diabetes mellitus (t dm) coincides with the deposition of fibrillar material in the islet of langerhans in the pancreas that is a clinical hallmark of more than % of patients suffering this disease. the main component of the pancreatic amyloid deposits is a -residues polypeptide hormone called islet amyloid polypeptide (iapp) or amylin. in this work we have examined, by means of cd spectroscopy and tht-fluorescence, the conformational polymorphism of both full-length - hiapp, and the related fragment hiapp - , and compared the results with the respective rat counterparts. moreover, the cytotoxic activity was determined toward different pancreatic β-cells lines in the attempt to correlate iapp's fibrillogenic properties with the ability to mediate cell death. together the results suggest that β-sheet conformational transition, that generally preludes to fibril formation, is not a prerequisite for eliciting toxicity toward β-cells cultures. interestingly, confocal microscopy indicated that both hiapp - and hiapp - can enter the cell and might exert their toxic action at intracellular level. acknowledgments: this work was supported by miur, firb-merit project rbne hwlz. due to its physiological functions, s proteasome is considered the target molecule in overcoming several diseases [ ] . its core particle s has three types of active sites: chymotrypsin-, trypsin-and caspase-like. many natural and synthetic compounds were tested for their ability to inhibit proteasome. a recent report describing the inhibition of s by the serine proteases inhibitor -bovine pancreatic trypsin inhibitor -was considered by us with great attention [ ] . our scientific interest is focused on peptide inhibitors and their interaction with serine proteases. sunflower trypsin inhibitor (sfti- ) is the smallest and the most potent peptide inhibitor in the bowman-birk family. owing to its size and the rigid structure (disulfide bridge and "head to tail" cyclisation) sfti- is willingly chosen as the lead structure in the search for new inhibitors [ ] . its sequence is shown below (lys the p residue responsible for specificity): & gly-arg-cys(& )-thr-lys -ser-ile -pro -pro-ile-cys(& )-phe-pro-asp& since native sfti- is not able to inhibit s [ ] , we have designed its monocyclic analogues (with disulfide bridge only) with lys or arg in position (p ) and at least one basic amino acid (lys or arg) in positions (p ') and/or (p '). all analogues inhibit chymotrypsin -(ic at the range of ÷ μm) and caspase-like (ic at the range of . ÷ μm) activities in vitro, whereas their activity towards trypsin-like specificity is much weaker. in several rat tissues, our view on ras has changed. metabolism of the ang-( - ) may represent alternative pathway of ang ii formation, importantly, independent on renin and ace activity , . ahmad et al. have described metabolism of ang-( - ) by human atrial tissues and showed that ang ii is formed mainly by chymase. this renin-inependent ang ii production could explain the "resistance" regarding use of ace inhibitors in patients with hypertension or diabetic nephropathy. noteworthy, the role of ang-( - ) in circulation is still unclear and there are no information about possible pharmacological modulation of its metabolism. in our study, we compared the ex vivo metabolism of angiotensinogen (fragment - ) in hypertensive (shr) and normotensive (wky) rats in organ bath of aorta and heart using lc-ms method . surprisingly, we identified ang-( - ) formed via reninindependent pathway to be a main product of angiotensinogen metabolism in rat aortic tissue and heart. in this setting, ang-( - ) appeared to be not only prevalent metabolite of angiotensinogen, but also served as a substrate for generation of ang i and ang ii. as compared to wky rats, formation of ang ii, from ang-( - ), was much higher in shr aortas but not in the heart. the functional consequences of these findings require further investigation. this study was supported by the grant n n polish ministry of science and higher education. the lysosomal cysteine protease cathepsin c (cat c), also known as dipeptidyl peptidase i (dppi), activates a number of granule-associated serine proteases with proinflammatory and immune functions by removal of their inhibitory n-terminal dipeptides. activity of this protease is associated with several pathologies in human body [ ] . in this work the characterization of cat c specificity using combinatorial chemistry methods will be described. the main goal of this work was to determine of substrate specificity of the prime region of this enzyme. the chemical synthesis and deconvolution of two libraries will be described. the hemostatic mechanism has the crucial role to prevent loss of blood from injured blood-vessels. this loss is prevented by the integrity of the vessel walls, by platelets aggregation or by blood coagulation, which in normal conditions is limited onto the local trauma of the vessel wall. in generally, the blood coagulation mechanism is important for maintaining vascular integrity and thus for the precaution of an organism from bleeding, which may also occur by blood coagulation caused by thrombin production. the diversion rate of this production leads to an expansion of thrombin to the general blood circulation. thus, when thrombin generation is not controlled by the mechanisms of inhibition, a widespread undesirable intravascular thrombosis is occurred. the whole process of platelets adhesion requires the presence of clotting factor viii (fviii), a necessary for the blood coagulation cascade glycoprotein, which takes part in the intrinsic pathway and acts as a coenzyme for the activation of factor ix, a serine protease depended on the thrombin production. the target of the present research is the synthesis of biologically active cyclic, head to tail, peptides, analogs of the sequence - of a subunit of fviii, which are potentially capable to block fviiia-fixa complex, reducing the thrombin production and thus the blood coagulation. the synthesized peptides are investigated for their inhibitory activity and tested for clotting deficiency by measuring the chronic delay in the activated partial thromboplastin time (aptt) and the reduction of the % value of the fviiia, which they generate in samples containing recombinant fviiia, in vitro. the blood coagulation is part of an important host defence mechanism, which under pathological conditions results in inappropriate intravascular coagulation when thrombin is produced. clotting sequence is the result of a cascade of two biochemical pathways, intrinsic pathway, so called because all components are present in blood, and extrinsic pathway, in which tissue factor is required in addition to circulating components. the activated form of factor viii (fviiia) is a key component of the fluid phase of the blood coagulation and plays an important role formatting a trimolecular complex with factor ixa, ca + and negatively charged phospholipids of the cells membrane. this complex is called tenase and participates in activation of prothrombin, which acts on fibrinogen to generate fibrin monomer, polymerized rapidly to form fibrin clot. the fviii is comprised of a heavy (a -a -b) and a light (a -c -c ) peptide chain, both cleaved by proteases at three sites, resulting in alteration of its covalent structure and conformation. its deficiency is known as haemophilia a. our research effort is focused on the synthesis, identification and biological evaluation of peptide analogs, expected to inhibit selectively the increasing of thrombin production. their sequence is based on the regions in which the fviii interacts with fix, specifically on the sequence - of the a subunit. the synthesized peptides are examined for their activity and tested for clotting deficiency by measuring the chronic delay in the activated partial thromboplastin time (aptt) and the reduction of the % value of the fviiia, which they generate in samples containing recombinant fviiia, in vitro. inhibitor with the following structure: ac-llllrvkr-nh , which has potent effects on the proliferation of prostate cancer cells. the potency and stability of this compound was subsequently enhanced by substitution of arg residue in position p with its conformationally restricted mimetic - -amidinobenzylamine (amba). nevertheless, the specificity toward pace was significantly reduced by this modification. thus, in order to improve its selectivity without sacrificing inhibitory potency we decided to use positional scanning approach. in this study we present synthesis of two series of peptide libraries, which were designed by substitution of leu in the p , p position of our control peptide (ac-llllrvkr-amba) with each of nineteen amino acid residues in order to verifying its influence on activity and selectivity of the resulting analogues. all peptides were synthesized by a combination of solid phase peptide synthesis and solution synthesis and tested for their inhibitory potency against furin and pace . the p -p fragments were synthesized by fmoc/tbu spps strategy on hydrazinobenzoyl or acid labile chlorotrityl chloride resin. then coupling of the -amidinobenzylamine · hcl was performed. the best modifications were combined to give as several multipoint substituted inhibitors. we believed that our work, will provide new important information about structure-activity relationship of these class of analogs in order to obtain potent and highly specific pace inhibitor. institute for research in biomedicine, parc cientific de barcelona, barcelona -spain a bacterial toxin-antitoxin (ta) system is composed of two genes organized in an operon encoding a toxin and an antitoxin that regulate the growth and death bacterial cell under various stress conditions. the operon parde encode a ta system formed by pare toxin and its antitoxin pard. pare is a kda protein that inhibits dna gyrase activity and thereby blocks dna replication. however the pare-gyrase interactions and the gyrase activity inhibition mechanism have not been explored. as an approach for understanding of this mechanism and to elucidate the pare region responsible for protein-protein interactions we have designed and synthesized a series of linear analogues of pare and investigated the ability of peptides to inhibit dna topoisomerases activity. so, based on structural data inferred from pare three-dimensional model , peptides were synthesized by solid-phase method. four peptides (parelc , parelc , parelc and parelc ), showed complete inhibition of dna gyrase supercoiling activity, by gel electrophoresis assay , with an ic of to μmol.l - . in addition, intrinsic fluorescence and fluorescence anisotropy assays showed that inhibition process must occur by interaction with the gyra subunit. differently of wild type pare, the peptide analogues were able to inhibit the dna relaxation of topoisomerase iv with lower ic values. interesting was that only parelc displayed inhibition of the relaxation activity of human topoisomerase ii. our results suggest a new class of molecules with simultaneous inhibitory activity in dna gyrase and topoisomerase iv. furthermore, we have obtained the first example of a synthetic peptide from a bacterial toxin with inhibitory activity on human topoisomerase ii. institute of experimental endocrinology, slovak academy of sciences, bratislava, slovakia the renin-angiotensin system (ras) has long been recognized as an important regulator of systemic blood pressure and electrolyte homeostasis. our understanding of ras has experienced remarkable change over the past two decades. besides, angiotensin ii, the new biologically active peptides [e.g. ang-( - ), ang-( - ), ang iv, ang-( - )] and pathways [e.g. angiotensin converting enzyme -ace ] have been described ; some of them, like ang-( - ) may oppose many actions of ang ii. importantly, despite all components of classical ras are found in adipose tissue , the data about fat formation of various angiotensins remain scarce. in our study, we compared the ex vivo metabolism of angiotensinogen, ang-( - ) and ang i in hypertensive (shr) and normotensive (wky) rats in organ bath of retroperitoneal and periaortic fat tissue using lc-esi-ms method. additionally, qpcr measurements of mrna expression of main enzymes involved in ang i metabolism were performed. both in the periaortic and epidydymal fat, the formation of ang-( - ) was higher than production of ang ii. fat tissue formation of two main ang i conversion products, ang ii and ang-( - ), differed significantly between shr and wky rats. compared to wky rats, the formation of ang-( - ) in periaortic fat tissue was decreased in shr. in opposite, in epidydymal fat tissue formation of ang-( - ) and ang ii was higher in shr. interestingly, there were no differences in aorta formation of ang ii and ang-( - ) between shr and wky rats. our results suggest that in hypertension visceral fat production of angiotensin peptides is increased, while generation of "beneficial" ang-( - ) in periaortic fat is decreased. however, the functional importance of such finding require further investigation. department of chemistry and biochemistry, university of washington, usa phospholipases a (pla ) are a superfamily of enzymes involved in various inflammatory diseases. in particular, human secreted giia spla is an attractive target for the development of novel medicines. we have shown that oxoamides based on γ-or δ-amino acids are potent inhibitors of cytosolic giva pla . very recently, we have demonstrated that a long chain -oxoamide based on (s)leucine displays inhibition of human and mouse giia spla s (ic nm and nm, respectively). a combined experimental/computational study was undertaken to further understand the role of the α-amino acid of -oxoamides for the inhibitor-enzyme binding. the crystal structure of giia spla s reveals a highly conserved ca + -binding loop and a catalytic dyad consisting of his /asp . -oxoamides based on hydrophobic αamino acids showed better binding score prediction compared to polar α-amino acid derivatives. a number of new -oxoamides based on α-amino acids were synthesised and tested for their inhibitory activity against giia, gv and gx pla . the -oxoamide based on (s)-valine displayed potent inhibition of giia spla (ic nm) in accordance with the predicted docking score. docking results reveal that (s)-valine-based inhibitor forms key interactions with the active site of the enzyme. the carboxylic group participates in a hydrogen bonding with gly and lys , and -carbonyl group with gly . furthermore, both carbonyl groups are in the proximity with ca + . the side chain of (s)-valine adopts a suitable orientation to interact with tyr and lys . the long aliphatic -oxoacyl chain is accommodated in the hydrophobic region of the active site and creates proximal contacts with leu , ile , his and phe . the search for novel classes of pharmaceutical molecules with enhanced therapeutic power has been the subject of numerous research groups all over the world. moreover, systems of immobilization and controlled release which are adapted to these new classes of molecules, has proven to be an area of extreme importance to provide the same therapeutic efficacy. using solid-phase chemistry a series of ccdb toxin analogous peptides were synthesized and were synthesized and tested against the capacity of inhibition of bacterial enzymes dna gyrase and topoisomerase iv (topo iv). subsequently those peptides were detained in drug delivery systems (dds) to be tested against the inhibition of growth of different bacterial species. in this data we could observed that the analogue ccdbsg could inhibit only dna gyrase and not the topoisomerase iv. in the other hand the analogue ccdbsg presents a hard inhibition potential against topo iv specially because of their structural difference. is possible conclude that topoisomerase iv presents the tertiary structure very similar to dna gyrase, but those mechanisms of action must be clearly distinct . in the in vitro studies, as expected, results revealed that the drug delivery systems are the key to the power efficiency of peptide analogues against the bacterial growth inhibition which cannot be observed when the peptides are free in solution. some of the different lipid compositions of the dds are demonstrating to be more efficient in the membrane cell transverse and this data previously assumes that it is possible to apply different types of dds to promote the peptide molecules transport across the cellular membranes according to several specific therapies. with this studies we have obtained more knowledge about the interaction system of enzyme-toxin and hopes which helps in future studies to development a new antimicrobial molecules class. it is urgent to develop less toxic and more efficient treatments for leishmaniases and trypanosomiases. we propose to target an ancestral form of the proteasome, the hslvu protease, which is present in the parasite's single mitochondrion, essential for the growth of these organisms and has no analogue in the human host. originally discovered in eubacteria, this complex is constituted by two central hexameric hslv protease rings sandwiched between two hexameric hslu atp-ase rings. as hslv shares a similar enzymatic mechanism with the host proteasome, we propose to inhibit the assembly of the complex in order to be selective. according to studies on bacterial hslvu, , the c-terminal segment of hslu is essential in hslv activation and in complex assembly, therefore representing a privileged target. we produced recombinant hslv, which is inactive alone, and showed that a synthetic c-terminal hslu peptide was able to induce the digestion by hslv of a fluorogenic substrate that we developed. with this enzymatic test in hands, we started the characterization of the interaction of the c-terminal portion of hslu with hslv. we will present the results obtained with various series of analogues of the original c-terminal hslu peptide, including truncated forms, ala scan, constrained analogues and multivalent constructions. helped by molecular modelling studies, the aim is to establish structural requirements, which could lead to high affinity and stable ligands able to inhibit the interaction between the hslu and hslv rings, obligatory for the degradation of proteins by the hslvu complex. finally, we checked that hslv was inhibited by classical active-site directed proteasome inhibitors like bortezomib. f. babos a,d , e. szarka b , gy. nagy c , z. majer d , g. saŕmay b , a. magyar a , f. hudecz a,d citrullinated filaggrin peptide (ccp) were detected in ra sera and anti-ccp positivity is widely used for diagnostic purposes. identification of new epitopes of filaggrin would be useful in the diagnosis of anti-ccp seronegative patients. in order to achieve optimal immune recognition of biotinylated epitope peptides it is important to analyse the effect of the labelling moiety on antibody binding. for these studies -as well as -mer peptides with nor c-terminal biotin were synthesised manually by spps, using fmoc/ t bu strategy. biotinylation was performed by using biotin, biotinyl- -aminohexanoic acid or , , -trioxa- , -tridecanediamino succinic acid linker modified biotin. labelled peptides were used in an indirect elisa, on neutravidin pre-coated plates and the binding was detected by anti igg-hrp. to examine the role of the presence/position of biotin in the secondary structure of the peptides, electronic circular dichroism (cd) method was used. we found that the ccp + serum samples specifically recognized the c-terminally biotinylated -mer filaggrin peptides, while showed no binding with the n-terminally biotinylated compounds. in case of the -mer epitope peptides there was no difference between the recognition of nand c-terminal biotinylated analogues. data presented suggest that the position of the biotin in case of the short filaggrin epitope peptides markedly influence the serum antibody binding. upon activation process, they are released from the granules and then involved in immunoresponse of the organism. when out of the cell those enzymes remain in free form or become associate with the cell membrane. the physiological role of this proteases is manifestated in several processes such cytokine and chemokine processing, platelet activation, and degradation of extracellular matrix's proteins [ ] . in this work results of the specificity of two members of nsps pr and hne evaluated using the combinatorial chemistry methods will be presented . both enzymes share primary specificity and to obtain the selective substrate that will be recognized only by one enzyme, the prime sites should be investigated. the general formula of the designed library is as follows: where in positions x ', x ' and x ', the set of proteinogenic amino acids (except cys) was introduced. abz is -amino benzoic acid served as donor of fluorescence and -nitro-l-tyrosine as acceptor. eukaryotic proteasome is a highly organized protease complex comprising a catalytic s core particle (cp) and two s regulatory particles (rp), which together form the s structure. the main function of this large intracellular protease is to degraded ubiquitine labeled proteins. the catalytic particle of the proteasome displays three distinct enzymatic activities: trypsin-like, chymotrypsin-like and glutamyl-like. the increase activity of the proteasome is associated with several disease including cancer [ ] . the main aim of this work is to synthesized the cell permeable fret displaying peptides that will selective cleaved by single proteasome activity. additionally each peptide when independently cleaved by the proteasome subunit, should emit the fluorescence energy in a different spectral region. our intention was designing substrates which would allow to monitor simultaneously (in a single experiment) and independently of three proteasome activities in this report, we will describe the chemical synthesis of several peptides modified at on cand n-termini by synthetic fluorescent amino acids the general formula of these peptides is as follows: where x is a non proteinogenic amino acid that serve as a donor of fluorescence, y amino acid that is a acceptor of fluorescence. the obtained fluorescent peptides were examined for their ability to cross the cell membrane. also kinetic parameters (k cat, km, kcat/km) with proteasome will be presented. approximately dubs are encoded in the human genome and are involved in a variety of regulatory processes, such as cell-cycle progression, tissue development, and differentiation. recently, several groups have introduced various methods for linking ubiquitin to different substrates via nonhydrolyzable isopeptide bonds, which resist the action of dubs. using these methods, one could explore the function and the mechanism of dubs and apply them in activity based profiling. here we present a new and convenient strategy for preparing nonhydrolyzable ubiquitinated peptides and proteins by nmethylating the isopeptide bond. using this method we prepared several nonhydrolyzable ubiquitinated peptides with different lengths derived from ubiquitinated h b and examined their affinity to different dubs. f.i. nollmann, c. dauth, d. reimer, h.b. bode* goethe universität, frankfurt, germany bacteria of the genus xenorhabdus and photorhabdus are gram negative gamma proteobacteria that live in symbiosis with nematodes of the genus steinernema. undergoing their partly entomopathogenic life cycle these bacteria not only produce antibiotics , and insecticides but also several different small molecular compounds and peptides. for the most part the biological benefits of these secondary metabolites have not fully been understood yet. with the help of inverse feeding experiments, hr-ms and nmr as well as molecular engineering we were able to characterize and/or isolate some of these peptides. since they are mostly produced in trace amounts, we synthesized them in order to make them accessible to continuative testing. given that not only linear but also highly methylated or cyclic peptides are produced, the synthesis was quite challenging. nevertheless, we were able to establish in our laboratory a general synthesis route for cyclic peptides and depsipeptides , as well as highly methylated hydrophobic linear sequences. testing several of these peptides has revealed activity against insect cells and against the causative organisms of neglected tropical diseases. cyclotides are a large class of plant peptides defined by a head-to-tail cyclized backbone and three conserved disulfide bonds in a knotted arrangement. these unique structural features confer them with remarkable stability and due to a range of bioactivities they are extensively investigated as templates in drug discovery . based on the use of oldenlandia affinis in traditional african medicine for its uterotonic principle we investigated crude plant extracts and semi-pure cyclotide fractions for the ability to induce uterine contractions using a collagen-gel contractility model . pharmacological analysis of the effects led to the identification of the oxytocin receptor, a representative of the g-protein coupled receptor (gpcr) family, as a molecular target for cyclotides. mass spectrometry-based sequence analysis of 'active' fractions revealed cyclotides with high similarity to the human oxytocin (h-ot) peptide that exhibited weak binding to the human oxytocin receptor. we further analyzed synthetic cyclotide-derived small ot-like peptides and grafted the h-ot sequence into the stable cyclotide frame. these peptides showed increased binding and activation as compared to native cyclotides. these findings may open new avenues for the discovery of gpcr ligands from natural peptide sources. gpcrs are promising drug targets and ~ % of currently used drugs act via binding to these receptors. natural combinatorial peptide libraries are likely to play an important role in identifying novel gpcr ligands . particularly plant cyclotides cover a large chemical space based on their high sequence diversity. together with their range of bioactivities and unique stable structure suggests that cyclotides are of current and future interest for drug discovery and development. acknowledgements: this work is funded by the austrian science fund fwf (p ). drosha and dicer are two key endonucleases for biogenesis of micrornas (mirnas) that regulate target mrna. drosha converts pri-mirna to ~ nucleotide (nt) pre-mirna in nucleus and dicer converts pre-mirna to linear ~ nt single-stranded mirnas in cytosol. even though dicer is potentially important to control availability of mature trans-acting rnas in cytosol, the enzyme itself does not seem to be the suitable target controlling mirna processing due to the lack of its substrate specificity. nature, however, might be intelligent enough to differentiate a variety of pre-mirna, so that a certain specific pre-mirna is converted to mature mirna in case it needs. therefore, other component(s) in the enzyme complex could be involved in recognition of auxiliary proteins from out sources to give extra specificity. we have synthesized trp-containing amphiphilic peptides against several pre-mirna. peptide b showed a picomolar binding affinity and a large specificity against pre-let a- . in vitro mirna processing, dicer activity was also selectively enhanced in the presence of this peptide. on treatment with this peptide on hct colon cancer and p ec cell lines, let a- mirna was more processed than reference mirnas. the toxicity of furan is known to rely on its selective oxidation in the liver by cyt p enzymes transforming it into the very reactive butenedial, which quickly reacts with proximate nucleophiles. this principle was used in our laboratory to develop a high yielding dna interstrand crosslinking methodology. in view of the demonstrated site-selectivity, the method further holds promise for sitespecific crosslinking dna to its binding proteins, which is highly relevant in the study of transient protein-dna interactions. furthermore irreversible dna binding can be achieved through such a covalent linkage, which is potentially useful for new generation therapeutics. the reactive furan moiety can in principle be incorporated either in the dna or in the protein. in the former case, a furan modified nucleotide was built into an oligonucleotide positioning the furan moiety at the periphery of the dna, to avoid interstrand crosslinking. for the latter approach, we initially chose to synthetically access a furan modified dna binding protein mimic. next to a previously described non-covalent gcn mimicking dimer, we have also investigated a new type of steroid-based dipodal dna binders. synthesis of the latter constructs has proven challenging in view of the immobilization of two peptide chains with helix forming tendency at close distance on the template. results, showing the power of microwave assistance will be discussed. in an alternative approach, a full length protein was modified with furan by amber suppression based on the structural similarity between a furan modified amino acid and pyrrolysine. pharmaceutical institute, university of bonn, an der immenburg , bonn, germany human matriptase- is a kda protein with trypsin like specificity. this protein exhibits a domain organization similar to family of membrane-bound serine proteinases known as type ii transmembrane serine proteinases. among many ascribed function in human body, this enzyme is a potent negative regulator of hepcidin, the peptide involved in iron homeostasis [ ] . matriptase- has a similar fold as other tmsp members, however their detailed specificity still remain unclear. the aim of this study was to determine the substrate specificity of this physiological important enzyme using combinatorial chemistry approach. in order to characterize the matriptase- specificity, the tetrapeptide library with c-terminal amide of aminocoumarin (acc-nh ) that serve as a fluorophore, was synthesized. its general formula is given below: x -x -x -x -acc-nh , where in position x -x the set of proteinogenic amino acid residues are present, whereas in position x lys or arg was introduced. deconvolution of such library was performed using iterative approach in solution. the results obtained indicate that matriptase- display diverse p -p specificity as compare to matriptase- . the most efficient hydrolyzed amino acid residue in position p appear to be ile, that is followed by arg in p and ser in p . the arg in position p is % faster hydrolyzed then lys. for selected substrates, the kinetic parameters (kcat, k m ) were determined. amyotrophic lateral sclerosis (als) is a chronic progressive disease. it is characterized by degeneration of upper or lower motor neurons, but its pathogenesis is still unknown and no effective treatment currently exists. it is known that antibodies to gangliosides have been found in some als patients, and these antibodies are also well known to be present in the patients affected by a variety of autoimmune diseases including multiple sclerosis. up to now anti-gangliosides antibodies are detected in clinical immunology laboratories using isolated non consistent antigen mixtures. therefore, we are interested in developing reliable and univocally characterized synthetic antigens for efficient antibody detection. csf (glc) is a family of structure-based designed glycopeptides that we previously developed as multiple sclerosis (ms) synthetic probes. these n-glucosylated peptides are able to detect specific autoantibodies in the sera of an antibody-mediated form of ms. autoantibody recognition was favored because of the exposition of the sugar amino acid on the tip of type ' β turn structures. aim of this study is the introduction, in the type ' β turn peptide structure, of the sugar moiety specific for anti-gangliosides antibody recognition by synthesizing specific building blocks. these building blocks are amino acids carrying glycans mimicking the biological activity of complex oligosaccharides. we selected sialic acids (in particular the n-acetylneuraminic acid -neu ac) because they are involved in a significant number of biological events. neuraminic acid and its derivates are widely distributed in animal tissues and in bacteria, especially in glycoproteins and gangliosides. therefore, we synthesized fmoc-l-asn(neu ac)-oh and fmoc-l-ser(neu ac)-oh. these building blocks will be introduced in the type ' β turn structure for the detection of anti-gangliosides antibodies in als. as a distinct pattern of ms could involve an antibodymediated demyelination, identification of autoantibodies as specific biomarkers is a relevant target. even if interesting data focused on the diagnostic and prognostic role of the detection of antibodies to myelin oligodendrocyte glycoprotein (mog) in adults' serum, its value remains dubious due to many other contrasting results. our research group identified csf (glc), an nglucosylated peptide, able to detect disease-specific autoantibodies in the sera of a statistically significant number of ms patients. , since this synthetic antigen may be considered as a mimic of aberrant post-translational modification (i.e. n-glucosylation) of myelin protein(s) triggering autoimmunity in ms, our goal is to obtain the extracellular domain of mog properly glucosylated thanks to a simplified native chemical ligation approach. for this purpose, the n-glucosylation will be introduced in a synthetic peptide fragment following the building-block approach by spps. the other protein fragment bearing an n-terminal cysteine will be expressed in e. coli after introduction of a selective point mutation into mog. finally, our aim is to test the semi-synthetic protein by sp-elisa to study the ability to detect autoantibodies in ms patients' sera and to find a potential cross-reactivity with csf (glc). this peptide is an endogenous ligand of the opioid receptorlike (orl ), previously referred to as "orphan" receptor, structurally and functionally related to the classical opioid receptors. also the hexapeptide ac-ryyrwk-nh is shown to be a selective ligand for the nop receptor with marked analgesic effect. with a view to developing ligands for the nop receptor with more potent analgesic activity, new series of the ac-rfmwmk-nh and ac-ryyrwk-nh , modified at position and respectively with newly synthesized β tryptophan analogues were synthesized . the aim of the present study was to examine the effects of naloxone (nal) and jtc- (nop receptor antagonist) in the analgesic activity of newly synthesized hexapeptide analogues. all peptides ( μg/kg), nal ( mg/kg) and jtc- ( , mg/kg) were injected intraperitoneally (i.p.) in male wistar rats. antinociceptive effects were evaluated by two nociceptive tests -paw-pressure (pp) and hot-plate (hp) and statistically accessed by anova. the results will be discussed compared to the referent compound in both tests used and mechano-and thermo-receptors are involved. [ ] . socs and socs have many similarities as well as some intriguing differences. both can block signalling by direct inhibition of jak enzymatic activity yet apparently require different anchoring points within the receptor complex. while the primary socs interaction is with a critical py residue within the jak catalytic loop [ ] it interacts also with py residues in the ifnαr and ifn r subunits in a jak -independent manner; the socs -sh domain also interact with y in jak , albeit with slightly lower affinity, but subsequent studies demonstrated a high affinity interaction with py residues located within receptor subunits [ ] . mutagenesis studies identified small regions at the n-termini of the socs and socs -sh domains, and at the c-terminus of the socs -sh domain, which were critical for phosphotyrosine binding. in order to gain insights in molecular discriminants for the interaction of both socs and socs toward jak and tyk we designed and synthesized peptides encompassing regions involved in proteins recognition. we set up a spr assay to evaluate the affinities of complexes formation. then through an alascanning approach we have designed new peptide sequences containing un-natural amino acids that are able to better recognize wild sequences and whole proteins. cellular experiments on stat activation signaling suggest their potential application as modulators of disorders involving socss overexpression. targeting proapoptotic death receptors (drs) to trigger apoptosis in cancer cells is a promising anticancer therapeutic approach. trail (tnf-related apoptosis inducing ligand) is a transmembrane homotrimeric protein belonging to the tnf family that triggers selective tumour cell apoptosis upon binding to its cognate receptors dr and dr . several strategies are being developed to exploit the unique cancer selectivity of the trail-dr pathway in therapy, including the use of recombinant trail targeting dr or dr . [ ] recently, a disulfide-bridged macrocyclic -mer peptide (derived from phage display) that binds selectively to dr has been identified. [ ] oligomeric versions of this macrocyclic peptide display increased binding avidity to the receptor and exhibit the capacity to activate the trail apoptotic pathway both in vitro and in vivo. [ ] however, disulfide bonds are susceptible to reduction and scrambling in vivo potentially resulting in the loss of the desired biological activity. among alternative linkages with increased redox stabilities, lanthionine thioethers, in which one of the sulfur atoms of the disulfide bond is removed have previously been introduced into biologically active peptides with some success. [ ] disulfide bridges can undergo a -elimination in alkaline conditions, followed by a michael addition to give a thioether bridge. optimization of this reaction led to the desulfurized analogue of the dr -binding peptide. the native dr -binding peptide and its desulfurized analog have been compared for their structural (nmr conformational analysis) and biological properties (affinity to dr and signaling pathways). the apelin/apj complex has been detected in many tissues and is emerging as a promising target for a number of pathophysiological conditions. in the central nervous system, apelin/apj was detected in brain regions involved in spinal and supraspinal control of pain, such as the amygdala, hypothalamus, dorsal raphe nucleus and spinal cord. we propose the hypothesis that apelinergic agonists represent a potential new approach to pain modulation and that the synthesis of stable analogues would lead to compounds with antinociceptive properties. there is currently little information on the structure/activity relationship (sar) of the apelin hormone. in an effort to better delineate sar, we synthesized analogs of apelin- modified at selected positions with unnatural amino acids, with a particular emphasis on the c-terminal portion. analogs were then tested in binding and functional assays by evaluating gi/o mediated reduction in camp levels and by assessing β-arrestin recruitment to the receptor. the plasma stability of new analogs was also assessed. several were found to possess increased binding and higher stability compared to the parent peptide. there is compelling evidence that the neuropeptide rfa and its cognate receptor gpr , are involved in the control of food intake and bone mineralization. among the gpcrs whose structures have been solved, gpr exhibits the highest sequence homology with the beta adrenergic receptor. the aim of this work was to experimentally characterize predicted ligand-receptor interactions by site-directed mutagenesis of gpr and design of point-substituted rfa analogs. starting from the x-ray structure of the beta -adrenergic receptor, a d molecular model of gpr has been built. the bioactive c-terminal octapeptide rfa( - ), kggfsfrf-nh , was subsequently docked in this gpr model and the ligandreceptor complex was submitted to energy minimization. in the most stable complex, the phe-arg-phe-nh part was oriented inside the receptor cavity whereas the n-terminal lysine remained outside. a strong intermolecular interaction was predicted between the arg residue of rfa and the gln residue located in the third transmembrane helix of gpr . in order to study this interaction, we have investigated the ability of rfa and arg-modified rfa analogs to activate the wild-type (wt) and the q amutant receptors transiently expressed in cho cells. the platelet receptor αiibβ plays a critical role in the process of platelet aggregation and thrombus formation. upon platelet activation its conformation changes leading to an increased affinity for fibrinogen. the αiibβ activation is regulated by "outside-in" and "inside-out" signaling. among the protein-protein interactions, which contribute to «inside-out» signaling, the most important is that of talin with the β cytoplasmic tail. it has been recently suggested that talin-mediated αiibβ activation relies on the cooperative interaction of the membrane proximal (mp) and the membrane distal (md) β regions with talin f domain and that the -n ply -motif of β , which can be phosphorylated at y , plays a critical role in this process. to evaluate the interaction of talin with the β tail of integrin we designed and synthesized three peptides corresponding to the md and mp parts of β in their carboxyfluoresceinlabeled form (md: cf-r akwdtannplyke -nh , cf-n nplykea -nh and mp: cf-k llitihdrke -nh ). emission and anisotropy fluorescence spectroscopy was used to quantitatively assess the affinity of these peptides for talin. furthermore, to challenge the role of the y phosphorylation in talin-α iib β interaction we also studied the binding of talin to the modified analogues of md, cf-r akwdtannpl(ptyr)ke -nh and cf-n npl(ptyr)kea -nh . our experiments revealed that the md and mp parts of β bind tightly to talin and that y phosphorylation has an inhibitory effect on this binding. functionalized oligoprolines as multivalent scaffolds in tumor targeting p. wilhelm, h. wennemers* eth zurich, zurich, switzerland oligoprolines are known to be structurally well-defined molecular scaffolds. in aqueous media, even short chain lengths of six proline residues adopt a polyproline ii helix (ppii). this secondary structure is a highly symmetric helix where every third residue is on top of each other in a distance of about . Å. [ ] the incorporation of azidoproline (azp) allows facile and versatile functionalization either via copper-catalysed azide-alkyne cycloaddition (cuaac) or an acylation that followed a staudinger reduction. [ ] based on the structural integrity of the oligoproline scaffold, targeting vectors can be conjugated via coppercatalysed azide-alkyne cycloaddition in defined distances. recent studies on radiolabeled oligoproline-bombesin conjugates, to target the gastrin-releasing peptide receptor (grp-r), showed in vitro and in vivo superior internalization in prostate cancer cells compared to the established monovalent ligands. [ ] a facile route to synthesize alkynylated ligands has been developed successfully. we are currently expanding this concept to the integrinligand c(rgdyk) as well as to [tyr ]-octreotide, which binds to somatostatin-receptors. the monovalent analogue of the latter, dota-[tyr ]-octreotide (dotatoc), is well established for diagnosis [ ] and therapy [ ] of somatostatinpositive tumors such as neuroendocrine tumors. the cu i -catalyzed azide-alkyne addition (cuaaa), the useful variant of "click chemistry," has emerged as a powerful technique for specific addition. that chemistry is also commonly used for conjugation, and cyclization of peptides. it is known that cyclization can increase the metabolic stability of peptides, as well as enhance potency or selectivity. another useful application of the cuaaa, which we are reporting, is the n-terminal crosslink of two synergic peptides to gain their potency. cuaaa reaction is performed on solid phase (merrifield resin) where one of the peptide components with azido group on the linker ( azido-hexanoic acid) is "clicked" with second peptide component in solution, made by fmoc strategy in partially protected form containing at n-terminal side alkyne group (fmoc-l-propargylglicine). cuaaa coupling is performed in dmf/t-buoh/h o with presence of cui and sodium ascorbate when reacting mixture was degassed. linked peptides are cleaved finally from resin and purified. as an application example we picked two endothelin active peptide analogues: bq derivative (a highly potent and selective eta antagonist) and irl- derivative angiogenesis is a key step in the transition of tumors from a dormant state to a malignant state. the vascular endothelial growth factor (vegf) is a major contributor to tumor angiogenesis. its pro-angiogenic activity is mainly mediated through binding to two tyrosine kinase receptors located predominantly on the surface of endothelial cells: vegfr- and vegfr- . vegf binding to these receptors triggers the activation of different signal transduction pathways responsible for the proliferation, survival and migration of endothelial cells . vegf/vegfr system constitutes a target to stop tumour growth. an attractive approach is the development of peptides, or small-molecules, with a high affinity for the extracellular domain of the receptors to prevent vegf binding. based on the x-ray structure of vegf and the d domain of vegfr- , cyclic peptides had been developed in our group . such peptides, mimicking simultaneously the - loop and helix · of vegf, can bind to d domain of vegfr- and inhibit receptors phosphorylation and thus map kinase pathway . we describe here our strategies to optimize peptidic antagonists of vegfr- . chemical modifications are made in order to better mimic peptide conformations and to increase their receptor binding affinities. we introduce a hydrophobic functional group at the c-terminal of the original cyclic peptide , some of such modified peptides reveal improved vegfr- binding affinity. otherwise, as the helix · presents most of the important residues in vegfr binding according to alanine-scan in the literature , we try to stabilize the helical conformation by insertion of aib residues or by peptide cyclisation. the peptides affinities are evaluated by an elisa test developed previously . institute of chemistry and biochemistry, freie universität berlin, thielallee , d- berlin, germany new polypeptide was isolated from the azemiops feae viper venom by combination of gel filtration and reversephase hplc and called azemiopsin. its amino-acid sequence (dnwwpkpphqgprpprprpkp) was determined by means of edman degradation and mass spectrometry. it consists of residues and does not contain cysteine residues. according to circular dichroism measurements, this peptide adopts a β-structure. peptide synthesis was used to verify the accuracy of the determined sequence and to prepare sufficient peptide amount for biological activity studies. azemiopsin efficiently competed with α-bungarotoxin for binding to torpedo nicotinic acetylcholine receptor (nachr) (ic . ± . m) and with lower efficiency to human α nachr (ic ± μm). ala scanning showed that amino-acid residues at positions - , - and - are essential for binding to torpedo nachr. in biological activity azemiopsin resembles waglerin, a specific blocker of muscle-type nachr from tropidechis wagleri venom. however the sequences of these peptides are markedly different, and azemiopsin is the first natural toxin to block nachrs that does not possess disulfide bridges. laboratory of peptide science, nagahama institute of bio-science and technology, nagahama, shiga - , japan while neutrophils infiltrate into damaged sites immediately after tissue injury, endogenous factors which induce their acute transmigration and activation have not been thoroughly elucidated. for the candidates, we recently identified two novel neutrophil-activating cryptides, mitocryptide- (mct- ) and - (mct- ), which were hidden in mitochondrial cytochrome c oxidase and cytochrome b, respectively [ ] [ ] [ ] . in addition, the presence of many neutrophil-activating peptides other than mct- and - was observed during their purification. these findings suggest that neutrophils are regulated by many unidentified peptides. here, we purified a novel neutrophil-activating octadecapeptide whose primary structure was identical to mitochondrial cytochrome c ( - ) from porcine hearts. we named this functional peptide as mitocryptide-cyc (mct-cyc). the structure-activity relationships of cytochrome c on β-hexosaminidase release from neutrophilic differentiated hl- cells demonstrated that cytochrome c ( - ) was the most potent cryptide among cytochrome c-derived peptides. since cytochrome c is known to be involved in the apoptotic process, our present results suggest that cryptides produced from cytochrome c play an important role in scavenging toxic debris from apoptotic cells by neutrophils. anthracis spores are very resistant and can remain dormant in soil for decades. therefore, an effective detection system for b. anthracis is urgently needed. recently, it was found that one of the components of the b. anthracis exosporuim is a collagen like protein whose carbohydrate portion is composed of the tetrasaccharide with the highly specific monosaccharide upstream terminal, named anthrose. since anthrose was not found on other bacterial spores, including those closely related to b. anthracis, this monosaccharide is an attractive target for the development of new b. anthracis detection and identification methods. peptide cyclization represents particularly interesting approach for the design of artificial receptors for anthrose, because cyclic peptides provide the possibility of having a spherical lipophilic binding site of appropriate size and shape for a particular carbohydrate substrate. the presence of hydrogen donor/acceptor groups within a three-dimensional structure permits carbohydrate substrates to be encapsulated, thereby allowing their binding in water. in order to determine whether the cyclic peptide receptor can selectively detect the anthrose, we have successfully prepared cyclic peptide combinatorial library (total peptides) by the process of divide, couple and recombine ("tea-bag" technology) using standard fmoc solid-phase peptide synthesis. prepared combinatorial library is screened for anthrose binding in fluorescence-based assay, and individual cyclic peptides with enhanced affinity toward anthrose are identified by the positional scanning deconvolution process. cyclization of linear sequences is a well-known approach used to restrict the flexibility of peptides. cyclization often increases selectivity of peptides towards one specific receptor type, increases metabolic stability and generally increases lipophilicity, which often improves the bloodbrain barrier permeability of peptides. in our previous study [ ] we have reported on the synthesis of a cyclic endomorphin- (em- ) analog, tyr-c(d-lys-phe-phe-asp)-nh , which elicited analgesia after peripheral administration. encouraged by the fact that this analog was able to cross the blood-brain barrier we designed and aliskiren is the first orally active, direct renin inhibitor to be approved for the treatment of hypertension. its structure and conformational analysis were explored using molecular dynamics (md) simulations. for the first time, md calculations have also been performed for aliskiren at the receptor site, in order to reveal its molecular basis of action. it is suggested that aliskiren binds in an extended conformation and is involved in several stabilizing hydrogen bonding interactions with binding cavity (asp / , gly ) and other binding-cavity (arg , ser , tyr ) residues. of paramount importance is the finding of a loop consisting of residues around ser that determines the entrapping of aliskiren into the active site of renin. the details of this mechanism will be the subject of a subsequent study. additionally molecular mechanics poisson-boltzmann surface area (mm-pbsa) free energy calculations for the aliskiren-renin complex provided insight into the binding mode of aliskiren by identifying van der waals and nonpolar contribution to solvation as the main components of favorable binding interactions. adamantyltripeptides and phospholipids in liposomal bilayers . now, we were primarily interested to study incorporation profile of mannosylated adamantyltripeptides. we have demonstrated that the adamantyl moiety, due to its liphophilic properties, penetrates into the lipid core of the bilayer while the hydrophilic part with the mannosyl moiety is exposed on the liposome surface. after concanavalin a (con a), a lectin, which specifically binds α-d-mannosyl residues, was added to the liposome preparation, increase in liposome size and appearance of aggregates has been observed. the enlargement of liposomes was ascribed to the specific binding of the con a to the mannose present on the surface of the prepared vesicles. the afm analysis revealed that the adamantyltripeptide molecules grouped into small domains that raise above the bilayer surface. the molecule size and molecular geometry, as well as the hydrophilic and hydrophobic surfaces in the structure of mannosylated adamantyltripeptides, are responsible for arrangement of molecules in the lipid bilayer. this approach might be a useful model for investigation of specific protein interactions with membrane receptors. also, the adamantyl moiety may be considered as a potential membrane anchor for different carbohydrate or other molecules of interest, which could be bound on it and thus exposed on liposome surfaces and as such used in targeted drug delivery. the assay is carried out in a well format p and images are captured throughout the course of the assay, thus we can not only determine a ligand's propensity to induce internalization, but also its efficacy and internalization rate. addition of test compound, followed by the standard agonist at a later interval, enables differentiation between agonist or antagonist activities. in the positional scanning format [ ] , while the possibility of agonists and antagonists working against each other within a mixture exists, the effects are minimized in screening the whole library as there are as many arrangements of the sub-libraries as there are defined positions. therefore while an agonist and antagonist might be present in a particular mixture in one sub-library they will be in different mixtures in all other sub-libraries. we have used this assay format to simultaneously screen for novel agonists and antagonists against the orexin receptor. assay development and library screening will be presented. [ ] . since the pro residue in position of em is very important in the proper conformational alignment of the two aromatic residues tyr and phe in em molecule at the receptor site, it is possible that structural modification around the pro residue yields compounds with unique biological properties and improved metabolic stability. in the present study, we synthesized seven em analogues containing isopro or constrained residues with oxopyrrolidine or oxopiperadine ring, instead of pro residue in position . all peptide analogues were synthesized solid phase method. incorporation of oxopyrrolidine and oxopiperadine rings were carried out on a solid support by the methods of gellerman, et al. [ ] and mohamed, et al. [ ] , respectively. opioid receptor binding activity for μ and δ-receptors using the development of resistance to mainstay cancer therapies has become a major limitation for the treatment of many cancers. there is an urgent need to develop new antineoplasic agents with innovative anticancer approaches. to overcome resistance to cancer therapies, our attention has turned to proteins that regulate multiple signalling pathways essential for tumour survival. among the few known nodal proteins upregulated in cancer cells and involved in many hallmarks of cancer, we are interested in survivin. an essential regulator of cell proliferation and apoptosis, survivin is sharply overexpressed in cancer cells and plays a major role in resistance. being a small protein, its bioactivity is relies mainly on protein-protein interactions (ppi) with different partners. a critical point for its multiple functions in cancer is its association with hsp , which is required for its stability. a nonapeptide from survivin called shepherdin has been shown to modulate the interaction of survivin with hsp by binding to hsp and to induce death of tumour cells. unfortunately, shepherdin is not cell permeable, has low proteolytic stability and shows poor bioavailability, limiting its use as anticancer therapeutic agent. to improve pharmacological properties of shepherdin, cyclic and peptidomimetic analogs of shepherdin have been synthesized followed by structure-activity relationship studies. in hsp binding studies, some cyclic hexa-and heptapeptidic analogs showed increased affinity compared to shepherdin. the synthesis of cyclic and peptidomimetic analogs and the results from the binding assays and the conformational analyses will be presented. the hexapeptides with formula ac-ryyr/kw/ir/k-nh have been identified as shortest peptide sequence with high nop receptor affinity, selectivity and marked analgesic effect. it was found that the following peptides act as partial or full agonists or antagonists of nop receptor in different in vivo and in vitro systems. these hexapeptides were used as chemical templates in sar studies , . the aim of the present study was the synthesis and the biological screening of new analogs of ac-rfmwmk-nh and ac-ryyrwk-nh , modified at position and respectively with newly synthesized β -tryptophan analogues . these non natural amino acids were prepared using reaction of asymmetric friedel-crafts alkylation of various indoles with a chiral nitroacrylate to provide optically active β-tryptophan derivatives. the four newly synthesized ligands for the nociceptin/orphanin fq (n/ofq) receptor (nop) have been prepared by solidphase peptide synthesis-fmoc-strategy. these compounds will be tested for agonistic activity in vitro on electrically stimulated smooth-muscle preparations isolated from vas deferens of wistar rats. bacterial infections are a common problem associated with dermal wounds. these infections can prolong or impair wound healing. hydrogel materials that display inherent activity against bacteria can be used to directly treat accessible wounds to prevent or kill existing infection. in this work, we describe the design and utilization of injectable gels prepared from self-assembling β-hairpin peptides having a high content of arginine. these gels were found to be extremely effective at killing both gram-positive and gramnegative bacteria, including multi-drug resistant p. aeruginosa. importantly, no added antibacterial agents are necessary since the nanostructure of the gel, itself, is the active agent. using self-assembling peptides for material construction allows facile structure-activity relationships to be determined since changes in peptide sequence at the monomer level are directly transposed to the bulk material's antibacterial properties. structure-activity relationships studies show that arginine content largely influences the hydrogel's antibacterial activity, and influences their bulk rheological properties. these studies culminated in an optimized gel, composed of the peptide pep r. pep r gels prepared at . wt % or higher concentration, demonstrate high potency against bacteria, but are cytocompatible towards mammalian mesenchymal stem cells. the general mechanism by which pep r exerts its action was explored and it is suggested that involves membrane disruption that occurs when cells come in contact with the gel's surface. atomic force microscopy (afm) was used to study the effect of the gel on the cell envelope morphology of e. coli. rheological studies indicate that the gel is moderately stiff and displays shear-thin recovery behavior, allowing its delivery via simple syringe. they are intimately involved in the molecular process leading to the delicate nano-patterned silica shells of diatoms. deciphering the mechanisms of silica-biogenesis in diatoms will inspire the development of novel routes for the biomimetic synthesis of silicon-based materials under mild conditions and expand the scope of biotechnological applications, e.g. for immobilization of enzymes in silica matrices. we synthesized silaffin peptides derived from c. fusiformis that carry posttranslational modifications such as phosphorylation or polyamines linked to lysine side chains. a distinct alteration of silica precipitation activity depending on the particular modifications of the silaffins emerged. these modified silaffin peptides were covalently linked to recombinant proteins by expressed protein ligation leading to stable protein-silaffin conjugates. using egfp as model protein, we could show that egfp-silaffin conjugates can induce biomineralization of silica and ensure an efficient and homogeneous immobilization of egfp into silica particles, superior to simple co-biomineralization approaches. moreover, a significant stabilization of immobilized egfp against denaturing agents was observed. we established a method for controlled immobilization of biomolecules based on covalent attachment of silaffin peptides with well-defined silica precipitation properties. currently this method is applied to the immobilization of biotechnological relevant enzymes in order to test their activity and the stabilization effect. herein, we present the covalent functionalization of multiwalled cnts (mwcnts) with organocatalysts based on proline or proline derivatives carrying either a dipeptide or a sulfonamide moiety. two different approaches were followed, namely, covalent grafting of the organocatalysts either at the tips or at the sidewalls of the cnts. for the former approach, mwcnts were oxidized in order to introduce carboxylic units at their tips and make them easily dispersed in aqueous solutions. then, oxidized mwcnts readily reacted with proline-based derivatives carrying a free amino unit yielding the corresponding hybrid materials. for the latter approach, the functionalization methodology based on in-situ generated aryl diazonium salts was followed. in this context, mwcnts were modified with aryl units carrying free amino terminal groups, which were subsequently conjugated with proline-based derivatives carrying a free carboxylic unit. all newly formed hybrid materials were fully characterized with complementary spectroscopic (atr-ir, raman), thermal (tga) and microscopy (tem) techniques. the catalytic evaluation of the activity of the cnt-based organocatalysts in aldol reactions is in progress. financial support from gsrt/ΕΣΠΑ - ΣΥΝΕΡΓΑΣΙΑ through ΣΥΝ- - -ΝΑΝΟΚΑΤΑΛΥΣΗ project is acknowledged. novel organogels based on self assembly of rationally designed pseudopeptides c. pappas, n. sayyad, a.g. tzakos, i. plakatouras section of organic chemistry and biochemistry, department of chemistry, university of ioannina, ioannina, gr- , greece self-assembly is becoming a rather intriguing way to build an array of nano-and micro-structured materials. low molecular weight organogelators can self-assemble into various architectural types in organic solvents through weak intermolecular interactions. such organogelators have potential applications in the generation of novel materials for nanobiotechnology . herein, we report the synthesis of rationally designed pseudopeptides and the conditions to form organogels. the obtained gels are responsive to temperature, and the sol-gel process is thermoreversible. the architecture of the constructed organogels was characterized via tem and spectroscopic techniques. diffusion ordered nmr spectroscopy (dosy) was further utilized to determine differences in the molecular shape of the different pseudopeptides. applications of the resulted compounds in nanotechnology will be reported. since , organocatalysis has met such a great rate of expansion that is nowadays considered the third major branch of modern asymmetric catalysis along with the transition metal catalysis and biocatalysis. after the seminal work of list, lerner and barbas on the enantioselective aldol reaction between acetone and -nitrobenzaldehyde catalyzed by proline, it became clear that amino acids and peptides could serve as an abundant pool full of potential to develop novel organocatalytic motives. following our recent report that the combination of a prolinamide with a thiourea group having as a spacer a chiral diphenylethylenediamine leads to an efficient organocatalyst for the aldol reaction, we recently considered the possibility to couple the prolinamide unit with an urea moiety. one of our main interests was the substitution of the diphenylethylenediamine spacer by a gem diamine derived from an α-amino acid. the gem diamine is easily synthesized via a curtius rearrengement of the corresponding acyl azide. after synthesis and evaluation of a number of potential catalysts, the prolinamide derivative bearing a gem diamine derived from (s)-phenylalanine and an aryl urea moiety proved to provide the best results in the reaction between cyclic ketones and aldehydes. utilizing mol% of our organocatalyst, the aldol products were obtained in high to quantitative yields (up to %), high to excellent diastereoselectivities(up to > : ) and high to excellent enantioselectivities (up to % ee). peptide self-assembled monolayers are of current interest to study physicochemical properties of modified metal (e.g. au) surfaces. rigid peptide scaffolds could enhance the interaction between gold surfaces and labels by reducing and precisely monitoring the distance between the supported monolayers and gold. the c α -tetrasubstituted αamino acid -amino- , -dithiolane- -carboxylic acid (adt) , which contains a cyclic disulfide system, is interesting in this respect because it may allow the parallel binding of the peptide helical chain to the metal surface. adt occurs in nature and has been utilized in medicinal chemistry and in a model compound of [fefe] hydrogenase. we synthesised a series of constrained helical peptides, based on the ala-ala or the ala-aib sequence, containing one or two adt residues. these peptides were functionalised with spectroscopic or opto-electronic labels. among the large number of reactions involving the formation of carbon-carbon bond, the addition of ketones to nitroolefins is a powerful tool for the synthesis of γ-nitrocarbonyl compounds, useful intermediates for pharmaceutical industry. our recently reported primary amine-thioureas based on tert-butyl esters of natural amino acids exhibit excellent performance for the michael reaction of ketones with nitroolefins providing the products quantitatively and almost stereospecifically (> % ee). , using this methodology, enantiopure baclofen and phenibut (analogs of gaba) have been synthesized. polymersupported organocatalysts constitute a great challenge for the michael reaction. in the current study, we report the immobilization of amine-thiourea catalysts containing ( s, s)or ( r, r)-diphenylethylenediamine and tert-butyl aspartate, on various polymer supports, either directly or through spacer units. the solid-supported catalysts evaluated in the reaction between acetone and βnitrostyrene and highlighted the importance of the choice of the polymer as well as the presence of the spacer or not. the direct attachment of the primary amine-thioureaaspartate to a crosslinked polystyrene-divinyl benzene resin containing a uniform distribution of aminomethyl groups provides a supported catalyst that affords the product of the reaction between acetone and β-nitrostyrene quantitatively and in high enantioselectivity ( % ee a. theodorou, g.n. papadopoulos, c.g. kokotos* laboratory of organic chemistry, department of chemistry, university of athens, athens, greece after the pioneering report that proline can catalyze efficiently the intermolecular aldol reaction between acetone and a variety of aromatic aldehydes, it became evident that amino acids and peptides can afford a plethora of different structural scaffolds for novel catalysts. along the first decade of its life, organocatalysis has grown to such an extend that now it is considered the third major branch of asymmetric catalysis. recently, researchers have paid special attention to other amino acids rather than proline. some primary amino acids have already been applied to a number of transformations with success. usually improved catalytic properties are observed when derivatives of primary amino acids are utilised. we have undertaken a study on the application of simple and cheap primary amino acids and amino acid derivatives, either commercially available or easily obtained, as organocatalysts for the asymmetric α-amination of aldehydes. in the present work, we report that the use of simple derivatives of primary amino acids like phenylalanine and aspartic acid can efficiently catalyze this transformation leading to products in high to quantitative yields and enantioselectivities up to % ee. the majority of the organocatalysts developed up to now for asymmetric organic transformations employ more than one functionalities in the catalytic mechanism that act through either covalent or non-covalent interactions. for example, proline employs the pyrrolidine nitrogen and the carboxylic acid group, while chiral thioureas combine the thiourea functionality with a tertiary or a primary amino group. we have recently shown that an amide of proline with a diamine carrying a thiourea group is a very good catalyst for the enantioselective aldol reaction. trying to improve the activity, we have found that a tripeptide-like thiourea having as building blocks (s)-proline, ( s, s)diphenylethylenediamine and (s)-di-tert-butyl aspartate provides the products of the reaction between ketones and aromatic aldehydes in high to quantitative yields and high stereoselectivities (up to : dr and % ee). a number of structural modifications of the catalyst were undertaken in order to understand the role of the hydrogen bond donors of the catalyst, i.e. the prolinamide hydrogen and the two hydrogen atoms of the thiourea group. we have come to the conclusion that the importance of the hydrogen bond donors of the catalyst follows the order: thiourea hydrogen originated from aspartate › amide hydrogen › thiourea hydrogen originated from diphenylethylenediamine. g eldrug s.a., patras , greece a convenient and facile synthesis and in vitro biological evaluation of n-substituted -butylimidazole derivatives as potent angiotensin ii (ang ii) receptor type (at ) antagonists have been reported in the present study. a series of imidazole based compounds bearing the biphenyl moiety at the n- position, a halogen atom at the c- and polar substituents such as hydroxymethyl at the c- position were synthesized. , these compounds were evaluated for binding to human at receptor and for ang ii antagonism in vitro on isolated rat uterus. in particular, butyl- -[[ ΄-( h-tetrazol- -yl)biphenyl- -yl]methyl]imidazole derivatives complexed with the at receptor and showed high binding affinity. these analogues were also found to be active in the rat uterotonic test. importantly, their binding affinities and potencies were comparable to those of losartan. these results indicate that the hydroxymethyl at the c- position of the imidazole ring is favorable for high affinity binding and antihypertensive activity and in line with the activities of the losartan counterparts. experimental findings are in good agreement with docking studies, which were undertaken in order to investigate ligand/at receptor interactions. z-leu-glu-his-asp-aluc, suc-leu-leu-val-tyr-aluc) are good substrates for bioluminescence assays, for example in the detection of caspase activity during apoptosis . these substrates generally offer significant advantages, such as increased sensitivity, ease of use, and high throughput screening capacity. luciferase-based assays are typically -to -fold more sensitive than the comparable fluorescent assays (rhodamine , -amino- -methylcoumarin (amc) and -amino- trifluoromethylcoumarin (afc)). the synthesis of different type peptide-amino-luciferin conjugates and their precursors have been published and some of them are commercially available. however, because of their high price the in vivo application of these conjugates is limited. to solve this problem we successfully worked out a new, easier and more convenient and economical method for the preparing these derivatives starting from -chloro-benzothiazole. moreover this products have excellent purity (> %) and adequate yield ( - %). major health problems arising from bacterial resistance towards existing antibiotics make discovery of antibacterial drugs with new mechanisms of action pertinent. although proof of concept for a novel antimicrobial approach using peptide nucleic acid (pna) antisense targeting of essential bacterial genes was obtained a decade ago, this technology is still limited by the lack of carriers that facilitate effective bacterial delivery and confer optimal pharmacokinetic properties to the prospective drugs. [ , ] in the past two decades, parallel efforts of exploiting naturally occurring antimicrobial peptides (amps) as drugs have been made. the cationic amp subclass appears to be directly involved in the innate immune response towards microbial infections. [ ] so far only few cell-penetrating peptides, with activity on mammalian cells, and other membrane-active peptides, have been investigated as potential vehicles for bacterial delivery. for instance, cationic amps with an internal target appear not to have been investigated for bacterial delivery of antibiotics. the aim of this project is to develop highly potent genetic antibiotics by exploiting naturally occurring antimicrobial peptides as potential delivery vehicles for antisense peptide nucleic acid oligomers. the amps are chosen from amps reported to act via intracellular targets, and thus must possess an inherent ability to permeate bacterial cell membranes without direct killing of the bacteria. faculty of chemistry university of gdansk, gdansk, poland azt ( '-azido- ' '-dideoksythymidine), a modified nucleoside used in antiretroviral therapy and peptide plant hormone -systemin were used as substrates of , -dipolar cycloaddition (click chemistry). systemin is -aa peptide defense hormone released in response to plant (tomato, tobacco) damage or pathogen attack. we examinated whether systemin's fast movement through plant tissues could be used for cargo (azt) transport. the huisgen cycloaddition also known as , -dipolar cycloaddition is a chemical reaction belonging to the larger class of cycloadditions. reaction between organic azide and alkyne appended substrates allows the synthesis of the desired conjugate in high purity and yields irrespective of the sequences and functional groups on either of the two substrates [ , ] . conjugate of azt-systemin has been synthesized by click chemistry, using systemin modified at n-terminus with propiolic group and azt. the conjugation was catalyzed by cu(i). the reaction was fast, efficient and regioselective. its progress was easily monitored by capillary electrophoresis (ce). ce was also applied for characterization of systemin and azt-systemin stability and movement throughout tomato leaf and stem. despite the fact that systemin moves rapidly through tomato tissues, our calorimetric (itc) studies showed that the peptide does not interact with liposomes-cell membrane model. universitätsklinik für nuklearmedizin, inselspital, bern, switzerland regulatory peptides (e.g., somatostatin, bombesin) have been shown to be suitable vectors for the specific delivery of radioactivity to tumors for diagnostic and therapeutic applications in nuclear oncology. a potential drawback of such vectors is their inherent instability in vivo. thus, new strategies are needed for the stabilisation of radiopeptides in order to improve their bioavailability and, consequently, increase their accumulation in the targeted tissue. it has been suggested that , , -triazoles, readily obtained by cuaac, are suitable amide bond surrogates which are resistant to proteases. in the present study, we report the synthesis and pharmacological evaluation of radiolabelled, triazole-containing analogues of the gastrin releasing peptide receptor (grpr) targeting peptide bombesin (bbn). to study the effect of backbone modifications in the minimal grp-binding sequence, we synthesized a series of analogues of [nle ]bbn ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , in which each amide bond is individually replaced by a , -disubstituted , , triazole. after radiolabelling of the peptidomimetics, their binding affinity and internalization kinetics were determined using pc- cells. metabolic stability was evaluated in blood serum. a number of the novel tumor-targeting peptide analogues presented exhibit both a retained high affinity (nm) towards the grpr and an improved serum stability. first preclinical data on the in vivo evaluation of the most promising candidate will be presented. to the best of our knowledge, this is the first report of the systematic replacement of amide bonds with , , triazoles within the binding sequence of linear, high affinity peptides. the methodology can be applied to a variety of peptide vectors and thus, holds great potential for the development of novel, stabilized peptide-based radiopharmaceuticals. dna is the molecular target for many of the drugs that are used in cancer therapeutics, and is viewed as a nonspecific target of cytotoxic agents. although this is true for chemotherapeutics, other agents that were discovered more recently have shown enhanced specificity. the development of new site-specific dna binders, which are associated with the recognition of the dna major groove, are based on the design of transcription factor mimics that bind the dna as a dimer , and prevent specific genes from being transcribed. these could ultimately result in interesting biomedical applications as designed genome interfering agents or diagnostics. in order to approach this biological constructs, we choose the bzip leucine zipper transcription factor as a model to mimic. as the entire structure cannot be synthesized without expensive, complicated and time-consuming biotechnological methods, the substitution of the dimerization domain by a less complex scaffold is the first step in the design. thus, we consider a steroid based scaffold as a candidate. the specific choice of the steroid scaffold as substituent is inspired by its known ability to enhance proteolytic stability of attached peptides, by its conformational properties ensuring correct positioning of the two appended chains and by its potential to increase bioavailability. this transcription factor binds specific dna sequences by dimerization and inserting short α-helices into the dna major groove. in order to attach the peptides to the scaffold, different strategies were studied. firstly, applying the well-known click chemistry, functionalizing the scaffold with an alkyne moiety, the peptide with an azide and viceversa. secondly, via the unknown resin to resin transfer reaction (rrtr), which has not been applied on peptide chemistry so far. this unprecedent methodology consists on the reaction of a peptide, which is attached on a safety-catch resin, with a second resin bearing a nucleophilic amino terminus resulting in amide bond formation. during the process, the peptide on solid support undergoes cleavage. an hexapeptide was synthesized on a preloaded safetycatch resin. deoxycholic acid derived scaffold with orthogonally protected amines was attached to tentagel resin that acts as acceptor resin. rrtr experiments were performed at both c and c positions of the deoxycholic acid derivative. in addition, this convergent strategy can be applied to other different peptide conjugated systems. we recently described a new kind of cyclized peptide in which the cyclization is performed between the side-chains of two diaminoacyl residues via a diversely substituted guanidine bridge. we showed that the degree of bridge substitution could impact on the orientation of the bridge inside the cycle and therefore the peptide conformation. we prepared two series ( and atoms cycle size) of cyclic enkephalin analogues to assess the potential effect of this kind of bridge on the biological activity. the compounds were synthesized on the solid support via the formation of a thiourea bridge and with the variable substituent being introduced at the last step before cleavage. it is noteworthy that the synthesis afforded at least two stable and separable conformers for each analogue of the shortest cycle series. generally, one major and one minor species were recovered. but in the case of di-substituted compounds with a cyclic moiety (pyrrolidine or piperidine substituents), three significant species were obtained. analogues were submitted to various biological assays (binding to μ and δ opioid receptors and functional assays). we observed a significant variation in affinity and selectivity for the receptors as a function of the degree of bridge substitution. a structural analysis by d nmr has been undertaken and correlated the variation in activity with a variation in conformation. the origin of the multiple conformers observed for the analogue with a pyrrolidine susbtituent was also investigated. this kind of cyclization could represent a useful tool to easily modulate the conformation and biological activity of a unique peptide sequence. the t-cell response is triggered by the formation of the trimolecular complex between the major histocompatibility complex (mhc), the immunodominant myelin protein epitopes and the t cell receptor (tcr). herein, we report the design and synthesis of non-peptide analogues with the ability to mimic the immunodominant epitope - of mbp , . the mimetics were designed to block the formation of the trimolecular complex and therefore the t-cell activation , . more specifically, indole analogues were synthesized with substitution at positions and or . these molecules contain a carboxyl or an ethyl ester group in position and a benzylamino or phenylamino group in position or . the synthesis of the indole ring was achieved by fischer reaction followed by catalytic hydrogenation, reductive amination or arylation and ester hydrolysis. the synthesized molecules were purified using liquid chromatography, and they were identified by mass spectrometry and h-nmr. laboratory of peptide science, nagahama institute of bio-science and technology, nagahama, japan amyloid β peptide (aβ), the main component of senile plaques in the brain of alzheimer's disease (ad) patients, is formed by proteolysis of amyloid precursor protein (app). as β-secretase (bace : β-site app cleaving enzyme ) triggers aβ formation by cleavage at the aβ domain nterminus, it is a molecular target for ad therapeutic intervention. previously, we reported potent pentapeptidic and non-peptidic bace inhibitors containing a substrate transition-state mimic. although these inhibitors exhibited potent inhibitory activities, their molecular-sizes appeared a little too big (mw> ) for developing practical drugs. in this study, we designed a series of small molecular peptides, with bace inhibitory activity, lacking the p -p ' region on the basis of the conformational structure bound in bace . design and synthesis of new '-peptidyl-trna analogues, in particular "hydrolysable" analogues, which represent covalent conjugates of peptide-nucleic acid (pna) with "stop-peptides," were carried out. such compounds are of interest as tools to study the ribosome functioning and as inhibitors of protein biosynthesis. ( aminoethyl)glycine pna models '-end trna sequence cca in designed structures. computer simulations showed the formation of watson-crick pairing of the pna cytosine residues with s rrna nucleotides g and g involved in interactions with peptidyl-trna during its specific binding in p site of the ribosomal peptidyl transferase center (ptc). short "stop-peptides" were planned for conjugation with pna. these peptides form stable complexes with the ribosomal tunnel (rt) that leads to ribosome stalling and translational arrest. structures of "hydrolysable" 'peptidyl-trna analogues that could form peptide bond with amino acid residue of aminoacyl-trna in a-site of ptc included '-deoxyriboadenosine instead of the pna adenine containing residue. such conjugates would permit to identify the chemical nature of specific sites localized in rt and responsible for interactions with amino acid residues of the nascent polypeptide chain. pna and "stop-peptide" as well as pna-"stop-peptide" conjugates were prepared by solid phase synthesis on sasrin polymer using fmoc/bhoc(boc) strategy. synthesis of "hydrolysable" conjugates included modification of the 'hydroxyl of '-protected '-deoxyadenosine by n-blocked "stop-peptide", deprotection of the '-hydroxyl, its conjugation with n-protected pna and removal of protecting groups from the resulted conjugate. the binding of the new '-peptidyl-trna analogues with ribosome will be tested by chemical probing and in the cell free translation system. this study was supported by the russian foundation for basic researches (grant - - -a). a close structural similarity of endomorphin- and another atypical opioid peptide, morphiceptin, which both have a phe residue in the third position, encouraged us to study antinociceptive activity of these two peptides and their analogues. in order to improve the affinity and chemical stability of these opioid peptides, we have designed, synthesized, and analyzed novel analogues. the first modification included endomorphin- and morphiceptin analogues, where halogenated phenylalanines in position or were incorporated as surrogates of the native phenylalanine. another important modifying element is non-protein amino acid canavanine (cav) and its analogue (sarg). it is well documented that cav and sarg exhibit strong analgesic activity. two new morphiceptin analogues were synthesized by introducing cav and sarg in position . we further characterized their antinociceptive activities by the paw pressure (pp) test. the experiments were carried out on male wistar rats ( - g), treated with i.p. doses of mg/kg. e eldrug s.a., patras , greece the renin angiotensin system (ras) has been a prime target for the therapy of cardiovascular diseases. angiotensin ii type (at ) receptor mediates vast majority of biologically detrimental actions. non-peptide at receptor blockers are presently the most specific means to block the ras enzymatic cascade. the dupont group was the first to develop losartan (dup ), an orally effective angiotensin ii receptor blocker, which is metabolized in vivo to the more potent antagonist exp . herein, we report on the preparation of e-urocanic acid based analogs, focusing our attention on the introduction and structural modifications of the substituents on the imidazole ring as well as the modifications on the acrylic side chain. in particular, we have designed and synthesized a series of urocanic acid analogs bearing the biphenylmethyl tetrazole moiety at the n- of the imidazole ring. additionally, the rigid acrylic chain was lengthened by esterification resulting in the ethyl ester and on the other hand the latter was readily converted to the corresponding acrylic alcohol or aldehyde which may proved to be effective structural elements for enhancing biological activity. finally, a lipophilic alkyl chain such as the n-butyl group was introduced at the -position of the ring which may possibly enhance the antihypertensive activity. docking studies and biological evaluation of the synthesized analogs are being undertaken. university of athens, department of chemistry, laboratory of organic chemistry, , panepistimiopolis zografou, athens, greece the backbone modification of bioactive peptides with replacement of a scissile peptide bond in enzymatic hydrolysis is a well-established strategy for developing protease inhibitors. in particular, for zinc metalloproteases, which contain a zinc atom in their active site, several successful modifications have been reported over the past years. phosphinic pseudopeptides are among the best candidates when addressing the challenge to potent and selectively inhibit zinc proteases. a thorough search in the literature revealed the absence of any reference regarding thiophosphinic pseudopeptides. we thought that this class of compounds would add a valuable tool in the field of zincbinding groups. in the present study, we describe in detail the first synthesis of a new class of phosphorous compounds, thiophosphinyl dipeptide isosters (tdis). we prepared several fully protected thiophosphinate pseudodipeptides of the general formula pg-phe-Ψ[p(s)(ox)ch ]-gly-pg' starting from the corresponding phosphinate pseudodipeptide using lawesson's reagent. selective deprotection of these compounds was also studied and the results are disclosed. these compounds can be used as building blocks for the synthesis of longer thiophosphinic pseudopeptides after suitable deprotection and elongation as well as transition transition state-mimicking inhibitors for several zinc metalloproteases. in the last decade, trypsin inhibitor sfti- isolated from sunflower seeds [ ] has become one of the most studied peptidic inhibitors of serine proteases. owing to its small size and a strong trypsin inhibitory activity (ka = . × m - ), sfti- is considered to be a very attractive template for designing proteinase inhibitors with the potential use as pharmacological agents [ ] . it could also serve as an affinity probe for the isolation of trypsin like (sfti- ) or chymotrypsin like ([phe ]sfti- ) proteinases. following this idea, we decided to synthesize a set of cell-permeable monocyclic sfti- analogues with a fluorophore moiety attached at their n-termini. the presence of the fluorophore in the molecule enabled us to show that the analogues can cross the cell membrane. the cell penetration assay was performed using multiple cell lines (hela cells and human fibroblasts cell line ( br. n) was obtained from european collection of cell cultures (ecacc)). for all the obtained peptidomimetics, we determined the association constants with cognate proteinases. selected peptides were also used as a probes for the detection of inhibitor -proteinase complex, which was achieved by the means of gel filtration chromatography equipped with fluorescence detector and acrylamide native gel electrophoresis. the functional reconstruction of folded protein surfaces with peptide-based mimics is an enormous scientific challenge. the majority of proteins show activity through a small area of their folded surface: "the binding site". however, linear peptides are too flexible and seldomly adopt the correct d-structure of the binding site spontaneously. therefore, they show limited or no activity at all . crucial for activity is to control the secondary (αhelix, β-sheet and/or β-turn) and tertiary structure (relative orientation of subdomain structures). we present the development of a new type of watersoluble scaffolds that have the potential to control both secondary and tertiary structure of discontinuous (i.e. double-loop) protein mimics. the new scaffolds contain a first pair of reactive functionalities to constrain the linear peptide conformation via a 'clips' reaction , stabilizing the secondary structure. next to this, a second functionality allows for ligation of two dissimilar constrained peptides to form a discontinuous binding site mimic via oxime-ligation or click-reaction. these ligations offer the ability to position different peptide loops in d, thus mimicking the tertiary structure of the native protein. most unique to our approach is the fact that all chemical conversions are performed in aqueous media, using side-chain unprotected peptides . growth hormone-releasing peptide (ghrp- ) is a synthetic hexapeptide (his-d-trp-ala-trp-d-phe-lys-nh ), which interacts with two kinds of receptors: growth hormone secretagogue receptor a (ghs-r a) and cluster of differentiation (cd ). the latter is a membrane glycoprotein member of the class b scavenger family, and decreases the internalization of oxidized lipids into macrophages, as well as causes inhibitory effects on angiogenesis associated with binding to thrombospondin. to increase activity and selectivity for the cd receptor, different analogues of ghrp- were synthesized. in particular, substitution of trp in ghrp- by aza-amino acids has given selective analogs, due likely to induction of a β-turn secondary structure. for aza-peptide synthesis, a submonomer solid-phase approach has proven effective to introduce side chains onto the semicarbazide residue. studying influences of benzylidene, benzhydrylidene and fluorenylidene residues during the alkylation of the semicarbazide, superior conversion was observed with fluorenone derivative, and mild alkylation conditions employing et noh as base have improved yields and minimized racemisation. our presentation will focus on the improved submonomer synthesis method for optimization of selective and potent cd- ligands with antiatherosclorotic and anti-angiogenic effects. for instance, the integrin αvβ , vitronectin receptor, is expressed in a number of cell types and has been shown to mediate adhesion of osteoclasts to bone matrix, vascular smooth muscle cell migration, and angiogenesis. integrin αvβ also play a significant role in tumor growth, invasion and metastasis, and is a receptor for the extracellular matrix proteins with the exposed arginine-glycine-aspartic (rgd) tripeptide sequence. rgd has been shown to be potent antagonist of the integrin αvβ , and has excellent anti-angiogenic properties including its suppression of tumor growth in animal models. in this context, drug design based on the rgd structure may provide new treatments for diseases such as thrombosis, osteoporosis, and cancer. we designed and synthesized series of short rgdmimetics containing the sequence xaa-gd, where xaa is arg-mimetic. as promising candidates we have chosen canavanine (cav) and canaline (can) instead of the basic residue arg. in order to improve antitumor activity of the parent molecule, c-terminal modifications were also applied. their cellular uptake was determined on human breast (mcf ) cancer cell lines. furthermore, the in vitro cytostatic effect was evaluated by mtt assay on human liver hepatocellular carcinoma (hepg ) and human breast (mcf ) cancer cell lines after , and hours of treatment. in the case with the human tumor cell lines (hepg , mcf ) and c-modified analogues, statistically reliable results were achieved for the most of concentrations used. acknowledgements: this work was supported by bulgarian ministry of education and science, project my-fs- / . microwave assisted solid phase synthesis of urea and urea/amide based foldamers k. pulka, c. douat-casassus, g. guichard* european institute of chemistry and biology, university of bordeaux -cnrs umr , pessac, france foldamers are fully arti cial molecules that structurally and functionally mimic variety of biopolymers. among them, aliphatic n,n'-linked oligoureas with proteinaceous side chains can adopt extremely robust helical folds stabilized by intramolecular three-centred h-bonds. owing to their resistance to enzymatic degradation, diversity of side chains and structural predictability urea-based foldamers represent unique scaffolds to elaborate functional mimetics of α-polypeptides. of note, heterogenous oligo(urea/γamides) backbones obtained by substituting nh groups by ch display very similar folding propensities. in our laboratory we are investigating the solid phase synthesis of urea and urea/γ-amide oligomers. urea bonds are incorporated into the growing chain by reaction of active succinimidyl carbamates. previously we have applied two different strategies involving fmoc-or bocchemistry, but both methodologies suffer some limitations. therefore a new strategy (compatible with the use of tfa sensitive linkers and side chain protecting groups) featuring azide as a masked amine group has been developed. the synthesis of new azido protected succinimidyl carbamate building blocks is reported. they were obtained in steps from α-amino acids ( - % overall yield). the staudinger reduction with pme was successfully applied to restore the amine group after urea formation on solid support. in addition, microwave irradiation has been found to dramatically accelerate the synthesis. overall, this azide-strategy combined with microwave irradiation was found to be very effective for the solid phase synthesis of oligoureas and related hybrids, surpassing previously developed approach utilizing fmoc chemistry. these antibiotics should have a mechanism different from currently used antibiotics to circumvent existing resistance mechanisms . previous results have shown that "genetic" antibiotics operating by gene silencing in bacteria via rna interference may be successful new candidates. efficient silencing requires efficient crossing of cell membrane. this step can be alleviated using cell penetrating peptides (cpp) as carrier of drug candidates, such as peptide nucleic acids (pnas) which inherently have poor internalization properties . the aim of this study is to elucidate mechanisms of uptake in bacteria using pna-cpp conjugates, which previously have shown promising antibacterial effects . the fate of the pna and cpp parts of the conjugates, once inside the cell, is investigated regarding localization and possible degradation within the cell. furthermore, a method for toxicity testing of pna-cpps is being developed using histamine release in rbl- h cells as a quantitative measure of allergenicity of pna-cpps. the prospect of this information is to define boundaries within which cpps can be found, thereby rationally designing novel efficient antibacterial biomolecular drug delivery systems. oxytocin and its fragments have the potential to influence behavioral and cognitive functions, including their disturbances in some brain disorders. therefore, there is an interest to synthesize new peptide-steroids chimeras for potential therapeutic use. oxytocin analogue was synthesized in solution by coupling azido-phenylalanyl residue or p-azidopegylated handle to the n-terminal end of oxytocin molecule. its c-terminal fragment pro-leu-gly-nh (mif- ) was elongated at proline residue by the same type of azido handles as well. both peptides were marked for fluorescent detection of their possible binding on brain slices. peptide chimeras with the suitable steroids were prepared via azide click to the triple bond on the modified steroid counterpart like ( α)- -hydroxypregn- -en- -yn- -one, -norchol- -en- -yn- β-ol. steroidyl-peptides were then used in the trials using rat-brain slices. the sites of the peptide-steroids chimeras bound to the brain tissue were identified with the aid of fluorescent microscopy. the suitable chimeras will be tested for their penetration through blood brain barrier for the pharmacological effects. indicating that the orientation of the n-butyl group is of primary importance. docking studies revealed that the highly active analog affords an additional hydrophobic binding feature compared to losartan which fits to an extra hydrophobic cavity. these results may contribute to the discovery of new biologically active molecules by a convenient and cost effective synthetic strategy. the context of pain research, the co-administration of opioid agonists and nk antagonists previously led to an enhanced antinociceptive potency, and recently largent-milnes and co-workers have shown that a hybrid opioid-nk octapeptide was able to attenuate tolerance development, related to sustained opioid treatment. our group has prepared a compact opioid agonist-nk antagonist peptidomimetic chimera dmt-d-arg-aba-gly-nme- ', '-bn(cf ) that served as a lead structure. we report a solid phase method for the synthesis of the amino- , , , -tetrahydro- -benzazepin- -one (aba) structure, which is used as a central unit in the investigated dual ligands. this method allowed the rapid assembly of new bifunctional ligands containing the aba structure. variations of the d-arg , gly and n-benzyl substituents were made. the introduction of d-cit , a gly → β-ala substitution and the removal of the trifluoromethyl substituents in caused considerable shifts in receptor binding. the obtained structure-activity relationships will be presented. hence, a promising approach for the treatment of dmd is the use of drugs to force ptc readthrough. (+)-negamycin is a dipeptidic antibiotic containing a hydrazide structure. although (+)- was not clinically developed due to some toxicity, it was recently reported that (+)- restore dystrophin expression in the muscles of mdx mice, an animal model of dmd. therefore, (+)- is a promising therapeutic candidate for diseases caused by nonsense mutations. based on our own efficient total synthetic method of (+)- , structure-activity relationship (sar) study was perfromed to discover derivatives with a potent readthrough-promoting activity. we found a derivative, ( r)- -hydroxy- -aminohexanoyl-glycine exhibited not antimicrobial activity but a similar readthrough activity to (+)- , suggesting that the ptc readthrough mechanism can be distinguished from the antimicrobial mechanism. moreover, we synthesized -epi-negamycin and found that this analog exhibited a similar activity to (+)- in in vitro readthrough assay. this result hence prompted us to synthesize a -dehydro-derivative, e.g., -dehydro- -epinegamycin , which is a natural product with little antimicrobial activity. surprisingly, we found that showed a higher in vitro readthrough-promoting activity than (+)- . this result suggests that mother nature independently evolved readthrough-promoting products like suppressor trna, in distinction from aminoglycosides, which show both antimicrobial and readthrough-promoting activities. agricultural university of athens, athens, greece high interest has been paid to synthetic structural motifs that promote specific conformations because of their importance for the development of new therapeutic peptidomimetics. in addition, such motifs may show catalytic activity for asymmetric organic transformations. during the last two decades, various synthetic structural motifs that promote reverse turns have been studied. following our interest on chiral prolinamide-thioureas that present interesting organocatalytic activity, we have undertaken a combined experimental/computational study to understand the structural features that may stabilize a reverse turn in short-length peptidomimetics containing a thiourea functionality. compounds with the sequence r-pro-diphenylethylenediamine-thiourea-asp(obut)-obut (r: boc or fmoc, or boc-ala), were synthesized and studied by nmr spectroscopy (tocsy, h- c hsqc, noesy, roesy spectra) for the sequential assignment and the exploration of the dipolar connectivities. sampling of the conformational space was driven by the noe intensities while molecular dynamics simulations were further applied to the consistent with the experimental data conformers in order to monitor the stability of the formed hydrogen bonding interactions in the course of time. energy refined produced conformers were subsequently modified by applying all combinations of d-and l-amino acids at each site in a stepwise manner. the modelled structures were studied in silico aiming to explore the combinations of heterochiral residues which would promote a folded structure and would favour the potential of β and γ turn motif. the most promising combinations were chosen for synthesis and subsequent nmr characterization. in this research project we will deal with chemical strategies to produce suitable surface modifications in order to induce multidirectional cellular migration along gold surfaces. to achieve this objective we want to use and characterize self-assembled monolayers (sams) of thiolated dna chains (dna-sh) adsorbed on gold surfaces through the hybridization with complementary modified single-stranded pnas. pna is a structural dna mimic obtained by polymerization of n-( aminoethyl) glycine monomers that replace the ribose-phosphate backbone characteristic of natural nucleic acids. it is an achiral, uncharged, and relatively rigid biopolymer of high biological and chemical stability, and it can bind complementary dna strands with higher affinity than the corresponding dna sequences.for all these reasons we have chosen pna as a key molecule to promote and assist the movement of cells. by producing a chemical gradient of dna-sh along a gold surface in the presence of a chemotactic molecule it will be possible to obtain and control a directed cellular migration. the norwegian structural biology centre and the centre for theoretical and computational chemistry, department of chemistry, university of tromso, troms , norway renin is a highly selective aspartic protease which catalyzes the hydrolysis angiotensinogen, a protein secreted from the liver, to the decapeptide angiotensin-i. angiotensin-i is further processed by the relatively nonspecific angiotensin converting enzyme (ace) to give the octapeptide angiotensin-ii, a potent vasoconstrictor and the dominant peptide produced by the reninangiotensin system. renin catalyses the rate determining step in the formation of angiotensin-ii, and has for several decades been an established therapeutic target for drug development in relation to hypertension. in the search for renin inhibitors, substituted piperidine derivatives have been identified as promising, - and piperidines have proven to be efficient scaffolds for the development of novel non-peptide aspartic protease inhibitors, particularly towards renin. [ ] [ ] [ ] we herein describe a series of -triazolyl substituted piperidine derivatives that have been synthesized from n-boc protected trans- -ethynyl- -hydroxy piperidine and tested as novel renin inhibitors. piperidine derivatives containing a -substituted , , -triazol- -yl substituent were found to be most active and molecular docking experiments provides a rank order that is in very good agreement with experimental data. the cxcr /sdf- axis is involved in many biological processes such as hematopoiesis, immune cell migration, as well as in cancer metastasis. cxcr also mediates the infection of t-cells with x -tropic hiv functioning as a coreceptor for the viral envelope protein gp . cxcr , as a pharmaceutical target, is of utmost importance but the lack of synthetic agonists has seriously slowed down drug development. it has been recently described by our research group , that grafting the sdf- n-terminus onto a side-chain of the inverse agonist t . generated high affinity synthetic agonists as well as partial agonists for the chemokine receptor cxcr . to remain stable towards proteases and act as useful pharmaceutical tools, the pk-adme properties need to be improved with a gradual transition to peptidomimetic structures. medicinal chemistry witnessed major advances with the discovery of small synthetic molecules that mimic the natural peptidic substrates. these small molecules do not undergo proteolytic degradation, an advantage they hold over natural counterparts. in order to improve stability against proteases, part of the sdf- chain was replaced with variable lengths of polyethylene glycol and unnatural amino acids at differents positions. here, we have produced a series of compounds, most of which showing nanomolar affinities for cxcr and some are displaying partial agonistic properties. tlrs are the innate immunity receptors that recognize the epitopes found on surfaces of various cells and therefore they initiate and sustain the atherogenic inflammatory response [ , ] . we assume that the use of small stat mrna−binding pna−inhibitors to manipulate the activity and expression of stat could prove an attractive therapeutic strategy in treatment of atherosclerosis. to that end we synthesized a specific stat mrna−binding pna inhibitor as well as a non-specific pna to compare their inhibition of gene expression. in our work we developed effective method of synthesis of pna−peptides conjugates by means of "click chemistry". determination of optimal conditions for conjugation (connection of pna with the peptide) will allow for the design of compounds useful in gene therapy. the specificity of pna hybridization to complementary dna fragment was verified by capillary electrophoresis (ce). as an artificially synthesized somatostatin analogue, tyr octreotate (toca) can specifically bind to somatostatin receptor (sstr), which are usually over-expressed on many tumor cells. carbohydration of n-terminus of toca has resulted in improved pharmacokinetics and tumor targeting ( ) . f is an ideal nuclide for positron emission tomography (pet) imaging; there may be significant uses of f labeled glucitol-toca and its analogues as tumor probes for the diagnosis of sstr-positive tumors. in order to explore a novel pet probe for diagnosis of sstrpositive tumors, we designed a synthetic route to synthesize n-gluc-lys(nota)-toca, which uses , , -triazacyclononane- , , -triacetic acid (nota) as the chelating reagent. n-gluc-lys([al f]nota)-toca is radiosynthesized quickly and efficiently using the chelation reaction of al f complex and n-gluc-lys(nota)-toca. the aim of this study is to develop an efficient method for the synthesis of monomers of triazolic nucleic acid (tna), a new class of artificial nucleic acids. but- -yne- , -diol and nucleobases derivatives will be substrates of the monomers synthesis. tna oligomers could be used as specific inhibitor of tar rna hiv- , the regulatory rna structure crucial for hiv replication. "click chemistry" based on , -dipolar cycloaddition will be used to conjugate an alkyne and azide derivatives of monomers subunits. a ru (ii) complex will be used as a catalyst of internal alkyne (but- -yn-based) cycloaddition. the reaction gives exclusively of , , -trisubstituted derivative of triazole ring . the monomers will be characterized using rp hplc, capillary electrophoresis (ce) and h and c nmr. the resulting monomers containing fmoc-protected amino group and a free carboxyl group will be used for the classical spps method to synthesize tna oligomers. tna sequences will be designed against tar's bulge and an external loop. through the recognition that the repertoire of polypeptide conformations can be greatly expanded by the creation of structures incorporating β-amino acids. moreover, the numerous advantages of hybrid (mixed α-and β-) backbone peptidomimetics with respect to homogeneous ones were quite recently outlined. we describe here various β-amino acid-based β-hphe-β-hphe dipeptide derivatives, also conformationally constrained, and their application to the synthesis and biological evaluation of hybrid analogues of the opioid endogenous peptide endomorphin- (em- ). the opioid system mediates a wide variety of pharmacological and physiological processes, including pain perception and modulation. the amidated tetrapeptide em- has been shown to be μ-opioid receptor (mor) agonist exhibiting a very high μ-receptor affinity and selectivity, and it is an important model in the search towards new analgesics. structural investigation of em- reveals the high conformational freedom of the phe side chains and also the inherent flexibility of the peptide backbone, indicating many probable bioactive conformations, ranging from βturns to extended conformations. with the aim of better clarify the relevant role of the proper spatial orientation of the aromatic rings and in particular of the benzyl side chains at position and , h nmr studies, molecular modelling, and molecular docking to a homology mor model of our hybrid analogues are currently under way. the lantibiotics represent a class of antimicrobial peptides, in which the unusual amino acids dehydroalanine and dehydrobutyrine and the intramolecular thioether bridges (lanthionines) are important structural features for bioactivity.the lipid ii -nisin complex is responsible for pore-formation since the c-terminal part of nisin is inserted into the bacterial cell membrane which ultimately results in cell leakage and collapse of vital ion gradients. in order to increase the metabolic stability of nisin, the oxidationsensitive thioether bridges can be replaced by metabolically stable dicarba moieties, as successfully demonstrated by the synthesis of nisin ab(c) analogs containing alkane/alkene bridges [ ] . to obtain more insight into the importance of the cross-bridged de-ring structure (i→i+ , i+ →i+ connectivity) on nisin's bioactivity, we synthesized a series of all four diastereomers of the crossed alkene-bridged de-ring mimic, using ring-closing metathesis. all four diastereoisomers were obtained by hplc and structurally characterized by nmr spectroscopy. an orthogonal protection scheme was used, to enable the independent n-or c-terminal modification of the bicyclic hexapeptides with azide/alkyne functionalities. via cu(i)-catalyzed cycloaddition chemistries, alkyne-functionalized natural abc-fragments of nisin, which were obtained by tryptic digestion of full length nisin followed by hplc purification, have been conjugated to synthetic de-ring mimics to obtain novel nisin derivatives and their affinity toward lipid ii and pore-forming capacity have been studied. herein, we report on the details of the synthesis and characterization of the geometric isomers of the synthetic de-ring mimics, and their use as synthons in cu(i)-catalyzed click chemistry to obtain newly designed nisin hybrids as potential novel peptide antibiotics. università di ferrara, dipartimento di biochimica e biologia molecolare, ferrara, italy mirnas play an important role in regulation of gene expression, being involved in numerous processes such as cell proliferation, cell differentiation, apoptosis and also in the progress of diseases as cancer and cardiovascular disorders. mirnas associated to diseases recently become targets for the development of new drugs based on antisense oligonucleotides or analogues complementary to the chosen mirna, in order inhibit the binding of the mirna to its mrna target. therapeutic silencing of mirna has been also observed in several animal disease model. in this work we propose a new approach to interfere in the mirna function, based on peptide nucleic acid (pna) oligomers designed to be complementary to selected regions of the mirna precursor (pre-mirna). as the pre-mirna bases belonging to the stem are not perfectly complementary, we hypothesized that the mismatched duplex of the pre-mirna could be opened by pnas inhibiting of its maturation into mirna. two pna sequences, targeting respectively the "sense region" and the " ' end region" of the pre-mir were designed. pnas were conjugated to different carrier peptides, hiv-tat, r , k and two nuclear localization signal (nls and binls), in order to increase their cellular uptake. to verify the ability of the designed pnas to give strand invasion on the pre-mirna, we conjugated also pnas to the thiazole orange, a probe which lights-up upon hybridization the development of privileged molecular scaffolds efficiently mimicking reverse turn motifs has attracted remarkable interest when structural constraints are exploited to increase both binding and selectivity of model peptides. one of the successful approaches to restrict peptide conformation is the disubstitution in the α position of an α-amino acid, leading to a conformational constraint and a stereochemically stable quaternary carbon center. in particular, spirocyclic scaffolds are able to provide, upon the attachment of appropriate functional groups, useful high-affinity ligands, relevant to the field of drug discovery. at present, we are interested to spirocyclic tryptophan (trp) analogues, in order to develop new reverse turn nucleating moieties able to be inserted into pharmacologically relevant peptidomimetic compounds. among peptides sharing a tryptophan-containing β-turn motif of which the trp residue is critical for binding, we looked at the hormone peptide somatostatin, acting in various organ systems as a neuromodulator and a neurotransmitter, as well as a potent inhibitor of various secretory processes and cell proliferation. somatostatin and its analogue octreotide (sandostatin® drug, clinically used for the treatment of endocrine tumors and acromegaly) are thought to interact with the sst - receptors mainly by inserting a β-turn substructure, carrying a lysine (lys) and a trp side chain into a pocket of the g protein-coupled somatostatin receptor. we report here the preparation and structural characterization of a new , , , -tetrahydro-β-carboline (thbc)-based spirocyclic lactam as type-ii β-turn model compound and the application of its core structure to the synthesis of a somatostatin mimetic, whose biological evaluation is under way. the analogues of sfti- modified in the p position by, βand γ-amino acids and n-substituted β-alanines r. lukajtis, a m. filipowicz, a a. legowska, a d. debowski, a a. lesner, a k. rolka a a faculty of chemistry, university of gdansk, - gdansk, poland serine proteinases play very important roles in many physiological processes in humans, such as: food digestion, fertilization of the ovum, blood clotting and dissolution of blood clots, immune response. however, their uncontrolled activity can evoke serious pathological conditions. therefore, serine proteinase inhibitors are considered to be a promising class of therapeutic agents. trypsin inhibitor sfti- , on which we focused our attention in the last decade, is an attractive template for the design of such compounds. its primary structure is shown below: & gly-arg-cys(& )-thr-lys -ser -ile-pro-pro-ile-cys(& )-phe-pro-asp& the inherent feature of natural peptides and proteins is their low stability towards proteases, which seriously reduces their bioavailability. there is a growing need for the development of artificial biopolymers with diverse side chains, capable of mimicing peptide function. β-and γpeptides are an interesting class of peptidomimetics with significant chemical and biological properties. the present communication describes the chemical synthesis and inhibitory activity of a series of trypsin inhibitor sfti- monocyclic analogues (with disulfide bridge only) modified in p position by βand γamino acids and n-substituted β-alanine (β-peptoid units). the following mimetics of proteinogenic lys or phe were used: β hlys, β hphe, γ hhlys, γ hhphe, βhnlys, βhnphe. all compounds were synthesized manually on solid support. β-peptoid monomers were introduced into the peptide structure by two steps method [ ] . newly obtained sfti- analogues modified in p position by β-derivatives of lys and phe were able to inhibit bovine β-trypsin and bovine αchymotrypsin, respectively, whereas the remaining ones (except for [βhnphe ]sfti- ) appeared to be inactive. the notion that early soluble aß intermediates are endowed with cytotoxic effects suggests that a major effort should be directed toward the inhibition of amyloid aggregation at very early stages. inhibiting aß self-oligomerization could, therefore, provide a useful approach to treating and controlling the pathogenic pathways underlying alzheimer's disease (ad). likely, agents that target the basic molecular recognition process preceding the formation of early intermediates are the most valuable candidates. we have conjugated a trehalose moiety to the known ß-sheet breakers pentapeptides lpffd. trehalose has received a special interest because it has been found to be effective in the treatment of neurodegenerative diseases associated with peptide or protein aggregation. the glycosidic moiety was covalently linked to different regions of the peptides' primary sequence, including the n-terminus or c-terminus or the aminoacid side chain. this new class of peptides showed an increased resistance to proteases. in this work, the inherent ability of these peptides to recognize and bind the monomeric form of recently reported a d-amino acid-containing hiv protease inhibitor with a sulfonyl group showed an activity enhancement against drug resistant viruses. x-ray crystallographic study of the derivative revealed existence of four bridging water molecules. we suggest that the additional indirect interactions through water molecules induced the inhibitor's flexibility in binding conformation, keeping the affinity with the mutated proteases. oxalyamide, so-called oxamide, has two carbonyl oxygen atoms as hydrogen bonding acceptor similar to sulfonyl group, which is promising to interact with water molecules. to increase the numbers of bridging water molecules, we built-in two oxamide structures to both terminals of pseudo-symmetric compounds with hydroxymethylcarbonyl-hydrazide isostere. the derivatives were tested for inhibitory activity using wildtype hiv protease and a highly mutated protease with lopinavir resistance. we found that the loss of potency against the mutated protease was relatively small in the oxamide derivatives. the molecular dynamic simulations suggested the ability of bridging water formation of the two oxamide groups. optimization of the pna-synthesis using different bases for fmoc-deprotection s. rawer , k. braun , r. pipkorn life technologies, darmstadt, germany dkfz, heidelberg,germany pna (peptide nucleic acids) are considered as highly sensitive and specific tools for antisense strategies especially conjugated with cell penetrating peptides. individual designed shuttle systems can be applied in cancer diagnostics and possible therapy ( ) . it is, however, undisputed that proper pnas' syntheses prove to be a challenge for coupling and fmoc-deprotection. due to the structure-formation the success of the synthesis depends strongly from parameters, like activator's quality and deproctection kinetics correlating to the length of the pna polymer spps product. using the example of the spps pna synthesis' results of the coding sequence of c-myc human exon ii, different bases, acting as fmoc-deprotection reagents, are compared and analyzed aiming at optimizing the pna synthesis strategy ( ) peptidoglycans are central structural components of the cell wall of bacteria. several plant receptors are known to recognize peptidoglycan fragments. it is believed that these receptors form part of the defense mechanism against bacterial infections in several plant species. peptidoglycans consist of long chains of alternating β( - )linked glcnac and murnac moieties that are crosslinked by short, non-ribosomal peptides. these peptides consist of several d-amino acids and the symmetrical (r,s)diaminopimelic acid (meso-dap). in particular, the latter complicates the synthesis of peptidoglycan fragments due to the requirement for individually addressing the two pairs of functional groups. some chemical syntheses of peptidoglycan fragments have been reported [ ] [ ] [ ] [ ] , hhich involved multi-step formation of an orthogonally protected dap moiety, and elaborate oligosaccharide synthesis. here we present a new and simple approach to peptidoglycan synthesis which is based on the use of commercially available building blocks for the dap and oligosaccharide components. this allows easy access to a range of peptidoglycan fragments for structure-activity studies. the introduction of solid-phase peptide synthesis (spps) and the subsequent refinement of resins, linkers, coupling reagents and amino acid protecting groups allowed access to a wide range of peptides. therapeutic peptides, in particular, have benefitted from the maturation of spps, as complex peptides can be synthesized more efficiently in comparison to conventional solution phase synthesis. however, peptides containing multiple disulfide bonds often still remain difficult to make due to a lack of orthogonal cysteine protecting groups that can be used in routine spps. the cysteine protecting group s-tertbutyl mercapto (s-tbu) is commercial and orthogonal to other cysteine protecting groups. removal of the protecting group is facilitated by reducing agents (e.g. thiols or phosphines) and is stable to tfa and piperidine, hence compatible with fmoc/o-tbu peptide synthesis. however, the protecting group cannot be used in routine spps due to long deprotection times ( - h) . in certain cases it has been shown to be impossible to remove due to proximity of bulky protecting groups and sensitivity to certain sequences. additionally, reports of desulfurization of s-tbu protected cysteine to dehydroalanine, by the use of prolonged exposure to reducing agents, show the limitations of this protecting group. the concept of cysteine protecting groups labile to reducing agents is promising due to orthogonality to other cysteine protecting groups and the limitations of s-tbu initiated an investigation into novel reductive cysteine protecting groups. herein, we introduce s-tmp as a novel cysteine protecting group that is very labile to reducing agents. the increased lability, in comparison to s-tbu, allows utilization of reducing agent labile protecting groups in routine peptide synthesis of disulfide containing peptides. as modern automated spps protocols allow the assembly of larger and increasingly complex peptides, a precise control of the coupling reactions is a crucial prerequisite in peptide synthesis. monitoring the progress of synthesis allows the detection of undesirable products caused by side reactions, incomplete couplings or deprotections. although different methods have been developed for monitoring spps, we observed that the use of colorimetric test or continuous-flow uv absorbance of the reaction column effluent was not informative enough to identify difficult steps in the synthesis. in this study we demonstrate the usefulness of the combination of a mw-assisted mini-cleavage protocol and the uplc-esi-ms analysis for monitoring the quality of the reaction steps. as a proof of concept, based on this strategy, we monitored the synthesis of pthrp( - )nh (synthesised by fmoc/tbu rt-spps, liberty™, cem), characterised by a cluster of arginine residues in the - region. by the use of mw irradiation during the mini-cleavage protocol, we optimized time for mini-cleavages particularly in case of multi-arginine containing peptides, protected by pbf group. the results obtained by uplc-esi-ms showed that the complete removal of the pbf groups from the arginine sidechain residues required h at rt. on the other hand, the mw-assisted mini-cleavage monitoring let us to obtain final results just in min, confirming that the use of microwave irradiation in mini-cleavages is an efficient strategy to monitor also difficult peptide couplings, such as multiarginine peptides. identification of some deletion sequences was helpful to recognise critical couplings in order to adopt more efficient coupling strategies and therefore to optimise the final yield and purity of the crude peptide. development of green sustainable chemistry is currently regarded as a challenge in science and technology to reduce the use of organic solvents and utilize less toxic solvents instead. water and aqueous-based solvent systems represent an increasingly significant choice for replacing traditional solvents in synthetic chemistry. until recently, peptide synthesis in aqueous solution has remained largely unexplored. this is because the most common building blocks are sparingly soluble in water and are considered inappropriate for in-water peptide synthesis. we have developed a method for solid-phase peptide synthesis in water, which utilizes water-insoluble fmoc-amino acids that are converted to water-dispersible nanoparticles. in this way, the solubility problem is overcome. our technology, which uses suspended nanoparticle reactants for the coupling reaction, offers many advantages in terms of reaction efficiency over inwater synthesis using water-soluble or non-disperse reactants. however, there are two main problems with this nanoparticle approach; (i) slow reaction rates compared to general peptide synthesis in ordinary organic solvents (ii) poor yields for the synthesis of long peptides because the protected peptide chains on the resin have a tendency to aggregate in water. mw assisted spps is particularly attractive because of the widespread availability of the new technology, including automated peptide synthesizers. a trial of mw assisted inwater solid-phase synthesis using non-disperse boc-amino acids has been reported by albericio previously. currently, fmoc-amino acids are routinely used as building blocks for solid-phase peptide synthesis. with this in mind, we have developed a mw irradiation procedure aimed at reducing reaction time and increasing reaction yield for in-water solidphase synthesis using water-dispersible fmoc-amino acid nanoparticles. and we demonstrated in-water solid-phase synthesis of difficult sequence peptide with mw irradiation. m. lebl, z. flegelova spyder institute praha, czech republic cotton was shown as a convenient solid phase support earlier - , but did not find wide acceptance by the peptide community. we decided to try its application as (i) a support of choice for the synthesis driven by combination of capillary forces and gravity, (ii) support for synthesis utilizing in situ neutralization boc based protocol, (iii) support for combinatorial synthesis based on easy labeling and physical separability of cotton substrate, and (iv) support for multisupport synthesis. -we have built a simple synthesizer in which the cotton carrier (functionalized thread) is placed inside the capillary tubing and the appropriate reagents are introduced by connecting the inlet with appropriate reagents. the speed of "pumping" the reagents is driven by the difference between the elevation of the inlet and outlet of the capillary tubing. -we have shown that boc solid phase synthesis utilizing in situ neutralization is compatible with cotton substrate and provides high quality products. combining with the fact that cotton by itself acts as the self-association breaking agent, makes cotton a suitable carrier for synthesis of "difficult" sequences. -labeling of individual solid support particles can be easily based on the length of the cotton thread pieces, number and positions of knots, or their attachment to a secondary carrier. in addition, it is possible to synthesize peptides differing by the partial structure (alternative linkers, terminal modifications, etc.) in a mixture of classical resin with labeled cotton carriers, which are easily separable at the end of the synthesis. . use of microwave irradiation provides peptides in a fraction of time compared to conventional methods, and the peptides are also often generated in higher yield and purity. while microwave technology is particularly suited for the synthesis of "difficult" to synthesize peptides, this tool can routinely be used for the synthesis of a wide variety of peptides without the need for extensive method optimization. the focus of this study is to demonstrate how a peptide can be synthesized on a small scale (for example μmol) up through larger scales (> mmol) with ease. as a biologically relevant model peptide the last residues of the human platelet factor protein (hpf - ) was selected due to its significant antimicrobial activity. however, the problem of developing a robust fmoc thioester method is that the deprotection of the fmoc group with base at each cycle is not compatible with an active ester at the c-terminus. many ingenious approaches have been developed to generate the required thioester peptide. , , the most popular has been to use an nacylsulfonamide as a base and acid stable (safety-catch) linker for peptide synthesis. alkylation of the sulfonamide after peptide assembly makes the linker labile to cleavage with nucleophiles. whilst popular, it has been plagued by notoriously low yields which originate from the incomplete acylation of the resin-bound sulfonamide with the c-terminal residue, incomplete alkylation of the sulfonamide and the incomplete thiolysis of the resin-bound protected peptide. in this poster we describe the development of a novel dual linker strategy , involving anchoring of the sulfonamide linker to a standard acid-labile resin. this variation overcomes many of the current limitations of the sulfamylbutyryl linker approach and provides a simpler and scalable method for peptide ligation via fmoc spps. m. ziovas, d. tataraki, p. manousou, n. parveri, f. satoglou, d. gatos and k. barlos department of chemistry, university of patras, patras, greece solid phase peptide synthesis is traditionally performed by the attachment of the c-terminal amino acid through its α-carboxyl function on a suitable solid support and elongating peptide chain towards the amino terminal of the peptide by adding sequentially the amino acid residues in the gradually growing peptide chain. several thousands of publications and patents describe this methodology and its application for the production of peptide pharmaceuticals. in contrary to the attachment of the c-terminal carboxyl function, attachment of amino acids and peptides through an amino acid side-chain on suitable resins and their application in spps is described in a small number of publications and patents. most of these publications describe the attachment of the amino acids through a side-chain carboxyl group of asp and glu. in the present work peptides were synthesized very efficiently in high yield and purity by anchoring of side-chain hydroxyl, amino or thiol groups of amino acids, amino acid amides, amino alcohols or small peptides on resins of the trityl or benzhydryl type. several peptides of pharmaceutical interest, such as exenatide, octreotide, pramlintide, calcitonin, bivalirudin, insulin b-chain and others were produced as examples of this technology, either by the step-by-step procedure or by fragment condensation in solution and on solid phase. step given that some of these diseases are caused by a mutation and/or malfunction of an essential protein, a better understanding of the structure and function of such proteins will allow us to prevent, slow down or even cure these diseases. to increase our knowledge, the synthesis of the target protein, a fragment involved in its activity or interacting peptides that modulate the protein activity is often required. in some cases the preparation of a protein analogue that improves its efficacy is envisage. however, conventional solid-phase peptide synthesis methods have some limitations when attempting to achieve these complex sequences of considerable length. using novel technologies, such as a microwave-assisted solid phase synthesis, commonly found in many peptide synthesis labs, here we performed the step-wise solidphase synthesis of a protein holding more than residues (d-vegf). this synthetic achievement indicates the suitability of this approach for synthesis of long proteins or their analogues. the detailed synthesis of the enatiomeric version of vegf and the selenomethionine substituted analogues of huprp ( - ), proteins involved in angiogenesis and prion protein amyloidoses respectively, are described as study cases, where the use of microwaves allow us to obtain them in a fast and efficient manner. therefore, the development of novel peptide analogues with enhanced in vivo stability could potentially provide therapeutic alternatives. the pharmacological evaluation of a bioactive peptide [des-gly ,tyr (ome),d-leu ,aze-nhet ]gnrh, analogue , is presented herein. in vitro (kidney mouse membranes) and in vivo (clinically relevant pharmacokinetic mouse model) bioassays were coupled to liquid chromatographytandem mass spectrometry. analogue , an agonist of the gnrh receptor with a binding affinity in the nanomolar range, caused testosterone release in mice that was acutely dose-dependent, an effect blocked by cetrorelix. repeated dosing studies in mice demonstrated that analogue was well tolerated and had potency similar to that of leuprolide, based on plasma and testis testosterone reduction and histopathological findings. analogue also shared with leuprolide similar significant antiproliferative activity on androgen-dependent prostate cancer (lncap) cells. on the basis of pharmacokinetic advantages, we expect that analogue or analogues based on this new design will be therapeutically advantageous for the treatment of cancer and endocrine disorders. cortexin is a polypeptide drug isolated from cattle and porcine brain cortex. cortexin is effective in monotherapy and in combination with traditional methods of treatment. cortexin produces tissue-specific, regulatory, and reparative effects on the brain cortex and contains active low-molecular-weight neuropeptides (< kda) penetrating through the blood-brain barrier. cortagen is a tetrapeptide h-ala-glu-asp-pro-oh (geropharm) produces nootropic and neuroprotective effects. oleylcortagen is a lipophylic analog of cortagen c h o-ala-glu-asp-pro-oh was created for increased proteolytic stability and increased penetrating through the blood-brain barrier. the main aim of our investigation is the analysis of psychopharmacological profile of peptide preparations in comparison with piracetam. have been shown oleylcortagen ( mg/kg) and piracetam ( mg/kg) possess activating effect on motor and research components of behavior in «open field» test. two of peptides (oleylcortagen, cortexin) decreased period of immobilisation and demonstrated antidepressant effects on rat behavior in the porsolt's test, on other hand cortagen demonstrated depressant action. therefore, the significant psychoactivating properties are typical for oleyl-cortagen, cortexin. the mechanism of the action of these peptides can be explained from the viewpoint of the regulatory cascade. they produce a direct information impact on cell structures of the brain, and then promote release of the regulatory peptides, which in turn, induce the release of the next group of peptides. neurology, queen square, london wc n bg, uk one of the hypotheses of alzheimer's disease neuropathology involves beta-amyloid (βa) binding with proteins on neuronal cell surface which leads to cell lysis and amyloid plaque formation. according to the latest data α -type of the nicotinic acetylcholine receptor (achr) and the prion protein can be the target for betaamyloid toxicity [ , ] . aggregated βa causes many pathological changes in cultures of mixed neurons and astrocytes such as sporadic cytoplasmic intracellular ca + -signal, activation of reactive oxygen species (ros) production and cell death. in the present work we demonstrated the ability of affinity purified antibodies to synthetic fragment - of α -subunit of the achr (achrabs) or to peptide - of the prion protein (prpabs) to protect cells from βa induced cell death. we also showed that both antibodies did not block βa induced ca + -signal in astrocytes. however, preincubation of cortical co-culture of neurons and astrocytes with achrabs or prpabs significantly reduced the rate of caspase activation and the rate of βainduced ros production via modulating nadph-oxidase. more detailed research of involvement of α -type achr revealed that α-bungarotoxin was also very effective in the inhibition of caspase activation and superoxide production. the observed positive effect of antibodies to α -type achr or to the prion protein gives an additional explanation regarding the involvement of these proteins in ad pathology and provides new approach into an anti-ad vaccine design. capturing and macrophage-aided clearance of amyloid beta by surface modified proteineous particles m. richman, s. rahimipour department of chemistry, bar-ilan university, ramat-gan , israel imbalanced homeostasis and oligomerization of amyloidβ (aβ) peptide in the brain are hallmarks of alzheimer's disease (ad). microglia and macrophages play a critical role in ad progression by clearing aβ from the brain or inducing inflammation. recent evidence suggests that the phagocytic pathway of aβ may be defective in ad microglia/macrophages that contributes to the build-up concentration of aβ in the brain. , therefore, efforts have been directed toward developing treatments that trigger these cells to clear aβ through alternative mechanisms. we have recently demonstrated that protein microspheres modified at their surface with multiple copies of an aβrecognition motif can strongly bind aβ, inhibit its aggregation and directly reduce its toxicity by sequestering it from the medium. here, we describe how the aβ-bound microspheres can stimulate microglial cells and be phagocytosed through a mechanism that is distinct from that of aβ. the phagocytosis was mostly effective with microspheres having diameter size of about . - mm and introduction of polyethylene glycol to the surface of the microspheres changed the kinetics of the phagocytosis. moreover, while aggregated aβ induced a significant inflammatory response that was manifested by the release of tnf-α from the microglial cells, the aβ-bound microspheres dramatically reduced the amount of the released cytokine. our data suggest that surface-modified microspheres could be utilized to detoxify other pathogenic or misfolded proteins that their accumulation may lead to genesis of other diseases. vasoactive intestinal peptide (vip) and its derivatives have been thought to be promising drug candidates for airway inflammatory diseases. however, the therapeutic potential of vips is highly limited because of rapid metabolic degradation and systemic side effects following systemic administration. previously, to overcome these drawbacks, our group developed a novel vip derivative, [arg , , , leu ]-vip-grr (ik ), with improved metabolic stability ( ), and respirable powder (rp) formulation of ik (ik -rp) for pulmonary administration ( ) . these attempts successfully led to enhanced pharmacological effects of ik in the airway system and reduced systemic exposure; however, further chemical modification of ik with a focus on metabolic stability might provide better clinical outcome. the present study aimed to design a pegylated vip derivative with improved metabolic stability and to develop its respirable powder (rp) formulation for inhalation therapy. ik was chemically conjugated with peg ( kda, p k), the physicochemical and biochemical properties of which were characterized by cd spectral analysis, binding assay, and metabolic stability. the rp formulation of pegylated ik (ik /p k) was prepared with a jet mill, and in vitro inhalation performance and in vivo pharmacological effects in antigen-sensitized rats were also evaluated. the cd spectral analysis demonstrated that peg conjugation had no impact on the solution structure of ik . although receptor-binding activity of the ik /p k (ic : nm) was estimated at ca. -fold less than that of ik (ic : . nm), metabolic stability for the ik /p k was highly improved. according to the laser diffraction and cascade impactor analyses, ik /p k-rp had fine in vitro inhalation performance. insufflation of ik /p k-rp ( μg of ik /p k) in antigen-sensitized rats resulted in marked attenuation of inflammatory events, as evidenced by significant decrease of inflammatory biomarkers and granulocyte recruitment in pulmonary tissue at h after antigen challenge. from these findings, pegylation of vip derivative, as well as its strategic application to the rp formulation, might be a viable approach to improve its therapeutic potential for treatment of airway inflammatory diseases. the previous studies have shown that trkb (tropomyocin receptor kinase) acts as an oncogenic agent and its binding to bdnf (brain derived neurotrophic factor) activates signaling angiogenesis of tumor proliferation [ ] . for finding the most stable and potentially effective peptides against the trkb, we applied the following protocol. at the first step of this protocol we designed a peptide library by using sequence tolerance method in rosetta . package, then peptide energy optimization performed by backrub protocol for finding the most stable peptides. the five best peptides in energy optimization selected based on backrup scores by using r package [ ] . d-structure prediction of the selected peptides was performed by using molecular dynamic in hyperchem software. docking of peptides with trkb receptor was carried out in haddock software. we used cyclotraxin, a selective trkb inhibitor as positive control for this protocol. cyclotraxin and the peptides were compared by anova or t-test. the peptides are going to be tested against the trkb in an in vitro model. dirucotide (mbp - ) is a synthetic peptide analog of , that consists of amino acids and tested in a phase trial were failed to reach his tolerance level on previous phase ii in rpms patients. one of the major disadvantages of peptide therapy is the activation of proteolytic enzymes, leading to peptide degradation. to address this problem cyclic peptide analogues have been synthesized. thus we synthesise a linear and cyclic analogues of dirucotide. the two analogues were synthesized by changing the amino acid residue at position from to the synthesis of the linear peptide, as well as of the cyclic one, was carried out by the fmoc/tbu methodology, utilizing the -chlorotrityl chloride resin (cltr-cl). the purification was achieved using semi-preparative rp-hplc and the identification was assessed by analytical rp-hplc and by mass spectrometry (esi-ms). the linear and cyclic peptide analogues will be used in human t-cell cultures to test their immunogenicity in patients versus healthy controls. in the first approach a reporter moiety was introduced to diagnose and treat cxcr related diseases. therefore, an anchor point to attach additional molecules to the ligand was elucidated. using sar studies to optimize the linker from the ligand to the detectable moiety the excellent receptor affinity could be retained. in a final step the reporter moiety was introduced to give a ligand for diagnosis via pet imaging and for possible endoradiotherapeutic applications. originating from the dimeric motif present in many active cxcr ligands several dimers were prepared using a monomeric ligand identified in the prior study. comparison of monomers and dimers yielded a possible subsite binding mode explaining why the dimers exhibit enhanced affinity using a model derived from the origins of the monomer. several peptidomimetic modifications were introduced to the ligand to reduce the peptidic structure. in a conformationally guided approach introduction of a peptoid motif could enforce a single active conformer that was enhanced through subsequent modifications. this yielded a compound times better than the original cxcr antagonist which is the most affine cxcr ligand reported so far. our previous studies have demonstrated that pace represents a potential therapeutic target for the treatment of prostate cancer . moreover, we have developed potent and selective pace inhibitor, ( -fold specificity over furin), known as multi-leu or ml peptide, which has a significant inhibitory effect on the proliferation of prostate cancer cell lines. peptide-based drug candidates can be limited by their poor metabolic stability and low bioavailability. thus, we performed structure-activity relationship studies to improve the pharmacokinetic properties of our ml inhibitor. we have designed and synthesized new ml peptide analogs having various chemical modifications. first, based on our previous results, we combined the most effective modifications of position p (d-amino acids) and p the arginine mimetic amidinobenzylamine (amba) to improve overall properties of our leading compound. second, the n-terminus of the resulting analogs was modified with a fatty acid, in order to enhance their cell permeability properties. third, we modified the inhibitors with a peg moiety to increase their stability and bioavailability. we tested the inhibitory activity, stability in plasma, cellular uptake, and cytotoxicity of each inhibitor. the results of this study demonstrate that the presence of the n-terminal extension (c or peg ) does not affect activity of our inhibitors. on the other hand, we show that introduction of the peg moiety does not increase cytotoxicity of ml analogs. it is interesting to note that the pegylated analog ac-peg -d-leu-lllrvkr-amba has better cell-permeability activity than its counterpart without peg unit. this combination of pharmacological properties makes our new ml analogs promising candidates for the development of potential anti-prostate cancer agents. [ ] peripheral coadministration of rf with opioid analgesics led to confirm the involvement of npff receptors as a part of the antiopioid system. indeed, rf was able to reverse the opioid induced hyperalgesia in rat (randall selitto test). then, a complete structure-activity relationships analysis was performed with rf , assessing the involvement of each moiety for affinity and selectivity towards both npff receptors. a first exploration of the n-terminus part of rf (> synthesized derivatives) led to replace the hydrophobic adamantane moiety by a hindered aromatic group, providing a subnanomolar npff ligand with more than two log-units selectivity against npff . then, the removal of the cterminal amide function led to reduce the dipeptide arg-phe-nh into an arginine derivative. in spite of an initial loss of affinity, optimization of the phenethyl moiety at the cterminus part of arginine led to non-selective nanomolar ligands for both npff & receptors. next, we applied efficient methodologies in order to synthesize non-natural analogs of arginine, leading to various compounds exhibiting selectivity for either npff or npff receptors. in particular, compound rf was identified as a selective npff antagonist (npff , ki = nm; npff : ki > μm). lacking of analgesia properties, oral administration of rf ( mg/kg per os) to rats was able to fully reverse fentanylinduced hyperalgesia. rf is the first orally available npff antagonist capable of reversing opioid induced hyperalgesia at low dose. moreover, this result allows identifying for the first time npff receptor as a key-partner of the anti-opioid system. administration of multiple antiplatelet agents has become the mainstay in the treatment of acute coronary syndromes in everyday clinical practice. we have previously reported significant antiplatelet effects of novel synthetic peptides' single administration on experimental carotid artery thrombosis in rabbits . in the present study we sought to investigate the peptides' effects when administered in marginally effective doses (significantly lower than those utilized in the past), in animals that had previously received low doses of aspirin. the peptides when co-administered with aspirin preserved the carotid artery's blood flow, in contrast to the total artery occlusion observed in animals receiving aspirin and placebo. blood flow at min after electrical stimulation was reduced to . ± . % and . ± . % in the ymesradr and psrcdcr-nh groups respectively (p< . vs aspirin and control). thrombus weight was significantly reduced in animals receiving ymesradr and psrcdcr-nh versus aspirin and control ( . ± . mg and . ± . mg, vs . ± . mg and . ± . mg respectively, p< . ). platelet aggregation was significantly inhibited in the ymesradr and psrcdcr-nh groups by . ± . % and . ± . % for adp (p< . vs aspirin and control), and . ± . % and . ± . % for aa (p< . vs aspirin and control), respectively. blood loss did not significantly differ among the various groups. administration of novel synthetic peptides, even at marginally effective doses, in animals previously treated with low doses of aspirin results in enhanced antiplatelet effects in an experimental model of arterial thrombosis. the study of peptide metabolism is particularly important when examining anticancer peptides; it can provide pivotal clues for the evaluation and improvement of stability of a peptide drug leading to enhanced pharmacokinetics and efficacy. gnrh analogues are used for the treatment of prostate cancer. as with other peptides a drawback is their short half-life due to their metabolic degradation. in order to examine the stability of these analogues we have developed several in vitro peptide stability and metabolism assays using specific tissues, isolated membranes, cancer cells that are analyzed subsequently using lc-ms/ms based approaches. such in vitro studies are followed up with pharmacokinetic studies in mice in order to establish the correlation between in vitro and in vivo approaches. the kidney is the main metabolic organ for peptide metabolism and for that reason we employed a peptide stability and metabolism assay previously described by our group using isolated mouse kidney membranes for the evaluation and comparison of different gnrh analogues. we tested gnrh analogues in other tissues such as mouse plasma, which is the "distributing" tissue for these drugs and mouse brain homogenate, a tissue known for its abundance in peptidases and relevance for centrally mediated effects. stability studies were also performed in cancer cells. in all cases lc-ms/ms based assays were developed for measurement of peptide drug and the resulting metabolites. for triptorelin, and a series of gnrh analogues, degradation and metabolite formation was studied by our mouse kidney membranes assay. studies of coadministering the peptide of interest with inhibitors that are presumed to block the metabolism in mice are ongoing. the vulnerability of gnrh analogues was verified after incubation with plasma and brain homogenate and by metabolite identification we obtained clues about the key cleavage sites. the described in vitro/in vivo protocols provide valuable information that could lead to the synthesis of more stable anticancer peptides with improved anticancer efficacy. in this work, we explored the use of a high-throughput synthesis and screening approach with peptide arrays to identify and structurally optimize shortened hiv- fusion inhibitors. the peptide array technology involves miniaturized synthesis of immobilized peptides, followed by affinity testing with a five-helix bundle, as a mimetic of the fusogenic gp protein. this exercise resulted in the identification of a class of truncated peptides which demonstrates a surprisingly high antiviral potency, despite the absence of the pocket-and the lipid-binding domain. the propensity of these peptides to adopt the bioactive α-helical conformation in solution as determined by circular dichroism, could be the key factor for this unexpected potency. these peptides are promising leads for the treatment and prevention of hiv. the pathological role of platelets in cardiovascular disease (cvd) is well established. platelets secrete adp to recruit additional platelets to a thrombotic site. we have previously identified novel cell-permeable peptide modulators of platelet function by using a bioinformatic screen based on patterns of evolutionary conservation in transmembrane proteins . in this study we further explored peptides derived from cadherin cell adhesion molecules. we explored a range of overlapping peptides derived from different cadherins varying from - amino acids long. peptides are synthesized and analyzed in a high-throughput platelet adp secretion assay. peptides ( . - μm) were assessed in the presence and absence of thrombin receptor activating peptide (trap; μm). we identified cadh- and proteins, but not cadh or in human platelets using western blotting and mass spectrometric analysis. peptides derived from paralogous juxta-membrane, cytoplasmic regions of these cadherins are potent modulators of platelet secretion. by systematically deleting amino acids from c or n-terminus of active peptides, we established that the minimal functional sequence for biological activity was a short six-residue motif, which corresponds to the known catenin-binding region of cadherin tails (kepllp) . peptides alone have no biological activity. however, they potentiate the response induced by the platelet agonist trap at low doses. thus we have identified a cadherin-derived peptide that can modulate platelet secretion events. this highlights a previously unknown role of cadherins in the regulation of platelet function. agents that interfere with cadherin signaling in platelets might have a therapeutic role in cvd. ageing of the brain leads to impairments in cognitive and motor skills, and is the main risk factor for several common neurological disorders such as alzheimer's disease (ad) and parkinson's disease (pd). altered protein handling (proteolysis, repair system, chaperones) forms a basis for a large number of protein conformational disorders. extra-and intracellular, as well as intranuclear accumulation of abnormal proteins, in the form of protein inclusions and aggregates, and dysfunction of the quality control mechanisms are common in all these disorders. alterations in protein homeostasis occur with age, causing molecular changes such as protein misfolding and aggregation. many biologically active proteins lack stable secondary and tertiary structure, these are called intrinsically disordered proteins (idps), some of them (e.g. β-amyloid, α-synuclein) are coupled to neurodegenerative disorders. idps exist as assemblies of rapidly fluctuating structures undergoing coupled folding and aggregation process. protein aggregation is characterized by polymorphism, where a mixture of soluble oligomers, amyloid fibrils and amorphous aggregates is the final product. soluble oligomers are inevitable formed during the self-association process and might initiate the neurodegenerative cascades of ad, pd and similar diseases. the emerging consensus that protein misfolding (leading to idps) is the cause of several neurodegenerative disorders now offers the opportunity to develop a general therapy. soluble oligomers with id regions are potential drug targets. recently short peptide fragments and small peptidomimetic molecules have been found also in our laboratory; these molecules bind to the id regions of β-amyloid and are putative drug candidates. precise control of bleeding is ensured by anticoagulant mechanisms, which under normal conditions prevail over coagulants mechanisms. disrupting the balance between procoagulant and anticoagulant systems due to congenital or acquired defects leading to thrombotic disorders. anticoagulants are substances that inhibit blood clot formation. their action consists in inhibiting the synthesis of prothrombin, the substances forming thrombus as well as some coagulation factors. many peptides and proteins with different molecular weight, such as antistasin (ats), ghilantens, hirudin, etc. showing high anticoagulant activity are isolated from salivary glands of ticks and leeches. they inhibit the action of serine proteinases from blood coagulation cascade. this creates an opportunity for targeted synthesis of low molecular weight analogues of some of these proteins, which can be used in the prevention and treatment of thrombotic disorders. ats is competitive, slow-binding inhibitor of factor xa. it is well known that blood coagulation could be blocked at different stages of the coagulation cascade through inhibition of various enzymes. therefore, it is interesting to determine the place of action of newly synthesized antithrombotic agents in the blood coagulation cascade. this can be done by determining the inhibitory constants of newly synthesized peptides on different enzymes of this cascade. herein we report on our kinetic investigation of newly synthesized peptide amides, analogues of isoform of ats . ki, km, vmax and type of inhibition on the factor xa, thrombin and plasmin were determinate. some interesting differences between type of inhibition of ats, free acids and amide analogues of ats were revealed. to evaluate the relative anti-platelet aggregation activities of each peptide, the lebetins were chemically synthesized and fully characterized. here we described the synthesis, the solution structure of lebetin g -g from the venom of vepera lebetina by h bidimensional nmr and the relation structure-activity. this peptide has been demonstrated to be associated with a potent anti-platelet aggregating activity. the g -g three dimensional structure consists in a compact β-bulged hairpain core from which emerges one loop and the cterminus and the n-terminus. we report on an approach whereby ligands are designed to bind and stabilize the - region of aβ in an α-helical conformation. these ligands reduce aβ toxicity to cells in culture and to hippocampal slice preparations. in addition, when these inhibitors are administered to drosophila melanogaster expressing human aβ ( - ) in the central nervous system, a prolonged lifespan, increased locomotor activity, and reduced neurodegeneration is observed . stabilization of the central aβ α-helix appears to counteract polymerization into toxic assemblies and indicates that this approach holds promise for the development of orally available compounds against alzheimer's disease. encouraged by the above results we are currently developing a second generation of designed ligands. this involves synthesis of different new peptoids and unnatural amino acids. additional support for the concept comes from recent molecular dynamics simulations that also uncover details of the mechanism of unfolding of the aβ central helix as well as retardation of the folding in presence of ligands designed to interact with the native helical conformation . synthesis and methodology for new ligands, which includes synthesis of novel amino acids, will also be presented. triostin a is a well-known natural product with antibiotic, antiviral, and antitumor activities. it inhibits rna synthesis by bifunctional intercalation into dna base pairs through its two quinoxaline units showing cpg selectivity. triostin a must adopt an altered conformation upon dna bisintercalation that is substantially different than its preferred native x-ray or solution conformation. this fact suggests that the destabilizing conformational change in the cyclic octadepsipeptide counteracts much of the gains derived from a second intercalation. nonetheless, the wide range of pharmacological activities exhibited by this compound prompted interests in identifying novel and additionally potent lead compounds with improved pharmacokinetic properties for clinical applications. herein, a library of twelve simplified triostin a analogues has been synthesized by solid-phase peptide synthesis. the introduction of the key quinoxaline units was carried out in solution. the analogues' conformation corresponds to the staple form that bisintercalator cyclic (depsi)peptides adopt when binding to dna and, in addition, some of the synthesized compounds showed improved solubility. our library was evaluated for its antiproliferative activity against four human cancer cell lines and one analogue showed greater cytotoxicity than triostin a and even comparable activity to doxorubicin, a very commonly used drug in cancer chemotherapy nowadays. surprisingly, little is known about its mechanism of degradation in solution or the degradation products. a recent study identified monomeric polysulfides and dimeric degradation products, postulated to derive from β-elimination followed by deamidation and dimerization. we recently reported that degradation of oxytocin and its analogues in aqueous solution at ph . produced monomeric polysulfides with up to sulfur atoms as well as dimeric products. unexpectedly, incubation of ot or of various analogues modified in position resulted in identical dimeric degradants. we concluded that β-elimination via breakage of the c-s bond of cys must be a key step of the process, and that the resulting Δala residue would have to undergo further modifications to yield the same dimeric products independently of the substitution of the n-terminal nitrogen. here we further clarify the degradation mechanism and propose a structure for the dimers. we postulate that hydrolysis of the Δala residue yields an n-pyruvoyl linear peptide, which loses one sulfur atom and subsequently forms dimers, which we found are linked by one disulfide bridge and one non-reducible bond. the putative linear n-pyruvoyl oxytocin intermediate was synthesized and found to degrade to the same dimers as the ones in the incubations of ot. a [u- c]cys ot analogue gave degradation products with c-nmr spectra consistent with a non-stereospecific aldol-type condensation. detailed experimental procedures, structures of the degradants and the postulated mechanism of ot degradation in near neutral solutions will be presented. inserm u paris, france αiibβ is the main platelet integrin and is responsible for platelet aggregation. a lipid-modified peptide corresponding to αiib intracellular sequence - (palmitoyl-k-l eeddeege , pal-k- - ), is platelet permeable and inhibits human platelet aggregation induced by thrombin . ymesradr, a peptide corresponding to the extra-cellular sequence - of αiib, is a platelet activation and aggregation inhibitor . the aim of the present study was to investigate the cooperativeness of the intracellular and extra-cellular peptides on platelet aggregation and their effect on the phosphorylation of fak and erk. pal-k- - together with the extracellular ymesradr peptide, at concentrations lower than their ic values, showed cooperative inhibition of platelet aggregation. the peptide combination inhibited also fibrinogen and pac- binding to activated platelets. fak phosphorylation is a postaggregation event related to outside-in activation of the receptor. the combination of peptides inhibited fak phosphorylation. erk phosphorylation is independent from platelet aggregation, and is enhanced by rgd-peptide inhibitors. the combined peptides inhibited erk phosphorylation. ovarian cancer (oc) is considered a rare disease and represents the fifth most common cause of death from cancer in women. the standard first-line treatment consists of a combination of paclitaxel and carboplatin (ddp) or carboplatin alone. in the case of progressive disease or drug resistance to platinum-based agents, either alone or in combination, investigational compounds should be used ( ) . the mechanisms behind acquired resistance to ddp and its derivatives are not clear yet, although it is evident that the process is multifactorial, including enhanced dna repair processes. some peptides designed from the interface subunit of the human thymidylate synthase (ts) have been identified recently ( ), as effective anticancer agents against sensitive and resistant oc cells. one of them was also able to recover the cellular sensitivity towards cisplatin in resistant oc cells in the μm range. to improve its potency and selectivity structural studies have been performed in combination with cellular assays aimed at understanding the mechanism of action. a label-free quantitative proteomic approach has been undertaken to study the effects of the peptide on the proteins involved in the modulated metabolic pathways, in particular those involved in the folate metabolism. structure-activity relationships (sar) have been performed to improve the lead peptide pharmacodynamics. all the compounds have been assayed and a protein profile set was studied to mark and validate their behavior as inhibitor of oc cell growth. hepatitis c is a liver disease provoked by a virus known as hcv. the disease is insidious. hcv causes anorexia, nausea, vomiting, fever, fatigue and jaundice. in about % of sufferers the disease is short, but others become chronic. in the chronic form in about % of cases the final result is cirrhosis of the liver and in the remaining % it leads to liver cancer. hcv is a very serious problem today. about % of people infected with hcv worldwide, i.e. about million are residents of europe. million people carry the disease as a chronic illness with the potential to develop into cancer in their liver. all these people represent a "reservoir" for storage and distribution of hcv. ribavirin, the nucleoside analog -β-d-ribofuranosyl- , , triazole- -carboxamide, known by the trade name virazole (also known as rebetron in combination with interferon-α), exhibits antiviral activity against a variety of rna viruses (paramyxoviruses, flaviviruses, etc.) as well as some dna viruses. in humans ribavirin is currently used in combination with interferon-α to treat hcv infections. this lack of strict specificity and a broad spectrum of activity are due to its multifunctional mechanism of action against viruses. these characteristics have made ribavirin a drug of substantial research interest. unfortunately, ribavirin shows a significant toxicity, causing bleeding in accumulation [ ] . herein, we report a total synthesis of modified in position ' of ribose residue, ribavirin in order to be further linked to cell penetrating peptides (cpps). in addition the synthesis of some cpps as well as bonding between two parts of final hybrid molecules will be reported. in our case the design of new hybrid molecules is done in order to: (a) vectorize ribavirin into liver cells; (b) transport ribavirin molecule trough cell membrane and (c) decrease toxicity of ribavirin. to obtain oligomeric aβ peptide, our laboratory uses a precursor depsipeptide of aβ. this precursor, termed as "iso-aβ" has an ester bond between the side chain of ser and gly . at physiological ph this ester bond becomes an amide bond via an o→n acyl shift. binding partners by which the oligomeric aβ mediates its toxic effect has not been yet investigated in the proteome or subproteome level. we used protein array technology to study the interaction of oligomeric aβ with recombinant human proteins, immobilized on a protein chip. aβ binding proteins were identified with the aid of a monoclonal aβ antibody. altogether proteins were found to interact with our aβ-oligomers. these proteins were grouped according to their function. one of the major groups contained proteins participating in translation. these proteins were found in ribosomes. to prove our proteomic results ribosomes from rat hippocampus were isolated. elisa experiment revealed that aβ binds to ribosomes in a dose-dependent manner. using the sequence of the aβ-binding proteins a homology search was performed to find oligopeptides, that possibly bind to aβ. based on these sequences a peptide chip containing hexapeptides was prepared. aβ interacting peptides were identified with a monoclonal antibody. several peptides were synthesized and tested on mtt assay. two out of four compounds inhibited the toxicity of aβ on rat hippocampal slices. summarizing our results aβ binding proteins and peptides were identified. knowledge about aβ binding proteins can help to understand the pathogenesis of ad, such us the possible involvement of ribosomes. oligopeptides can be lead compounds of future drug development. huge proteolytic complex named proteasome catalyzes protein degradation in every eukaryotic cell. it consists of subunits forming four stacked rings and one or two regulatory caps. two inner rings of the proteolytic part contain three catalytic β-subunits that possess different substrate specificity. higher vertebrates can express γinterferon-inducible immuno-β-subunits. proteasome plays an essential role in continual turnover of intracellular proteins and in antigen processing. autoimmune diseases such as multiple sclerosis and its murine model eae are believed to rise from breakdown of tolerance of the immune system. it assumed that immunoproteasome could play an important role in autoimmune diseases. several classes of chemicals proved to be inhibitors of proteasome and the most active are boronate peptide derivatives. these inhibitors totally inactivate proteasome and result in full stop of intracellular protein turnover and cell death via apoptosis. another class of inhibitors, epoxy ketones, was shown to be more selective for immunoproteasome and could be used not for full stop of proteasome function, but for fine tuning of altered proteasome functioning. we examined properties of several inhibitors of four different classes, namely peptide boronate bortezomib, peptide aldehyde mg , lactam lactacystin, and peptide epoxyketones epoxomicin, mg ek, uk and pr- . for inhibition experiments we used proteasome isolated from eukaryotic cell lines cho, nso and hek, treated and non-treated with γ-interferon, as a model cells contatinig constitutive and immunoproteasome. the upregulation of proteasome immunosubunits was revealed in cho and nso cells treated with γ-interferon. the ic values for all studied inhibitors were obtained, and ki in some cases were calculated. the epoxyketones were shown to selectively inhibit in submicromolar concentrations the proteasome sample which contain high amount of immunosubunits. in order to find an effective antimalarial, this study refers to some angiotensin ii (aii) analogues which were considered the important physicochemical characteristics described by silva et al. to verify the biological activity against plasmodium gallinaceum and to understand the hydrophobic cluster influence, explained by tzakos et al. these analogues were synthesized and characterized as described by silva , as well as the biological assays and comprises, to verify: the hydrophobic cluster activity -a) drvyhipf; b) drvypr; c) ryhipf and d) fphiyvrd; the importance of these residues in aii molecule -e) rypf; the importance of aromatic residues -f) yhpf and the action of these hydrophobic residues, when interacting with the parasite membrane -g) vipf. it was observed that in a ( % of bioactivity), the phenol group of tyr is close to imidazole group of his that could promote a hydrogen bond formation. besides that, could occur van der waals interactions between ile and phe residues due its proximity and non-polar characteristic. these interactions could not be effective in native aii ( %) , because ile residue promote a steric influence on the organization of his and tyr residues that not exist in b ( %). in c ( %) and e ( %) analogues, the influence of the arg residue could promote a cation-π interaction with tyr residue and the cluster may have suffered slight destabilization and its antiplasmodial activity was compromised subtly. in d ( %), the electrostatic change, obtained with the total inversion can have disordered its interaction with parasite membrane, since it is not related to membrane receptors, because d-aii presented % of biological activity. moreover, hydrophobic and aromatic residues importance was confirmed through the results obtained % and % of activity, with g and f, respectively. we conclude that hydrophobic cluster modifications and interactions of amino acid side-chain influences in the biological activity. closed joint-stock company "vertex", st-petersburg, russia creatine (cr), a small molecule synthesized in the kidney, liver and pancreas plays important role in atp synthesis, replenishing its store even in the absence of oxygen. cr is able to protect brain cells against ischemic damage; however it has poor ability to penetrate the blood-brain barrier without specific carrier protein. thus, synthesis of stable hydrophobic derivatives capable of crossing the bbb by alternative pathway is of great importance for the treatment of different neurological diseases including stroke, traumatic brain injury and hereditary crt deficiency. here we describe the synthesis and biological activity of new hybrid compounds -creatinyl amino acids. originally the title compounds were synthesized by guanidinylation of sarcosyl peptides. however, for large scale synthesis better results can be obtained using direct cr conjugation with amino acid or peptide derivatives by isobutyl chloroformate method. addition of equivalent amount of ptoluenesulfonic acid as lipophilic counterion ensures efficient cr dissolution in dmf along with its simultaneous protection towards intramolecular cyclization. it excludes the application of expensive guanidinylating reagents and permits to simplify the synthetic procedure. purification of final product and its conversion into appropriate salt form can be achieved by iec followed by crystallization from organic solvents. synthesized creatinyl amino acids and peptides exhibited significant biological activity in different assays including platelet aggregation test, ischemic stroke and nano -induced hypoxia model. one of the most effective compounds -creatinyl-glycine ethyl ester increases life span of experimental animals more than two times in hypoxia model and has neuroprotective action in brain stroke model when applied both before and after ischemia. these data evidenced that creatinyl amino acids can represent promising candidates for the development of new drugs useful in stroke treatment. the efficient recognition and destruction of tumor cells via specific cellular markers is a major goal in cancer therapy. various growth factor receptors such as egfr, hgfr, vgfr and their downstream signaling networks have been proven to be effective molecular targets, as they are frequently involved in cancer proliferation and metastasis. downregulation of these receptors and/or blocking their signaling pathways have clear anti-tumoral effects. drugs based on monoclonal antibodies (mab) targeting such cell surface receptors have attracted a lot of attention as a new generation of therapeutics. however, their production is costly and identifying new, variable routes to modified molecules with similar properties is currently a major focus. , here we present an approach to chemically synthesize a molecule that combines the mode of action of antibodies with the advantages of smaller, chemically accessible molecules. these "synthetic antibody" (sab) molecules contain a chemoattractant that activates the innate immune response and resembles the fc domain of a typical antibody. specificity is imparted by two binder peptides that assume the function of the variable antibody domains and bind to a cell surface target. the fc and fab domains of the sab molecules are connected via polyethylene glycol linkers. sab molecules are prepared by solid phase synthesis, a flexible technique that allows fast production, full control of their properties and targeting two different cell surface receptors (bispecific tumor targeting). they are currently tested in vitro and in vivo for their effect on the innate immune system, general toxicity and selective binding to cancer cells. the key enzyme in the processing of polyproteins translated by viral rna genome of sars-cov is a kda protease called c-like protease ( cl protease). sars cl protease is a cysteine protease containing a cys-his catalytic dyad, and cleaves precursor poly proteins at as many as conserved site involved a conserved gln at the p position and a small amino acid (ser, ala, or gly) at the p' position. due to its functional importance in the viral life cycle, sars cl protease is considered to be an attractive target for drug design against sars. recently, we found tetrapeptide aldehyde, ac-thr-val-cha-his-h, showed high inhibitory activity with ic value of nm toward cl-r i mutant protease , . to compare the inhibitory activity of small compounds with those containing active functional groups, we synthesized serine-derivatives within the essential functional groups and evaluated its inhibitory activity. the synthetic scheme was started from fmoc-ser(tbu)-oh, following modification of c-terminal carboxyl group with p , n-terminal amine with p and side chain alcohol with p functionalities. steps overall reaction led to obtain novel serine derivatives for the small molecular inhibitors of sars cl protease. the assay with cl r i mutant protease was examined to evaluate the inhibitory activity of the synthetic serine derivatives. then, molecular docking study of complex of cl protease with the ligand was carried out. docking simulation experiment with r i (pdb id: aw ) and the inhibitor, which has the best activity in the serine derivatives, indicated that p fitting s ' pocket. at the result of assay, p , p and p positions of the inhibitor should be modified by benzoyl group, cyclohexyl group and cinnamoyl group, respectively. their bioactivities are underpinned by their distinctive structure with exceptional stability, thus making cyclotides exciting, not only for agricultural and pharmaceutical purposes, but also as a template in drug design. in all of the reported activities, cell membranes seem to be the primary target for cyclotide activity. to unravel the importance of lipid membranes on the reported activities of cyclotides, a set of cyclotides belonging to möbius and bracelet subfamilies were compared in their mode of action. the lipid selectivity and membrane affinity were compared with their efficiency against different target cells (e.g. red blood cells, bacteria, hiv particles). we have found that the bioactivity of cyclotides is dependent on the lipid composition of the target cell membrane and independent of a protein chiral receptor. in particular, all the native cyclotides tested target the cell membrane through specific binding to phospholipids containing phosphatidylethanolamine (pe)headgroups, but the membrane binding affinity is further modulated thorough non-specific peptide-lipid hydrophobic interactions, which are dependent on the specific cyclotide. in addition, the bioefficiency of cyclotides broadly correlate with their ability to target and disrupt the cell membrane. overall, we have shown that even with a common specificity for membranes containing pe-phospholipids, a fine selection was found across the family. in particular, each cyclotide inserts and disturbs the membrane in a distinct way, which explains the diversity of this family but also their distinct activities. the observation that all the tested cyclotides have a preference for a specific lipid makes this family truly intriguing and brings insights to optimize the use of the cyclotide template in drug design. malaria is a disease that affects around million people causing . - million of deaths annually. based on our previous studies, angiotensin ii (aii) presented antiplasmodial activity against plasmodium gallinaceum, but due to pressure activity, it cannot be used as an antimalarial drug. in an attempt to increase antiplasmodial activity and reduce hypertensive activity, we synthesized by solid phase method, cyclic analogues of aii with i-(i+ ) and i-(i+ ) lactam bridge scaffold using asp and lys residues. the bridge was more effective when inserted next to n-terminal extremity , probably this insertion, on another portion of the peptide, provides a change in the conformation of the molecule and its hydrophobic cluster formed by tyr, ile and his , which may have influence in the peptide-membrane interaction. thus, we have focused in the n-terminal extremity, testing new analogues, using glu/asp/orn/lys residues as bridgeheads components in i-(i+ ) lactam bridge scaffolds, which showed that antiplasmodial activity is increased using glu residue and that larger lactam rings are better to biologically active. therefore, new restrict peptides by i-(i+ ) and i-(i+ ) lactam bridge were designed, using glu residue as bridgehead element, but the same effect was not verified, getting a maximum of % of bioactivity. on the other hand, we promoted an increase in the hydrophobic character of the molecule, replacing the asp residue of aii sequence by fmoc-glu and asp(ofm), in order to improve the interaction of these compounds in the sporozoite membrane. the replacement by fmoc-glu provided a decrease of activity, while that asp(ofm) kept the aii activity, because there are changes of charge in the peptide, which may have modified the conformation in physiological medium. this kind of approach may offer the basis for development of new drugs and chemotherapy against malaria. animal venoms are complex chemical cocktails, comprising a wide range of biologically active reticulated peptides that target with high selectivity and efficacy a variety of membrane receptors such as ion channels or g-protein coupled receptors. venoms can therefore be seen as large natural libraries of biologically active molecules that are continuously selected and highly refined by the evolution process. the vision associated with the venomics project is to investigate in depth the enormous structural and pharmacological diversity of venom peptides through the development, integration and implementation of a novel research paradigm combining cutting-edge "omics" technologies in a high-throughput workflow. this new paradigm enclosed in venomics aims at replicating in vitro the diversity of venoms to generate original peptide banks to be used in drug discovery programs. herein, we show the different strategies we adopted for efficient solid phase synthesis and folding with an easy purification of peptides rich in cysteine and containing posttranslational modifications (ptm). angiogenesis depends on the adhesive interactions of vascular cells. the adhesion receptor integrin av b was identified as a marker of angiogenic vascular tissue. the αν β integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis, mainly by interacting with matrix proteins through recognition of an arg-gly-asp (rgd) motif. inhibition of the αν β integrins with a cyclic rgd peptide impairs angiogenesis, growth and metastasis of solid tumours in vivo. the aim of this study was to investigate the effects of replacement of a-amino acids by aza-β -amino acid analogs in cyclic rgd-peptides as αν β -integrin antagonist on angiogenesis, microcirculation, growth and metastasis formation of a solid tumour in vivo. the selectivity profile of these antiadhesive cyclopeptide is rationalized by a special presentation of the pharmacophoric groups. we synthesized cyclic rgd peptidomimetics that include aza-β -amino acid residues. modifications were added to the rgd skeleton in order to optimize the peptide activity. then, we investigated the pharmacokinetics activity of these pseudopeptides in hek (human embryonic kidney ) and endothelails cells huvec (human umbilical vein endothelial cells) cell by analyzing cell viability and protein involved in the angiogneisis processes. since tenascin c is a factor expressed highly in the tumorassociated matrix, targeting it would be a desirable first step for targeting the tumor-specific microenvironment in fact, a high level of tenascin c expression has been reported in most solid tumors, including lung cancer, colon cancer and glioblastoma. therefore, the targeted binding of tenascin c in tumor stroma would inhibit tumor metastasis by modulating cancer cell growth and migration. we isolated a peptide that bound to tenascin c by phage display peptide library selection, and the selected peptide specifically recognized tenascin c protein in xenograft mouse tissue. we also observed exclusive staining of tenascin c by the selected peptide in tumor patient tissues. moreover, the peptide reduced tenascin c-induced cell rounding and migration. we propose that the tenascin c targeting peptide may be useful as a specific anti-cancer diagnostic and therapeutic tool for most human solid tumors. radiolabeled pansomatostatins are expected to enhance hsst - tumor-uptake and to broaden clinical indications as compared to currently established sst -prefering radioligands. previous experience has revealed [ in-dota ,dtrp ]ss- ([ in]at s) as a true pansomatostatin analog, exhibiting however poor in vivo stability. in order to enhance metabolic stability, we introduced a second disulfide bridge to the at s motif by formation of extra / -amino acid (aa) or / -aa ring generating at s and at s, respectively. the orthogonally protected sequences were assembled on the solid support, deprotected and cleaved from the resin with tfa. the first cys -cys (at s) or cys -cys (at s) cyclization was performed in dmso, while the second was completed with iodine oxidation after in situ deprotection of cys (acm) and cys (acm). during hsst - +-autoradiography, at s showed unexpected total loss of sst - affinity, whereas at s showed high affinity (ic in nm) to all hsst - (hsst = . ± . ; hsst = . ± . ; hsst = . ± . ; hsst = . ± . ; and hsst = . ± . ). consistent with this finding, only at s stimulated sst internalization during immunofluorescence-based internalization assays, showing agonistic properties for sst . furthermore, [ in]at s internalized rapidly and specifically in sst + ar - j and hek -hsst +-cells. hplc analysis of min ex-vivo mouse blood samples revealed that > % [ in]at s remained intact. after injection in scid mice bearing ar - j and hek -hsst + tumors [ in]at s specifically localized in the rsst a+ ( . ± . %id/g vs. . ± . %id/g + nmol tate at h postinjection (pi)) and in hsst + implants ( . ± . %id/g vs. . ± . %id/g + nmol ke at h pi). this study has shown that introduction of an extra disulphide bridge in at s confers high metabolic stability. however, in a / member ring combination it leads to total loss of affinity. the reasons for this effect are currently investigated by nmr conformational studies. transporter compounds are useful tools to solubilise and increase the delivery of therapeutic molecules in the human body. one system to improve the cellular uptake of such therapeutic molecules are cell-penetrating peptides (cpps). these short peptide chains are either polycationic (containing several arg and lys) or show a more amphiphatic structure. it is known that the multivalency effect -the presentation of several copies of a cpp motif on a single molecule -can increase the cellular uptake. peptide dendrimers represent a group of tree-like, multivalent macromolecules, which are synthesized for different chemical and biological applications in our group. we now combine linear cpps with peptide dendrimers to get a well defined branched molecule made up of only natural amino acids. in our systematic study of peptide dendrimers decorated with different cpps we found that the potency of the single cpp as a transporter for small molecules can be increased and that these peptides show usually low cytotoxicity. additionally we designed new dendritic cell penetrating peptides with similar activities like linear cpps. all compounds are covalently linked to fluorescein for visualization with flow cytometry and confocal analysis. the results show that the peptides can transport efficiently a hydrophobic cargo into the cells. chemical stability of esters of acyclovir with amino acid and cholic acids k. chuchkov, r. chayov, i. g. stankova* south-west university "neofit rilski", blagoevgrad, bulgaria amino acid esters of antiviral drugs are a very good solution for improving oral bioavailability of the actual medicine. one of the most effective and tolerant prodrugs is valine ester of acyclovir -valaciclovir. taken orally exhibits three to four times higher bioavailability of acyclovir. the chemical stability of amino acids ( -fphenylalanine) (r,s) and bile acids (deoxycholic acid and chenodeoxycholic acid) esters of acyclovir was studied in experimental conditions simulating some relevant biological medias (ph . and . , °c).the chemical stability experiments revealed that the examined amino acid ester of acyclovir were relatively unstable in acidic ph, but bile acid ester is stable in the same ph. the examined amino acid and bile acid esters of acyclovir in neutral ph are relatively stable. in ph , all of tested compounds are more stable than valacyclovir (t / = h) -the first effective prodrug of acyclovir. in acidic ph acyclovirdeoxycholat and acyclovirchenodeoxycholat are more stable than valacyclovir. acyclovirchenodeoxycholat is the most promising anti-ebv prodrug candidate with high activity and satisfying chemical stability. cell-penetrating peptides (cpp) have become efficient tools for the cellular internalization of bioactive molecules due to their ability to cross the plasma membrane of diverse cells and cell lines. [ ] we recently reported that the cpp sc , which consists of the residues - of the c-terminal region of the cationic antimicrobial peptide cathelicidin (cap ), is an effective carrier peptide for small organic molecules like fluorophors and toxic peptide sequences into various cell lines [ ] . however, in general linear peptides are more susceptible to proteolytic degradation than their cyclic analogs [ ] . therefore, we investigated the cyclization of cpp derived from sc by means of cui-mediated azidealkyne cycloaddition (cuaac) [ ] . furthermore, we examined their conformation and proteolytic stability as well as their internalization efficiency and toxicity against various cell lines, in comparison to their linear equivalent and to other cpp. looking for the proper prodrug: a peptidomimetic approach to identify and inactivate bacterial mono-adp-ribosyltransferase toxins m. beich-frandsen, r. jørgensen division of microbiology and diagnostics, statens serum institute, copenhagen, denmark mono-adp-ribosylation is an endogenous posttranslational modification in eukaryotic cells, simultaneously utilized as virulence strategy by deadly secreted bacterial toxins. many bacterial toxins have been found to act as mono-adp-ribosylating enzymes, targeting anything from g-proteins to the actin skeleton. the diphtheria toxin from c. diphtheriae and exotoxin a from p. aeruginosa, both target the diphthamide-group of a unique modified histidine in eef , inhibiting protein synthesis by ribosome mimicry , . we aim to inactivate these nad+-utilizing toxin enzymes by nad-conjugated peptidomimetics, in a target-specific prodrug-approach. the adp-ribosylation reaction follows a random third-order s n mechanism. in the proposed model for the transition state of the reaction, the cleavage of the n c -nn bond of nad + releases strain and generates a oxacarbenium ion intermediate with a positively charged nicotinamide (n)ribose, subject to a nucleophilic attack from the substrate , , . adp-ribosylating toxins are commonly characterized by a artt-motif involved in substrate recognition . studies suggests conformational rearrangement of the residues surrounding the substrate binding site to be required for optimal geometry of the initial glycosidic nc -nn bond cleavage within nad + . subtype specific nad-conjugated peptides, designed based on previous structural analysis of the adpribosylation reaction, act as substrate for the enzymatic adp-ribosyl-transfer, and hereby attach covalently to and inactivate the nad + -utilizing toxin. relying on previous structural studies, and established ligand-binding and kinetic data, an initial peptide library, designed by bioinformatics and evaluated for specificity of common targets in-silico identifies initial leads. lead-scaffolds are implemented in rational peptide-design, based on high-resolution structural-and biophysical studies of multiple peptide-enzyme complexes, to identify possible prodrug-strategies for enzyme inactivation. nanoparticles play a crucial role in medicine for their potential application as in vivo carriers of active principles [ ] . liposome display unique pharmacokinetic properties slowly releasing drugs loaded in the inner aqueous cavity. in the last years we have developed supramolecular aggregates labeled by bioactive peptides able to recognize overexpressed receptors on tumour cells membrane delivering doxorubicin chemiotherapeutic drug [ ] . neurotensin(nt), a amino acid peptide, has dual functions of neurotransmitter or neuromodulator. the cterminus short fragment - preserve the activity but the half life of wild type form in vivo is very short. nt receptor type (nts ) is overexpressed in severe malignancies such as small cell lung cancer and colon, pancreatic, and prostate carcinomas. we have designed new amphiphilic molecules containing in the hydrophobic moiety two aliphatic chains and in the hydrophilic moiety a the bioactive portion able to aggregates with phospholipid molecules achieving liposome. we have synthesized neurotensin wild type sequence, the truncated form and the tetra-branched neurotensin(nt - ) or a truncated form(nt - ) tetrabranched peptides(nt ) adopting an opportune synthetic strategy on solid phase. all liposome were formulated adding the neurotensin amphiphilic monomer in ratio : with dopc in order to evaluate the capability to recognize selectively receptors overexpress on cell membrane surface. the liposomes size was determined by dynamic light scattering measurements, values for the hydrodynamic radius(rh). the selective internalization and cytotoxicity of fully doxorubicin loaded liposomes as compared to pure dopc liposomes, was tested in ht human colon adenocarcinoma and te human rhabdomyosarcoma cells. recently, small interfering rna (sirna), one kind of rna interference (rnai) technology represent the most common and, to date, the most effective method to inhibit target gene expression in human cells. it is also a common recognition that non-toxic delivery of sirna is an urgent problem for the therapeutic application of sirna. for the efficient gene silencing in vivo, prolonged circulation of sirna with take efficient and non-toxic cellular uptake and resistance against enzymatic degradation are indispensably required. ) telomerase activity has been regarded as a critical step in cellular immortalization and carcinogenesis and because of this, regulation of telomerase represents an attractive target for anti-tumor specific therapeutics. in this paper, we present the efficient and non-toxic cellular uptake of sirna using novel amphiphilic peptides and the application to silencing of htert in human cancer cell lines. in the present study, we investigated the intracellular delivery of sirna using some amphiphilic peptides and the silencing effect of sirna targeting htert mrna in human cancer cell lines, jurkat, hela and k . the complex of sirna and a specific amphiphilic peptide or its hybrid with an intracellular transport signal peptide could be effectively taken up into cells. the complex also showed a high silencing effect against htert mrna. moreover, the combination of sirna-nes conjugates and the amphiphilic peptides improved silencing effects up to . %. the amphiphilic peptides and their hybrids showed almost no cyto-toxicity and protected sirna against intracellular nuclease digestion in % fbs (half life time was over h). tumor targeting with the decapeptide gonadotropinreleasing hormone (gnrh) or its analogues is based on the discovery that gnrh receptors are overexpressed in many tumor cells, compared with their expression in normal tissues. using these peptides as carriers/targeting moieties in a conjugate with therapeutic agents can increase the selectivity and the stability of the conjugates, or eliminate the toxic side effects of the drug. gnrh-iii (<ehwshdwkpg-nh ) is especially favoured as a targeting moiety because of its antiproliferative effect and weak endocrine activity in mammals. the aim of our work was to synthesize novel short-chain fatty acid (scfa) acylated daunorubicin-gnrh-iii bioconjugates with enhanced enzymatic stability, cellular uptake, in vitro and in vivo antitumor activity. ser in position of gnrh-iii was replaced by lys and its ε-amino group was acylated with various fatty acids. gnrh-iii( lys(x), in conclusion, glu - /dglu - substitution in gi-derived radiopeptides enhanced stability and reduced renal values by ~ . fold. on the other hand, removal of the (glu) sequence in mg suppressed renal uptake but undermined stability. both modifications, however, compromised tumor targeting, demonstrating that more efficacious structural changes in the gi motif are warranted to improve the in vivo profile of resulting radioligands. synthesis and characterization of a mtc-labeled rhodamine-conjugated angiotensin peptide as a potential cardiac function imaging agent s.m. okarvi king faisal specialist hospital & research centre, cyclotron and radiopharmaceuticals department, riyadh , saudi arabia angiotensin ii (ang ii) is an -amino acid peptide that has been known to play a vital role in cardiovascular system. the actions of ang ii have been implicated in many cardiovascular conditions, such as coronary heart disease and heart failure. in an effort to develop a cardiac imaging agent with enhanced targeting capacity, we attached ang ii to rhodamine (rh), a lipophilic cation that specifically accumulates in the myocardium. the rh conjugated ang ii was evaluated for its potential as a heart imaging agent. rh-lys-gly-cys-asp-arg-val-tyr-ile-his-pro-phe-conh was prepared by solid-phase peptide synthesis according to fmoc/hbtu methodology. nhs-rh was attached to the ang ii peptide via the free amino group of lys residue by manual synthesis. rh-ang ii conjugate was labeled with tc- m by the stannous-tartrate exchange method. in vitro stability was determined in human plasma and in excess of cysteine. in vivo biodistribution and biokinetics were performed in balb/c mice and sprague dawley rats. the rh-ang ii conjugate radiolabeled efficiently with tc- m (> % labeling efficiency) as determined by hplc analysis. tc- m-rh-ang ii exhibited good chemical stability against cysteine transchelation and sufficient metabolic stability in human plasma. in mice, the bioconjugate displayed efficient clearance from the blood and excreted mainly through the renal route with some excretion by the hepatobiliary pathway. the uptake in the heart was . ± . % id/g as early as min post-injection; whereas, the uptake in the lungs, liver, stomach and kidneys varied between - % id/g. in rats, the bioconjugate displayed relatively better pharmacokinetic characteristics, with low uptake in the major organs (< % id/g). the uptake in the heart ( . ± . % id/g) was found to be higher than the uptake in the blood and muscle, resulting in good heart-to-blood and heart-to-muscle uptake ratios. this initial study towards the development of an effective cardiac imaging agent advocates that the use of hybrid conjugates appears to hold a great promise as a new and attractive approach for rapid and efficient imaging of heart. in humans two isoforms of gnrh are exist, gnrh-i (<ehwsyglrpg-nh , where <e is pyroglutamic acid) and gnrh-ii (<ehwshgwypg-nh ). in contrast to gnrh-i, exact function of gnrh-ii is not well identified. however, it was indicated that gnrh-ii derivatives have more potent antiproliferative activity on human endometrial and ovarian cancer cells in vitro than gnrh-i analogs. some kinds of tumor cells (e.g. breast, prostate, colon) produce gnrh-i and gnrh-ii, and express their receptors. however, the presence of functionally active gnrh-ii receptors on cancer cells is still not clear - . on the other hand, gnrh-ii analogs, both agonists and antagonists, induce apoptosis in many different cell types. in contrast to gnrh-i agonists, gnrh-ii agonist [d-lys ]gnrh-ii does not activate nfκb, therefore does not protect the cancer cells from apoptosis [ ] [ ] [ ] . in the regulation of apoptosis, not only the pro-and antiapoptotic proteins play a role , but other molecules also attend (e.g. ck , casein kinase ii enzyme) . our aim was to synthesize new proapoptotic peptide or enzyme inhibitor containing [d-lys ]gnrh-ii based derivatives and investigate their in vitro cytotoxic, cytostatic and apoptotic effects. the sythesis of [d-lys ]gnrh-ii, which is one of the most potent gnrh-ii agonist, was carried out on solid phase using fmoc/tbu strategy. different proapoptotic peptides or enzyme inhibitors were attached directly to the gnrh-ii derivative. in vitro cytostatic and cytotoxic effects of gnrh-ii conjugates were investigated by mtt-assay using different human breast (mcf- and t -d) and colon carcinoma (ht- ) cell cultures. early apoptotic effect of gnrh-ii conjugates was studied by flow cytometry on mcf- , t- d and ht- cell lines. according to our results, these conjugates might be potential candidates for cancer treatment, because the combination of a targeting moiety (gnrh derivative) with proapoptotic peptides or enzyme inhibitors and drug molecules might have synergistic activity resulting in highly potent multifunctional therapeutic agents. department of pharmaceutical and pharmacological sciences, university of padua, padua, italy melanoma is the most deadly skin cancer with an increasing incidence. its therapy continues to be a challenge since, regardless of the treatment used, longterm survival is quite uncommon. melanoma cells are characterized by high levels of glutathione s-transferase p - (gstp - ) isozyme. this enzyme is also highly expressed in solid tumours and drugresistant cells, where a role for this protein in the regulation of cell proliferation has been described. in the past, lyttle et al. synthesised glutathione (gsh)linked anticancer prodrugs activated by physiological concentration of gsts. structure and mechanistic studies showed that in the gst-gsh complex, the gst tyr residue placed near the s atom of the cys in gsh, is in the phenoxide form to facilitate the deprotonation of the sulfhydryl group in gsh, making it able to react with electrophilic species. by taking advantage of this active-site geometry, we synthesised two tyrosinase activated melanoma prodrugs ( -methoxyphenol and n-acetyl- -s-cysteaminylphenol) linked to gsh. in these compounds an ethoxycarbonyl group was placed between the gsh sulphur, oxidate to sulfone, and the tyrosinase-activated prodrugs. in this way the tyr phenoxide (tyr in gstp and tyr in gstm isozyme) would be able to abstract one of the acidic methylene protons linked to the sulfone moiety. such a deprotonation should trigger a beta-elimination, resulting in the release of melanoma prodrugs. the stability of the obtained putative prodrugs has been evaluated in different ph buffers and in rat liver and kidney cytosolic fractions. synthetic peptides represent first choice biomolecules, with respect to proteins and mab, for the monitoring of population variability and receptor functionality associated to a tumor pathology due to their favorable pharmacokinetic profile. it has been demonstrated that the surface molecule integrin alfa-v beta- could be an ideal target for the interaction with melanoma directed radiolabeled peptides. this integrin is indeed overexpressed both in tumor cells and in tumor vessels endothelium, while it is not expressed in the majority of normal tissues. cyclic peptides with the sequence arg-gly-asp (rgd), are known to bind the integrin alpha-v beta- with high affinity and selectivity and could conveniently target a radiotherapeutic to melanoma cells. we previously demonstrated that the cyclic rgdfk peptide interact with high affinity to a human melanoma cells. the aim of this work was to conjugate the mtc to the peptide by using the [ m tc(n)(pnp)] + system (pnp=aminodiphosphines) to obtain a radiolabeled peptide useful in melanoma imaging. , spectrum. a nh(i)→βch(i- ) cross peak more intense than the nh(i)→βch(i) cross peak is observed when the peptide adopts the . -helixl conformation. by contrast, an opposite trend of intensities of the same noe cross peaks indicates the occurrence of a -helical conformation. by using this method, we were also able to determine that polar solvents, such as mecn or meoh, may induce a helical structure in deg homo-peptides which otherwise adopt the . -helix conformation in cdcl . this finding allows us to rapidly switch these peptide series from "short" ( -helix) to "long" ( . -helix) by changing the solvent only, thus making them extremely attractive tools. school of biological sciences, university of auckland, auckland , new zealand naturally occurring anti-freeze proteins (afps) enable organisms like polar fish to survive the freezing temperatures of their natural habitat. as well as being cryoprotective, afps exhibit properties of ice recrystallization inhibition (ri). the ability of afps to influence the size, morphology and aggregation of ice crystals can be used in food technology, where the growth of ice crystals in frozen foods is of primary concern. chemical synthesis of afps enables access to pure material for structural and functional studies. our new research programme in the 'food and health' area, prompted us to develop tailor made anti-freeze peptides for potential applications in the frozen food industry. 'multiple salt-bridge analogues' of the naturally occurring winter flounder afp were synthesized and evaluated for their antifreeze activity in comparison to the natural sequence. details on the molecular modelling, structural characterization and anti-freeze activity analysis on the synthetic afps will be presented. several disorders are now believed to be conformational diseases caused by misfolding in the structure of specific proteins leading to the gradual aggregation and deposition of these abnormal proteins. attempts to develop effective therapies for these proteopathies (such as alzheimer's, parkinson's and huntington's disease) have been directed toward reducing the production of the proteins, inhibiting their aggregation, or promoting their removal. conformation-dependent antibodies that specifically target misfolded proteins are proving to be useful tools to study the pathogenesis and as possible treatments of these diseases. we have previously shown that active immunization with tetrapalmitoylated aβ - peptide embedded in liposomes (aci- ) was capable of eliciting a conformation-specific immune response in mice. the antigenic peptide was shown to mimic the aggregated β-sheet conformation of the pathological a β peptide on the surface of the liposomes by cd, ssnmr and tht assay. we have also reported the modulation of the conformation of aβ - derived sequences by means of different lipidation patterns, lipid chain lengths and liposome surface charge. here we present the characterization of this panel of liposomal peptide constructs by atr-ir and correlate the results with previous studies by cd. in summary, atr-ir secondary structure analysis of a battery of amyloid-derived lipopeptides allowed to study the key parameters responsible for the adopted conformation on the lipid bilayer. the presented strategy to control the conformation of liposomal peptide immunogens could be applied to the rational design of vaccines against a range of protein misfolding diseases, such as alzheimer's disease. cyclic lipodepsipeptides (clps) are secondary metabolites produced by various bacteria, most prominently bacillus and pseudomonas. these often display potential antimicrobial properties, frequently ascribed to the ability to disrupt cellular membranes or form ion-pores. clps are a structurally diverse group of molecules always consisting of an oligopeptide chain cyclized by a lacton bond and a fatty acid moiety. they are divided into several groups based on sequence similarity. one such group is the viscosin group, which contains clps produced by pseudomonas that all feature nine amino acids and a conserved sequence pattern of hydrophobic and hydrophilic residues. recently, we have extensively described the self-assembly properties of one viscosin group member, pseudodesmin a. , we have shown that in non-polar organic solvents this clp forms anisotropic supramolecular structures of unrestricted size, reminiscent of the ability to form ionpores in the non-polar cellular membrane environment. here, we report on how the most prominent structural variations within the viscosin group affect the conformation and self-assembly properties. since the well-defined monomer conformation of pseudodesmin a is key to this self-assembly, the most striking variation within the viscosin group is the variability of the leu stereochemistry. for the first time, a conformational study will be reported for clps featuring an l-leu residue, such as viscosin and viscosinamide. the results of this study contribute to a deeper understanding of the structure-function relationship of clps in general. effective design of peptide mimicking protein helical segments is a great challenge. an intriguing approach to improve the helical content in a peptide sequence is achieved through the "peptide stapling" strategy using a hydrocarbon cross-linker (staple) . in this study we have investigated the effect of the introduction and location of staples on secondary structure of sequences derived from a reported peptide, mimicking cullin binding region to kctd , , to gain an insight into the designing of new molecular entities able to modulate the kctd oncosuppressor activity. indeed, kctd plays a crucial role in the ubiquitination of hdac by acting, in complex with cullin , as an e ubiquitin ligase, thereby affecting hedgehog activity and medulloblastoma growth. in the designed peptides the stapling has been achieved by ring-closing olefin metathesis between two s- -( 'pentenyl) alanine residues incorporated at i and i+ positions and into different sites within the peptide chains. the stapling position has been chosen on the basis of cullin -kctd complex model. circular dichroism studies have highlighted that the stapled peptides exhibit remarkable and different changes in secondary structure contents in comparison with the linear precursors and the unmodified sequence. a nmr conformational analysis will allow the determination of the conformational preferences of the stapled peptides at atomic resolution with also relevant implications for the kctd-cullin recognition. based on our findings from protease-mediated ligation of cleavage-sensitive peptides and proteins using ionic liquids (ils) as additives and from chemical acylation reactions of peptides in ils, we hypothesized direct and unique interactions between biomolecules and ils as the molecular reason for il-mediated structural effects. to verify this, we performed systematic structural investigations of suc-ala-xaa-pro-phe-pna and ala-xaa-pro-phe model peptides dissolved in aqueous imidazolium-based ils using high resolution magic angle spinning (hr-mas) nmr spectroscopy. highly resolved nmr spectra are obtained that allow for complete h resonance assignments of the peptides as solutes and the ils as solvents. the changes in the proton chemical shifts Δδ( Η) of a peptide on dissolution in ils arise from direct interactions between both, including il-induced structural/conformational changes of the peptide molecules. , the strength, position, and type of these interactions are influenced by the nature of the amino acid residues in the peptide sequence as well as the nature and type of the il anions. downfield shifts indicate strong interactions between the ils and the peptide backbone, whereas upfield shifts point to hydrophobic interactions and stacking effects with the il imidazolium ring. the pattern of Δδ( Η) along the peptide chain reveals the sites and the type of interactions. the extent of Δδ( Η) implies a significant effect on the structure of the peptides. different Δδ( Η) were obtained for cis-and trans-configured peptide isomers and point to a conformer-specific il/peptide interaction. calcitonin is a -amino acid polypeptide hormone, which takes part in calcium metabolism in bones. it belongs to a protein family whose members may form amyloid fibrils. these fibrils (or pre-fibrillar aggregates) are related to serious diseases known as amyloidoses, whereas amyloid-like fibrils may have natural functional roles in many organisms. the amyloid form of human calcitonin is related to medullary thyroid carcinoma. using our online consensus prediction tool for 'amyloidogenic propensity' of protein molecules "amylpred", a novel hexapeptide of human calcitonin was predicted as a novel, possible amyloidogenic peptide. we investigated experimentally its ability to form amyloid fibrils, utilizing tem, x-ray fibre diffraction, atr ft-ir spectroscopy and polarized light microscopy. analysis of the results shows that this peptide self-assembles into amyloid-like fibrils and that amyloid fibrillogenesis is mediated via nuclei of liquid crystalline nature, known as spherulites. we thank the university of athens for the financial support. school of pharmacy, university of norwich nr tj ; inserm u , université de rouen, mont saint aignan rfa, a rfamide neuropeptide, is the ligand of gpr and the system rfa/gpr is involved in feeding behaviour [ ] . a structural study by nmr showed that rfa, in a membrane mimetic medium, possesses a helix p -r -linker k -g -turn f -r motif. sar studies on truncated fragments of rfa indicate that, in addition to the turn motif, additional residues are required to maintain a correct biological activity ( rfa ec = , nm; rfa - ec = , nm; rfa - ec = , nm; rfa - ec = nm) [ ] . our aim is to synthesize new gpr ligands using an iterative process combining rational conception, peptide and modified amino acid syntheses, biological and structural analyses. structural and biological results will allow us to find new modifications and conceive more potent analogues. using nmr, we demonstrated that rfa - encompasses a nascent helix and a turn while rfa - possesses only the c-terminal turn structuration. the destabilization of the helical structure on fragments with a shorter n-terminal region could explain the decrease of the biological activity of rfa fragments. we decided to focus on the importance of a stabilized helix on the biological activity. we chose the hydrogen bond surrogate method which stabilizes a helix by replacing the main hydrogen bond with a covalent link without modifying the side-chains [ ] . syntheses, nmr characterization and molecular modelling of analogues will be discussed. acknowledgments: this work is supported by erdf funding through the is:ce-chem project and interreg iv a france-(channel)-programme. firstly, it induces a constraint by restricting the ı o-dihedral angle to values around - °. secondly, the xaa-pro peptide bond is subject to cis-trans isomerization characterized by an increased cis population and an activation energy that is low when compared to the other amino acids. besides, the five-membered ring of the pro residue can adopt two main distinct conformations (up-puckered or downpuckered) that are almost equally abundant in peptides and proteins. a variety of mimics and analogs have been designed in order to control the conformation of the peptide backbone and/or to alter the cis/trans ratios and the rotational barriers for cis-trans isomerization. in this presentation, nmr studies and theoretical calculations have been performed on model peptides ac-ser(Ψpro)-nhme, (s,s)-ac-ser(Ψh, cf pro)-nhme, and (r,s)-ac-ser (Ψ cf ,hpro)-nhme. their thermodynamic and kinetic features have been analyzed in chloroform, dmso, and water, allowing a precise description of their conformational properties. we found that trifluoromethyl c δ -substitutions of oxazolidine-based pseudoprolines can strongly influence the cis-trans rotational barriers with only moderate effects on the cis/trans population ratio. in chcl , the configuration of the cf -c δ entirely controls the ψ-dihedral angle, allowing the stabilization of γ-turn like or ppi/ppii-like backbone conformations. moreover, in water and dmso, this c δ -configuration can be used to efficiently constrain the ring puckering without affecting the cis/trans population ratio. theoretical calculations have ascertained the electronic and geometric properties induced by the trifluoromethyl substituent and provided a rational understanding of the nmr observations. in contrast to formation of the native state, aggregation of misfolded protein molecules into so-called amyloid fibrils is a strongly irreversible, kinetically-controlled process which may lead to structural variants of fibrils composed of chemically-identical protein chains. this poorly understood aspect of polymorphism of amyloid fibrils has important implications in vivo in the course of amyloidassociated neurological disorders -e.g. in the form of prion strains in creutzfeldt-jakob disease. in this paper, we examine cross-seeding behavior of a model amyloidogenic polypeptide -bovine insulin (bov-ins) and recombinant lys b -arg b insulin (kr-ins). fibrils of bov-ins and kr-ins exhibit characteristic "fingerprint" features in the amide i' infrared region. these spectral traits are transferred to daughter generations of fibrils upon cross-seeding of native bov-ins with kr-ins fibrils and vice versa. this study highlights an interesting aspect of a non-anfinsenian behavior of aggregating insulin: the memory effect. our results indicate that fine infrared features in the amide i region of fibrils from closely related proteins may not reflect different amino acid composition but rather the fact that a particular amino acid sequence favors an amyloidogenic pathway that determines a certain and spectrally-distinct stacking mode of β-strands. an encounter with an alien seed on the amyloidgenic pathway would hijack the yetnative monomers and force them to follow the assembly pattern (and resulting ir characteristics) of the preformed fibril. phosphorylation and oglcnacylation are dynamic intracellular protein post-translational modifications that often are alternatively observed on the same protein serine and threonine residues. phosphorylation and oglcnacylation commonly occur on residues in natively disordered regions of proteins, and often have opposing functional effects. in the microtubule-associated protein tau, hyperphosphorylation is associated with protein misfolding and aggregation as the neurofibrillary tangles of alzheimer's disease, whereas oglcnacylation is stabilizing. a series of peptides derived from the prolinerich domain (residues - ) of tau was synthesized, with the free ser/thr hydroxyls, with phosphorylated ser/thr, with oglcnacylated ser/thr, and with the dietylphosphorylated ser/thr. phosphorylation and oglcnacylation were found by cd and nmr to have opposing structural effects, with phosphorylation favoring polyproline helix (ppii) formation, with oglcnacylation opposing ppii, and with the free hydroxyls intermediate. phosphorylation of serine and threonine residues in prolinerich sequences was found to induce ppii in other proline-rich sequences, with significant conformational restriction at phosphoserine and phosphothreonine residues observed by nmr. notably, serine and threonine residues are common in proline-rich domains of proteins, and ppii is a common conformation observed for phosphorylated ligands in protein-protein interactions. the ser/thr diethylphosphate was found to be a practical mimic of oglcnacylation, as an easily synthesized neutral amino acid with similar sterics to oglcnac and strongly opposing ppii. dept. of biology, university of padova, padova , italy we recently established that designed hairpin peptides interfere with the amyloidogenesis of both human pancreatic amylin and α-synuclein ( α-syn), two 'unstructured' polypeptides associated with human diseases. in the case of α-syn, the most potent 'amyloid inhibitors' act by diverting α-syn to non-amyloid aggregates. the binding sites for these agents and nonhairpin peptides of similar sequence have been determined by d-nmr-monitored titration studies using universally n-labeled α-syn. in the case of the hairpins that produce non-amyloid aggregates from α-syn, initial binding occurs in the non-amyloidogenic c-terminal segment of α-syn and the hairpin peptide is incorporated in the resulting aggregates. nmr studies also serve to define binding interactions and conformational changes that may be essential early steps in aggregation and pre-amyloid state processes. mt-ii is a long acting, non-selective super-agonist of melanocortin gpcr receptors, characterized by lactam bridge between residues i and i+ stabilizing a type-ii β turn structure. the recently introduced cui-catalyzed azide-alkyne , -dipolar cycloaddition, presents a promising opportunity to develop a new paradigm for an orthogonal bio-organic and intramolecular side chain-toside chain cyclization. we introduced this [ , , ]triazolyl-mediated cyclization in a pthrp model peptide, and demonstrated that i-to-i+ side chain-to-side chain cyclization offers a powerful approach for generating stable helix mimetic structures. , , in the current study we explored the potential of the i-to-i+ side chain-to-side chain , -disubstituted [ , , ] triazolyl-bridge to stabilize -turn conformations. we designed a series of [ , , ]-triazolyl containing cyclo-heptapeptides, derived from the model peptide lactam mt-ii, differing in the size of the bridge, the location and orientation of the [ , , ]-triazolyl moiety. conformational and biological studies, performed on this series demonstrate that [ , , ] triazolyl-mediated side chain-to-side chain cyclization results in the conformational stabilization of type-i β turn generating heterodetic cyclopeptides with enhanced in vitro potency and improved receptor subtype selectivity. this study confirms the versatile nature of the [ , , ] triazolyl-mediated side chainto-side chain cyclization. moreover the chemical accessibility, functional compatibility, and metabolic stability suggest that the [ , , ] international associated laboratory samuel de champlain, regional platform for cell imaging of haute-normandie (primacen), institute for medical research and innovation (irib), university of rouen, mont-saint-aignan, france scorpion toxins are polypeptidic molecules capable of selectivity binding to ion channels with the highest affinities and represent natural peptide blockers that are good tools to characterize new or particular types of ion channels. astroglial cells appear to express all ion channels species even voltage-gated ion channels previously suspected to be present only in excitable cells; but these channels are poorly studied and their function are not still completely elucidated.thus, the purpose of the present study was to investigate for the first time, the global effect and the potential protective effect of one scorpion toxin acting on kv channels, the pi ,on h o -induced oxidative stress and cell death in rat astrocytes. pi is a -residue toxin crosslinked by disulfide bridges, isolated from the venom of pandinus imperator and is one of the most potent and selective inhibitor of shaker b and kv . channels . in our study, pi was obtained by solid phase peptide chemical synthesis(spps) and named spi . the identity and homogeneity of spi were also verified by hplc, mass spectrometry analysis(maldi-tof) and an enzyme-based cleavage to determine the assignment of spi half-cystine pairings.the synthetic spi was found to be indistinguishable from the natural toxin. incubation of cultured astrocytes with graded concentrations of h o for h provoked a dose-dependent reduction of the number of living cells as evaluated by flow cytometric analysis and lactate dehydrogenase assay. interestingly, pre-treatment of astrocytes with very low concentrations of spi ( - to - m),dose-dependently prevented cell death induced by h o , and the effect of spi was accompanied by a marked attenuation of ros accumulation. also, spi stimulated sod and catalase antioxidant activities in a concentrationdependent manner,and blocked h o -evoked inhibition of sod and catalase activities. in addition, at low concentration ( - to - m) the toxin exhibits no toxic effect on astrocytes.taken together these data indicate that the spi acts as a protective agent on astrocytes from oxidative assault and then might have therapeutic value in the potential treatment of a number of neurodegenerative diseases. [ ] . in order to select small active peptides, the library was kept at the minimum size and was built using only selected d-amino acids to avoid structural redundancies. to further increase the chemical diversity [ ] a set of different carboxylic acids were coupled to the n-terminus. the iterative screening led to the identification of an n-terminally modified tetrapeptide with a very high radical-scavenging activity. by analyzing several peptide variants, we found that antioxidant properties were strongly sequence-and structure dependent, because scrambled or proline-scan variantswhose secondary structures are deeply altered -are much less effective. data on peptide structure-activity relationship will be presented. an investigation of the in vitro effects produced in cellular models of oxidative stress are also in progress. structural analysis of peptide-analogues of zp proteins with amyloidogenic properties: insights into mammalian zona pellucida formation. n.n. louros, v.a. iconomidou, s.j. hamodrakas* department of cell biology and biophysics, faculty of biology, university of athens, athens , greece a filamentous structure surrounding mammalian oocytes, called zona pellucida, is responsible for species-specific sperm binding. this extracellular porous structure is composed of - glycoproteins, termed zp - , which assemble into filamentous polymers that are cross-linked by disulfide bonds. all zp proteins present a unit with high sequence homology at their c-terminal end, designated as the zp domain. this structural module is found in a family of extracellular proteins with various functions and from a wide variety of organisms (zp domain proteins). it is a binary structure of approximately amino acids, divided into two domains, the n-terminal region named zp-n and the c-terminal region known as the zp-c domain. the zp-c block is associated with protein function, whereas the zp-n domain is responsible for protein polymerization. structural analysis revealed a possible polymerization model based on backbone interactions between specific parts of the zp-n units. peptide-analogues of the aforementioned segments were synthesized, since the ''amylpred'' application, an 'amyloidogenic determinant' prediction algorithm developed in our lab, predicted them as being potentially 'aggregation prone'. our experimental data clearly indicate that these peptides do actually fold and self-assemble into amyloid-like fibrils. therefore, all the derived data suggest that mammalian zona pellucida is a novel functional amyloid, similar to its biological equivalent, silkmoth chorion that has been established to be a natural, protective amyloid. we thank the university of athens for the financial support. biostrucutres and bioimmaging institute of national research council, naples, italy the definition of the molecular basis of human diseases is one of the most important goals of structural biology. nucleophosmin (nmp ) is an ubiquitously expressed protein is a nucleolar chaperon and has a key role in several biological processes from ribosome biogenesis, to cell-cycle regulation and is a frequently target of oncogenic mutations in acute myeloid leukaemia (aml) [ ] . these mutations occur predominantly at the c-terminal domain of npm compromising its stability and causing the aberrant translocation of npm to the cytosol. this domain is structurally characterized by the presence of three alpha helices, and the folding of cter-npm proceeds via an extended nucleus and the denatured state retains significant malleable structure at the interface between the second and third helices [ ] . here, we have further investigated the structural determinants in the folding process of the c-term npm domain and on the basis of available structural and composition data, several peptides covering the three helices were designed and synthesized. their conformational properties were investigated by cd and nmr spectroscopy: these studies demonstrated that once removed from the protein architecture helix and helix substantially miss fully ordered conformations while helix retain a high helical content also in mutated variants. our observations may help structure-based drug-design inserm u university of evry-val d'essonne, bâtiment maupertuis, evry, france sl (stop-like protein with mass of kda) is a neuronal protein with a calmodulin-binding and microtubulestabilizing activity. microtubule binding is inhibited by calmodulin , . the lack of the stops gives rise to defects in synaptic plasticity, as observed in schizophrenia . to study the sl protein's structure and its interaction with calmodulin, we have used a residue peptide derived from sl , containing the microtubule binding motif mn. the structure of the peptide has been analyzed using circular dichroism (cd) spectropolarimetry. the cd spectra showed that the peptide's secondary structure consists in a predominantly β-sheets, while the tertiary structure consists in rather folded d-structure. the temperature dependence of cd indicated that the secondary structure of the peptide remained very stable between °c and °c, while the tertiary structure changed at about °c. the interactions between the sl peptide and calmodulin have been studied using the isothermal titration calorimetry (itc). the results showed that calmodulin binds the peptide with the affinity of m- , in a ca + -independent manner, the reaction being driven by entropic contributions. our results might contribute to understanding the microtubule stabilizing activity of the sl protein. peptides , . based on the structure in polar environment a transition from -helical to an α-helical conformation at the water/lipid interface in bilayer membranes is hypothesized the efficient method development for the analysis of proteins, peptides, and synthetic oligonucleotides with a novel hybrid reversed phase column f. becker * , n. shoji * , a. matsui * , c. yokoyama * , t. sato * , t. takai * , and n. kuriyama * * ymc europe gmbh, * ymc co., ltd. the recent development of bio-pharmaceutical industry has been remarkable and an effective analytical method with higher sensitivity, superior selectivity, and increased speed has been required in the characterization of peptides, proteins and oligonucleotides by highperformance liquid chromatography (hplc). the method development of reversed phase hplc requires optimization of several conditions, such as bonded-phase, column efficiency, solvent type, ph and temperature. especially ph and buffer type is a most important parameter to control retention of biomolecules which have multiple ionic functional groups. furthermore, temperature often becomes a key tool to achieve better peak shapes and resolution for larger molecular-weight compounds. although silica based reversed phase columns have been widely used for separation of biomolecules, they have low stability under alkaline conditions and a limited usable ph range. to improve the chemical stability at an expanded ph and temperature range , we have developed a new type of organic/inorganic hybrid reversed phase column, ymc-triart c and ymc-triart c . the novel technologies of manufacturing particles and surface modification provide outstanding chemical stability and excellent peak shape for many types of compounds under a variety of mobile phase conditions. in this poster, we will show characteristics of this new hybrid column, and some example cases of efficient method development in separation of the biomolecules such as peptides, proteins and synthetic oligonucleotides. the fully-extended peptide conformation ( . -helix) is the longest possible for a single α-amino acid as it is characterized by an axial translation per residue of about . Å. therefore, it is extremely attractive for its application as a molecular bridge. however, it is known that the stability of this type of helical structure (relative to the competing, much shorter -helix) appears to be dramatically governed by the choice of the c-terminal functionality (only esters seem to be appropriate). in this work, we examined the series pyrac-(deg)n-o-(pno )bzl [where pyrac is the fluorophore -pyrenylacetyl, deg is cα,α-diethylglycine, n= - , and o-(pno )bzl is para-nitrobenzoxy] with the deg homo-peptides as fully-extended bridges in a static fluorescence study. to perform a very stringent comparison with our ft-ir absorption and nmr results, the solvent used was chcl . the trend observed in the quenching efficiencies for the shortest members of the series, the (deg) - peptides, steadily decreases from . (n= ) to . (n= ), and . (n= ). this finding is compatible with the conclusion that these three peptides are all essentially fully-extended in chcl solution. however, the quenching efficiencies for the longest deg homo-peptides. . for n= and . for n= , are not further reduced. the inevitable conclusion is that the tetra-and pentapeptides populate at least two different conformations (most probably, the . -helix and the helix), in which the probe dyads are located at different distances. notably, these results fit well with those extracted from our ft-ir absorption and nmr studies in the same solvent of low polarity. we conclude that a combination of terminally aromatic groups, as in the deg homo-peptide series investigated in this work, is not eligible for the fabrication of a stable fully-extended conformation. the surfactant protein c (sp-c) is a -residue highly hydrophobic peptide with two covalently linked fatty acyl chains that adopts a mainly helix structure while associated with the membrane. however, it misfolds into a β-rich structure under certain environmental conditions, which suggests that sp-c is in a metastable state . the slow α→β transitions are likely to be caused by the membranedissociation, deacylation and exposure to the neutral ph of the alveolar subphase, leading to the formation of amyloid aggregates associated with pulmonary alveolar proteinosis. we have been studying the ph-dependent conformational behaviour of both the acylated and deacylated isoforms of sp-c in a membrane mimetics chloroform/methanol/water mixture using a constant-ph molecular dynamics method [ ] [ ] [ ] . our simulations identify the increase of ph as a promoter of peptide-peptide interactions, which may contribute to the α→β transitions observed at neutral and alkaline ph conditions in the experimental results . we also observe that the loss of structure is more prone to begin at the c-terminal region and that the β motifs start to form between the n and cterminal disordered regions of the sp-c. in addition, the presence of the acyl groups results in a slightly higher structural stabilization of the sp-c helix and in a decrease of the n-terminal plasticity at acidic and neutral ph. since a comprehensive conformational analysis and the elucidation of the mode of association between sp-c and the membrane are essential to understand the interactions involved in structure stabilization and in some specific functions of the peptide, we are now studying both sp-c isoforms in a membrane environment. these studies will not only be the most relevant for the in vivo situation but will also answer to some questions left open by the previous work. were rather small. vp plays a very important role in controlling resorption of water by the distal tubules of the kidney and in regulating the osmotic pressure of blood in mammals. vt, while exhibiting both oxytocin and vasopressin activities, has not changed over the last million years until appearance of the mammals. currently, it occurs naturally in the non-mammal vertebrates [ ] . here we report on studies of arginine-vasopressin (cyfqncprg-nh , avp) and arginine-vasotocin ([ile ]avp, avt) in a mixed dodecylphosphocholine (dpc) and sodium dodecylsulfate (sds) micelles. dpc micelles are considered good models of eukaryotic cell membrane [ ] , while the sds micelle improves peptide/micelle solubility [ ] . for these studies we use d nuclear magnetic resonance (nmr) supported with molecular modelling methods. conformation-activity considerations relationships and the role of the peptide-micelle interactions on the structure of the analyzed peptides will be described and discussed. vibrational and chiroptical spectroscopic investigation on diamide peptide models k. knapp, a m. hollósi, a e. vass, a zs. majer a a eötvös lor nd university, institute of chemistry, laboratory for chiroptical structure analysis, budapest, hungary understanding of peptide folding has great importance in many fields of chemistry. since amino acid residues are the simplest building blocks of peptides, the investigation of their conformational properties could help us in understanding the structures of peptides or in designing peptides of specific d structure. describing the conformational building blocks of even the simplest peptide models (e.g., amino acid diamides) can serve as a good starting point to decipher higher-level structural properties of their polymers. among the simplest compounds which include the essential structural elements of polypeptide backbone are the n-methylamides of n'acetylamino acids. these compounds have been the subject of extensive spectroscopic analyses (ftir, vcd, ecd, nmr) [ , ] . the conformational landscape of these model compounds were analyzed by solution phase vcd and ecd spectroscopy and density functional theory (dft) computations. this work reports systematic vcd and ecd spectroscopic studies in different solvents on synthesized diamide models (ac-α/βhomo-xxx-nhch , xxx = ala, pro, phe). vcd and ecd data, combined with quantum chemical calculations performed at the b lyp/ - ++g** and - ++g** level of theory was particularly useful for identifying the preferred dipartimento delle scienze biologiche, università di napoli "federico ii", napoli, italy c dipartimento di scienze ambientali, seconda università di napoli, caserta, italy β-hairpin is an important secondary structural element used by many proteins for biomolecular recognition, and thus is an attractive tool for mimetic design . aβ -hairpin motif consists of two antiparallel strands linked by a turn region. in this work we aimed to stabilize a β-hairpin conformation by an interstrand covalent bridge, made through a click chemistry reaction, which yields , -disubstituted , , , triazole, starting from alkyne and azide reactive groups. in particular, we explored the conformational stability of a series of β-hairpin peptides containing a , -disubstituted , , triazole bridge with variable length. we employed the well structurally characterized trpzip peptide as a convenient b-hairpin model system. as alkyne amino acids were introduced propargylglycine (pra), homopropargylglycine (hpg) or bishomopropargylglycine (bpg), while the following azide amino acids were inserted: lβ-azidoalanine (dap (n )), l-γ-azidohomoalanine (dab (n )), and l-δ-azidoornitine (orn (n )). all peptides were synthesized and purified. successively, the linear unprotected peptides were cyclized in solution by the cu(i)-catalyzed azide alkyne cycloaddition (cuaac) reaction and purified prior to the spectroscopic characterization. linear and cyclic peptides were analyzed by a combination of cd and nmr techniques. have been investigated. their behaviour has been compared with that of proline in homochiral and heterochiral dipeptide sequences including l-or dphenylalanine. nmr and ir studies as well as x-ray diffraction analysis provide evidence that the additional β-phenyl substituent does not disrupt the tendency of proline to occupy the i+ position of a β-turn. moreover, the β-turn conformation is stabilised, with reference to l-pro, when l-cis(βph)pro or l-trans(βph)pro are combined with d-phe, that is, in the heterochiral sequences. the pyrrolidine conformation is significantly affected by the presence of the β-phenyl group. the puckering modes that alleviate most the steric hindrance introduced by the bulky phenyl substituent are preferred. the l-cis(βph)pro residue shows a marked propensity for the cγ-endo/cβexo half-chair arrangement whereas the trans derivative exhibits a higher flexibility, with different pyrrolidine shapes being accessible. interactions between the aromatic ring of (βph)pro and that of the contiguous l-or d-phe residue are observed in all the dipeptides crystallised. in solution, they seem to occur only for the heterochiral sequences. this intramolecular aromatic-aromatic interaction may be responsible for the higher β-turn ratio observed for such sequences and also seems to affect the conformational preferences of the pyrrolidine ring. in fmoc chemistry the trt and acm groups are widely accepted as a protecting group for cys. upon incorporation of these cys derivatives onto the growing peptide chain, however, considerable base-catalyzed racemization of cys is known to always occur at the activating and coupling steps with phosphonium or uronium reagents such as pybop or hbtu, respectively. in addition to this, racemization of the c-terminal cys esterified to resins also occurs during the repetitive n α -fmoc deprotection using piperidine. in order to find an s-protected cys derivative applicable for fmoc chemistry that can efficiently suppress racemization of cys both when its incorporation is conducted by phosphonium or uronium reagents and when it is linked to a resin via ester linkage, we introduced the methoxybenzyloxymethyl (mbom) group into the thiol function on cys. the mbom group was found to substantially supress racemization of cys ( . %) during its incorporation mediated by phosphonium or uronium reagents in comparison with the trt and acm groups ( . % and . %, respectively). even after a -h treatment of the peptide resin, in which its c-terminal cys was linked to a trt-type resin, with % piperidine in dmf, the mbom group caused a significant reduction in the level of racemization with the c-terminal cys ( . %) while trt and acm groups led to a considerable extent ( . % and . %, respectively). next, we demonstrated the usefulness of the mbom group on cys by applying it to ncl chemistry involving the coupling of a peptide thioester and a cys-peptide. in the synthesis of the latter peptide, however, a diastereoisomer with racemization of the nterminal cys tends to be difficult to separate from the intact peptide as its chain length increases. thus, this measure using fmoc-cys(mbom) can facilitate the avoidance of ambiguities in the quality of the synthesized peptides. labeled peptides are important tools in chemical biology to obtain information on their biological function in, for example, cellular communication processes as well as to study the molecular basis of diseases. the functionalization of bioactive peptides with biophysical reporter groups like strongly absorbing chromophores, is most often performed via post-synthetic chemoselective biocojugation reactions, involving amino, thiol, or azide specific reactions. an alternative approach for the installation intense chromophoric organic molecules, is the coupling of nonchromophoric precursor molecules to obtain chromophoric properties in the final product. recently, we have shown that the [ + ] cycloaddition-cycloreversion reaction between peptide-functionalized electron-rich alkynes and electrondeficient cyanovinyl derivatives results in the formation of a new class of π-conjugated peptidic donor-acceptor chromophores. , the chromophoric properties of the final product can be tuned by varying the p-electron conjugation pattern. herein, we describe the synthesis of the versatile building blocks, n-a-( -ethynylphenyl)-n-a-(methyl)-glycine and n-a-( -( , , -tricyanovinyl)benzoyl)-glycine, their subsequent application in spps to functionalize peptides with d-p-a chromophore precursor molecules, and their uvvis absorption characteristics. as a typical example, alkyne-functionalized aβ peptides, val-ala-ile and lys-leu-val-phe-phe-ala-glu, have been reacted with a series of cyanoolefins to obtain far-red intense imaging chromophores for amyloid-rich peptide aggregates. a novel method for regioselective disulfide formation in mini-proteins by kinetic oxidation control t. lindner, u. haberkorn, w. mier department of nuclear medicine, university hospital heidelberg, im neuenheimer feld , heidelberg, germany rigid frameworks are essential for the development of peptide-based alternatives to immunoglobulins. [ ] disulfideconstriction covalently stabilizes tertiary structures, which allows further miniaturization of protein-derived drugs by chemical synthesis. while the amino acid assembly steps in solid phase peptide synthesis are highly efficient, regioselective formation of multiple disulfide bonds is still a challenging and time demanding task. even after applying laboriously optimized folding conditions and extensive purification, a successful synthesis of the correctly folded isomer is not guaranteed. [ ] to circumvent these problems, a procedure that takes advantage of the different reactivity of free vs. acmprotected thiols during iodine oxidation was developed. this protocol to sequentially form two disulfide bridges in a one-pot reaction was applied to different peptides, such as (a) min , a mini-protein scaffold for phage-display assisted drug design; [ ] (b) α-conotoxin imi, a neurotoxic peptide isolated from the venom of marine cone snails; [ ] and (c) endothelin- , a peptide involved in blood pressure regulation. [ ] the presented technique tolerates precarious residues within the to-be-folded-peptide, such as fluorescent dyes or metal chelators as used for bioactivityassays. compared to the individually optimized literature procedures, this novel approach offers significant improvements: overall yields > % are obtained in first attempts and stepwise formation of the disulfide bridges is performed within a few hours instead of days. in recent thirty years, c-terminal modified peptides have been proved to have greater potential as apis (active pharmaceutical ingredients) due to their increased chemical and enzymatic stability and improved pharmacodynamic properties - . a prominent example, octreotide - , an octapeptidoalcohol, has witnessed as a potent anti-cancer agent targeted for gastro-entero carcinomas. in view of synthetic methodology, peptidoalcohol can not be directly prepared by standard spps protocol becouse of the c-terminal structure released from resin are not alcohol but always peptidoacid or peptidoamide. to overcome this problem, a novel protocol of shortened n- coupling cycles on merrifield resin and then the ammonolysis of peptedyl resin by an aminoalcohol as the c-terminal residue getting peptido-alcohol as targetting product has been devoloped in our lab. because of the cleavage treatment of peptidyl merrifield resin is not under acidic condition, such as hf or tfmsa, but ammonolysis, some side-chain producting groups(spg) related to boc chemistry like bzl, clz, tos…; must be avoided in sequence assembly. therefore a hybrid orthogonal protection (hop) of boc/fmoc protocol was adopted for the sake of producing naked peptidyl (without any spg) resin before ammonolysis. fifteen peptidoalcohols with different terminal alcohols were conveniently prepared, most of them released form resin with very good yields. due to its cyclic structure, proline is the coded amino acid with a more restricted conformational flexibility. the incorporation of additional groups into the pyrrolidine ring is a useful means to produce new amino acids that combine the conformational properties of proline with sidechain functionality. this is the case of β-phenylproline, (βph)pro, that can be regarded as a proline-phenylalanine hybrid in which the orientation of the aromatic substituent is dictated by the conformation of the five-membered ring and the cis or trans configuration of the phenyl group relative to the carbonyl moiety. accordingly, cis(βph)pro and trans(βph)pro combine the conformational properties of proline with an aromatic side-chain functionality that is rigidly oriented with respect to the peptide backbone, and this may be useful in the design of biologically active peptides and other applications relying on specificallyoriented side-chain moieties. we have developed synthetic procedures for the preparation of the cis(βph)pro and trans(βph)pro stereoisomers in enantiomerically pure form. the methodology is based on the preparation of racemic precursors of each amino acid and their subsequent hplc resolution on chiral columns. multigram quantities of the target amino acids have been isolated in optically pure form and suitably protected for use in peptide synthesis. the importance of peptide cyclization for studying peptide conformation, creating new structures, or for developing peptide therapeutics is well established. in particular, sidechain lactam bridges linking two amino acid residues that are several residues apart in the linear sequence or headto-tail backbone peptide cyclization enable rigidification of the structure and improvement of in vivo stability. native chemical ligation (ncl) is now an established method for producing backbone-cyclized peptides or proteins. the application of ncl to the synthesis of sidechain cyclized peptides is less frequent. head-to-side-chain cyclization by ligating a c-terminal thioester with a cys residue located on a lysine side-chain was used by few authors. the alternative tail-to-side-chain cyclization mode is rare, probably due to the difficulty of installing a thioester group on amino acid side-chains such as aspartic or glutamic acids the reaction of a bis( -sulfanylethyl)amido (sea on ) group with an n-terminal cysteine residue in water and at neutral ph results in the formation of a native peptide bond. [ ] oxidation of sea on results in a cyclic disulfide called sea off having a , , -dithiazepan- -carbonyl structure. [ ] sea off is a self-protected form of sea on . we show here that bis( -sulfanylethyl)amido side-chain acid(dab), ornithine and lysine were selected as building block; a and n,n'-cbz- -amidinopyrazole ( b) were selected as guanidinylating reagents for specific situation. for synthesis of n-terminus local cyclo-guanidine peptide, designated peptides were assembled on acid labile solid support such as rink amide resin by fmoc strategy. then either fmoc-dab(boc)-oh, fmoc-orn(boc)-oh or fmoc-lys(boc)-oh was incorporated respectively at n-terminus. fmoc was removed followed by guanidinylating by b and then peptide was cleaved by acid. by neutralizing with nmm in acetonitrile solution, side chain amino group and a-guanidine would form , or membered local cycloguanidine. the remaining cbz could be removed by hydrogenation. for synthesis of backbone side chain cyclic peptide, bis-fmoc-daa was introduced in the peptide previously on resin followed by removal of fmoc. selective guanidinylate side chain aminogroup by a followed by peptide assembling with an insertion of orthogonal protected daa at - aa apart from first daa. for synthesis of a-nh sidechain cyclic peptide, first daa should be introduced with orthogonal protected form. b was used to guanidinylate a-nh . after cleavage and neutralization of those two kinds of intermediates, guanidine-bridged marco-cyclic peptide was formed. the resin is also a multipurpose tool for the synthesis of carboxylic acids, esters and thioesters. when the synthesis is completed, the fully protected peptide hydrazide resin is oxidized with either n-bromosuccinimide (nbs) or copper(ii) acetate in pyridine. the resulting acyl diazene resin is then cleaved by peptide displacement at the c-terminus with amine. the fully deprotected peptide amide is finally obtained by treatment with trifluoroacetic acid (tfa). in our approach, we used a -fmoc-hydrazinobenzoyl am novagel resin to synthesize a peptide-substituted amide in the c-terminus. first, the oxidative cleavage was carried out with nbs in pyridine and a nucleophile [a protected (aminomethyl) benzimidamide (amba)]. however, the yield of the reaction was very poor. in the next step, we applied copper(ii) acetate in the presence of pyridine and amba. following optimization, the efficiency of the process was significantly improved. herein we discuss the conditions needed to obtain a reasonably high efficiency of the oxidative cleavage in the synthesis of our c-terminal modified peptides using the aryl hydrazine resin linker. blood vessels on tumor tissues, similarly to integrin receptors. this observation suggests cd as a selective target for targeted delivery of drugs and nanoparticles to tumor neovasculature using ngr peptides as homing motif. in our work, new cyclic-ngr peptides containing a thioether linkage were prepared. the influence of their structure on the speed of succinimide ring formation and deamidation was evaluated and compared with the previously published data on cyclic-ngr derivatives containing amide bond or disulfide bridge in the cycle (c[kngre]-nh and c[cngrc]-nh ). to avoid the deamidation under the conditions used for cyclization, the synthetic routes were optimized. the influence of the ph, ionic strength and temperature of the solution on their chemical stability was investigated. the structure of the cyclic peptides was investigated by circular dichroismand nmr-spectroscopy. receptor binding ability and the influence of the cyclic peptides on the cell adhesion and motility were also evaluated. this work was supported by grants from the hungarian national science fund (otka nk and k ) and the national innovation office (bio_surf, om- / ). clickable peptides and their attachment to oligonucleotides m. wenska, m. alvira, r. strömberg department of biosciences and nutrition, karolinska institutet, novum, se- huddinge methodology for the ready conversion of peptides into "clickable" azido-peptides with the possibility of selecting either n-terminus or c-terminus connection is presented. synthesis of peptide-oligonucleotide conjugates (poc's) include conjugates of oligonucleotides with peptides known to be membrane penetrating and nuclear localization signals. a general procedure, based on a new activated alkyne linker, for the preparation of poc's has been developed. with this linker, conjugation is effective at room temperature in mm concentration and submicromolar amounts. this is made possible since the use of a readily attachable activated triple bond linker speeds up the cu(i) catalyzed , -dipolar cycloaddition ("click" reaction). the main scheme for conjugate preparation involves sequential conjugation to oligonucleotides on solid support of i) an h-phosphonate based aminolinker ii) the triple bond donor p-(npropynoylamino)toluic acid (pata) and iii) azido-functionalized peptides. the method gives excellent conversion of oligonucleotide to the poc on solid support, and only involves a single purification step after complete assembly. the procedure which makes use of a low concentration of copper ions leads to a product with very little copper left (similar or less than in drinking water). the synthesis is flexible and can be carried out in non-specialist laboratories without the need for specific automated synthesizers since it has been designed to utilize commercially available oligonucleotide and peptide derivatives on solid support or in solution. comparison of alternative deprotection reagents to piperidine for the synthesis of a poly-alanine peptide on the tribute® peptide synthesizer m.a. onaiyekan,* j.p. cain, c.a. chantell, m. menakuru protein technologies, inc. tucson, az, usa in peptide synthesis, piperidine is a common agent for fmoc removal. however, piperidine is a controlled substance which requires special handling and cannot be used in some countries. therefore, it would be useful to identify alternative deprotection reagents to piperidine for fmoc removal. it is well known that poly-alanine sequences have a high propensity to aggregate after the fifth residue. in this application, (a) k-oh was synthesized using the tribute®'s intellisynth uvmonitoring and feedback system to compare the efficiency of fmoc removal by piperidine vs. three alternative bases (pyrrolidine, cyclohexylamine, and tertbutylamine) in the last cycles of the synthesis. it was found that pyrrolidine produced a higher purity product with fewer deprotection repeats and shorter deprotection times per cycle than piperidine, proving it to be a highly efficient, viable alternative to piperidine for fmoc removal. the endogenous tripeptide gpe also nammed "glypromate" is made up by the three n-terminal residues (glycine-proline-glutamate) of the insulin-like growth factor (igf ). this tripeptide is a partial glutamate antagonist and showed good results in different neuroprotective in vitro and in vivo experiments. , gpe also binds to glial cells regulating neurotransmitter levels in the brain. , however, gpe suffers from poor lipophilicity and a short half-life in vivo. that's why there is a need for more lipophilic and protease resistant analogues of gpe. in this poster we present the synthesis of trifluoromethylated analogues of gpe based on the or -cf -pseudoproline residues. introduction of fluorine atoms on bioactive compounds is known to deeply modify their physico and biochemical properties increasing lipophilicity and resistance to protease. thus, developing a trifluoromethylated analogues, we intend to increase the bioavailability of gpe, keeping the benefit of its neuroprotective properties. our research team is strongly involved in the synthesis of trifluoromethylated alpha-amino acids. recently we published the synthesis of -trifluoromethyl- , oxazolidines derived from fluoral and (l)-serine and we demonstrated that these five membered ring -cf pseudoprolines are hydrolytically stable and can be considered as proline analogues. that's the reasons why we are interested to replace the proline residue of gpe by those trifluoromethylated compounds. the development of original coupling conditions and the detailed synthesis of two pseudoprolines analogues of gpe will be presented in this poster. modifications. in combination with automated spps, unprecedented access to large peptides and small proteins for biological research has been achieved. we demonstrate the application of this methodology to the synthesis of a variety of peptides on the prelude® peptide synthesizer. exploring the space of fluorine-labeled α-amino acids for solid state f-nmr structure analysis of peptides: rational design, synthesis and applications p. solid state f-nmr is a powerful method to study membrane-active peptides, as it can reveal their conformation, orientation and dynamics when embedded in biomembranes. for this purpose the native peptide has to be selectively labeled with a suitable f-containing amino acid at several different positions. the resulting battery of singly f-labeled analogues is then analyzed by solid state f-nmr. the main limitation to this approach currently lies in the poor arsenal of available f-labels. we have therefore rationally designed and synthesized several specific amino acids bearing a cf -reporter group, which fulfil all strict criteria to a "proper" f-label. , to allow a geometry-based structure calculation, the cf group has to be rigidly attached to the peptide backbone. we thus rigidified the side chain using either a [ . . ]bicyclopentane moiety, a cyclobutane ring, or the intrinsic proline framework. this way, suitable cf -labeled analogues were created as substitutes for bulky hydrophobic amino acids (leu/ile/val/met), for aromatic residues (phe), for polar side chains (ser/thr), and for proline (pro). by now we have applied the developed f-labels for a comprehensive structure analysis of more than ten different membrane-active peptides (gramicidin s, pgla, mag , kigaki, sap, temporin a, bp , etc). recently, several new activators have been introduced into the market, and they were evaluated along with some older activators for their ability to synthesize a range of peptides with shorter and longer reaction times on the symphony® peptide synthesizer. it was found that hdmc, pyclock, comu, hctu, and hatu worked well at shorter reaction times ( x min), but pyoxim and tffh only worked well at longer reaction times. the performance of pybop at shorter reaction times was poor only for more difficult sequences. these results are important for selecting an appropriate activator for fast spps applications. the plant cyclotides form the largest family of cyclic peptides . they contain a signature motif referred to as the cyclic cystine knot, which is derived from the cyclic backbone and three inter-knotted disulfide bonds. intriguingly, cyclotides can be boiled, treated with chemicals or enzymes without disrupting their overall fold. thus, they are sometimes labeled as ultra-stable proteins. in addition, cyclotides are tolerant to mutations, and as a scaffold they can successfully accommodate foreign bioactive epitopes of variable sequences . cyclotides share many of these properties with another disulfide containing cyclic plant peptide, the sunflower trypsin inhibitor (sfti- ) . emerging evidence indicates that cyclotides and sfti- are valuable not only as peptide stabilizing scaffolds; in combination with their cell penetrating properties, these disulfide rich cyclic peptides have significance as intracellular drug carriers. although both peptides are genetically encoded, studies to ascertain the exact mechanisms of their biosynthesis are currently on going. thus, the synthesis of cyclotides and sfti- are currently restricted to chemical means. we have recently adapted a fmoc-spps method for cyclic peptide synthesis, via n-acylurea intermediates with the assistance of microwave irradiation. this method is a safe and convenient alternative to boc-spps and has the ability to be automated conveniently. using this method, parent scaffolds as well as several cyclotide and sfti- analogues with potential antimicrobial and matrix metalloprotease activities were synthesized. with the rising interest in the cyclization concept as a tool to impart stability on unstable peptides, the cyclic peptide synthesis method adapted herein is anticipated to have numerous applications. fixed configuration. the nonnatural oligomers have an extended conformational space and are supposed to adopt non-canonical secondary structures . in addition, the backbone modification makes these molecules more stable towards proteolytic degradation. the majority of proteins in nature are post-translationally modified, and the most abundant modification is the protein glycolysation, which introduces wide structural variety to proteins. glycoproteins have an important role in the biological recognition process, such as immunodifferentiation, cell adhesion, cell differenciation and regulation cell growth . new aza-β -amino acids bearing either an azide instead of amine on lys and orn chain or an alkyne group will be described and used in solid phase synthesis to finally performed a click chemistry to cyclize pseudopeptides or to introduced a glycosylated function . true for a series of peptides that display strong corticotropin releasing factor (crf) antagonistic activity. seminal studies by rivier et al. have shown that the incorporation of a lactam bridge in the crf-sequence resulted in an enormous increase in activity and potency, due to stabilization of the bioactive a-helical conformation of the peptide; and the newly designed peptide was called astressin. based on the astressin sequence, we started a truncation and deletion study to arrive at astressin analogs with a reduced size but still remain active as crf antagonists. this study resulted in the smallest active crf antagonist, astressin( - ). this sequence was further optimized by the introduction of novel covalent constraints, other than the well-known lactam bridge. as a first approach, the alkene/alkane bridge, which can be introduced via ring-closing metathesis via alkenesubstituted amino acid side chains, and as a second approach, the triazole bridge ('click' macrocyclization), via either a cu(i)-or a ru(ii)-catalyzed cycloaddition reaction between azide-and alkyne-derivatized amino acid residues were explored. herein, we will present the details of the synthesis of the alkene-, azide-, and alkyne-functionalized amino acids, their use in spps, and the optimized approaches for macrocyclization. furthermore, the peptides have been characterized by hplc, nmr, lcms, and studied by circular dichroism spectroscopy to obtain insight into the helical propensity of the peptides in relation to the cyclic constraint. the synthesis of a nitronyl nitroxide, c α -tetrasubstituted αamino acid (a class of sterically restricted amino acids that promote the formation of peptide β-turns and helical structures) was achieved by derivatisation of racemic amino- -cyano-indan- carboxylic acid [aic(cn)]. racemic boc-aic(nn)-oh was prepared by bis(alkylation) of ethyl isocyanoacetate under phase transfer conditions with , -(bis)bromomethyl benzonitrile as alkylating agent, followed by acidic hydrolysis, n α -boc protection, and saponification of the ester function. resolution was achieved through formation of the diastereomeric amides of (s)-phenylglycinol with chromatographic separation and mild acidic hydrolysis. reduction of the nitrile group to an aldehyde was carried out with raney nickel in the presence of sodium hypophosphite. condensation with , -diamino- , -dimethylbutane gave the corresponding tetramethylimidazolidine, which was oxidised with chloroperbenzoic acid to the desired nitronyl nitroxide. the uv-vis absorption and epr spectra of the amino acid were recorded and its magnetic properties were examined. in order to develop the synthesis of this peptide using the fmoc solid-phase peptide synthetic methodology, orthogonally protected β-hydroxyaspartic acid was needed. more precisely we wish to dispose of ( r, r)-n -fmoc- -tbdm-silyloxy-aspartic acid α -allyl ester instead of the recently reported dmab ester - indeed, in preliminary assay using this protective group we experienced difficulties during the final cyclisation step . the synthesis was developed starting from inexpensive l(+) dimethyltartrate and extended to the others stereoisomers of the β-hydroxyaspartic acid. structure. for that, we chose to replace proline by silaproline to afford polysilaproline. this study shows the comparison of two polyamino acids: polyproline and polysilaproline polymers. homopolypeptides were synthesized by polymerization of corresponding amino acid n-carboxyanhydride . multicomponent reactions (mcrs) represent a chemical process involving at least three reactants for the formation of several covalent bonds in one operation . by definition mcrs are chemo-and regioselective, convergent stepefficient procedures and take place with high atom economy. the copper(i)-catalyzed , -dipolar cycloaddition of organic azides and terminal alkynes (cuaac) reported by meldal and sharpless has been involved in various fields of chemistry and biochemistry research. however only few reports describe the implementation of cuaac and mcrs. , recently our research focused on a novel threecomponent reaction based on a cu(ii)-triggered aminolysis of peptide hydrazide resin and an azide-alkyne cycloaddition sequence. copper(ii)-induced oxidative aminolysis of hydrazides generates cu(i), catalyst of the azide-alkyne cycloaddition. this feature was exploited to design a solid phase detaching three-component reaction. the mcr process requires a peptide hydrazide resin, an amino azide linker and an alkyne, resulting in the formation of peptide modified at the c-terminus through an amino , , -triazole linker. this method can potentially be applied to the synthesis of a large variety of peptide derivatives starting from fmoc-spps assembled peptidyl resins. furthermore, it is not practical to compare hplc spectra from different resin samples (e.g., before and after reaction) directly. a comparison by analyzing the same (mg) amount of resin would involve tedious sample preparation that is extremely error-prone and would be impractical because factors resulting from the increase or decrease of the molecular weight of the resin-bound compounds may have a significant influence on the results. the use of internal reference compounds allows rapid assessment of reactions performed on solid supports. the internal reference compound is bound to the resin together with the substrate and cleaved with the products after completion of the reaction. commercially available compounds can be used for this purpose, or likewise, the reference compound can be generated from the substrate by partial capping of a functionality. the peak integration of the reference compound in the hplc-uv spectra can be correlated directly to those of the rest of the compounds present in the reaction mixture and therefore a quantitative interpretation of the spectra with respect to conversion and yield is possible. here we demonstrate the proof of principle as well as the accuracy of this method. modifier proteins such as ubiquitin are conjugated to protein substrates in cells and thereby mediate various biological processes. of high interest, is the ubiquitin fold modifer (ufm , residues) which has structural similarity to ubiquitin but has no sequence similarity. unlike ubiquitin, ufm has not been extensively studied and little is known about its biological role. to understand ufm 's biological functions, access to pure, homogeneous natural and modified ufm protein is essential. chemoselective ligation techniques are suitable for providing such proteins. recently, a variation of the α-ketoacid-hydroxylamine (kaha) ligation was developed, which utilizes the chemoselective reaction between a c-terminal peptide αketoacid and a n-terminal -oxaproline. this modified form of the kaha ligation furnishes a native peptide bond and a homoserine residue. this ligation is useful for the synthesis of proteins from two unprotected protein segments in aqueous buffers. for the synthesis of larger proteins, a sequential ligation strategy is necessary. using ufm as the model system we have developed a sequential ligation procedure using kaha ligation with -oxaproline. applying the new sequential ligation strategy we have prepared ufm by total chemical synthesis. we have also prepared a cterminal thioester surrogate of ufm protein, which is suitable for conjugation to proteins of interest. the syntheses required the development of a bifunctional peptide segment bearing an α-ketoacid and an orthogonally protected -oxaproline. the preparation of the protein segments, their intermediates, the deprotection, and sequential kaha ligations towards the syntheses of ufm protein and c-terminal modified thioester ufm protein will be discussed. affinity and biological activity, we have designed and synthesized new analogues by multiple n-methylation of hut-ii( - ) backbone amide bonds. all the peptides were performed by a novel synthetic approach, in which the introduction of n-methyl groups occur during regular solidphase peptide synthesis. on these new ligands we evaluated the binding affinity and biological activity at the ut receptor and performed preliminary nmr conformational studies. since that time a number of different machines have been used to automate peptide synthesis. modern machines are following two general setups; the so called "single approach" and the "parallel approach". in the single approach, the machine is developed to synthesize one or few peptides simultaneously. the user is able to optimize the synthesis conditions on each single peptide and each single coupling step. the maximum product quality regarding purity and yield is the major task of this approach. in the parallel approach, the machine is developed to synthesize a huge number of different peptides in the same single setup and time-frame. the user always has to find a synthesis protocol appropriate for the needs of each peptide to reach the maximum quality, knowing that there will be always a number of failed peptides. as a result you will find both types of peptide synthesizers in laboratories all over the world: the single machine, for the complicated peptides, and the parallel machine, allowing generation of multiple peptides with standardized protocols for each. the tetras is the first instrument combining the advantages of both machine types and allows the user to synthesize up to different peptides in parallel. each peptide can have its own individual synthesis protocols, separate of all others. the user can combine different synthesis scales, peptide lengths, and activator reagents in one run. finished peptides can be removed and new peptides can be started while the tetras is still running. the tetras allows the user to establish an uninterrupted production shop using one instrument only. siemion, i. z.; peptide res the peptides: analysis, synthesis and biology monitoring peptide folding in membrane-active peptides: a time-resolved spectroscopic study e. gatto a cordopatis p. st european peptide symposium sar studies of triazolyl-containing cyclopeptides: a defined -turn structure increases potency and selectivity to melanocortin receptor subtypes c. testa, a,b proc. natl. acad. sci proc. natl. acad. sci usa multicomponent reactions microglobulin: a "difficult" protein s. abel, m. beyermann the protein ß -microglobulin constitutes the noncovalently bound light chain of the major histocompatibility complex class i (mhc) and plays an essential role in the dialysisrelated amyloidosis. [ , ] to examine the amyloid fibrils of the ß -microglobulin (ß -m) via infrared spectroscopy we intended to synthesize -c-labeled ß -m. [ ] due to the two cysteine residues in positions and we used the native chemical ligation (ncl) strategy for assembling the -mer protein. this necessitates the synthesis of three segments which was accomplished on solid phase using the fmoc/t-bu chemistry. the preparation of the segments had to be optimized with respect to aggregation, aspartimide and piperidide formation, trifluoracetylation, and s-tert-butylsulfonium formation. additionally, ncl steps had to be optimized, because of "internal" thioester formation, dimerization and the formation of side-products of the activated n-terminal segment peptaderm inc., krakowskie przedmie cie str. , warsaw, poland immunosuppressors, such as cyclosporine a (csa) and tacrolimus®, are routinely used in prevention of graft rejection after organ transplantation and in therapy of some autoimmune diseases, including skin inflammation. a naturally occurring in linseed oil cyclolinopeptide a (cla, c(-pro-pro-phe-phe-leu-ile-ile-leu-val) possesses a strong immuno-suppressive activity, comparable at low doses with that of csa , but is much less toxic. we synthesized new cla analogs, containing instead of one proline residue its six-membered mimics, pipecolic acid (pip): c(-pip-pro-phe-phe-leu-ile-ile-leu-val) ( ) and c(-pro-pip-phe-phe-leu-ile-ile-leu-val) ( ). the incorporation of pipecolic acid residue led to different conformational behavior of the nonapeptide cycle. nmr experiments in cdcl solution showed that cla analogue with the pipecolic acid residue in position was much more flexible than cyclopeptide . the new peptides were devoid of toxicity up to μg/ml with regard to human peripheral blood mononuclear cells (pbmc), did not inhibit tumor necrosis factor alpha production in blood cell culture, but exhibited dosedependent, anti-proliferative actions for phytohemagglutinin a-activated pbmc. since peptide was more potent it was tested for growth inhibition of l- lymphatic leukemia. the peptide was found to strongly inhibit the cell growth even at low concentration ( % inhibition at μg/ml). hiv- has emerged as the largest and the most devastating public pandemic in our days, affecting approximately million people worldwide . development of an effective, safe and preventive hiv vaccine remains an urgently needed priority. epitopes for hiv-specific antibodies in elite controllers, a subgroup of long term non progressors, encompassing segments of mper of gp and for the v loop of gp were identified using the phage display technology . immunization experiments with epitopes conjugated to an artificial sequential oligopeptide carrier (soc ), formed by four repeats of the tripeptide lys-aib-gly in tandem, or to the palmitoyl group are currently in progress. all syntheses were performed on a rink amide resin following the fmoc technology. conjugation of epitopes to the soc carrier was realized via a chemoselective ligation approach, which generates an oxime bond between the h n-o-groups of the modified lysine residues and the aldehyde group of each epitope institute of immunology and experimental therapy, polish academy of sciences, - wrocław, poland peptaderm inc., krakowskie przedmieście , - warszawa, poland nonproteinogenic amino acids have been a tool to modify the structures of natural peptides since a long time . bioactive peptides involved in a physiological and biochemical processes cannot be applied in the therapy because of their instability in physiological conditions. that's why the synthesis of their stable active analogues is a challenge for medicinal chemistry nowadays. -trans-hydroxyproline (hyp) is an important building block of natural collagen. it is responsible for the stabilization of collagen super helix, forcing the trans amide bonds configuration with preceding amino acids . at the same time the impact of trans- -hydroxyproline on the conformation other than the collagen peptide chains of biologically important compounds is little known. it is known that immunosuppressive activity of cla is comparable with cyclosporine a and is associated with the presence of the tetrapeptide fragment pro-pro-phe-phe containing pro-pro cis amide bond. now we present synthesis, conformation and biological activity of new analogues of cyclolinopeptide a (cla), containing -transhydroxyproline instead of proline residues in position or . we expected that the introduction of the hydroxyl group in the pyrrolidine ring might influence the biological activity and conformation of the native peptide due to its hydrophilic character and hydrogen bonding ability. the linear precursors of modified cla analogues were prepared manually by standard solid-phase procedure "step by step" on wang resin using fmoc/tbu strategy and tbtu as coupling reagent. the cyclizations of linear peptides have been made under high dilution conditions by means of edc/hobt coupling reagents. the biological activity of newly synthesized compounds as well as the conformational study will be evaluated. dip. di scienze ambientali, seconda università di napoli, caserta, italy nmr spectroscopy is a powerful method to perform structural studies on peptides. to completely fulfill the potential of nmr, peptides labeled with stable isotopes ( n, c, h) are essential. peptides are easily prepared on solid-phase but chemical synthesis becomes prohibitively expensive when applied to the incorporation of isotopes. an alternative cost-effective strategy is the recombinant expression of peptides in e. coli as fusion constructs with carrier proteins. the main problem of this approach is the need of chemical reagents or proteases to cleave the target peptide from its fusion partner after purification. proteases may determine the heritage of extrasequence amino acids at the peptide n-or c-terminus, while chemical reagents require harsh reaction condition that may modify target peptides. an interesting solution is represented by the use of inteins as fusion partner. inteins are protein elements that can catalyze their self-excision from a flanking sequences in mild conditions, by adding nucleophilic agents such as thiols or simply by shift of ph and temperature, bypassing the use of proteases or chemical reagents. we used the self-cleaving mxegyra mini-intein as fusion partner for the preparation by recombinant means of two isotope labeled peptides, hplw and qk. , the two peptides target vascular endothelial growth factor receptor (vegfr) and have been described to modulate vegf-dependent angiogenesis. our expression and purification scheme allows to obtain homogeneously isotope labeled peptides. the availability of isotope labeled hplw and qk opens the way to nmr studies aimed to characterize the folding dynamics of the two peptides and their structures in complex with vegfr. an nmr method to discriminate between the fullyextended and different helical conformations in a spacer peptide c. peggion*, m. crisma, f. formaggio, c. toniolo icb, padova unit, cnr, department of chemistry, university of padova, padova, italy the ideal fully-extended, α-peptide conformation, also known as . -helix, is characterized by φ = ψ = ω = °t orsion angles. the repeating motif of this foldamer is a pentagonal (pseudo)cyclic structure (called c ), stabilized by an intraresidue h-bond. the n-h and c=o groups in the . -helix are not involved in intermolecular h-bonds. multiple c conformations were observed in homopeptides made up of c α,α -dialkylated glycines with both side chains longer than a methyl. this is the case for c α,αdiethylglycine (deg), the residue studied in this work. it is known that deg homo-peptides can adopt the . -helix or the -helix depending on environmental factors and nand/or c-terminal moieties. , in this communication, we introduce an nmr method to discriminate between the . -helix and the -helix based on the observation of cross-peak intensities in the noesy human serum amyloid a (saa) is a highly conserved apolipoprotein produced by the liver under inflammatory conditions accompanying e.g. atherosclerosis, cancer and amyloidosis [ ] . it is also known that saa α isoform has the amyloidogenic properties [ ] . till now it is little known about structure of human saa, as it hampers structural studies due to its facile aggregation. the analysis of protein sequence and cd data together with theoretical studies revealed a typical globular structure of the protein [ ] . the c-terminal sequence of saa contains three proline residues, which probably are responsible for the unordered structure. recent in vitro studies involving saa and human cystatin c (hcc) revealed direct interactions between the ( - ) fragment of saa and the ( - ) sequence of hcc. the results of elisa test for the ( - ) saa fragment have shown that it binds to hcc very well. the nmr studies for the wild ( - ) sequence found an unordered structure in phosphate buffer. based on these data we decided to check how the point mutations pro→ala in ( - ) saa fragment could influence the peptide's structures. we synthesized four peptides with pro→ala point mutations and we performed cd experiments at different conditions. the results show that two of them contain disordered structure and two α-helical structures. in this project we analyze the solution structures of these peptides at the atomic resolution using d nmr supported with molecular dynamics. design and conformational analysis of stapled peptides mimicking cullin binding region to kctd . i. de paola, a l. pirone, a e. pedone, a s. di gaetano, a l. vitagliano, a r. fattorusso, b g. malgieri, b l. zaccaro*, a acknowledgement: this study was supported by eu within the european regional development fund (poig. . . - - / - ). model of angiotensin ii bound to the at receptor in the lipid bilayer environment m.t. matsoukas, t. tselios* department of chemistry, university of patras, gr- , patras, greece the renin-angiotensin system () plays a major role in blood pressure regulation. a sequence of enzyme reactions leads to the release of angiotensin ii which interacts principally with the type- angiotensin ii receptor (at ), a -residue, which belongs to the g protein-coupled receptor family. in the present study, the human at d model was constructed using modeler for the sequence alignment and loop refinement tools. on this basis, the crystal structure of bovine rhodopsin, (pdb code u ), was used as a d template. the gromacs software and amber sb forcefield were utilized for molecular dynamics calculations [ ] in order to evaluate the binding mode of angiotensin ii. the role of the critical amino acids of the binding site v , n , l , a , k , s , h , n and y is being studied. moreover, newest information on the role of the nd extracellular loop by unal et. al. [ ] have been implemented on the model, therefore we propose the contribution mechanism of the residues f -q for binding of angiotensin ii to the at receptor for activation and signaling. a. stavrakoudis department of economics, university of ioannina, greece one key step in the immune response against infected or tumor cells is the recognition of the t-cell receptor (tcr) by class i major histocompatibility complexes. it has been found [ , ] that such peptide/mhc complexes can interact with antibodies as well. this happens mainly in the central part of the peptide in class i complexes [ ] , or at the cterminal of class ii complexes [ ] . in some cases, the same peptide/mhc complex has been found to interact with both tcr and antibodies [ ] . in these study a series of supermolecular complexes have been studied with stateof-art molecular dynamics simulations [ ] the dipeptide kyotorphin (tyr-arg, kyo) plays a role in pain modulation in the mammalian central nervous system (cns), and is one of the most investigated neuropeptides. the tyr-arg motif exists widely throughout the brain not only as kyotorphin, but also as the n-terminal part of several endogenous analgesic peptides , . also, this peptide is very rapidly degraded by aminopeptidases . one of the successful strategies in the design of neuropeptides with enhanced stability and improved delivery to the cns is that with the use of non-protein amino acids, like canavanive (cav), a structural analogue and antimetabolite of arginine (arg trichogin ga iv (noct-aib-gly-leu-aib-gly-gly-leu-aib-gly-ile-lol, in which noct is n-octanoyl and lol is leucinol) is an antimicrobial lipopeptaibol, a unique group of membraneactive compounds of fungal origin, characterized by a high content of the nonproteinogenic ca,a-disubstituted glycine aib (a-aminoisobutyric acid). owing to the gem-dimethyl substitution on the c a atom, aib exhibits a strong propensity to induce β-turns and /α-helical conformations in peptides. we have previously reported on a fluorescent analog of trichogin ga iv, the primary structure (and acronym) of which are: fmoc-aib-gly-leu-aib-gly-gly-leu-toac-gly-ile-leu-ome (f t ) where fmoc is fluorenyl- -methyloxycarbonyl, toac is , , , -tetramethylpiperidine- -oxyl- -amino- -carboxylic acid, and ome is methoxy. the double substitution of an energy donor (fmoc) at the n-terminus and an acceptor (toac) in the trichogin sequence enabled us to make use of time-resolved optical spectroscopies, spanning from the nanosecond to the microsecond time regime, to investigate the conformational propensity and the dynamical features of f t . experimental and computational results indicated that the d-structural and dynamical properties of f t are characterized by a transition from an elongated helix to a more compact conformation mimicking a helix-turn-helix motif. to further investigate the role of the flexible gly -gly central motif we synthesized a new trichogin analog having the gly residue substituted by aib: fmoc-aib-gly-leu-aib-gly-aib-leu-toac-gly-ile-leu-ome (f a t ) experimental and computational results indicated that also the f a t peptide populate two conformations, the dynamics of which were studied at different temperatures using time-resolved spectroscopic measurements. this replacement was demonstrated to stiffen the peptide backbone by reducing the flexibility around the crucial -gly -gly -dipeptide unit. the antigen α β , a member of the integrin family, is involved in the migration of lymphocytes through endothelium to the site of inflammation. thus, α β antagonists may be useful tools for the treatment of various inflammation disorders such as asthma and inflammatory arthritis. in addition, recent studies indicate that α β integrin promotes angiogenesis by allowing the invasion of myeloid cells into tumors, while α β antagonists prevent monocyte-induced angiogenesis, macrophage colonization of tumors and tumor angiogenesis. aiming to the discovery of novel α β antagonists, a series of new peptide analogues cyclized through cysteine disulphide bonds were synthesized and tested in vivo against angiogenesis in chicken embryo chorioallantoic membrane (cam model) . sar results indicated that: yr-c(cdpc)-conh promoted angiogenesis at the higher studied concentration and showed slight inhibition at the lower one, sal-r-c(cdpc)-oh, sal=salicylic acid, showed important inhibition of angiogenesis at dose-dependent manner, yr-c(cdpc)-oh and sal-yr-c(cdpc)-oh both showed no activity on angiogenesis. nmr spectroscopy was applied for the sequential assignment as well as for the elucidation of specific conformational features. experimental noe data were further imposed as distance constraints to a thorough conformational search by applying molecular dynamics simulations. energy refined produced conformers were used as template for the generation of the pharmacophore model associated with the antagonistic activity. such studies are intended to drive a rationalized design and development of this class of inhibitors. hynes r. o. cell, , , - . scaffold discovery by phylomers: a novel cd l specific scaffold derived from glycyl trna synthetase s.r. stone [ ] , k. hoffmann [ , ] , n. milech [ , ] , p. t. cunningham [ ] , m. kerfoot [ ] , s. winslow [ ] , y-f, tan [ ] , m. anastasas [ ] , c. hall [ ] , m. scobie [ ] , p.watt [ ] , and r. hopkins [ , ] [ ] drug discovery technology unit, telethon institute for child health research, roberts road, subicao, , western australia [ ] phylogica pty ltd, roberts road, subiaco, , western australia biopanning of phylomer phage display libraries against human cd l yielded a cluster of highly specific overlapping peptide fragments, from three bacterial genomes, corresponding to the highly conserved catalytic domain from the tetrameric gα β class of glycyl trna synthetases. structural analysis of the overlapping peptide fragments described a scaffold consisting of a central βsheet, comprising anti-parallel β-strands, flanked by nand c-terminal α-helices. further structural analysis revealed that these key structural features, which also encompass the crucial atp-binding motifs of the catalytic domain, are conformationally conserved across both tetrameric gα β and dimeric gα glycyl trna synthetases, yet importantly, there is only limited sequence conservation across these classes. given the identical function of the described domain and it's structural conservation, we postulated that members of the dimeric gα class would display similar cd l specific binding as the tetrameric gα β class, despite the sequence dissimilarity. to test this hypothesis, structurally equivalent peptide fragments of representative bacterial, archaeal and eukaryotic genomes comprising the dimeric gα class were tested for cd l binding in a process we termed ortholog scanning. the results showed that both archaeal (p. horikoshii) and eukaryotic (h. sapiens) structurally equivalent peptides bound to cd l with reasonable specificity and inhibited the cd :cd l interaction with comparable ic 's to the primary gα β class sequences. similar results were also observed for the representative bacterial gα class peptides. that the sequentially diverse orthologous peptides display cd l specific binding has important implications to the affinity enhancement strategies to develop the scaffold as a therapeutic agent, and in improving its "drug-like" properties. we have initiated an investigation related to the effect of radical species upon structures of some peptide segments. in the proposed experimental protocol, aqueous peptide solution was submitted to gamma ray irradiation in controlled - kgy doses. the generation of peptide analogues, possibly induced by reactive oxygen species were examined by electrospray triplequadrupole tandem mass spectrometry (collision induced dissociation approach) and amino acid analysis of crude and/or purified by-products. noteworthy, the gamma irradiation process induced, regardless of the peptide sequence, a non-linear and progressive degradation of all peptides assayed. furthermore, these peptides could be classified in some different classes according to their halflife dose. for instance, the vasoactives angiotensin ii (aii), ang ( - ), bradykinin (bk) and some related peptides were more stable than the melanocyte-stimulating hormone α-msh, substance p or the bk 's ( - ) b receptor fragment (lvyvivgkrfrkksrevyqai). usually, the most prominent derivatives generated from this experimental protocol revealed that they are likely induced by oxidation process, yielding a variation of + da in their molecular weight. the main source of peptide modifications seems to lie either on the phe (hydroxyl group insertion at o-, m-or p-positions of its aromatic side chain) or met oxydation. in the former case, only phe and not phe is oxidized in the bk structure whereas substance p generates an analogue bearing metsulfoxide without modifying its phe , residues. thus, collectively, these findings clearly stress the complexity of factors involved in peptide structural modifications induced by gamma ray-type strong electromagnetic irradiation experiment. an additional target of this approach lies indeed, in the production of unusual peptides for further structure-function investigations. university of bern, bern, switzerland linear peptides are typically poor drug candidates due to their low bioavailability and rapid proteolysis. these limitations can be overcome by rigidifying their structure through head-to-tail or side chain-involving cyclizations. cyclic constraints may also increase biological activity by stabilizing secondary structures and by reducing the entropic penalty of binding to a protein target. the use of multiple branching amino acids in a peptide sequence, like diamino acids (as used in peptide dendrimers ) or amino diacids, allows to design peptides resembling polycyclic alkanes, a type of topology only rarely found in nature (e.g. amatoxins and lantibiotics). bicyclic homodetic peptides such as "norbornapeptides" (bicyclo[ . . ]heptapeptides) were prepared using an orthogonal protection scheme: the first cyclization is performed on resin after selective deprotection of a glutamic acid residue, whereas the second ring closure is achieved by amide bond formation at high dilutions. these peptides are structurally well-defined and cover an almost pristine area of peptide topological space. their conformational rigidity was investigated by means of d-nmr and x-ray crystallography and may offer a platform to design drugs tackling protein-protein interactions. the interaction of peptide ligands with protein receptors face peculiar challenge in recognizing binding surfaces due to availability of a multitude of conformations. therefore it is essential to constrain the peptide conformations for the recognition of receptors and thus finding the bioactive conformation. the cell surface receptor protein family integrins recognize "rgd" sequence which is present in different proteins. to determine the bioactive conformation required to bind with receptor αiibβ , the peptide sequence "riprgdmp" from kistrin was inserted into cdr loop region of rei protein (rei-rgd ). it helps out in finding the possible bioactive conformation of peptide by restricting the sampling space. the activity of rei-rgd was studied and found that as the temperature increased rei-rgd showed a higher affinity towards the receptor αiibβ . the proposed mechanisms for the increased activity of rei-rgd at higher temperature were justified in either of two ways. the modified complex forces the restricted peptide to adopt a bioactive conformation or it unfolds the peptide in a way that opens its binding surface with high affinity for receptor. in this study we model the conformational preferences of "rgd" sequence in octapeptide "riprgdmp" at two different temperatures ( o c and o c) using multiple md simulations. we found that at higher temperature "rgd" sequence from "riprgdmp" adopt turn conformation, while a bend conformation was observed at low temperature. the analysis of various pharmacophoric parameters hint that the turn conformation of "rgd" sequences adopted at higher temperature could be the potential bio-active conformation, and helps out in designing of antagonists for cell surface receptor αiibβ . the -residue peptaibol antibiotic trichovirin i- a (tv) of the linear, covalent structure ac-aib-asn-leu-aib-pro-ala-val-aib-pro-aib-leu-aib-pro-leuol (ac, acetyl; aib, α-aminoisobutyric acid; leuol, l-leucinol) has been synthesized and very thin (~ μm) hair-like crystals were obtained from a methanolacetonitrile-water mixture. diffraction data were collected at k at the diamond light source england, using the microfocus beamline i and a x-ray beam focused to a size of μm full-width-half-maximum. two independent molecules (a) and (b) were located in the crystal's asymmetric unit . both chains assume complete turns of a curved right-handed helical conformation stabilized by intramolecular hydrogen bonds. up to now tv represents the longest right-handed -helix of a natural peptaibol sequence complementing those of synthetic, protected homooligo-aib- insulin is a protein hormone that plays a key role in regulation of blood glucose levels and, thus, has widespread impact on lipid and protein metabolism.insulin is known to act through binding to the insulin receptor (ir); however, the structure of the insulin-ir complex is not known. the crystal and nmr structures of insulin represent only inactive storage forms. it is widely acknowledged that insulin must undergo structural changes in the c-terminus of the b-chain upon binding to the ir. in addition, the n-terminus of the bchain may adopt two different conformations in hexamer, known as t-and r-states. the r-state of the n-terminus of the b-chain creates a long b -b central a-helix. the t-state of n-terminus is in an extended conformation. however, the biological relevance of the t/r forms remains elusive . in this study, we have focused on the synthesis of new insulin analogues modified at the nterminus of the b-chain and subsequently correlated their biological activities with their d-structures. the invariant residue glyb seems to be critical for the t/r transition. glycine can adopt wide range of dihedral angles (φ/ψ) and it occupies significantly diverse dihedral angles in t-and r-states. a-aminoisobutyric acid (aib) is an amino acid with a high helical propensity, which often folds into right-or left-handed α-helix. we have introduced aib at position b , b and b with the aim to induce the r-state of the hormone. in contrast, as d-pro and nmeala are not able to adopt the φ/ψ angles of the right-handed α-helix we have introduced these amino acids at position b to obtain the t-state of insulin. peptide dendrimers are tree-like molecules formed by alternating functional amino acids with branching diamino acids such as lysine. unfortunately these molecules have not yielded to structural characterization and little is known about their molecular-level structure. computational methods seem to be an adequate tool to address these issues.herein we present a comprehensive structural characterization of peptide dendrimers using molecular simulation methods. multiple long molecular dynamics (md) simulations were used to extensively sample the conformational preferences of several third-generation peptide dendrimers, including some known to bind aquacobalamine. we used several conformational analysis procedures (clustering, energy landscapes and multivariate analysis) to analyze conformational changes that can be correlated with particular structural trends.the results point to a high conformational flexibility of these molecules, with no clear "folded state", although two markedly distinct behaviours were identified. some dendrimers favour mainly loose conformations, while others prefer more compact configurations. through a series of computational mutations we investigated the influence of the presence and placement of charged residues in dendrimer topology, finding that electrostatic interactions among charged residues are a major determinant in structure acquisition by peptide dendrimers. these conclusions bring new insight into the conformational behaviour of these systems and may provide better routes for their functional design. acid-mediated prevention of aspartimide formation in solid phase peptide synthesis t. michels, a r. dölling, b u. haberkorn a , w. mier*, a a department of nuclear medicine, university hospital heidelberg, heidelberg, germany; b biosyntan gmbh, robert-rössle-straße , berlin, germany aspartimide formation is one of the major obstacles that impede the solid phase synthesis of large peptides and proteins. the main reason for aspartimide formation is the piperidine-catalyzed fmoc cleavage of peptides containing aspartic acid. several side chain protecting groups have been developed but the complete prevention of aspartimide formation can only be achieved using n-( hydroxy- -methoxybenzyl) (hmb) as backbone protecting group. however, hmb-protected building blocks are difficult to synthesize and only the dipeptide containing glycine (fmoc-asp(tbu)-(hmb)gly) is commercially available. until now, no cost effective strategy to suppress this side reaction has been developed. formally, aspartimide is the result of an attack of an amidate species at the carbonyl carbon of the otbu protected side chain carboxylate of aspartic acid, which might be prevented by protonation of the amidates with piperidinium ions. in this work the suppression of aspartimide formation by adding small amounts of organic acids to the deprotection agent piperidine was studied. this procedure was shown to efficiently prevent the formation of aspartimide side products in several peptides, i.e. pres - -y, a -mer peptide derived from the hbv surface antigen and a peptide parathyroid hormone (pth) fragment. the testing of a series of different acids covering a broad range of pka values showed that this effect is virtually independent of the acid strength. since aspartic acid is found in most oligopeptides, the authors recommend to generally add % (v/v) formic acid to piperidine based fmoc cleavage mixtures. decomposition of the resin linkers during tfa cleavage of peptides in fmoc-strategy leads to alkylation of sensitive amino acids . this side product formation is a crucial drawback, especially during the synthesis of biologically important cys-containing peptides on wang support. through a battery of approaches ( h-nmr, uv and lc/esi-ms) we detected an unexpected alkylation of the sulfhydryl group of cysteine side-chain residues by the phydroxyl benzyl group from the wang resin linker. herein, we present the feasibility for s-alkylation of cys-containing peptides from wang linker decomposition. this sidereaction occurs during the final tfa cleavage of the peptide from the solid support, while the position of the cysteine residue within the peptide sequence as well as the resin's substitution influence the extent of cys-alkylation. the stephan angeloff institute of microbiology, bulgarian academy of sciences, sofia, bulgaria influenza viruses cause epidemics and pandemics all over the world. therefore, the development of virus resistance to drugs, leads to search for novel derivatives and approaches to chemotherapy for human influenza infection. antioxidant therapy is known to be one potential approach. the application of combination therapy of antioxidants with antiviral drugs could reduce the complications and lethal effects, caused by an influenza virus . in our study, amino group of neuraminidase inhibitor -oseltamivir, which belong to second generation anti-flu drugs, was covalent conjugated with known antioxidantscysteine, histidine. tryptophan and etc. the study of the role of the modified by antioxidants oseltamivir on proliferation of influenza virus is in progress. recently we reported a short synthesis of or -membered cyclic guanidine via intramolecular reaction of alkyl diamine with n,n'-cbz-methylisothiourea( a). here we report a further application of synthesis two types of cyclo-peptide guanidine-bridged cyclopeptides utilizing this mechanism -n-terminus local cyclo-guanidine peptide and backbone guanidine-bridged marco-cyclic peptide. three n,n'protected diaminoacids (daa) including , -diaminobutyric p . antifreeze glycoproteins (afgps) are found in the deep sea teleost fish in arctic and antarctic oceans. these biomolecules are able to inhibit the growth of ice crystals and depress the freezing temperature of the blood serum in fish enough to keep them from freezing in their sub-zero environments while the melting temperature remains unchanged . despite afgps have been consider as a potent cryopreservation, obstacles to develop afgps as medicinal and industrial application are mainly due to the lack of access to pure form from natural sources and the problem of understanding how afgps inhibit ice crystal growth. as a result, a considerable progress toward the design and synthesis of afgp analogues has been made several groups . in the course of the studies on the structure-activity relationships of afgps, we are interested in peptoids as mimics of α-peptides and synthesized monoglycosylated peptoid analogues by substituting the glyco-thr residue as afgps mimics. in this presentation, we will show our studies on how the insertion of peptoid residue into afgp backbone affects the afgp activity by measuring both thermal hysteresis (th) and ice recrystalliztion inhibition (iri). [ ] , both for diagnosis and endoradiotherapy. for this application the peptides can be attached with chelating agents that bind radioactive metals such as ga, ga or in for imaging or therapeutic radiometals such as y and lu. the chelating agent most frequently applied is the macrocyclic ligand , , , -tetraazacyclododecane-n,n',n'',n'''-tetraacetate (dota), it is commonly introduced as the tris(tbu ester). the cleavage of the tbu protecting groups on dota is known to be sluggish [ ] . several attempts have been made to synthesize dota with protecting groups that can be removed under mild conditions. however, these derivatives have not yet found widespread application. our new approach was to prepare a protecting group for dota-based prochelators that is convergently cleaved under the cleavage conditions of the amino acid protecting groups of the peptide. o-phenylisopropyl (opp) esters are more sensitive towards acid than tbu esters. deprotection occurs with % trifluoroacetic acid in dichloromethane [ ] . therefore, a synthesis of the prochelator dota-tris(opp ester) was developed. the copper-catalyzed azide-alkyne cyclization (cuaac), the most commonly recognized variant of "click chemistry," has emerged as a powerful technique for ligation, conjugation, and cyclization reactions of peptides. it is known that cyclization can increase the metabolic stability of peptides, as well as enhancing potency or selectivity by stabilizing an active conformation. one application of the cuaac that has generated interest is the use of this reaction to replace a disulfide bridge with the product triazole, which among other complementary properties may prevent in vivo redox chemistry. in this poster, we synthesize a new analogue of the cyclic cancertargeting peptide cngrc where we replace the disulfide bond with a triazole linkage using click chemistry and a fully automated, on-resin method using the single-shot delivery feature on the prelude® peptide synthesizer. unnatural amino acids including d-amino acids are manufactured mainly by the enzymatic process. however, one enzyme can produce only one amino acid due to its high specificity and it takes a long time and a lot of expenses to develop the appropriate enzyme itself. arca (alanine racemase chiral analogue) is an organic catalyst which can overcome these drawbacks and can produce almost all kinds of amino acids efficiently. the amine functionality of l-threonine is freely reacted with the aldehyde group of arca to form the corresponding imine, which is easily epimerized in the presence of organic base due to the acidity of the alpha proton of imine. the difference in the stability between the imines of the optical epimers rendered them to be shifted to d-allo-threonine derivative dominantly. once the epimerization reaction reached equilibrium, the reaction mixture was hydrolyzed under acidic condition to give d-allo-threonine and arca, which could be recycled repeatedly without significant loss in yield or purity to produce more d-allo-threonine from lthreonine in excellent yields. optimization of the reaction conditions with various bases and solvents is discussed and mass production of optically active d-allo-threonine including optical purification is described. the manufacuring process for the preparation of arca will be shared as well. our group is interested in the development of efficient synthetic routes for the preparation of enantiopure atrifluoromethylated amino acids (a-tfm-aas) starting from chiral cf -oxazolidines or imines and their incorporation into a peptide chain. these non-natural amino acids are very attractive compounds for the design of biologically active molecules, particularly peptides, due to the unique physical, chemical and biological properties impart by the cf group. as conformationally constrained cyclic amino acids have recently gained considerable interest, we are particularly focused on the preparation of pyrrolidine-type a-tfm aas. , incorporation of proline derivatives is known to restrict the amino acyl-proline cis/trans isomerization, to limit the protein folding and consequently to modulate the biological activity of peptides. based on these observations, mutter's group introduced pseudoproline building blocks (ψpro) into a peptide sequence as reversible protecting groups for ser, thr and cys. the ψpro residues proved to be versatile tools for overcoming the aggregation caused by hydrophobic interactions encountered during solid-phase peptide synthesis (spps). they also turned out to be inducers of βturns containing predominantly cis-amide bond and useful tools in peptide cyclization. here, we report the results obtained for the preparation of various hydrolytically stable trifluoromethylated pseudoprolines (cf -ψpro) as well as the methodological studies developed to optimize the synthesis of various c-and n-terminal cf -ψpro containing dipeptides. rennes, france protein strructure and function rely on a still not fully understood interplay of energetic and entropic constraints defined by the permutation of the twenty genetically encoded amino acids. many attempts have been undertaken to design peptide-peptide interaction pairs and synthetic receptors de novo by using special building blocks. a rational approach starting from hydrazine to create new building blocks based on a tailored metalchelating amino acid analogues was envisaged. to create chemical recognition units, which bind oligohistidine tags with high affinity and stability, several supramolecular entities containing one to three nitrilotriacetic acid analogue (ynta) moieties were synthesized. these new building blocks additionally contained an amino group or an acido group, which can be flexibly introduced into peptide in n or c-termini or into the peptidic chain by solid phase chemistry in fmoc/t-bu strategy. these multivalent chelators were characterized and the corresponding metalchelating peptides could act as metal sensors and synthetic receptors for histidine-tagged proteins. the potential of peptides as drug candidates is often limited by their pharmacokinetic properties. structural modification of the peptide backbone via n-methylation is a powerful medicinal chemistry tool that confers oral bioavailability to these molecules. n-methylation exerts a strong effect on the backbone conformation and, as a result, many n-methylated peptides show enhanced biological activity and higher receptor selectivity. another approach to increase the solubility of peptides is by conjugation of peg to a derivatizable functionality. by combining these two approaches we have developed n-oegylation. this novel form of peptide modification consists of the attachment of oligoethylene glycol (oeg) chains to the amide bonds. many bioactive peptides comprise one or more n-me amino acids which are essential for their activity. thus, we consider that replacement of a backbone n-me group by an oeg chain may imply a minimal structural perturbation and may lead to n-oegylated peptides with preserved biological activity. furthermore, our strategy is a promising way to improve the bioavailability of cyclic peptides that do not have any site where a peg could be attached. as a proof of principle, several n-oeg analogs of two bioactive cyclic peptides were synthesized in spps. first, we performed a full n-oeg scan of the sansalvamide a peptide. next, several analogs of cilengitide were prepared by replacing the n-me of valine by oeg chains of different length. depending on its size, the oeg residue was incorporated by using an n-oeg derivative as building block or, alternatively, using an n-substituted amino acid bearing an attachment site where a peg was conjugated post-synthetically.the biological activity of all the n-oegylated peptides was evaluated. some of the sansalvamide a peptide analogs exhibited cytotoxicity within the same range as the original peptide, which suggests that backbone amide groups may be useful oegylation sites in bioactive cyclic peptides. the modification of peptides is an important step in pharmacology to vary the affinity and the stability of peptidic drugs. whereas a wide range of strategies exists for the functionalization of the n-terminus and the side chains, facile variation of the c-terminus remains an important challenge. we consider peptidyl-phosphoranes as a promising platform to enable orthogonal and mild introduction of a great variety of chemical functionalities at the c-terminus. a convenient method for the synthesis of soluble peptidyl-phosphoranes has been presented by our group recently. in this, -bromo-acetyl bromide was coupled to a wang-resin followed by alkylation of triaryl-or trialkyl phosphine moiety. deprotonation to the phosphorus ylide and subsequent acylation with an fmocamino acid created the basis for assembly of the peptide by spps. final acidic cleavage produced a decarboxylated and unprotected, soluble peptidyl-phosphorane. from this point, a variety of orthogonal modification reactions at the peptides c-terminus is possible, e.g. click reaction with azides allow for the incorporation of triazoles as peptide bond mimetics. the wittig reaction opens up another interesting portal for c-terminal modification, as vinyl ketones are formed by reaction with aldehydes. the described chemistry was applied to modify caspase- inhibitors. in order to address the s site of caspase- , the commonly known devd inhibitor was varied at the c-terminus by introduction of different residues. the devd motif was synthesized as peptidyl-phosphorane and modified in wittig reactions. the resulting c-terminal vinyl ketones were obtained by the reaction of aliphatic and aromatic aldehydes. a small library was generated, and novel compounds were tested for their potential to inhibit caspase- . semmelweis university, department of biophysics and radiation biology, budapest, hungary considering the impact of uv irradiation on the structure and function of proteins , it is a matter of utmost importance to resolve the conditions of photolysis more deeply. we think that a protein, as a complex unit, gives multiple responses to all impacts therefore the analysis of these responses is a rather complex problem. the main goal of our research is the deeper understanding the tryptophan-mediated photolysis of disulphide bridges in bio-active proteins upon near-uv irradiation using cyclic peptide models, as small protein units, to define the caused functional damage. cation -pi interaction is increasingly recognised as an important noncovalent binding interaction which plays a role in establishing the final structures of proteins. within a protein, cation -pi interactions can occur between the cationic side-chains of either lys or arg and the aromatic side-chains of phe, tyr or trp . our earlier results with gla indicate that new covalent bonds are also formed between cys and lys during illumination, which is also a reason why the lys residue is planned to be included in the sequence of the models. our aim is to study whether the cation -pi interaction can have an influence on the ss-bridge splitting in small cyclic pentapeptide models. here we report about the conformational analysis, synthesis and spectroscopic investigation of lys-and arg-containing model peptides. the azide functionality is very popular mainly due to azidealkyne click chemistry used in many peptide ligation strategies. azido-peptides are usually prepared by incorporation of azide containing residues or azide functionalization of aldehyde resins affording c-terminal azido-peptides. alternatively, the n-terminus can be converted into an azide via a cu(ii)-catalyzed diazotransfer reaction using triflyl azide. recently, a number of safer, shelf-stable and easily prepared diazotransfer reagents has been developed, of which imidazole- -sulfonyl azide has been used to introduce azide moieties in proteins under copper-free conditions. typically, it has not been reported to be used on the solid phase. we provide a very easy, fast and efficient method for conversion of amines into azides on a solid phase support which in our opinion has major benefits over earlier reported methods making using of less stable reagents that require a metal ion catalyst. we demonstrate how the diazotransfer reaction can be performed on a solid phase support using the imidazole- sulfonyl azide reagent without the need for a metal ion catalyst. using a model peptide we studied the effect of stoichiometry, added base and solvent. in addition we examined the effect of the nature of the n-terminal residue on the efficiency the diazotransfer reaction. finally, we found that the optimal conditions to perform the reaction also depend on the nature of the solid phase support the reaction is performed on. the novo nordisk foundation center for protein research, university of copenhagen, copenhagen, denmark site-selective strategies for post-translational modification of peptides and proteins are essential tools for many areas of research in the life sciences, yet remain a chemical challenge due to the multiplicity of functional groups present. there are powerful chemoselective reactions, however, they aim at introducing only one functionality at each reaction site. here we present a one-pot, threecomponent dual-functionalization of peptides or proteins based on a , -dipolar cycloaddition between a functionalized malemide, an n-hydroxylamine and a peptide or protein with an n-terminal serine residue at the n-terminus, which is selectively oxidized to a -oxoaldehyde. most common moieties for labeling, e.g. fluorophors and peg-chains, are commercially available as maleimides. nitrones were easily obtained by condensation of peptide-aldehydes and primary nhydroxylamines under aqueous conditions. the chemoselective , -dipolar cycloaddition reaction between the peptide-nitrone, and a functionalized maleimide proceeded in aqueous solution at room temperature or with gentle heating, which provided the stable isoxazolidine product. we envision that this 'one site -two functions' method can be used widely to introduce two separate moieties. the method was used to introduce two separate ligands in a range of other peptides. for example, new multimodal molecular imaging techniques depend on facile chemical methods for site-selective dual-functionalization. we used our new methodology to synthesize a cyclic rgd-peptide for combined pet and optical molecular imaging. finally, the small protein ipb was successfully n-terminally modified, including with a peg-chain, using this new, general method. multiple sclerosis (ms) is the most known chronic, inflammatory, demyelinating disease of the central nervous system (cns), characterized by a progressive neurodegeneration, caused by an autoimmune response to self-antigens in genetically susceptible individuals. it is nowadays known that post-translational modifications may affect the immunogenicity of self-protein antigens, triggering an autoimmune response and creating neoantigens; in particular aberrant glycosylations affect various parts of the immune response and have profound effects on immune tolerance. in previous studies we demonstrated the value of the glycopeptide csf (glc) which, by virtue of the particular type i' β-turn structure, optimally exposes the minimal epitope asn(glc) to autoantibody recognizing in elisa in multiple sclerosis patients' sera . elisa assays allowed to conclude that the ability in detecting autoantibodies in multiple sclerosis sera was stricktly linked to saccharidic moieties and to conformation around minimal epitope of the antigenic glycopeptide. herein, taking advantage of such considerations, we focused our attention on the synthesis of a little library of lysine branched multiple antigen peptides (maps), containing the minimal epitope asn(glc), in an attempt to increase the antigenicity of linear peptide sequences . with this aim, we performed the spps of glucosylated maps via the building block approach, studying the role of different long spacers on the dendrimeric core, and the role of different peptide sequences around the sugar moiety, in order to optimize the synthetic process and to evaluate the influence on the affinity and specificity in sp-elisa. environmentally induced co-or post-translational modifications of autoantigens are hypothesized to break immune tolerance leading to self reactivity in pbc. it has been previously reported that the use of synthetic post-translationally modified peptides, introducing fmoc-l-lys(nε-(±)-α-lipoic acid)-oh, as peptidomimetics of natural neoantigens allowed to detect autoantibodies in the sera of patients affected by pbc, and they might be useful diagnostic tools that can be used in earlier stage patients and possibly to monitor disease activity. only the r-(+)-enantiomer of α-lipoic acid exists in nature and is an essential cofactor of four mitochondrial enzyme complexes. but it remains unclear if the tridimensional structure of the lipoic acid is of any importance in the interactions antibody-peptide during the indirect elisa tests. therefore, it is necessary to synthesize each peptide separately with one absolute configuration of the lipoic acid. herein, we describe the synthesis of the two diastereoisomers fmoc-l-lys(nε -(r)-α -lipoic acid)-oh and fmoc-l-lys(nε -(s)-α-lipoic acid)-oh that have to be used in fmoc/tbu spps as building blocks for the synthesis of post-translationally modified peptides. recently it has been reported the introduction of a new generation of cd diagnostics based on a unique antigen approach, consisting on human ttg cross-linked with gliadin peptides coated on the elisa plates . on the basis of experimental data obtained by mass spectrometry and indicating which are the fragments of these two proteins that are supposed to be involved in the antibody recognition, we were able to select the most representative ttg and gliadin fragments , to design and synthesize by fmoc-spps nine cross-linked eoepitopes. aim of our study was the characterization of autoantigenic epitopes by testing, in celiac patients' sera, the reactivity of these nine synthetic peptides. these neoepitopes were tested in elisa to evaluate the iga and igg response against ttg-gliadin adducts in celiac patients' sera in order to develop a new elisa test based on peptides as an even more powerful diagnostic tool in terms of specificity and sensitivity. more than analogs have already been described, wherein the hydroxy acid and the amino acid constituents were replaced by d-amino acids and/or n-methyl amino acids with preserved or altered side chains. for certain types of cancer cells, several of these analogs were found to be more active than the natural product itself. however, it does appear that many of these compounds have limited solubility in water. b here we report the synthesis of novel analogs of the sansalvamide a peptide bearing an n-oligoethyleneglycyl (oeg) chain attached to the different backbone positions. attachment of this chain is aimed to enhance the hydrophilicity of the original peptide. our synthetic strategy to modify the backbone with the n-oeg group relies on the use of n-oeg amino acids, which were synthesized in solution and then used as building blocks in spps. as expected, couplings to the n-oeg residues were found to require special conditions. methods for the coupling to nmethyl amino acids were applied and this enabled to obtain the different linear pentapeptides, which were cyclized in solution. both the synthetic strategies of these demanding peptides as well as the preliminar evaluation of their biological activity will be deeply discussed. glycoconjugates such as glycoproteins and glycolipids have important roles in cell functions, for example, intercellular recognition, cell proliferation control, and information transmission. in order to study the structurefunction relationship, synthesis of these glycoconjugates is essential. glycoproteins and glycopeptides are classified into two categories: n-and o-glycosylated derivatives. the n-acetyl-α-d-galactopyranosylated ser or thr derivatives [ser/thr(α-d-galnac)] are important intermediates for o-glycopeptide synthesis. however, the synthesis of ser/thr(α-d-galnac) derivatives by chemical glycosylation is difficult because of the decreased nucleophilicity of hydroxy function in the glycosyl acceptor due to an unfavorable hydrogen-bonding pattern between the oh and α-nh groups . several approaches to overcome this problem have been reported , . in addition, the o-glycosidic bond is cleaved easily in acidic conditions. in this study, we assumed that the formation of a cyclic structure containing an α-nh group would increase the reactivity of oh function. thus, we focused on the n, n'isopropylidene derivatives of ser/thr containing dipeptides . we found the reaction of mannopyranosyl trichloroacetimidate and the n, n'-isopropylidene dipeptide in the presence of tmsotf in dichloromethane produced the desired glycosylated dipeptide in good yield. however the selective intermolecular disulfide bond formation is a very difficult and complicated synthetic problem. in this work we report on synthetic approaches for the formation of conjugates with intermolecular thioether or disulfide bonds. for the disulfide bond formation, we use two activation approaches: i) activation of the four cys residues of the carrier testing two activating reagents, in both solid and liquid phase respectively and ii) activation of the cys containing bioactive molecule. as bioactive molecule we selected the r ppleed sequence derived from the intracellular part of the αiibplatelet integrin receptor. this region is critically involved in platelet aggregation and is a target of intervention for developing antithrombotic agents . the ac-[lys-aib-cys(ch co-αiib - )] -nh and ac-[lys-aib-cys(cys-αiib - )] -nh conjugates were synthesized and examined for their ability to inhibit platelet aggregation. the biological assays indicated that the synthesized conjugates penetrate the platelet membrane and inhibit human platelet aggregation, in contrast to the corresponding free peptide analogues. the molecules were reported to exhibit broad-spectrum cytotoxicity against the tumor cell lines. although these peptides contained the novel β-methoxytyrosine, lipton et al. reported the synthesis and cytotoxicity of desmethoxycallipeltin b, in which substitution of d-tyrosine for β-methoxytyrosine did not substantially affect the cytotoxicity of callipeltin b , . however, a structure-activity relationship study of the molecules has not been shown to date in detail. in the course of our recent research regarding the synthetic study of cyclic depsipeptides, we conducted studies on the synthesis of callipeltins supposed to be efficient structures for ccr inhibitors as anti-hiv drugs or anti-cancer agents. in the present study, we report the synthesis of cyclic depsipeptides of callipeltin b analogues consisting of l-, d-amino acids and/or n-methyl amino acids, for a structure-activity relationship study of linear-and cyclic depsi-peptides against hela cells . in the assay of synthetic peptides, all of the synthetic callipeltin b analogues exhibited no cytotoxicity. we supposed that dimethylpyroglutamic acid of callipeltin b was essential structure to show the cytotoxicity against hela cells. monash university, melbourne, australia protein-protein interactions represent a significant portion ( - %) of all interactions within the cell; as such these interactions are ideal targets for drug discovery. while difficult to target using small molecules, these interactions can be disrupted using a small section of the protein's binding partner. these short peptides must retain the defined secondary structure associated with the protein binding interface in order to inhibit their protein targets. as the secondary structure adopted by the parent protein is not always exhibited by its derived peptides, constraints are introduced as necessary to help define the structure of the peptide. inducing secondary structure reduces the energy required for organisation, decreasing the energy of binding and has the potential to increase stability with respect to degradation by proteases. solid phase peptide synthesis was used to make several small peptides corresponding to the structured sections of the binding partners for three protein-protein interactions. these peptides were designed to target heart disease, prostate cancer, and liver cancer respectively. secondary structure was introduced using lactam bridge constraints. for the αhelical peptides, a side chain constraint approach was used to nucleate helix formation. as hydrogen bonding between the c=o of the i th residue and the nh of the (i+ ) th residue stabilises native α-helices, constraints were introduced linking the side chains such that the residues were held in close proximity. for β-pleated peptides, an antiparallel β-sheet arrangement was achieved by introducing turn regions into the peptide in such a way that the β-strands were aligned. constraints were again introduced using lactam bridges between lys and arg or glu side chains. these peptides were characterised by nmr and cd spectroscopy to verify the correct secondary structure had been induced. göttingen, germany different properties can be combined in a single molecule by using a scaffold arranging functional groups in a predefined topology. the tasp (template-assembled synthetic proteins) concept describes templates to reinforce and direct the folding of designed molecules into a predetermined topology. [ ] due to their resistance to proteolytic degradation and their rigid basic structure, cyclic β -tripeptides are suitable carrier molecules for bioactive compounds; they are further known to form tubelike structures by stacking of the peptide rings leading to higher organization of functionalized peptides. [ ] with this scaffold different inhibitory systems were synthesized that feature cell penetrating and fluorescent properties. the signal transducer and activator of transcription (stat ) protein, which has been described as an oncogenic protein, was selected as the first target. [ ] a peptide sequence which targets the sh domain of stat was used in two different approaches. it was either directly attached to the cyclic β-tripeptide via a huisgen [ + ]cycloaddition or the peptide was incorporated into the inhibitor loop of the cystine knot microprotein omcoti-ii, which was also attached to the cyclic-β -tripeptide. [ ] further, sodium channels are addressed usingconotoxines. first, an alkyne functionalized conotoxin siiia was synthesized applying different folding methods. the alkyne linker will be used to attach a fluorophore or to functionalize a cyclic-β-tripeptide. using single molecule imaging the spatial distribution, local concentration and organization of the ion channels in neurons will be imaged. further, the cyclic-β -tripeptide templating effect will be used functionalizing with μ-conotoxines. those proteins are folding helper proteins. together with chaperones, they form receptor complexes. they catalyze the isomerization of prolyl bonds in various folding states of target proteins. indeed, their role has been implicated in refolding of denatured proteins, de novo protein synthesis and the biologically active conformation of proteins . among them, the fkbp subclass comprises the small ppi calstabin and . it is of interest to try to understand the way those proteins act, in order to help the overexpression of various types of membrane proteins, aiming at the renaturation, purification and crystallization attempts of receptors. we chose to work on calstabin because this short ( aa) protein has been described as a sub-family comprising isoforms (from ~ to ~ aminoacids), some of them not being fully described to date. the relative shortness of those proteins together with the fact that the two higher molecular weight ones are catalytically active as prolyl isomerases, facilitate the characterization of the synthetic proteins.in order to obtain the full length calstabin , a native chemical ligation (ncl) approach was chosen . an optimized stepwise elongation allowed the obtention of the c-terminal segment up to the cys . moreover, several methods were compared for the synthesis of peptide - opportunely functionalized at its c-terminus for the ncl.the ligation at thr site between peptide - featuring a bis( -sulfanylethyl)amino the chemical diagnostics of paintings is a relevant topic in the field of chemical sciences applied to the conservation and safeguard of cultural heritage. chromatography is a highly sensitive and suitable technique for accurate methods of analysis of the limited amount of sample material typically available from works of art. paint media deriving from proteins traditionally include egg, milk, animal and fish collagen glue. egg yolk (egg tempera), egg albumin (glair) and casein (a blend of related phosphoproteins commonly found in milk) are traditionally used as pigments binders. we propose the uplc-based amino acid analysis as diagnostics technique on non pre-treated or submitted to extraction processes model samples, showing that good results can be achieved with very scarce sample manipulation and great advantage. we applied the amino acids analysis carried out by the accq•tag™ ultra performance liquid chromatography to the standard and model samples. in particular, after protein hydrolysis ( h, °c, m hcl) of the samples, the amino acid derivatization by -aminoquinolyl-n-hydroxysuccinimidyl carbamate allowed a reproducible amino acids analysis characteristic of the protein type. the results obtained confirmed the reliability of the data achieved and demonstrated that the accq•tag™ ultra uplc method could be a powerful technique to be applied to the relevant field of protein binders diagnostics for paintings conservation. moreover a multivariate analysis that offers a wide variety of tools and methods mainly concerned with mathematical models for the representation of multidimensional data has been proposed and the high model efficiency has been established for sample containing mixture of proteins. reactions performed on solid supports, such as resin, are commonly monitored by hplc-uv after cleaving the products from the support. however, uv-absorption coefficients may differ between compounds, and therefore the relation of the area percentage values of the peaks may not directly reflect the molar concentrations of the corresponding compounds. it is for this reason that, for example, in solid-phase peptide synthesis it is difficult to calculate the yield of the coupling of a fmoc-amino acid or the removal of the fmoc-group because of its high absorbance. recently, we reported the identification of minimal phosphopeptides that specifically interact with the pbd of human plk , but not those of the closely related plk and plk . comparative analyses of the crystal structures of the plk pbd in complex with the minimal phosphopeptides revealed that the c-terminal spt dipeptide functions as a high-affinity to the interaction. in an attempt to obtain the adequate cellular permeability and stability in vivo, we have accomplished the peptide-peptoid hybrid or peptomers cyclization using various methods like formation of amide, thioether and triazole and screened the plk inhibition activity on the first cyclic peptomers liibrary using pbd-binding assay. based on our first screening results, we also carried out the detailed investigation to further increase the activity and also to understand the significance of peptoid mimics as plk inhibitors. the mode of interaction between the cyclic peptomers and pbd might provide a template for designing therapeutic agents that target plk . a synthetic amino acid long peptide corresponding to the minimal metacaspase catalytic domain induces cell death in leishmania major c. servis, h. zalila*, i. gonzalez, l. lozano, n. fasel department of biochemistry, university of lausanne, epalinges, switzerland despite a lot of controversy during the last decade, there is increasing experimental evidence that cell death (cd) is genetically programmed in lower eukaryotes.in the cd proteolytic cascade of plants and protozoa, caspases are likely replaced by metacaspases that are cysteine peptidases recognizing arginines or lysines in p position. metacaspases have been found to control cell death in plants. the human protozoan parasite leishmania major expresses a single metacaspase (lmjmca) harboring a central domain with the catalytic dyad histidine and cysteine as found in caspases. metacaspase could therefore be one of the executioners of the death pathway in leishmania.in this work we showed that, in stress conditions, lmjmca precursor forms were extensively processed into soluble forms containing the catalytic domain and this domain was sufficient to enhance sensitivity of parasites to hydrogen peroxide by impairing the mitochondrion function. we tested different lengths of the lmjmca catalytic domain and found that the overexpression of the polypeptide corresponding to amino acids - was sufficient to sensitize l. major mitochondria to oxidative stress.we synthetized an aa long peptide corresponding to the minimal metacaspase catalytic domain (aa - ) and showed that it has specific metacaspase activity in vitro.we are currently investigating its activity on possible target proteins, which have been identified in a yeast two-hybrid screen. identifying proteins involved in the metacaspase signaling pathway will shed light on the understanding of cd in leishmania and open new perspectives in drug target investigation to fight leishmaniasis and other major infectious diseases. s. alasibi, g. ashkenasy department of chemistry, ben-gurion university of the negev, beer-sheva, israel various factors can affect the conformations and folding states of protein molecules and as a consequence their activity. these factors include amino acid mutations, interactions with other macromolecules, binding to regulatory molecules, and also external changes such as ph jump or shining light. in order to control the folding states and to modulate the functions of peptides and proteins by light, photocleavable groups are usually incorporated into specific residues to mask critical interactions. for example, introducing caging groups into coiled-coil proteins recognition interface affects complex formation and template-assisted ligation reactions, in which the coiled-coils serve as templates to catalyze the condensation reactions between two short peptide fragments . our research group has been studying peptides replication networks, which were made of coiledcoil peptides and analyzed the response of such networks to light as external trigger . it was shown that even replicating networks made up of a small number of molecules can possess complex behavior, considering the wealth of catalytic pathways and transformations. hence, boolean logic operations can provide valuable means to analyze and interpret their behavior . herein, we describe the use of chemical inputs and uv light to manipulate peptides folding and functionality within new synthetic networks. these networks perform complex behavior and, as a result, selective product formation is used to implement boolean operations that have not been achieved before. institute of bioorganic chemistry of ras, moscow, russia earlier, we have shown that n-acylated amino acid nitriles and amides react with ethylene derivatives forming the amino-and -hydroxypyridines and pyrroles [ ] . a possible reaction mechanism is the geterodienic condensation of aminooxazole derivatives to dienophiles. the higher yields were observed when used the dicarboxylic acids as dienophiles and -amino or alkoxyoxazole as geterodienes. while the same reactions with the fullerene derivatives, as dienophiles, gave low yields. the nitrile groups of specified pyridines possess ability to react with amino groups of peptides and proteins even at room temperature. in view of high activity of nitrile groups such pyridines can form tetrapyridotetrazoporphyrins and self-condensation products giving appropriate dendrimers (possible due to mobile hydrogen in the th position). high molecular weight dendrimers were identified by massspectrometry, gel electrophoresis and dynamic light-scattering. catalytic oxidizing properties of tetrazaporphyrin derivatives and phtalocyanin were used in synthesis of cyclic peptides and for the s-s bonds formation. the transformation of peptides into heterocycles via an intramolecular reaction of nitrile groups was used to determine the sequence of some peptides, which favored the resistance of transformed compounds to hydrolysis and to the electron impact at mass spectrometry. diazotization of peptides and their derivatives facilitates identification of amino acid sequence by mass spectrometry due to the peculiarities of their fragmentation. in addition, an amino acid analysis of the diazotized peptide makes it easy to determine the n-terminal amino acid. we present here a multi-disciplinary approach combining x-ray crystallography, computational analyses, and immunological tests to identify epitopes of the oligopeptide-binding protein a (oppa bp) from the gramnegative pathogen burkholderia pseudomallei, the etiological agent of melioidosis. computational analysis on oppa crystal structure was used to design potential consensus epitopes, that once synthesized as free peptides (comp - ) were found to be immunoreactive against sera from melioidosis patients. notably, one of the predicted peptides allowed to distinguish between seropositive, seronegative and recovered groups, underlining its potential for diagnostic purposes. parallel experimental epitope mapping, based on proteolysis and mass spectrometry, allowed us to identify linear peptide epitopes (exp - ) localized in similar protein regions as comp - . moreover, the match between theoretical and experimental mapping of epitopes was improved by expanding our computational approach, i.e. including an energy based decomposition procedure to divide oppa bp into separate fragments. overall, our results illustrate the successful development of a novel integrated structurebased approach for the discovery, design and preparation of epitopes. nonetheless, given antigen crystal structures, our method is expected to be broadly applicable in the design and generation of new epitope candidates, as being confirmed by on going experiments on different antigens. the application of peptide thioacids as reactive intermediates and building blocks has received considerable attention recently. the chemical ligation reaction between thioacids and azides has been reported for the synthesis of small to larger peptides as well as for the modification of proteins. fmoc based methods for the preparation of peptide thioacids have to our knowledge not been extensively researched and a facile approach to their synthesis is desirable.we have recently shown that t-butyl thioesters are robuster than previously reported, and can be used for the fmoc based solid-phase preparation of peptide thioesters being also easily cleavable with thiolates. peptides attached via a -mercapto -methylpentanol (mmp) resin can be cleaved using -mercaptopropionitrile to obtain protected thioacid peptides with a ß-elminable cyanoethyl group.the thioacid peptides could then be obtained in situ after treatment with dbu ( . % in dmf) and further reacted with sulfonyl azides in the presence of , -lutidine in a one pot reaction. by treating the cyanoethyl peptide thioesters with (nh ) s in a sodium phosphate buffer (ph = ), various model penta-peptide thioacids could be obtained cleanly at room temperature in up to % overall yield based on initial coupling. these peptides were then further ligated with electron deficient sulfonyl azide functionalized peptides.larger peptide thioacids could also be obtained using this protocol. a mer derivative of penetratin- , a cell-penetrating peptide from the third helix of the homeodomain of the antennapedia protein, was prepared as a peptide thioacid in a % yield (based on coupling of the first amino acid). in this report, a sensitive, selective and rapid uplc-ms method was developed for the determination of the [lys-gly] -mog - peptide in order to control the conjugation of mannan with the [lys-gly] -mog - peptide. the separation was performed on an acquity uplc system with a beh c column packed with . μm particles. the total run time was min. calibration curve based on peak area ratio was linear at the concentration range of - μg/ml, with a detection limit of μg/ml. the method showed satisfactory reproducibility and confirmed the entire conjugation between oxidized mannan and peptide sequence. the development of simple, low-cost and fast methods for protein purification is of increasing importance both for academic and industrial applications. a very promising approach is inverse transition cycling (itc) that exploits the temperature dependent aggregation properties of elps. elastin-like polypeptides (elp) are artificial polypeptides composed of pentameric repeats (val-pro-gly-xaa-gly) derived from mammalian elastin. elps are characterized by a specific transition temperature (t t ) that depends on the amino acid composition of the pentarepeat; they are water-soluble below and aggregate reversibly above this narrow temperature range (t t ). these properties are transferred to target proteins by n-or cterminal fusion with elps. during itc these fusion proteins precipitate, while other components remain in solution. repeated cycles of heating and cooling allow simple recovery of the target protein.we synthesized various elps consisting of to pentameric repeats and including different guest residues. the transition temperature of all synthetic elps was determined using photometric assays and measuring turbidity. in order to test if elp properties can be efficiently transferred, we fused elp to a small recombinant protein (ras-binding domain, rbd) by expressed protein ligation. this approach will allow the incorporation of elps with unnatural amino acids and other chemical modifications into target proteins. currently we are focusing on biotechnologically relevant enzymes that constitute a major cost factor in industrial processes. the authors thank süd-chemie/clariant for their financial support. department of pharmacy, university of patras, rio, greece peptides penetrating the cell membrane, known as cell penetrating peptides (cpps), as well as their mimics, used as delivery agents to cells have been reported , . cpps can be natural sequences or artificial constructs designed to capture the features of natural formations. cpps are particularly important in the delivery of peptides, proteins, nucleic acids, small molecule drugs or imaging agents. incorporation of a heterocyclic motif into a peptide or peptide-like backbone introduces conformational constraints and/or latent reactivity related to the heterocycle's structural profile. heterocycle-based cpp mimics are, thus, promising candidates for therapeutics protected synthetic non-ionic peptides, which are for example synthetic intermediates for the production of api's, are often very hydrophobic and not soluble in most common solvents. they are thus difficult to purify by preparative rp-hplc, classically used for industrial production. it is then challenging to develop alternative purification chromatographic processes using suitable solvents and providing good yields, high purity and sufficient productivity. the technique of support free liquid-liquid chromatography , including both its hydrostatic (centrifugal partition chromatography or cpc) and its hydrodynamic (counter-current chromatography or ccc) declensions, are mainly involved in phytochemical studies but has also been applied to peptide purification . the previously developed biphasic solvent systems are not adapted to the purification of highly hydrophobic protected peptides. to overcome this problem, two new scales of biphasic solvents systems and a ternary biphasic solvent system were developed to overcome solubility problems often encountered with those peptides. the new systems composed of heptane/thf/ch n/dmso/water, heptane/me-thf/nmp/water, and cmpe/dmf/water were efficiently used for the cpc purification of a mer protected exenatide and a mer protected peptide intermediate of bivalirudin synthesis. the developed scales show a wide range of polarity and should be useful for general use in cpc for the separation of hydrophobic synthetic free or protected peptides. the progressive aggregation of β-amyloid peptide (β-ap) into insoluble amyloid fibrils ultimately leading to formation of toxic amyloid plaques is widely considered to be the central pathogenic cause of alzheimer's disease. in the last decade accumulating evidence suggests that soluble oligomeric non-fibrillar forms of β-ap are neurotoxic as well. consequently, inhibiting the aggregation of β-ap is one of the therapeutic strategies against alzheimer's disease and a number of small molecules have been identified as inhibitors of β-ap aggregation and neurotoxicity. among these, curcumin, the phenolic yellow pigment and active ingredient of the turmeric herb, is receiving special attention because of its rich pharmacology that includes in vitro and in vivo inhibitory action against alzheimer's disease insults. in the current work the interaction of β-ap( - ) with curcumin is investigated with fluorescence, cd, and nmr spectroscopies in water and water-methanol mixtures and at various β-ap( - ):curcumin ratios. in nmr studies in % methanol curcumin behaves like a macromolecular species with a change in the sign of its noe signal providing direct indication of its association with β-ap( - ). in % methanol the presence of β-ap( - ) results in great broadening of the h peaks of curcumin, indicative of a complete change in its solution state. additionally, the fluorescence of curcumin in % methanol shows a blue shift with enhanced intensity, observations consistent with a hydrophobic modification of curcumin environment upon interaction with β-ap. finally, in water the induced circular dichroism spectrum of curcumin in the near uv region provides clear evidence for the loss of symmetry of curcumin molecule due to changes in its microenvironment generated by interaction with β-ap( - ). our experimental findings support the direct interaction of β-ap( - ) with curcumin and establish its importance as a potential aggregation inhibitor of β-ap. [ ] . based on its sequence, we synthesized h-tyr-d-trp-nh- -ada ( -adamantane) (yo- ) and h-tyr-d-trp-nh- -ada ( -adamantane) (yo- ) and reported they had potent antiproliferative activity on cancer cells (a- and sw ), which were comparable to tt- and cycloheximide. a structure-activity relationship analysis revealed that lipophylicity of yo- and - could be responsible for their antiproliferative activity. now, we described the substitution of tyr of yo- and - by tyr(bzl), phe, -nal( -naphthylalanine), -nal ( -naphthyalanine) and the anticancer and dna polymerase inhibitory activities in order to explore the effect of hydrophobic substituent. among the compounds, yo- and - had the highest lipophilicity judging from their retention time and lipophilicity index (yo- : . min, . ; yo- : . min, . ). yo- and - exhibited strong dna polymerase inhibitory activity as well as antifroliferative activity on hct cells at m. these activities were greater than those of yo- and - . antiproliferative activity of the compounds containing -ada such as yo- , - , - , - and - , was comparable to that of the compounds containing -ada such as yo- , - , - , - and - . these findings suggest that the lipophilicity well correlates with dna polymerase inhibitory activity and antiproliferative activity on hct cells. further structureactivity relationship study is progressing in our group. multiple sclerosis (ms) is a chronic autoimmune disease of the central nervous system (cns) , . our aim was to immunologically control the attack of the myelin sheath in ms patients without the total suppression of the immune system. anthraquinones (mitoxantrone, ametantrone) are widely used in cancer therapy as immunosuppressants.mitoxantrone is also used to treat several forms of advancing ms, including secondary progressive ms, progressive relapsing ms, and advanced relapsingremitting ms . more specifically, mitoxantrone is an inhibitor of the type ii topoisomerase, which disrupts dna synthesis and dna repair in both healthy cells and cancer cells. herein, we report the synthesis of an anthraquinone type compound conjugated to the immunodominant - myelin oligodendrocyte glycoprotein (mog - ) for the selective immunosuppression of the encephalitogenic t cells in ms patients. the anthraquinone was synthesized by a friedel-crafts acylation of hydroquinone from phthalic anhydride, followed by reduction of the resulted quinizarine to its leuco form, addition of the appropriate diamine and air oxidation . the synthesized molecules were purified using liquid chromatography, and they were identified by mass spectrometry and h-nmr. the synthesis of the mog - was performed under microwave irradiation and its conjugation with the anthraquinone was performed in solution. the final analogue was purified by rp-hplc and identified by esi-ms. benzopyrans, diketopiperazines and , benzodiazepin- , -diones are well-known and widely investigated scaffolds, e.g. the latter showing anxiolytic and antiarrhythmic effects. now, we propose a new potential "privileged structure" containing a triazole moiety mimicking the cis-amide bond within the , -benzodiazepin- , -dione motif .molecules based on this [ , , ]-triazolo [ , -d] benzo- , diazepin- -one scaffold are synthesized and decorated via a modular approach on wang resin using α-amino acids, -ethynylaniline building blocks and n-alkylating agents resulting in five points of diversity. the methodology involves the attachment of α-amino acids onto a solid support, subsequent removal of the fmoc group followed by an optimized diazotransfer reaction of the resulting amine yielding a resin-bound azide. conversion of the latter into a , -disubstituted , , -triazole moiety is achieved quantitatively by addition of a range of -ethynylaniline building blocks using a ru(ii)-catalyst. the desired scaffold can be obtained in high crude purities (> %) in solution via an acid catalyzed one-step cyclisation-release strategy. solution-phase n-alkylation finally affords the fully diversified scaffold. interestingly, n-alkylation induces atropisomeric effects which can be studied via h nmr spectroscopy.taking into account future screening results of the synthesized libraries, a well-thought decoration of this scaffold leading to discovery of new lead molecules is within reach. peptide symposium in the wonderful small seaside town of porto carras. maurice manning and lajos balaśpiri were nominated as captains of the two teams, the rest of the world and europe. similar matches were organized at subsequent european peptide symposia. now, in greece, the two captains would like to hand over their roles to younger scientists [professors gabor mezö(hungary) and laśzlóÖtvös (usa)] to continue this tradition at the coming european and possibly american peptide symposia. it seems best to play in the free time (in the evenings after the excursions). necessary conditions: good weather; a nice large soccer field; a soccer ball, preferably new; and jerseys and shorts (different colours), organized as always by the organizer commettee. the captain of the winning team will receive a trophy at the end of the nd european peptide symposium. the teams will remember two earlier excellent referees: professor lajos kisfaludy in porto carras [ ] and the soccer professor + ferenc puskaś (hungary) in budapest ( ). in the poster session, the results from the past years will be presented in about - pictures. these pictures may possibly be bought free, % can be saved at the poster session. all participants are welcome at the new party in athens. conclusion will be presented by the players and fans in athens. the human lactoferrin-derived peptide, hlf - , was proven to be highly active against antibiotic-resistant bacteria . however, the clinical use of this antimicrobial peptide (amps) is hampered by the peptide low stability due to fast degradation or to peptide aggregation, as the use of higher peptide concentrations results on higher toxicity levels. amp immobilization onto a biomaterial surface could be the pathway to overcome these difficulties . the aim of this work is the development of an antimicrobial surface by covalent immobilization of hlf - onto the surface of chitosan thin films. chitosan ultrathin films were prepared through the spincoating of a . % chitosan solution in gold substrates. hlf - immobilization was performed through an ss bound between hlf - terminal cysteine and an n-acetyl cysteine previously coupled at chitosan films. surfaces were characterized using ellipsometry (thickness), infrared reflection absorption spectroscopy (irras) and x-ray photoelectron spectroscopy (xps). bacterial adhesion studies were performed using methicillin-resistant s. aureus (atcc ). chitosan films were incubated with this bacterial suspension at ºc for h and h. the viability of the attached bacteria was evaluated using live/dead® bacterial viability kit (baclight tm ) and fluorescence microscopy. hlf - peptide was successfully covalently immobilized onto chitosan thin films. both soluble and attached peptide presented a higher antimicrobial activity than the control chitosan. identified as a potent vasoconstrictor that binds with high affinity to ut receptor. the cysteine-linked cyclic region, hut-ii( - ), is responsible for the biological activity and has been widely used to elucidate the structure-activity relationship of hut-ii. with the aim to investigate the role of hydrogen bond and the effects of a peptide backbone constraint on binding key: cord- -bcw f b authors: nan title: abstracts: th ebsa european biophysics congress, august rd– th , budapest, hungary date: - - journal: eur biophys j doi: . /s - - -z sha: doc_id: cord_uid: bcw f b nan o- structure determination of dynamic macromolecular complexes by single particle cryo-em holger stark max-planck-institute for biophysical chemistry, goettingen, germany macromolecular complexes are at the heart of central regulatory processes of the cell including translation, transcription, splicing, rna processing, silencing, cell cycle regulation and repair of genes. detailed understanding of such processes at a molecular level requires structural insights into large macromolecular assemblies consisting of many components such as proteins, rna and dna. single-particle electron cryomicroscopy is a powerful method for threedimensional structure determination of macromolecular assemblies involved in these essential cellular processes. it is very often the only available technique to determine the d structure because of the challenges in purification of complexes in the amounts and quality required for x-ray crystallographic studies. in recent years it was shown in a number of publications that it is possible to obtain near-atomic resolution structures of large and rigid macromolecules such as icosahedral viruses. due to a number of methodological advances there are now also great perspectives for high-resolution single particle cryo-em studies of large and dynamic macromolecules. successful high-resolution structure determination of dynamic complexes requires new biochemical purification strategies and protocols as well as state of the art electron microscopes and high-performance computing. in the future cryo-em will thus be able to provide structures at near-atomic resolution and information about the dynamic behavior of macromolecules simultaneously. detection and rapid manipulation of phosphoinositides with engineered molecular tools tamas balla section on molecular signal transduction, program for developmental neuroscience, nichd, nih, bethesda, md , usa polyphosphoinositides (ppis) are ubiquitous lipid regulators of a variety of cellular processes serving as docking sites and conformational switches for a large number of signaling proteins. the localization and dynamic changes in ppis in live cells have been followed with the use of protein domain gfp chimeras. in this presentation we will show experimental systems that allow rapid manipulation of the levels of ppis in specific membrane compartments. we are also actively pursuing strategies that will allow us to map the distribution and possible functional diversity of the phosphatidylinositol (ptdins) pools within intact cells since they are the precursors of ppis. we will show our most recent progress in addressing this question: the use of a ptdins specific plc enzyme isolated from listeria monocytogenes together with a highly sensitive diacylglycerol sensor to determine the distribution and also to alter the level of ptdins in living cells. these studies reveal that a significant metabolically highly active ptdins pool exists associated with tiny mobile structures within the cytoplasm in addition to the known er and pm ptdins pools. we will show our most recent data on the consequences of ptdins depletion within the various ptdins pools on ppi production and on the morphology and functions of various organelles. the functionality of proteins is known to be intimately related to the motion of their constituents on the atomic/molecular level. the study of microscopic motion in complex matter is often reduced to the observation of some average mean square atomic displacement, a first, very partial characterization of the dynamics. the marked crossover in the temperature dependence of such quantities in hydrated proteins around k, the so called ''dynamic transition'' has been originally observed a quarter of century ago. the origin, nature and the key characteristics of the atomic motions behind this remarkable evolution of the mean square displacement in proteins remained controversial over the past decades. recent analysis of mö ssbauer, dielectric relaxation and neutron scattering spectroscopic data provide unambiguous evidence that this phenomenon is caused by the temperature dependence of a relaxation process spread over several orders of magnitude in the time domain, similarly to the b relaxation process observed in glasses. the review and critical analysis of the available data highlights the inherent ambiguities of commonly used data fitting approaches. emerging evidence from model independent observations tend to exclude some of the proposed mechanisms. microbial rhodopsins: light-gated ion channels and pumps as optogenetic tools in neuro-and cell biology e. bamberg, c. bamann, r.e. dempski, k. feldbauer, s. kleinlogel, u. terpitz, p. wood department of biophysical chemistry, max-planck-institute of biophysics, frankfurt, germany microbial rhodopsins are widely used in these days as optogenetic tools in neuro and cell biology. we were able to show that rhodopsins from the unicellar alga chlamydomonas reinhardtii with the transmembrane helix motif act as light-gated ion channels, which we named channelrhodopsins(chr , ). together with the light driven clpump halorhodopsin chr is used for the non-invasive manipulation of excitable cells and living animals by light with high temporal resolution and more important with extremely high spatial resolution the functional and structural description of this new class of ion channels is given (electrophysiology, noise analysis,flash photolysis and d crystallography). new tools with increased spatial resolution and extremely enhanced light sensitivity in neurons are presented. a perspective for basic neurobiology and for medical applications is given. extracellular signals consists of the induction of specific gene expression patterns and the re-organization in space and time of stereo-specific macromolecular interactions that endow the cell with its specific morphology. we develop quantitative experimental and computational approaches to derive and conceptualize physical principles that underlie these dynamics of signal processing and cellular organization. we have an experimental emphasis on functional microscopic imaging approaches at multiple resolutions to study the localization and dynamics of protein reactions/ interactions, maintaining the inherent spatial organization of the cell. we have a strong recursion between computation of molecular dynamics in realistic cell geometries as sampled by microscopy, and experiments that reveal the dynamic properties of networks in living cells. we investigate the cellular topography of activities that transmit signals from receptors at the cell surface. here we ask, how spatial partitioning of intracellular signalling activities is achieved by the causality structure of the signalling network, and how this partitioning affects signal response. this entails the experimental elucidation of connections between reactions and the determination of enzyme kinetic parameters in living cells. o- molecular photovoltaics mimic photosynthesis michael grä tzel laboratory of photonics and interfaces, institute of chemical science and engineering, station , ecole polytechnique fé dé rale, ch- lausanne, switzerland e-mail: michael.graetzel@epfl.ch the field of photovoltaic cells has been dominated so far by solid state p-n junction devices made e.g. of crystalline or amorphous silicon, profiting from the experience and material availability of the semiconductor industry. however, there is an increasing awareness of the possible advantages of devices referred to as ''bulk'' junctions due to their interconnected three-dimensional structure. their embodiment departs completely from the conventional flat p-n junction solid-state cells, replacing them by interpenetrating networks. this lecture focuses on dye sensitized mesoscopic solar cells (dscs), which have been developed in our laboratory. imitating natural photosynthesis, this cell is the only photovoltaic device that uses a molecular chromophore to generate electric charges from sunlight and that accomplishes the separation of the optical absorption from the charge separation and carrier transport processes. it does so by associating the molecular dye with a film constituted of tiny particles of the white pigment titanium dioxide. the dsc has made phenomenal progress, present conversion efficiencies being over percent for single junction and percent for tandem cells, rendering the dsc a credible alternative to conventional p-n junction devices. molecularly single-molecule imaging and tracking techniques that are applicable to living cells are revolutionizing our understanding of the plasma membrane dynamics, structure, and signal transduction functions. the plasma membrane is considered the quasi- d non-ideal fluid that is associated with the actinbased membrane-skeleton meshwork, and its functions are likely made possible by the mechanisms based on such a unique dynamic structure, which i call membrane mechanisms. my group is largely responsible for advancing highspeed single molecule tracking, and based on the observations made by this approach, i propose a hierarchical architecture of three-tiered meso-scale ( - nm) domains as fundamental organizing principles of the plasma membrane. the three tiers i propose are the following. [tier ] - nm compartments made by partitioning the entire plasma membrane by the membrane-associated actinbased meshwork (membrane skeleton: fences) and its associated transmembrane proteins (pickets). since the entire plasma membrane is partitioned by these structures, and the membrane skeleton provides important platforms for the molecular interactions and pools, membrane compartments are the most basic tier for the plasma membrane organization. [tier ] meta-stable - nm raft domains that can be turned into stable * - -nm domains (receptor-cluster rafts), based on ligand-induced homo-dimers of glycosylphosphatidylinositol (gpi)-anchored receptors (coupling with [tier ]) and facilitated by raft-lipid interactions. [tier ] protein complexes of various sizes ( - nm) and lifetimes. i will also talk about how domains of tiers and are coupled to the membrane partitioning (tier ). the concept of the three-tiered domain architecture of the plasma membrane and the cooperative interactions of different tiers provides a good perspective for understanding the mechanisms for signal transduction and many other functions of the plasma membrane. introduction: in the present study we investigate the effects of electromagnetic fields (emf) on the binding of norfloxacin (nrf) to human serum albumin (hsa) by fluorescence, three-dimensional fluorescence and uv-visible spectroscopic approaches. hsa is the most abundant protein in human blood plasma which works as a carrier that transports different materials in the body. nrf is used to treat variety of bacterial infections. it works by stopping the bacterial growth. methods: hsa, nrf and potassium phosphate buffer were purchased from sigma. fluorescence spectrofluorometer, uv-vis spectrophotometer, three-dimensional fluorescence and a home-built emf generator apparatuses were used. results: results obtained from this study indicated that nrf has a strong ability to quench hsa in nm. in addition, there was a slight blue shift, which suggested that the microenvironment of protein became more hydrophobic after addition of nrf. moreover, synchronous fluorescence demonstrated that the microenvironment around tyrosine (tyr) had a trivial increase. these, and the results of hsa-nrf in the presence of emf with khz, illustrates the same results inferred from quenching and blue shift. however, there was a significant decrease in k sv of nrf with hsa in presence of emf exposure. moreover, the binding parameters including the number of binding sites and the binding constant were calculated form hill equation. conclusion: it was shown that nrf could induce conformational changes in hsa both in the absence and presence of emf with no significant difference. yet, the affinity is decrease significantly in the presence of emf. the clinical implications are discussed in detail. characterization of the biochemical properties and biological function of the formin homology domains of drosophila we characterised the properties of drosophila melanogaster daam-fh and daam-fh -fh fragments and their interactions with actin and profilin by using various biophysical methods and in vivo experiments. the results show that while the daam-fh fragment does not have any conspicuous effect on actin assembly in vivo, in cells expressing the daam-fh -fh fragment a profilindependent increase in the formation of actin structures is observed. the trachea specific expression of daam-fh -fh also induces phenotypic effects leading to the collapse of the tracheal tube and lethality in the larval stages. in vitro, both daam fragments catalyze actin nucleation but severely decrease both the elongation and depolymerisation rate of the filaments. profilin acts as a molecular switch in daam function. daam-fh -fh , remaining bound to barbed ends drives processive assembly of profilin-actin, while daam-fh forms an abortive complex with barbed ends that does not support profilinactin assembly. both daam fragments also bind to the sides of the actin filaments and induce actin bundling. these observations show that the drosophila melanogaster daam formin represents an extreme class of barbed end regulators gated by profilin. electron spin echo studies of free chain-labelled stearic acids interacting with b-lactoglobulin rita guzzi, luigi sportelli, rosa bartucci dipartimento di fisica, università della calabria, rende (cs), italy b-lactoglobulin (blg) binds non-covalently fatty acids within its central calyx, a cavity in the barrel formed by the strands ba-bh. we present results of pulsed electron paramagnetic resonance (epr) spectroscopy on the interaction of blg with stearic acids spin-labelled at selected positions, n, along the acyl chain (n-sasl, n = , , , , ). d o-electron spin echo envelope modulation (eseem) fourier transform spectra indicate that all segments of the bound chains in the protein binding site are accessible to the solvent. the extent of water penetration decreases progressively on moving from the first segments toward the terminal methyl end of the chain. about % of the nitroxides in the upper part of the chain (n = , ) are h-bonded by a single water molecule and this fraction reduces to % at the chain terminus (n = , ). a lower fraction of the nitroxides are h-bonded by two water molecules, and it decreases from about % to a vanishingly small value on going down the chain. echo-detected ed-epr spectra reveal subnanosecond librational motion of small amplitude for both -and -sasl in the protein cavity. the temperature dependence of the librations is more marked for -sasl and it arises mainly from an increase in librational amplitude with increasing temperature. fusion peptides (fp) pertaining to the spike glycoprotein from severe acute respiratory syndrome (sars) coronavirus are essential for the fusion between viral and host cellular membranes. here we report a biophysical characterization of the interaction of two putative fps with model membranes. fluorescence and dsc experiments showed that both peptides bind stronger to anionic than to zwitterionic lipid membranes. esr spectra showed that toac-sars ifp rotational dynamics is modulated by lipid composition and ph as compared to the spectrum of this peptide in solution. however, stearic acid spin labels reported no changes on the dynamic structure of zwitterionic micelles, whereas the whole chain of anionic surfactants was perturbed by the peptides. finally, cd data revealed a predominant b-strand structure for sars fp and an a-helix for sars ifp in the presence of micelles, in contrast to their disordered structures in buffer. overall the results point out that electrostatic and hydrophobic interactions are both important to the energetic behavior of peptide membrane interaction. these findings might provide a useful rationale for the elucidation of one of the steps involved in the fusion process, and thus help understanding the more general way of action of fps at a molecular level. interaction of filamentous actin and ezrin within surface modified cylindrical nanopores daniela behn , and claudia steinem institute for organic and biomolecular chemistry, university of gö ttingen, tammannstraße , gö ttingen, germany, ezrin is a member of the ezrin-radixin-moesin (erm) protein family that acts as a dynamic linker between the plasma membrane and the actin cytoskeleton and is hence involved in membrane organization, determination of shape and surface structures and other cellular processes. the protein is highly enriched in microvilli of polarized epithelial cells, where it binds filamentous actin (f-actin) with its c-terminal domain, while the n-terminal domain is connected to the plasma membrane via specific binding to l-a-phosphatidylinositol- , -bisphosphate (pip ). nanoporous anodic aluminum oxide (aao) films provide similar dimensions as microvilli and are thus a versatile template to investigate the interaction of ezrin with f-actin within spationally confined areas. owing to their optical transparency, functionalized aaos can be used to measure the binding process of ezrin to a pip containing solid supported membrane by means of time resolved optical waveguide spectroscopy (ows). confocal laser scanning microscopy (clsm) will elucidate, whether f-actin binding to ezrin takes place within or atop the nanopores. furthermore, elasticity mapping of f-actin filaments by means of atomic force microscopy will allow determining binding forces and the lateral tension of the actin cytoskeleton. in vitro application of porphyrin photosensitisers on mcf , hela and g tumour cell lines binder s., kolarova h., bajgar r., tomankova k., daskova a. deparment of medical biophysics, faculty of medicine of palacky university, olomouc, czech republic tumour treatment presents a challenge to all scientists and clinicians. contemporary methods like radiotherapy, chemotherapy or surgery have many undesirable side effects. photodynamic therapy (pdt) seems to be one of alternatives which can be helpful in malignant cell therapy. pdt is not only limited to cancer treatment but is also used as an alternative for cardiovascular, skin and eye disease treatment. pdt employs photosensitive agents which need to be activated by light which is not harmful to a patient. the activated photosensitive agent provokes a formation of reactive oxygen species leading to cell damage or death. the phototoxicity of the two porphyrin photosensitizer (tmpyp, zntpps . h o) on the malignant cell lines (g , hela, mcf ) irradiated with the jcm - doses was evaluated by ros production assay, mtt assay and comet assay. our results indicate higher efficiency of tmpyp over zntpps . h o. as for the photodynamic effectiveness of the used photosensitizers on chosen cell lines we found that hela cell line is the most sensitive to phototoxic damage induced by tmpyp. p- nmr analysis of the respiratory syncytial virus m - protein structure and of its interaction with some of its targets c. sizun the respiratory syncytial virus (rsv) is a major cause of acute respiratory tract infections (bronchiolitis, pneumonia) in human and a leading cause of viral death in infants and immunocompromised patients. rsv genome is formed of a single non-segmented negative strand rna which transcription and replication is ensured by a specific rna-dependent rna polymerase complex formed of the large (l) polymerase subunit and of several cofactors. this complex has no cellular counterpart and represents an ideal target for antiviral drugs. among the cofactors, m - acts as an antitermination factor and increases the polymerase processivity. its central domains has been shown, in vitro, to bind the phosphoprotein p and genomic rna in a competitive manner. here we report the nmr structure of this central domain and its interaction with p and rna fragments. m - shares structural similarity with vp , a transcription factor of ebola virus. the binding surfaces for rna and p are distinct but overlapping. rna binds to a basic cluster located next to residues found to be critical for transcription both in vitro and in vivo by mutational analysis. we speculate that m - might be recruited by p to the transcription complex, where interaction with rna takes place, stabilized by additional elements. force spectroscopy at the membrane-cytoskeleton interface: interactions between ezrin and filamentous actin julia a. braunger , , ingo mey and claudia steinem institute for organic and biomolecular chemistry, georg-august-university of gö ttingen, tammannstraße , gö ttingen, germany, ggnb doctoral program: imprs - ezrin, a member of the erm (ezrin/radixin/moesin) protein family, provides a regulated linkage between the plasma membrane and the actin cytoskeleton. it contributes to the organization of structurally and functionally distinct cortical domains participating in adhesion, motility and fundamental developmental processes. ezrin is negatively regulated by an intramolecular interaction of the terminal domains that masks the f-actin binding site. a known pathway for activation involves the interaction of ezrin with phosphatidylinositol , bisphosphate (pip ) in the membrane, followed by phosphorylation of the threonine residue in the c-terminal domain. to date, it is unclear to what extent both regulatory inputs contribute to the activation. we developed an in vitro system that facilitates the specific analysis of the interaction forces between ezrin and f-actin by means of atomic force spectroscopy (afm). applying ezrin wild type and the pseudophosphorylated mutant protein ezrin t d, respectively, permits to monitor the individual influence of phosphorylation on the f-actin-ezrin interaction. thus, a thorough characterization of the acting forces at the ezrin-actin interface will elucidate the activation mechanism of ezrin. delivery system even more efficient, we have constructed nano-carrier by coating of ldl by polyethylene glycol (peg) . the hydrophilicity of peg should reduce the interaction of ldl with other serum proteins and consequently decrease the redistribution of loaded drug from ldl to the (lipo)proteins. dynamic light scattering was used for determination of hydrodynamic radius of ldl-peg particles. cd spectroscopy measurements didn't reveal structural changes of apoliprotein b- (ligand for ldl receptors on cell surface), after conjugation of peg with ldl. interaction of ldl-peg complexes with hypericin (hyp) a natural photosensitizer was studied by fluorescence spectroscopy. we have demonstrated accumulation of higher number of hyp in ldl-peg than ldl particles. however, the kinetics of hyp redistribution from hyp/ldl-peg complex to free ldl have similar parameters as those for the kinetics of hyp transfer between non-modified ldl molecules. we suggest that hyp molecules are mostly localized in the vicinity of the surface of the ldl-peg particles and they are prone to redistribution to other serum proteins. grant support: lpp- - , vega- - . modification of the head-group of aminophospholipids by glycation and subsequent lipid oxidation affect membrane's structure causing cell death. these processes are involved in the pathogenesis of aging and diabetes. non-enzymatic glycation forms in the first step a schiff base (sb), which rearranges to a more stable ketoamine, amadori product, which leads to the formation of a heteregenous group of compounds (ages). although several studies have been focused on identification of aminophospholipid glycation products, less attention has been paid to kinetic mechanism of the reaction. for that reason, in the present work, we compare the kinetic reactivity of polar head-group of phosphatidylethanolamine (pe) and phosphatidylserine (ps), the two target phospholipids components of mammalian cell membranes. the reaction of pe and ps's head-group with glycating compounds (glucose and arabinose) was studied in physiological conditions by using nmr spectroscopy. the obtained formation rate constants for sb are lower than those determined for the sb of the peptide ac-phe-lys with the same carbohydrates. it suggests that the phosphate group may delay the glycation process. moreover, the ps's head-group has a carboxilic group in the structure, which affects the stability of the sb. we developed ultrasensitive, elisa-like nanoimmuno assays suitable for proteomics/interactomics studies in low sample volumes. we exploit the approach of dna microarray technologies applied to proteomics [ ] , in combination with atomic force microscopy (afm) to generate functional protein nanoarrays: semisynthetic dna-protein conjugates are immobilized by bioaffinity within a nanoarray of complementary ssdna oligomers produced by afm nanografting (ng). a nanoarray of different antibodies or synthetic molecular binders can be generated in a single operation, once the dna nanoarray is produced. moreover, ng allows adjusting the packing density of immobilized biomolecules to achieve optimum bio-recognition. afm-based immunoassays with these nanoarrays were shown to achieve detection limit of hundreds of femto molar, in few nanoliters volumes, with very high selectivity and specificity [ ] . to detect the hybridization efficiency of our devices, we run a combined experimental-computational study that provides quantitative relations for recovering the surface probe density from the mechanical response (afmcompressibility measurements) of the sample. nucleoside analogues used as anticancerous drugs can be rapidly degraded within treated cells, constituting a major obstacle of their therapeutic efficiency. among the enzymes responsible for this degradation, the cytosolic 'nucleotidase ii (cn-ii) catalyses the hydrolysis of some nucleoside monophosphates. in order to improve the efficacy of anticancerous drugs and to define the precise role of cn-ii, new original inhibitors have been developed against cn-ii. virtual screening of chemical libraries on the crystal structure has allowed us to identify very promising candidates that turned to be competitive inhibitors of cn-ii. one molecule was included in the anticancerous treatment of tumoral cell lines in order to evaluate the potential benefit and could induce in fine a sensitization of certain anticancerous drugs. we also explore other inhibitors targeting the allosteric sites of this enzyme using a strategy that takes into account the dynamics of cn-ii. the chemical structures of the newly identified allosteric inhibitors as well as the atomic interactions with enzyme residues will be presented. the final goal of this study is to find molecules that can freeze the enzyme in a conformation for which its dynamics is severely limited and therefore its function. native mass spectrometry to decipher interactions between biomolecules sarah cianferani laboratoire de spectromé trie de masse bio-organique, université de strasbourg, iphc, rue becquerel strasbourg, france. cnrs, umr , strasbourg, france mass spectrometry is generally understood as ''molecular mass spectrometry'' with multiple applications in biology (protein identification using proteomic approaches, recombinant protein and monoclonal antibody characterization). an original and unexpected application of mass spectrometry emerged some twenty years ago: the detection and the characterization of intact biological noncovalent complexes. with recent instrumental improvements, this approach, called native ms, is now fully integrated in structural biology programs as a complementary technique to more classical biophysical approaches (nmr, crystallography, calorimetry, spr, fluorescence, etc.). native ms provides high content information for multiprotein complexes characterization, including the determination of the binding stoichiometries or oligomerization states, sitespecificities and relative affinities. recent developments of ion mobility / mass spectrometry instruments (im-ms) provide a new additional level for ms-based structural characterization of biomolecular assemblies allowing size and shape information to be obtained through collisional cross section measurements. these different aspects of native ms for structural characterization of biomolecular assemblies will be illustrated through several examples, ranging from multiprotein-complexes to protein/nucleic acid assemblies. complex coacervation is a process which may result by electrostatic interaction between charged polysaccharides. it depends essential on ph, ionic strength and biopolymers properties like ratio, concentration and charge density. in this case, the main work was to study the structural properties of a colloidal system of opposite charge -chitosan and gum-arabic by atomic force microscopy (afm). according to some of complexes show tendency to agglomerate. this depends on the molar ration of the macromolecules and their relative molecular weights. afm micrographs show, too, that some formation of irregular aggregates by both polymers were due to presence of noncharged polar monomers in chitosan molecule. at higher gum-arabic/chitosan ratios biopolymer concentrations, coacervates appear like a core-shell miccelar structure composed of hydrophobic core (charge neutralized segments) stabilized by the excess component (positive zeta potential) and non-charged segments of gum arabic. interaction of human serum albumin with rutin theoretical and experimental approaches Ícaro p caruso human serum albumin (hsa) is the principal extracellular protein with a high concentration in blood plasma and carrier for many drugs to different molecular targets. flavonoids are a large class of naturally occurring polyphenols widely distributed in plants. rutin (quercetin- -rutinoside) is the glycoside between flavonoids quercetin and disaccharide rutinose. like other flavonoids, rutin displays anti-inflammatory and anti-oxidant properties. the interaction between hsa and rutin was investigated by fluorescence spectroscopy, ab initio and molecular modeling calculations. fluorescence titration was performed by keeping the hsa concentration ( lm) constant and stoichiometrically varying the rutin concentration ( - lm) . the emission spectra were obtained in the range of to nm, with the excitation wavelength at nm. the obtained fluorescence data were corrected for background fluorescence and for inner filter effects. the stern-volmer quenching constant values were . and . m - at and k, respectively. from the modified stern-volmer association constants . (at k) and . m - (at k) were calculated the thermodynamic parameters dh = . kj mol - , dg k = - . kj mol - and dg k = - . kj mol - , and ds = . kj mol - k - . fluorescence quenching method was used also to study the binding equilibria thus determining the number of binding sites . and . , and binding constant . m - and . m - at and k, respectively. the efficient quenching of the trp fluorescence by rutin indicates that the binding site for the flavonoid is situated within subdomain iia of hsa. the distance r = . nm between the donor (hsa) and the acceptor (rutin) was obtained according to fluorescence resonance energy transference (fret). wavelength shifts in synchronous fluorescence spectra showed the conformation of hsa molecules is changed in the presence of rutin. the structure of rutin utilized in molecular modeling calculation was obtained by gaussian program. the optimization geometry of rutin was performed in its ground states by using ab initio dft/b lyp functional with - g(d,p) basis set used in calculations. the molecular electrostatic potential (mep) was calculated to provide the molecular charge distribution of rutin. the gap energy value between the homo and lumo of the rutin molecule was about . ev which indicates that rutin is classified as a reactive molecule. from molecular modeling calculation the interaction between hsa and rutin was investigated using the autodock program package. the three-dimensional coordinates of human serum albumin were obtained from the protein data bank (entry pdb code ao ) and of rutin were obtained from output optimization geometry of dft. the best energy ranked result shows that rutin is localized in the proximity of single tryptophan residue (trp ) of hsa that is in agreement with the fluorescence quenching data analysis. the effect of toxofilin on the structure of monomeric actin lívia czimbalek, veronika kollá r, roland kardos, gá bor hild university of pé cs, faculty of medicine, department of biophysics, pé cs, hungary actin is one of the main components of the intracellular cytoskeleton. it plays an essential role in the cell motility, intracellular transport processes and cytokinesis as well. toxoplasma gondii is an intracellular parasite, which can utilise the actin cytoskeleton of the host cells for their own purposes. one of the expressed proteins of t. gondii is the kda-sized toxofilin. the long protein is a monomeric actinbinding protein involved in the host invasion. in our work we studied the effect of the actin-binding site of toxofilin - on the g-actin. we determined the affinity of toxofilin to the actin monomer. the flourescence of the actin bound e-atp was quenched with acrylamide in the presence or absence of toxofilin. in the presence of toxofilin the accessibility of the bound e-atp decreased, which indicates that the nucleotide binding cleft is shifted to a more closed conformational state. the results of the completed experiments can help us to understand in more details what kind of cytoskeletal changes can be caused in the host cell during the invasion of the host cells by intracellular parasites. t bacteriophage, as a surrogate on non-enveloped viruses was selected as a test system. both tmpcp and bmpcp and their peptide conjugates proved to be efficient photosensitizers of virus inactivation. the binding of porphyrin to phage dna was not a prerequisite of phage photosensitization, moreover, photoinactivation was more efficiently induced by free than by dna bound porphyrin. mechanism of photoreaction (type i. versus type ii) and the correlation between dna binding, singlet oxygen production and virus inactivation capacity was also analyzed. dna binding reduced the virus inactivation due to the reduced absorbance and singlet oxygen production of bound photosensitizer, and altered mechanism of photoinactivation. as optical melting studies of t nucleoprotein revealed, photoreactions of porphyrin derivatives affected the structural integrity of dna and also of viral proteins, even if the porphyrin did not bind to np or was selectively bound to dna. anesthesia is a medical milestone (friedman & friedland, medicine's greatest discoveries, ) and local anesthetics (la) are the most important compounds used to control pain in surgical procedures. however, systemic toxicity is still a limitation for la agents as well as low solubility, as for tetracaine (ttc). approaches to improve la effects include macrocyclic systems formation, such as in cyclodextrins (cd). we have studied complexes formed between ttc and b-cd or hydroxylpropyl (hp)-b-cd through nmr and other (uv-vis, fluorescence, dsc and x-ray diffraction) techniques. at ph . a : stoichiometry of complexation was detected for both complexes, with association constants of m - and m - for ttc:b-cd and ttc:hp-b-cd, respectively. the nuclear overhauser nmr data disclosed trough the space proximities between hydrogens h h and h iat the aromatic ring of ttc -and hydrogens from the inner cavity of the cyclodextrins, allowing us to propose the topology of ttc:cd interaction. complex formation did not curb ttc association with model (liposomes) and biological membranes since the total analgesic effect (infraorbital nerve blockade in rats) induced by mm ttc increased % upon complexation. supported by (fapesp # / - , - ) brazil. p- itc as a general thermodynamic and kinetic tool to study biomolecule interactions philippe dumas , dominique burnouf , eric ennifar , sondes guedich , guillaume bec , guillaume hoffmann isothermal titration calorimetry (itc) is a powerful technique for thermodynamic investigations that is little used to obtain kinetic information. we have shown that, in fact, the shape of the titration curves obtained after each ligand injection is strictly governed by the kinetics of interaction of the two partners. a simple analysis allowed us to explain several facts (e.g. the variation of time needed to return to equilibrium during a titration experiment). all simplifications were further released to obtain a very realistic simulation of an itc experiment. the method was first validated with the binding of the nevirapine inhibitor onto the hiv- reverse transcriptase by comparison with results obtained by biacore tm . importantly, for more complex systems, the new method yields results that cannot be obtained in another way. for example, with the e. coli transcription-regulator thiamine pyrophosphate riboswitch, we could resolve kinetically and thermodynamically the two important successive steps: ( ) the binding of the tpp ligand and ( ) the subsequent rna folding. our results show that initial tpp binding is controlled thermodynamically by tpp concentration, whereas the overall transcription regulation resulting from rna folding is kinetically controlled. gfps, due to their tendency to dimerize at high concentration. we have characterized for the first time the selfassociation properties of cfp (cyan fluorescent protein), the fluorescent protein mostly used as fret donor. we found that the fluorescence quenching observed at high expression level in the cell cytoplasm and the fluorescence depolarization measured at high concentration in vitro are insensitive to the a k mutation, shown to dissociate other gfp dimers. both phenomena are satisfactorily accounted for by a model of non-specific homo-fret between cfp monomers due to molecular proximity. modeling the expected contributions to fluorescence depolarization of rotational diffusion, homo-fret within a hypothetical dimer and proximity homo-fret shows that cfp has a homo-affinity at least times lower than gfp. this difference is due to an intrinsic mutation of cfp (n i), originally introduced to increase its brightness and that by chance also disrupts the dimers. biomolecular recognition typically proceeds in an aqueous environment, where hydration shells are a constitutive part of the interacting species. the coupling of hydration shell structure to conformation is particularly pronounced for dna with its large surface to volume ratio. conformational substates of the phosphodiester backbone in b-dna contribute to dna flexibility and are strongly dependent on hydration. we have studied by rapid scan ftir spectroscopy the isothermal b i -b ii transition on its intrinsic time scale of seconds. correlation analysis of ir absorption changes induced by an incremental growth of the dna hydration shell identifies water populations w (po --bound) and w (non-po --bound) exhibiting weaker and stronger h-bonds, respectively, than those in bulk water. the b ii substate is stabilized by w . the water h-bond imbalance of - kj mol - is equalized at little enthalpic cost upon formation of a contiguous water network (at - h o molecules per dna phosphate) of reduced !(oh) band width. in this state, hydration water cooperatively stabilizes the b i conformer via the entropically favored replacement of w -dna interactions by additional w -water contacts, rather than binding to b i -specific hydration sites. such water rearrangements contribute to the recognition of dna by indolicidin, an antimicrobial -mer peptide from bovine neutrophils which, despite little intrinsic structure, preferentially binds to the b i conformer in a water-mediated induced fit. in combination with cd-spectral titrations, the data indicate that in the absence of a bulk aqueous phase, as in molecular crowded environments, water relocation within the dna hydration shell allows for entropic contributions similar to those assigned to water upon dna ligand recognition in solution. segmental-labeling expression of sh domains of cd ap protein to study interaction with their ligand i.f. herranz-trillo , j.l. ortega-roldan , n.a.j. van transient and low affinity interactions within the cell can be enhanced by the combination of more than one domain. up to now most of the effort has been put on the study of the regulation in the affinity and specificity of the binding to isolated single domains but little is known about the effect of the presence of a second or third domain. multiple examples of proteins containing tandem domains exist in the genome like the cin /cms family of adaptor proteins. in this family all three n-terminal sh domains are involved in a wide variety of different interactions, they share higher similarity among themselves than to any other sh domains, suggesting an overlapping specificities in binding. cd associated protein (cd ap) is an adaptor protein and belongs to this family, its n-terminus consists of three sh domains and the interaction of each one of them with its target(-s) might be ultimately modulated by the presence of its next-door-neighbor. in this work we present the expression and purification of the tandem cd ap-sh a/ sh b produced by segmental labeling techniques that allow us to express the domains with different isotopic label, improving the nmr signal and facilitating to study the interaction of the natural ligand in the presence of nextdoor-neighbor domain. there are plenty of molecules that exert their effects at the cell membrane. the evaluation of these interactions, frequently quantified by the nernst lipid/water partition constant (kp), helps to elucidate the molecular basis of these processes. we present here a recently derived and tested method to determine kp for single solute partitions using fpotential measurements. the concept was then extended to the interaction of supramolecular complexes with model membranes. a simultaneous double partition with an aqueous equilibrium is considered in this partition model. the results were validated by dynamic light scattering -dls, f-potential, fluorescence spectroscopy and laser confocal microscopy experiments. we evaluated the interaction of supramolecular complexes (peptides derived from dengue virus proteins with oligonucleotides) with luv to study our biophysical models. dengue virus (dv) infects over - million people every year and may cause viral hemorrhagic fever. no effective treatment is available and several aspects of its cellular infection mechanism remain unclear. the extension of the interactions of these complexes with biomembranes helps to elucidate some steps of dv life cycle. the aggregation of amphotericin b in the lipid membranes induced by k + and na + ions: langmuir monolayers study marta arczewska, mariusz gagoś department of biophysics, university of life sciences in lublin, poland the polyene antibiotic amphotericin b (amb) is currently the drug of choice in the treatment of fungal infections despite its undesirable side effects. according to the general conviction, the biological action of the drug is based on the formation of transmembrane channels which affect physiological ion transport, especially k + ions. this work reports the results of langmuir monolayers study of the effect of k + and na + ions on the molecular organizations of amb in the model lipid membrane. the two-component monolayers containing amb and phospholipid (dppc) have been investigated by recording surface pressure-area isotherms spread on aqueous buffers containing physiological concentration of k + and na + ions. the strength of the amb-dppc interactions and the stability of the mixed monolayers were examined on the basis of surface pressure measurements, the compressional modulus and the excess free energy of mixing. the obtained results proved a high affinity of amb towards lipids in the presence of k + than na + ions. the most stable mixed monolayers were formed with the : and : stoichiometry in the presence of k + and na + ions, respectively. this research was financed by ministry of education and science of poland within the research project n n . microcalorimetric study of antibiotic amphotericin b complexes with na + , k + and cu + ions arkadiusz matwijczuk, grzegorz czernel, mariusz gagoś department of biophysics, university of life sciences in lublin, poland amphotericin b (amb) as a metabolite of streptomyces nodosus is one of the main polyene antibiotics applied in the treatment of deep-seated mycotic infections. we presented microcalorimetric (dsc) study of molecular organization of amphotericin b in lipid membranes induced by na + , k + and cu + ions. the analysis of dsc curves indicates the influence of na + and k + ions on the main phase transition of pure dppc lipid. for the molar fractions of , , , mol% amb in dppc we observed the thermal shift towards higher temperatures in respect to pure lipid, both in the presence of na + and k + ions. this result may be connected with the changes in dynamic properties of the model membrane system. in case of amb-cu + complexes in aqueous solution at two ph values, . and . , the dsc measurements reported endothermic heat effect. this phase transition was related to the dissociation process of amb-cu + complexes. the formation of amb-cu + complexes are accompanied by changes of the molecular organization of amb especially disaggregation. these all observed effects might be significant from a medical point of view. this research was financed by ministry of education and science of poland within the research project n n . membrane proteins and peptides are acting in an environment rich in other proteins or peptides. aim of our study was to understand how such molecular crowding and resulting intermolecular interactions can influence the behavior of membrane proteins, using various antimicrobial peptides and membrane proteins as examples. in the case of antimicrobial peptides we have previously described a change in their alignment in the membrane at a characteristic threshold concentration. to understand whether this change is due to unspecific crowding or specific peptidepeptide interactions, we tested if this re-alignment depends on the presence of additional peptides. in most cases we found a similar re-orientation behavior irrespective of the added peptide type, indicating unspecific crowding. when pairing pgla and magainin- , however, we observed a distinctly different sequence of pgla re-orientation in the membrane, indicating a specific interaction between these two peptides, which correlates well with their known synergistic activity. a rather different effect of crowding was observed for the larger channel protein mscl, which was found to form clusters of functionally active proteins in the membrane. we propose that this clustering is caused by lipid-mediated protein-protein interactions. water, hydrophobic interaction, and protein stability j. raul grigera and c. gaston ferrara instituto de física de líquidos y sistemas bioló gicos (iflysib), conicet-unlp, la plata, argentina although there are several forces maintaining protein structure, it is well know that hydrophobic interaction is the dominant force of protein folding. then, we can infer that any factor that alters hydrophobic interaction will affect the protein stability. we have study by computer simulation a model system consisting in solution of lenard-jones particles in water (spc/e model) at different pressures and temperatures and analyzed the solubility i.e. the aggregation properties, of such a system. from the obtained data we are able to build up the phase surface determining the critical point. the computing results where compared with experimental data of binary mix of non polar substance in water and of protein denaturation, finding high coincidence on the critical point. since the behavior of our model system can only be due to hydrophobic effects, the coincidences with the denaturation of proteins allow us to conclude that the dominant factor that determine temperature and pressure denaturation of proteins is the hydrophobic interaction. the temperature and pressures at which the denaturation, as well the disaggregation of simple non-polar particles, starts agree with what we could expect based on the cross over line of the low to high density structure water transition. the functional reconstitution of a mitochondrial membrane protein into a lipid bilayer was studied using a quartz crystal microbalance. the xhis-tagged protein was immobilised via specific binding to a cu + terminated sensor surface, with a change in frequency indicating approximately % coverage of the sensor surface by the protein. a lipid bilayer was reconstituted around the protein layer, with a final change in frequency that is consistent with the remaining area being filled by lipid. incubation with a specific ligand for the protein resulted in a significant change in frequency compared to the interaction with the surface or lipid alone. the change is greater than expected for the mass of the ligand, indicating a possible conformational change of the protein, such as the opening of a channel and increased water content of the layer. electrical impedance measurements on the same system have provided additional evidence of protein-lipid bilayer formation, and it is intended that this system will be studied with neutron reflectometry to characterise potential ligand induced channel formation. valuable functional and structural information about this membrane protein was obtained by using surface sensitive techniques to study the protein in a biomimetic lipid bilayer. visualizing and quantifying hiv-host interactions with fluorescence microscopy jelle hendrix , *, zeger debyser , johan hofkens and yves engelborghs laboratory for biomolecular dynamics, university of leuven, belgium, laboratory for molecular virology and gene therapy, university of leuven, belgium, laboratory for photochemistry and spectroscopy (*present address), university of leuven, belgium protein-chromatin interactions are classically studied with in vitro assays that only provide a static picture of chromatin binding. fluorescence correlation spectroscopy (fcs) is a non-invasive technique that can be used for the same purpose. being applicable inside living cells it provides dynamic real-time information on chromatin interactions. transcriptional co-activator ledgf/p has well characterized protein and chromatin interacting regions. we studied ledgf/p in vitro and inside living cells with fcs and other techniques (luminescent proximity assay, spot/half-nucleus fluorescence recovery after photobleaching, continuous photobleaching). protein-protein interactions in living cells can be monitored with fluorescence cross-correlation spectroscopy (fccs) using fluorescent proteins as genetic labels. advantages over using fö rster resonance energy transfer (fret) are the independence from intermolecular distance and knowledge of absolute protein concentrations. we characterized fccs with fluorescent proteins in vitro and then studied the intracellular complex of ledgf/p and the hiv- integrase (in) enzyme both with fret and fccs. nucleus and its compartment nucleolus are a seat of enormous biosynthetic activity in human cancer cells. nucleolar proteins, e.g. b or c , play an important role in regulation of cell division and proliferation. one of the strategies how to intermit malignant cell proliferation is affecting, e.g. by drug treatment, a net of intracellular protein interactions to bring the cell on a way of apoptosis. a cytostatic agent actinomycin d initiates apoptosis in human cancer cells, as well as in normal peripheral blood lymphocytes. at the same time, translocation of b and c into nucleoplasm is observed in the treated cells. therefore interaction between nucleolar and apoptotic proteins comes into a question. co-immunoprecipitation, fluorescence microscopy and yeast two hybrid analysis are used to answer it. in co-immunoprecipitation experiments, tumor suppressor p showed up to be a promising candidate for the interaction. fluorescence deposits mostly constituted by variants of transthyretin (ttr), a homotetrameric plasma protein implicated in the transport of thyroxine and retinol [ ] . nowadays, the only effective therapy for ttr amyloidosis is liver transplantation. new therapeutic strategies are being developed taking advantage of our current understanding of the molecular mechanisms of amyloid formation by ttr [ ] . a significant effort has been devoted to the search and rational design of compounds that might decrease ttr tetramer dissociation, for example, through ligand binding at the thyroxine binding sites of ttr [ , ] . here, we use isothermal titration calorimetry (itc) to characterize the thermodynamic binding signature of potential ttr tetramer stabilizers, previously predicted by computerassisted methods [ ] . itc allows the measurement of the magnitude of the binding affinity, but also affords the characterization of the thermodynamic binding profile of a protein-ligand interaction. high affinity/specificity ttr ligands, enthalpically and entropically optimized, may provide effective leads for the development of new and more effective drug candidates against ttr amyloidosis. we have established a set of vectors to promote easy cloning of ecfp and eyfp fusions with any protein of interest. we exploit these fluorescent fusion proteins to study protein-protein interactions by fluorescence lifetime of ecfp. the decrease of ecfp lifetime reveals fret between ecfp and eyfp and hence the interaction between proteins in question. groel-groes chaperonin complex is required for the proper folding of eschericia coli proteins. bacteriophage t and its distant relative coliphage rb encode co-chaperon proteins (respectively gp and coco) that can replace groes in the chaperonin complex. gp is also required in the folding of the major capsid protein of the phage. prd is a large membrane-containing bacteriophage infecting gram-negative bacteria such as e. coli and salmonella enterica. it has kb long linear dsdna genome and the capsid has an icosahedral symmetry. the groel-groes chaperonin complex is needed in the assembly of prd . we have found evidence that prd protein p can work similar way as other viral co-chaperones and substitute groes in chaperonin complex. fluorescence lifetime studies between proteins groel and p reveals an interaction that backs up the theory. structural modification of model membranes by fibrillar lysozyme as reaveled by fluorescence study a.p. kastorna v.n. karazin kharkiv national university, svobody sq., kharkiv, , ukraine recent experimental findings suggest that protein aggregation, leading to the formation and depositions of amyloids play a central role in the neurodegenerative diseases, type ii diabetes, systemic amyloidosis, etc. in the present study we focused our efforts on investigation of the influence of fibrillar lysozyme on the structural state of model lipid membranes composed of phosphatidylcholine and its mixtures with cardiolipin ( mol %) and cholesterol ( mol %). to achieve this purpose, two fluorescent probes with different bilayer location, , -diphenyl- , , -hexatriene (dph) distributing in membrane hydrocarbon core and -lauroyl- -dimethylaminonaphthalene (laurdan) locating at lipid-water interface, have been employed. the changes in membrane viscosity under the influence of amyloid lysozyme were characterized by fluorescence anisotropy of dph. this fluorescence parameter was not markedly affected by fibrillar protein in all types of model membranes. the changes in emission spectra of laurdan were analysed by the generalized polarization value (gp). it was found that adding of amyloid lysozyme resulted in the increment of gp value. our data suggest that lysozyme fibrils cause reduction of bilayer polarity and increase of lipid packing density. isothermal titration calorimetry (itc) is the gold standard for the quantitative characterisation of protein-ligand and protein-protein interactions. however, reliable determination of the dissociation constant (k d ) is typically limited to the range lm [ k d [ nm. nevertheless, interactions characterised by a higher or lower k d can be assessed indirectly, provided that a suitable competitive ligand is available whose k d falls within the directly accessible window. unfortunately, the established competitive itc assay requires that the high-affinity ligand be soluble at high concentrations in aqueous buffer containing only minimal amounts of organic solvent. this poses serious problems when studying protein binding of small-molecule ligands taken from compound libraries dissolved in organic solvents, as is usually the case during screening or drug development. here we introduce a new itc competition assay that overcomes this limitation, thus allowing for a precise thermodynamic description of highand low-affinity protein-ligand interactions involving poorly water-soluble compounds. we discuss the theoretical background of the approach and demonstrate some practical applications using examples of both high-and low-affinity protein-ligand interactions. interaction of myoglobin with oxidized polystyrene surfaces studied using rotating particles probe m. kemper , , d. spridon , l.j. van ijzendoorn , m.w.j. prins , eindhoven university of technology, department of applied physics, eindhoven, the netherlands, dutch polymer institute, eindhoven, the netherlands, philips research, eindhoven, the netherlands the interaction of proteins with polymer surfaces is of profound importance for the sensitivity of biosensors. polymer surfaces are often treated in order to tune their chemical and physical properties, for example by oxidation processes. to get a better understanding of the association of proteins to treated polymer surfaces, we use the rotating particles probe (x.j.a. janssen et al., colloids and surfaces a, vol. , p. , ). in this novel technique, protein coated magnetic particles are in contact with a substrate and the binding is recorded for all individual particles using a rotating magnetic field. we investigate the interaction of myoglobin coated magnetic particles to spincoated polystyrene surfaces that have been oxidized with a uv/ozone treatment. the surfaces have been characterized by xps, afm and water contact angle measurements. we will demonstrate a clear influence of polystyrene oxidation on the binding fractions of the myoglobin coated particles. we interpret the results in terms of dlvo-theory: electrostatic as well as electrodynamic properties of the surfaces will be influenced by the oxidation. interact with monomeric and/or filamentous actins. twinfilin is a - kda protein composed of two adf-homologue domains connected by a short linker. in our work we studied the effects of the mouse twinfilin- (twf ) on the monomeric actin. we determined the affinity of twf to the atp-actin monomer with fluorescence anisotropy measurement (k d = . lm). the fluorescence of the actin bound e-atp was quenched with acrylamide in the presence or absence of twf . in the presence of twinfilin the accessibility of the bound e-atp decreased, which indicates that the nucleotide binding cleft is shifted to a more closed conformational state. it was confirmed with stopped-flow experiments that the kinetics of nucleotide-exchange of actin decreased in the presence of twf . we determined the thermodynamic properties of twf and investigated the effect of twinfilin on the stability of actin monomer with differential scanning calorimetry. the twf stabilized the stucture of the g-actin. our results can help us to understand the regulation of actin cytoskeleton in more details. magnetic np have attracted attention due to their potential of contrast enhancement of magnetic resonance imaging and targeted drug delivery, e.g. tumor magnetic hyperthermia therapy. potential nephrotoxicity of single i.v. administration of fe o np was studied in female wistar rats i.v. administered either placebo ( % v/v rat serum in . % nacl), suspension of tio np (positive control, bimodal / nm distribution), or fe o np (bimodal / nm distribution) in doses of . , . or . mg/kg. rats were sacrificed h, -, -and -days after np injection (n= - /each group). administration of np did not alter kidney size significantly; renal function of np administered rats as monitored by plasma creatinine and urea concentrations, creatinine clearance and protein excretion rate did not differ significantly in either interval from rats administered placebo. one week after administration significant rise in plasma ca, its urinary and fractional excretion was observed in rats administered mg fe o /kg. plasma mg levels rose in this group and weeks after administration. no significant changes in the expression of tnf-a, tgf-b, and collagen iv genes in renal cortex were revealed. no obvious nephrotoxic effects were observed in rats after a single i.v. dose of fe o np. study was supported by fp ec eu: nanotest (development of methodology for alternative testing strategies for the assessment of the toxicological profile of nanoparticles used in medical diagnostics.), grant no.: . biomimetic supramolecular assemblies for studying membrane interactions in vitro and in vivo s. kolusheva, r. jelinek ben-gurion university, beer-sheva, israel we designed a novel biomimetic sensor, composed of conjugated polydiacetylene (pda) matrix embedded within lipid vesicles. the system is capable of detecting various compounds occurring within lipid membranes through rapid colorimetric as well as fluorescent transitions. the colorimetric response of the sensor is correlated to the extent of compound-membrane binding and permeation and quantified binding sensitivity to lipid composition. we describe a new disease diagnostic approach, denoted ''reactomics'', based upon reactions between blood sera and an array of vesicles comprising different lipids and polydiacetylene (pda), a chromatic polymer. we show that reactions between sera and such a lipid/pda vesicle array produce chromatic patterns which depend both upon the sera composition as well as the specific lipid constituents within the vesicles. through attachment of chromatic polydiacetylene (pda) nanopatches onto the plasma membrane, real-time visualization of surface processes in living cells is possible. the ras protein is mutated in % of human tumors. ras acts as a switch, transmitting a growth signal in an active gtp-bound form and turning the signal off in an inactive gdp-bound form. the switch off is accomplished by gtp hydrolysis, which is catalyzed by ras and can be further accelerated by gtpase activating proteins (gaps). mutations which prevent hydrolysis cause severe diseases including cancer. we investigate the reaction of the ras gap protein-protein complex by time-resolved ftir spectroscopy. detailed information on the mechanism and the thermodynamics of the reaction was revealed: first, the catalytic arginine-finger of gap has to move into the gtp binding pocket, then cleavage of gtp is fast and h po hydrogen-bonded in an eclipsed conformation to the b-phosphate of gdp is formed. further, we performed for the first time atr-ftir spectroscopy of ras in its native environment, a lipid membrane. in this setup we are able to do difference spectroscopy of the immobilized protein. interactions with other proteins can be determined in a similar way as in spr experiments but with the additional information from the infrared spectra. galectins are a family of animal lectins that specifically bind b-galactosides and have gained much attention due to their involvement in several biologic processes such as inflammation, cell adhesion and metastasis. in such processes, several issues are still not clear including the mechanisms of interaction with different carbohydrates. galectin- (gal- ) is a tandem-repeat type galectin that contains two carbohydrate recognition domains (crd-i and crd-ii) connected by a linking peptide. in this study, we performed spectroscopic studies of the carbohydrate-recognition domains from human gal- . our goals are two-fold: ( ) to monitor conformational changes in each domain upon its binding to specific ligands and then to correlate the observed changes with structural differences between the crds and ( ) to investigate the interaction between the crds and lipid model membranes. to achieve such objectives we used a combined approach of spectroscopic techniques involving circular dichroism and electron spin resonance. overall the results obtained so far show that crd-i and crd-ii have distinct behaviors in terms of carbohydrate recognition and membrane binding. this may be due to specific differences in their structures and certainly suggests a non-equivalent role in protein function. hemoglobin influence on lipid bilayer structure as revealed by fluorescence probe study o.k. kutsenko, g.p. gorbenko, v.m. trusova v.n. karazin kharkov national university, kharkov, ukraine hemoglobin (hb) is a red blood cell protein responsible for the oxygen transport. its affinity for lipid bilayers represents interest for gaining insight into protein biological function as well as for some applied aspects such as development of blood substitutes or biosensors. hb influence on lipid bilayer structure was investigated using fluorescent probes pyrene and prodan. model membranes were prepared of phosphatidylcholine (pc) and its mixtures with phosphatidylglycerol (pg) and cholesterol (chol). hb penetration into membrane interior is followed by the increase of relative intensity of pyrene vibronic bands and decrease of prodan general polarization value suggesting an enhancement of bilayer polarity. this implies that hb incorporation into membrane interior decreases packing density of lipid molecules, promoting water penetration into membrane core. chol condensing effect on lipid bilayer prevents protein embedment into bilayer, thus decreasing membrane hydration changes as compared to pc bilayers. in the presence of anionic lipid pg hb-induced increase of bilayer polarity was found to be most pronounced, pointing to the modulatory role of membrane composition in hb bilayer-modifying propensity. we present optimized sialic acid-based mimics binding in the low nanomolar range. molecular interactions were determined with surface plasmon resonance (spr), characterizing the affinity and the kinetics of binding. furthermore, isothermal titration calorimetry (itc) was applied to dissect the standard free energy of binding (dg°) into the standard enthalpy of binding (dh°) and the standard entropy of binding (ds°). in order to pass the cell membranes, most of these medicines has to be administrated to patients as nucleoside pro-drugs and not directly as triphosphorylated forms. because of the poor phosphorylation of the nucleoside analogues used in therapy, it is important to understand and to optimize their metabolism. our aim is to understand how compounds of chirality l turn away -phosphoglycerate kinase (pgk) from its normal glycolytic function to be converted into the triphosphate forms. in order to elucidate pgk mechanism and substrate specificity, we have measured the kinetics of the different steps of the enzymatic pathways by rapid mixing techniques and studied the influence of the nature of the nucleotide substrate thereon. we first performed an extensive study with d-and l-adp (see poster by p. lallemand). we are now extending the studies to other nucleotide diphosphates (some of them used in therapies). changes in the nature of the nucleobase or deletion of hydroxyl group of the sugar affect the efficiency of phosphorylation by pgk, either by decreasing dramatically their affinity or by altering the phospho-transfer step itself. structural explanations are given based on docking data. probing drug/lipid interactions at the molecular level represents an important challenge in pharmaceutical research, drug discovery and membrane biophysics. previous studies showed that enrofloxacin metalloantibiotic has potential as an antimicrobial agent candidate, since it exhibits antimicrobial effect comparable to that of free enrofloxacin but a different translocation route. these differences in uptake mechanism can be paramount in counteracting bacterial resistance. in view of lipids role in bacterial drug uptake, the interaction of these compounds with different e. coli model membranes were studied by fluorescence spectroscopy. partition coefficients determined showed that lipid/antibiotic interactions were sensitive to liposomes composition and that the metalloantibiotic had a higher partition than free enrofloxacin. these results corroborate the different mechanism of entry proposed and can be rationalized on the basis that an electrostatic interaction between the metalloantibiotic positively charged species, present at physiological ph, and the lipids negatively charged head groups clearly promotes the lipid/antimicrobial association. oligomerization and fibril assembly of amyloid b peptide amyloid b peptide (ab) forms a large amount of extracellular deposits in the brain of alzheimer's disease (ad) patients and it is believed that this peptide is related to the pathogenesis of that disease. the most abundant monomeric form of physiological ab (* %) is constituted by amino acids and is benign, but by an unknown mechanism this endogenous material becomes aggregated and neurotoxic. increasing evidence suggests that membrane interaction plays an important role in ab neurotoxicity. in this work it will be studied the interactions of ab( - ) with ctac (cationic), sds (anionic), pfoa (anionic with fluorine atoms) and og (nonionic) amphiphiles in monomeric and micellar forms. the results demonstrated that ab( - ) forms fibrils with different morphologies in the presence of micelles. in addition, the presence of micelles accelerates the formation of fibrils and decreases the lifetime of oligomers. we present here the exploitation of the powerful approach of surface plasmon resonance imaging and mass spectrometry coupling for protein fishing in biological fluids such as human plasma at the same sensitivity. on one hand, multiplex format spri analysis allows direct visualization and thermodynamic analysis of molecular avidity, and is advantageously used for ligand-fishing of captured bio-molecules on multiple immobilized receptors on a spri-biochip surface. on the other hand, maldi mass spectrometry is a powerful tool for identification and characterization of molecules captured on specific surface. therefore, the combination of spri and ms into one concerted procedure, using a unique dedicated surface, is of a great interest for functional and structural analysis at low femtomole level of bound molecules. to reach these goals, particular surface engineering has been engaged to maintain a high level of antibody grafting and reduce non-specific adsorption. thus, various chemistries have been tested and validated towards biological fluids such plasma, keeping in mind the capacity of the in situ investigation by ms. finally, signal to noise ratio was magnified leading to the characterization of protein lag , a potential marker of breast cancer, in human plasma. atenolol incorporation into pnipa nanoparticles investigated by isothermal titration calorimetry mihaela mic, ioan turcu, izabell craciunescu, rodica turcu, national institute for r&d of isotopic and molecular technologies, cluj-napoca, romania e-mail: mihaela.mic@itim-cj.ro poly(n-isopropylacrylamide) (pnipa) is a thermo-sensitive hydrogel undergoing a volume phase transition at about of °c close to the body temperature. this volume phase transition is envisaged as a key property for drug binding and release. the purpose of our research is the thermodynamic characterization of the binding of atenolol by pnipa polymeric nanoparticles. the thermodynamic parameters which characterize the binding process are obtained using the isothermal titration calorimetry (itc) as the main investigation technique. when polymeric nanoparticles bind drug molecules, heat is either generated or absorbed depending on the amount of bond molecules and also on the exothermic or endothermic character of the binding process. the heat measurement allows the determination of binding constants, reaction stoichiometry and the thermodynamic profile of the interaction. itc technique has been used to investigate the binding properties of nanoparticles which shrink from the swollen to the collapsed state. the capacity of such nanogels to bind atenolol molecules is directly related to relevant differences between the binding properties in the swollen and in the collapsed state respectively. aggregation study of x-(alkyldimethylamonium)alkylaldonamide bromides p. misiak , b. ró _ zycka-roszak , e. woźniak , r. skrzela , k.a. wilk department physics and biophysics, wrocław university of environmental and life sciences, wrocław, poland, department of chemistry, wrocław university of technology, wrocław, poland sugar-based surfactants are of considerable research interest because they have improved surface and performance properties, reduced environmental impact, and have potential pharmaceutical and biomedical applications. x-(alkyldimethylamonium)alkylaldonamide bromides (c n gab) with different chain lenghs (n = , , , ) belonging to cationic sugar-based surfactants were newly synthesised. the aggregation processes of c n gabs were studied by means of isothermal titration calorimetry (itc), electric conductance method and molecular modelling methods. the critical micelle concentrations (cmc), the degree of micelle ionization (b), the enthalpies (dh m ) and the entropies (ds m ) of micellization as well as the contributions of the headgroups to the gibbs free energies (dg m (hy)) were calculated. the obtained values were compared with those for dodecyldimethylethylammonium bromide and literature data for analogical glucocationic surfactants. the latest compounds differ from c n gab surfactants by substitution of sugar chain by gluco ring. molecular modelling methods were used to relate the molecular properties of the compounds with their experimentally studied properties in solution. this work was supported by grant n n . every year over million people are infected with dengue virus (denv), transmitted by a mosquito (aedes aegypti). this enveloped virus, member of the flaviviridae family, has four distinct serotypes. it has a single stranded positive rna molecule with a single open reading frame that encodes a single poliprotein, which, after appropriate processing by viral and host proteases, gives rise to three structural proteins (c, prm and e) and seven non-structural proteins (ns , ns a, ns b, ns , ns a, ns b and ns ) [ ] . the surface of the immature virion is composed of e and prm heterodimers that are arranged as trimers protruding from the membrane [ ] . the virus is thought to enter the host cell via a receptor-mediated endocytosis, although, if any, the specific dengue receptors have not been described. once inside the cell, the acidified environment inside the endocytic vesicle triggers an irreversible trimerization of the envelope (e) protein, inducing the release of the nucleocapsid (composed of viral rna and multiple copies of c protein) to the cytoplasm, thus starting the infection process, where the poliprotein is translated and processed, originating all viral proteins. considering the structural proteins c and e, these are essential for the viral infection, specifically, protein c is thought to be involved in the viral assembly and specific encapsidation of the genome and protein e (a class ii fusion protein) plays a major role in the fusion process. as recently described by some studies [ ] , protein c is composed of four a helices connected by four short loops and has a highly hydrophobic region forming a concave groove that could interact with lipid membranes and a region with an increased concentration of positive charges, possibly interacting with the viral rna. as for protein e, it is composed of three b stranded domains. it is proposed that the fusion loop is located in domain ii of this protein and the putative receptor binding sites, considered essential for the viral entry, are supposedly located in domain iii. in this work, we describe the identification of the membrane active regions of both these proteins, considering both theoretical studies, hydrophobic moments, hydrophobicity and interfaciality values as well as experimental ones, namely fluorescence spectroscopy, where a fluorescent probe is encapsulated in model membrane systems, and differential scanning calorimetry [ ] . we have found one region in protein c and four regions in protein e with membranotropic activity. this is the first work describing experimentally the putative membrane interacting zones of both these proteins. this work was funded by grant bfu - -bm from ministerio de ciencia y tecnologia, spain, granted to jose villalaín. investigation of membrane-membrane interaction mediated by coiled coil lipopeptides gesa pä hler, andreas janshoff georg-august-university, tammannstrasse , gö ttingen, germany e-mail: gpaehle@gwdg.de specific cellular membrane interaction and fusion are crucial points in vivo which are in eukaryotic cells mediated by snare proteins. the definite mechanism behind these processes is still poorly understood, but the coiled coil formation of a snare core complex consisting of four a-helices seems to generate a fusogenic driving force. this offers the possibility to design a straightforward experimental setup to mimic the complex protein-mediated membrane-membrane interaction by using mere protein fragments or peptides attached to artificial lipid bilayers which self-assemble to a coiled coil structure. in our approach, two artificial three heptad repeat coiled coil forming peptides were synthesized and attached to maleimide functionalized membranes via an in situ-coupling reaction. thus, secondary structure changes, kinetic characteristics and binding energetics were monitored during coiled coil formation with real time ellipsometry, ir and cd spectroscopy. the lipopeptide mediated membrane-membrane interaction itself is investigated by colloidal probe spectroscopy and tirfm. these techniques and the setup of our model system allow screening the energetic and structural properties of variable coiled coil forming peptides, i.e. linker-modified or biologically inspired sequences. enzymatic reactions in nanostructured surfaces: unzipping and cutting the double helix pietro parisse , matteo castronovo , bojana lucic , alessandro vindigni , giacinto scoles and loredana casalis sincrotrone trieste s.c.p.a., trieste, italy, temple university, philadelphia, usa, protein-dna interactions are vital for living organisms. from viruses to plants and humans, the interactions of these two different classes of biopolymers control processes as important and diverse as the expression of genes and the replication, rearrangement, and repair of dna itself. to understand these processes at the molecular level, and to follow changes in cellular pathways due to different kinds of perturbations and/or diseases it is necessary the identification and quantification of proteins and their complex network of interactions. we have exploited the high spatial resolution given by atomic force microscopy to generate dna arrays of variable density by means of nanografting. on such nanostructures, we investigate the mechanism of different enzymatic reactions (from restriction enzymes to helicases). registering with high precision the height variation due to the action of the enzyme onto the engineered dna sequences (in the case of restriction enzymes) or taking advantage of the different mechanical properties of single and double stranded dna (in the case of helicases, where for the first time kinetic data were obtained on recq human helicase), we were able to monitor either the activity and/or the action mechanisms of these two important classes of enzymes. in this study an attempt has been made to investigate the structure, dynamics and stability of cyclic peptide nanotubes (cpnts) formed by the self-assembly of cyclic peptides (cps) using classical molecular dynamics (md) simulation and semiempirical quantum chemistry calculation employing pm . the structure and energetics of monomer and various oligomeric cpnts have been investigated by considering the (cyclo-[(d-ala-l-ala) ]) peptide as the model for cp. various geometrical parameters extracted from the md simulation reveal that the terminal residues are loosely hydrogen bonded to the inner subunits regardless of degree of oligomerization. the hydrogen bonds present in the inner core regions are stronger than the terminal residues. as the degree of oligomerization increases, the stability of the tube increases due to the hydrogen bonding and stacking interactions between the subunits. the results show that the binding free energy increases with the extent of oligomerization and reaches saturation beyond cpnt . in addition, hydrophobic and electrostatic interactions play crucial roles in the formation of cpnts. analysis of both structural and energetics of formation of cpnts unveils that the selfassembly of dimer, trimer and tetramer cpnts are the essential steps in the growth of cnpts. monolayers on a langmuir trough constitute a great biomimetic model to characterize protein-protein or protein-lipid interaction, where the physical state of the interfacial layer is completely controlled. we present here three studies performed on monolayers, with a wide panel of experimental (optical, spectroscopical, rheological) techniques. i) surface properties and conformation of nephila clavipes spider recombinant silk proteins (maspi and masp ) was studied at the air-water interface: we show that the mechanism of assembly of both proteins is different, although both proteins share the same sequence pattern and a close hydrophobicity. they both exhibit a certain propensity to form b-sheets that may be important for the efficiency of the natural spinning process. ii) the dystrophin molecular organization and its anchoring in a lipidic environment depend on the rod fragment used and on the lipid nature. moreover the interaction is guided by the lateral surface pressure. this lipid packing variation is essential to understand the role of the dystrophin during compression-extension cycle of the muscle membrane. iii) we evidence that non additive behavior of mixtures of food globular proteins leads to enhanced foaming properties or to self assembled objects. nucleolar-targeting peptides (nrtps) were designed by structural dissection of crotamine, a toxin from the venom of a south-american rattlesnake. at lm concentration, nrtps penetrate different cell types and exhibit exquisite nucleolar localization. the aim of this work was to pursue with the study of nrtps molecular mechanism for translocation, as well as to determine the ability of nrtp to delivery large molecules into cells. for the translocation experiments, rhodamine b-labeled nrtps were used and tested with giant multilamellar vesicles. confocal microscopy results show that there is an efficient translocation across model membranes. high levels of intracellular peptide were also seen in different cell lines and pbmc, soon after incubation with nrtp. furthermore, a conjugate of nrtp (nrtp c) bound to b-galactosidase was prepared by chemical synthesis and tested in hela cells. this conjugate maintains enzymatic activity and is stable at °c for several days. the work done so far with this new family of cell-penetrating peptides revealed strong interaction and translocation with lipid model systems. moreover, successfully cellular delivery of bgalactosidase was observed and quantified. interaction of zinc phthalocyanine with ionic and non ionic surfactants: uv-vis absorption and fluorescence spectroscopy for application in photodynamic therapy m. p. romero, s. r. w. louro physic department, pontifícia universidade cató lica de rio de janeiro puc-rio, brazil among the second-generation photosensitizer (ps) developed for the treatment of neoplastic diseases by photodynamic therapy (pdt), metallo-phthalocyanines (mpc) have been proposed as an alternative to the currently used ps in clinical application. unsubstituted mpc are not soluble in physiological solvents and their in vivo administration relies upon their incorporation into carriers or their chemical conversion into water-soluble dyes by the attachment of selected substituents. in this work, uv-vis absorption and fluorescence spectroscopy were used to study the ability of different micelles for dispersing zinc phthalocyanine (znpc). the following surfactants were tested: sds, ctab, hps, tween , tween , and pluronic f . znpc has low solubility in virtually all solvents, but dmf and dmso are observed to dissolve znpc in concentrations of the order of . and . mmol/l, respectively. stock solutions of znpc in dmf and dmso were prepared. micelles of the different surfactants containing znpc were prepared by dissolving in aqueous medium (milli-q water or phosphate buffer ph . ) small amounts of the stock solutions previously mixed with each surfactant. the amounts of each surfactant were calculated to give an average ratio of one znpc molecule per micelle in the final solution. the absorption and fluorescence spectra of znpc in the micellar systems were obtained, and were observed to change in time. immediately after dissolution the spectra are characteristic of monomeric znpc, suggesting formation znpccontaining nanoemulsions with the mixture of znpc-organic solvent in the hydrophobic region of the micelle. since dmso and dmf are miscible with water, the solvent diffuses out of the micelle and znpc stays inside the micelle in a monomeric or aggregated form. the different surfactants lead to different time evolution of znpc aggregation. aggregation lifetimes vary from one hour, in the case of pluronic f , to more than twelve hours, in the case of ctab and hps. it was observed that the ionic surfactants were more efficient than non ionic ones for monomeric delivery of znpc . work partially supported by cnpq, inami and faperj. nucleobase-containing peptides are an important class of molecules comprising both artificial (synthetic nucleopeptides) and natural (peptidyl nucleosides and willardiine-containing peptides) compounds characterized in many cases by interesting biological properties. , in this work, we report a spectroscopic study on the properties of a chiral nucleobase-bearing peptide obtained by chemical synthesis starting from commercial sources. the findings of this research strongly encourage further efforts in the field of the use of nucleopeptides as supramolecular assembling systems and open the way to novel drug delivery approaches based on nucleobase recognition. conformational plasticity. their structure depends tremendously on their local environment and confinment, and may accommodate several unrelated conformations, that are a strong challenge for the traditional characterizations of structure, supramolecular assembly and biorecognition phenomena. atomic force microscopy (afm) has been successfully exploited for both highly controllable nanolithography of biomolecules and for biorecognition studies, such as oriented prion protein -antibody interaction (sanavio et al., acs nano ( ) ( ): , bano et al. nano lett ( ) ( ): - ). here, we report different strategies for selective, oriented confinement of alphasynuclein at the nanoscale for sensitive and accurate direct detection, via precise topographic measurements on ultraflat surfaces, of biomolecular interactions in confined assemblies. a new class of cell penetrating peptides (cpps) was generated by splicing the ( - ) and ( - ) segments of crotamine, a toxin from crotalus durissus terrificus venom [ ] . as they localize preferably on the nucleolus, these novel cpps were named nucleolar-targeting peptides (nrtps). the extent of nrtp partition to zwitterionic (popc; popc:cholesterol : ) and anionic (popg; popc:popg : ) lipid vesicles was studied following the intrinsic tyr or trp fluorescence of the peptides. the partition curves into popc zwitterionic vesicles were characterized by downward slopes and higher partition coefficients (k p * - ). for pure popg, an upward curve and smaller partition coefficient point out for a different type of membrane-peptide interaction. popc:popg membranes present characteristics of both types of interaction. from red edge excitation shift and quenching experiments similar conclusions were attained. leakage assays ruled out lipid vesicle disruption by crotamine or nrtps. further studies on nrtp cellular translocation mechanism and large molecule delivery are currently in progress. dystrophin is essential to skeletal muscle function and confers resistance to the sarcolemma by interacting with cytoskeleton and membrane. we characterized the behaviour of dys r - , a five spectrin-like repeats from the central domain of human dystrophin, in the presence of liposomes and monolayers as membrane models. interaction of dys r - depends on the lipid nature, anionic or zwitterionic, with suvs, and on the lipid packing when comparing luvs to suvs. lateral pressure of lipid monolayers modifies the protein organization and leads dys r - to form a regular network as revealed by afm. trypsin proteolysis assays show that the protein conformation is modified following its binding to monolayer and suvs. label free quantification by nano-lc/ms-ms allowed identifying the helical amino acid sequences in repeats and that are involved in the interaction with anionic suvs. results indicate that dys r - constitutes a part of dystrophin that interacts with anionic as well as zwitterionic lipids and adapts its interaction and organization depending on lipid-packing and lipid nature. we provide here strong experimental evidence for a physiological role of the central domain of dystrophin on sarcolemma scaffolding through modulation of lipid-protein interactions. extracellular matrix proteins. overexpression of the mmps has been associated with a variety of diseases ranging from periodontal disease and arthritis to tumor invasion and metastasis. the majority of the more powerful synthetic inhibitors of mmps incorporate a hydroxamate group, but exhibit low selectivity and are toxic. in a recent modeling study, astaxanthin (ast), a carotenoid with potent antioxidant property, has been shown to be a potential inhibitor of mmp- function by occupying a binding site near the active center of the enzyme (bika di et al. ). in our ongoing project, we investigate the binding of ast to the catalytic domain of mmps using biochemical and ultimately crystallization to validate the proposed action of ast. along these lines, the catalytic domain of mmp- (cdmmp- ) was expressed in e.coli bl (de ) codon-plus and refolded using a novel effective refolding method. our results reveal that ast has a potent inhibitory effect on cdmmp- activity, however, determination of ic % or k i is difficult due to fast oxidation and structural instability of ast. ongoing work aims at optimizing the inhibition conditions and improving the refolding yield to allow analyzing structure and function of the ast-bound mmp- in more detail. hyaluronic acid (hyaluronan, ha) is a linear polysaccharide with a molar mass in the range of to da and is built from alternating units of glucuronic acid and n-acetylglucosamine. synthesized in the cellular plasma membrane, it is a network-forming and space-filling component in the extracellular matrix of animal tissues. here, we create hyaluronic acid films atop a porous alumina substrate, where they act as a barrier for macromolecular transport depending on their length and geometry. the geometry of the hyaluronic acid switches between a fully stretched and a mushroomlike state and is dependent on the concentration of hyaluronic acid. to bind hyaluronic acid selectively atop the nanoporous anodic aluminum oxide (aao), the aao is orthogonally functionalized by silane chemistry. by means of time resolved optical waveguide spectroscopy (ows) the transport of macromolecules, e.g. avidin, across the hyaluronic acid barriers can be recorded as a function of the pore diameter and hyaluronic acid concentration in a time resolved and label free manner. confocal laser scanning microscopy (clsm) provides an alternative method to investigate the orthogonal functionalization of the pores and to elucidate whether a molecule can cross the barrier at the pore entrance. we functionalized gold surfaces with a hydroxy-terminated self-assembled thiol monolayer exposing an adjustable fraction of biotin moieties. [ ] by in-situ acetylation or fluorination, surface properties could be fine-tuned to different protein immobilization scenarios. using streptavidin as a linker protein, immobilization of human abcc [ ] in liposomal and planar bilayer systems was possible. abcc -containing proteoliposomes doped with a biotinylated anchor lipid were successfully tethered to our streptavidin-coated surfaces. biotinylation of the extracellular glycosylation of abcc allowed direct immobilization with inside-up orientation and subsequent assembly of a lipid bilayer. outside-up orientation was achieved by exploiting the c-terminal histidin tag of recombinant abcc for immobilization via ni + and biotinnitrilotriacetate. all systems were thoroughly characterized by quartz crystal microbalance, atomic force microscopy and surface plasmon resonance techniques with respect to monitoring abcc mediated substrate transport in real time. because of its role in the apoptotic pathway, conformational transitions of cytochrome c (cyt c) have gain on interest. in native cyt c, met and his residues serve as heme axial ligands. cyt c interaction with the membrane causes the disruption of the iron-met bond. this allows the binding of others endogenous ligands forming alternative low spin species , or induces peroxidase activity through the formation of a five coordinated high spin iron specie. acquisition of this peroxydase activity by cyt c has been shown to be a key stage before its release from the mitochondria . in order to study these non native low spin species by checking the possible amino acids able to bind the human cyt c heme, different mutants have been designed and produced: h q, h n, and the double one h q/h n. sds in countries where seafood is an integrate part of the diet, fish are among the most common food allergen sources. the major fish allergen parvalbumins are abundant in the white muscle of many fish species. parvalbumin belongs to the family of ef-hand proteins and has a globular shape containing six helical parts. high pressure is known to unfold proteins. we performed high pressure ftir experiments, to explore the p-t phase diagram of cod parvalbumin, gad m , to test the possibility of its inactivation by high pressure treatment. the infrared spectrum of parvalbumin is characteristic for the helical conformation, in agreement with the crystal structure. a marked transition in the structure of the parvalbumin was observed with the central point of . gpa (at room temperature). the amide i position shifts to a wavenumber which is between the helical and the unfolded position. we assign this change to a native-molten globule transition. it was reversible as seen from the infrared spectra. it has been proven in the past that reflectometric interference spectroscopy (rifs) is a powerful measurement system for the detection of protein-protein interactions . we present here the development of a reflectometric sensor which allows for the detection of membrane-protein interactions in the micromolar regime. in this study we employ two different instrumental assemblies. the first installation enables direct detection and quantification of the interaction of membrane proteins with solid supported lipid bilayers. in the second assembly the original instrument is combined with an upright fluorescence microscope. the advantages of this installation are the direct optical control of the experiment as well as a smaller sensing area. the set-up allows for the detection of interactions on lipid-patches of just several micrometers in diameter. the aim of this work is an experimental system that enables the measurement of transport processes through lipid membranes. we attempt to achieve this by covering a closed porous substrate with a lipid membrane. the first steps to reach this goal were done by spanning membranes over anodized aluminum oxide substrates. initiation of actin polimerization in cells requires nucleation factors. a pointed-end-binding protein of f-actin -the lei-omodin -acts as a strong filament nucleator in muscle cells. the dynamical, structural and kinetic properties of a protein can provide important information to understand the intramolecular events underlying its function. we are interested in how does the leiomodin regulate the actin polimerization. our aim is to determine the dissociation constant of the actin-leiomodin complex, and study a possible side-binding effect of the leiomodin . the cardiac leiomodin of rattus norvegicus is a kda molecular weight protein, which contains a kda n-terminal, a kda leucin reach repeat (lrr) and a kda c-terminal region. the n-term and the lrr regions are together tropomodulin homologues. we expressed the wild type the n-term+lrr the lrr+c-term and the c-term protein fragments by using a ptyb vector that contains an amplicillin resistance gene. the expression of the proteins was carried out with the twin-cn (ne bio-labs) kit, which is a chitin-intein self-cleavage and purification system. the nucleation activity of leiomodin and the polimerization speed of actin in the presence of tropomyosin and leiomodin were studied by using pyrene-actin polimerization assay. we measured the stoichiometric, conformational and kinetic properties of the leiomodin-actin complexes with co-sedimentation assay, fluorescence spectroscopic and rapid-kinetic methods. the results showed that the rate of actin polimerization depended on the leiomodin concentration. the nucleator activity of leiomo-din was ionic strength dependent. the data also confirmed that leiomodin is a side-binding and pointed-end binding protein of f-actin. the binding of leiomodin to the sides of the actin filaments was slower than to the pointed-end of the f-actin. the structure of f-actin was changed by the sidebound leiomodin . these observations will contribute to the better understanding of the development and function of thin filaments in cardiac and other muscle tissues. leukemias are one of the most common malignancy worldwide. there is a substantial need for new chemotherapeutic drugs effective against these diseases. doxorubicin (dox), used for treatment of leukemias and solid tumors is poorly efficacious when administered systemically at conventional doses. therefore, in our study, to overcome these limitations, we used transferrin (trf) as a drug carrier. we compared the effect of dox and doxorubicin-transferrin conjugate (dox-trf) on human leukemic lymphoblasts (ccrf-cem). the in vitro growth-inhibition test, xtt assay, indicated that dox-trf was more cytotoxic for leukemia cells than dox alone. in our researches we also evaluated the alternations of mitochondrial transmembrane potential (dw m) , and production of reactive oxygen species (ros). we monitored the dw m using dye jc- ( , ', , '-tetrachloro- , ', , '-tetraethylbenzimidazolcarbocyanine) . the level of ros was studied using the fluorescent probe dcfh -da ( ', 'dichlorodihydrofluorescein diacetate). the results demonstrate that dox-trf induced, decrease of mitochondrial membrane potential and significantly higher production of ros compared with dox treated cells. moreover, all these results seem to be correlated with dna fragmentation analyzed by dna ladder. the tested processes were partially inhibited by the antioxidant, n -acetylocysteine (nac). the changes induced by dox-trf conjugate and free drug were suggest the different mechanism of action of dox alone and conjugated with transferrin. time-resolved detection of protein-protein interaction masahide terazima kyoto university, kyoto, - , japan revealing molecular mechanism of a protein reaction has been a central issue in biophysics. for that purpose, a variety of time-resolved spectroscopic methods have been developed. however, most of them can monitor only dynamics associated with an optical transition and it has been very difficult to trace processes without optical transition. we used the pulsed laser induced transient grating (tg) method to study spectrally silent reactions of various proteins in time-domain. here we will show studies on pixd. pixd is a kda short protein which consists of the bluf domain and additional short helices, and is involved in phototactic movement. the photochemical reaction studied by absorption spectroscopy revealed that this protein exhibits typical photochemistry of the bluf proteins. the red-shifted intermediate is generated within a ps. the spectrum does not change after this initial reaction, and returns back to the dark state with a time constant of s at room temperature. we studied the reaction of this protein by our method and found that the proteinprotein interaction is drastically changed during the reaction. the details and the biological meaning will be presented. human ileal bile acid-binding protein (i-babp) plays a key role in the enterohepatic circulation of bile salts. previously we have shown that the protein has two binding sites and triand dihydroxy bile salts bind with strong and moderate positive cooperativity, respectively. positive cooperativity is thought to be related to a slow conformational change in the protein. our current study is directed at the structural and dynamic aspects of molecular recognition in human i-babp using nmr spectroscopy and other biophysical techniques. as a first step in the investigation, n relaxation nmr experiments have been employed to characterize the backbone motion in the apo and holo protein on a wide range of timescales. our results show a moderately decreased ps-ns flexibility in the ligated protein, with most significant ordering near the portal region. in addition, the measurements indicate a slow ls-ms fluctuation at four distinct segments in the apo protein, a motion not observed in the doubly-ligated form at room temperature. our studies support the hypothesis of an allosteric mechanism of binding cooperativity in human i-babp. to shed more light on the molecular details of the binding mechanism, a site-directed mutagenesis study is in progress. cationic porphyrin-peptide conjugates were recently shown to enhance the delivery of peptide moiety to the close vicinity of nucleic acids but their interaction with dna is not yet studied. we synthesized two cationic porphyrin-peptide conjugates: tetra-peptides were linked to the tri-cationic meso-tri( -n-methylpyridyl)-mono-( -carboxyphenyl)porphyrin and bi-cationic meso- , -bis( -n-methylpyridyl)- , di-( -carboxyphenyl)porphyrin. dna binding of porphyrins, and their peptide conjugates was investigated with comprehensive spectroscopic methods. evidences provided by the decomposition of absorption spectra, fluorescence decay components, fluorescence energy transfer and cd signals reveal that peptide conjugates of di-and tricationic porphyrins bind to dna by two distinct binding modes which can be identified as intercalation and external binding. the peptide moiety does not oppose the interaction between the dna and cationic porphyrins. we compared the effect of complexation on structural stability of dna and nucleoprotein complex : hela nucleosomes and t phage. uv and cd melting studies revealed that the porphyrin binding increases the melting temperature of dna and destabilizes the dna protein interaction in the nucleosomes but not in the t phage. the wide nucleotide specificity of -phosphoglycerate kinase (pgk) allows its contribution to the effective phosphorylation (activation) of nucleotide-based pro-drugs against hiv. here the structural basis of the nucleotide-pgk interaction is characterised in comparison to other kinases, namely pyruvate kinase (pk) and creatine kinase (ck) by enzyme kinetic and structural modelling studies. the results evidenced favouring the purine vs. pyrimidine base containing nucleotides for pgk rather than for pk or ck. this is due to the exceptional ability of pgk in forming the hydrophobic contacts of the nucleotide rings that assures the appropriate positioning of the connected phosphate chain for catalysis. the unnatural l-configurations of the nucleotides (both purine and pyrimidine) are better accepted by pgk than either by pk or ck. further, for the l-forms the absence of the ribose oh-groups with pgk is better tolerated for the nucleotides with purine rather than pyrimidine base. on the other hand, positioning the phosphate chain of both purines and pyrimidines with l-configuration is even more important for pgk, as deduced from the kinetic studies with various nucleotide-site mutants. these characteristics of the kinase-nucleotide interactions can provide a guideline in drug-design. the role of the enzyme types atp-ases in the muscle contraction g. vincze-tiszay , j. vincze , e. balogh hheif, budapest, hungary, nové zá mky, slovakia the myofibrilla assuring muscle contraction gains energy to the slipping in mechanisms and the degree of efficiency of this process will decisively be determined by the velocity of recombination of the atp molecule. in this there play a particular part the na + -k + -atp-ase and mg ++ -atp-ase enzymes. chemical reactions taking place in the living organism are catalyzed by enzymes, so the recombination from adp to atp, too. this transport process can be modelled from the energetic point of view on the basis of the general transport theorem through the following formula: grad a x dx dt: from the point of view of muscle contraction it is of interest that, dependent from the type of the motions whether the length of time is very short, some seconds, or we can speak about a long lasting process. in the first case one can compare the decomposition of the atp with the avalanche effect while in the spot. its degree of efficiency is determined by the migration and linkage velocity of the ions. conclusion: the degree of efficiency of the muscle contraction is determined by the quantities of the two enzymes (na + -k + -atp-ase and mg ++ -atp-ase) as related to each other. [ ] . experiments with deletion mutants have shown that the aminoterminal domain contains a beta sheet with an ordered array of acidic residues, which mediates the attachment to basic calcium phosphates [ , ] . the inhibition is based on the formation of nanometer-sized, spherical mineral-fetuin-a colloids, denoted as calciprotein particles (cpps) [ , ] . the initially formed cpps show hydrodynamic radii in the range of nm and are only transiently stable. after a distinct lag time, they are subject to a morphological change towards larger prolate ellipsoids with hydrodynamic radii of - nm [ ] . in this context, we studied the role of fetuin-a in the formation and ripening of cpps. on the one hand, dynamic light scattering (dls) was used to study the influence of temperature, fetuin-a concentration and mineral ion supersaturation on the kinetics of cpp ripening [ ] . on the other hand, the protein fetuin-a was investigated by means of small angle x-ray scattering (saxs) and fluorescence correlation spectroscopy (fcs degradation of the mrna cap (mrna ' end) by dcps (decapping scavenger) enzyme is an important process of the gene expression regulation, but little is known about its mechanism. the biological role of dcps and its potential therapeutic applications, e.g. as a novel therapeutic target for spinal muscular atrophy, make it an interesting object for biophysical investigations. the ability of dcps to act on various short capped mrnas will be presented. we have examined the substrate specificity and binding affinity of the enzyme in a quantitative manner, employing experimental physics' resources, such as atomic force microscopy (afm) and fluorescence spectroscopy for enzyme kinetics and timesynchronized-titration method (tst ) . in this study we extended the application of mqd-ihc to investigate potential biomarkers associated with prostate cancer (pca) invasiveness and lethal progression. objectives: to establish a mqd-ihc protocol using qd light-emitting nanoparticles ) to detect the expression/activation of critical cell signaling proteins at the single cell level; ) to image the plasticity and lethal progression of human pca with specific emphasis on emt and c-met signaling; and ) to examine the utility of mqd-ihc in clinical pca specimens to determine its invasion ability and predict its metastatic capability. results: we analyzed the co-expression and activation of osteomimicry associated biomarkers: b -microlgobulin (b -m), phosphorylated cyclic amp responsive element binding protein (pcreb) and androgen receptor (ar) in , cells from localized pca tissue areas (gleason and ) of patients with known metastatic status. the overall median % triple positive for b -m + /pcreb + /ar + cells was . %. the median triple positive for the samples with metastatic potential was % compared with those without metastatic potential (median = %); p = . by a wilcoxon rank sum test. the results were confirmed in pca bone metastatic specimens. we also investigated the c-met signaling in castration-resistant human pca model or crpc xenografts and the clinical pca specimens and found that the downstream signal components including pakt and mcl- were activated. conclusion: to validate our findings, additional clinical specimens with confirmed survival data will be analyzed and the cell-signaling-network-based mqd-ihc will be automated by vectra image analysis system in a high throughput manner with the hope to predict the lethal progression of pca prior to clinical manifestation of distant metastases. protein ligand binding is an important field of biopharmaceutical research. there are many techniques for quantitative determination of the ligand binding. the combination of isothermal titration calorimetry (itc) and thermal shift assay provides a robust estimate of the binding constant. many binding reactions are coupled to the absorption or release of protons by the protein or the ligand, conformational changes of the protein and other processes. to correlate the structural features of binding with the energetics of binding one needs to carry out a detailed thermodynamic study of the binding reaction and to determine dependencies such as ph, buffer and temperature. here we present a detailed thermodynamic description of radicicol binding to human heat shock protein hsp and determined proton linkage contributions to observed binding thermodynamics. we calculated the pk a of the group responsible for proton linkage, the protonation enthalpy of this group and intrinsic thermodynamic parameters for radicicol binding. the intrinsic enthalpy of radicicol binding to hsp is one of the largest enthalpies observed for any protein -ligand binding. the structural features responsible for such large binding enthalpy and very favorable intrinsic binding gibbs free energy are discussed. neuronal systems and modelling o- optogenetic electrophysiology walther akemann, amelie perron, hiroki mutoh, and thomas knö pfel laboratory for neuronal circuit dynamics, riken brain science institute, japan the combination of optical imaging methods with targeted expression of protein-based fluorescent probes enables the functional analysis of selected cell populations within intact neuronal circuitries. we previously demonstrated optogenetic monitoring of electrical activity in isolated cells, brain slices and living animals using voltage-sensitive fluorescent proteins (vsfps) generated by fusing fluorescent proteins with a membrane-integrated voltage sensor domain. however, several properties of these voltage reporters remained suboptimal, limiting the spatiotemporal resolution of vsfpbased voltage imaging. a major limitation of vsfps had been a reduced signal-to-noise ratio arising from intracellular aggregation and poor membrane targeting upon long-term expression in vivo. to address this limitation, we generated a series of enhanced genetically-encoded sensors for membrane voltage (named vsfp-butterflies) based on a novel molecular design that combines the advantageous features of vsfp s and vsfp s with molecular trafficking strategies. the new sensors exhibit faster response kinetics at subthreshold membrane potentials and enhanced localization to neuronal plasma membranes after long-term expression in vivo, enabling the optical recording of action potentials from individual neurons in single sweeps. vsfp-butterflies provide optical readouts of population activity such as sensoryevoked responses and neocortical slow-wave oscillations with signal amplitudes exceeding % dr/r in anesthetized mice. vsfp-butterflies will empower optogenetic electrophysiology by enabling new type of experiments bridging cellular and systems neuroscience and illuminating the function of neural circuits across multiple scales. opsin molecules are a burgeoning new tool for temporally precise neuronal stimulation or inhibition. opsin properties are commonly characterized in cell culture or acute brain slice preparations using whole cell patch clamp techniques, where neuronal membrane voltage is fixed at the resting potential. however, in vivo, where neurons are firing action potentials, opsins are exposed to large fluctuations in membrane voltage and transmembrane ionic concentrations which can influence opsin function. in the case of implanted light delivery devices, stimulation light power varies as a function of brain tissue volume. we therefore investigated the stability of opsin properties across a variety of in vivo-like stimulation conditions. we find that off-kinetics of excitatory opsins vary significantly with holding membrane potential; channelrhodopsin (chr ) slowing with depolarisation and chief (chr /chr hybrid) in contrast, accelerating. new chr point mutation variants demonstrate stability across all membrane potentials. we additionally explore responses to initial and subsequent light pulses and find that chief has the unique property of accelerating kinetics after the first light stimulation. inhibitory opsins vary in sensitivity to light in a manner which correlates with their off-kinetics. slower opsins, such as mac (leptosphaeria maculans), have higher sensitivity at low light power densities, saturating early relative to fast inhibitory opsins such as arch (archaeorhodopsin) and nphr (halorhodopsin). we discuss the relative merits of stability versus versatility of opsins under variable stimulation conditions. it has been previously shown that overexpression of ndm ncrna in a sknbe -derived neuroblastoma (nb) cell line leads to cell differentiation, with a decrease of malignant potential. here we use the patch-recording technique to characterize the ionic channel apparatus of nb cells expressing ndm at its basal level (mock cells) or at . fold higher levels (s cells). the two cell lines shared very similar pools of functional k channels, but s cells displayed larger ttx-sensitive na currents and were able to generate action potentials, while mock cells were not. in addition, while mock cells barely express functional gaba receptors, in the majority of s rapid application of gaba elicited a current with a ec = . lm; this current was antagonized by bicuculline ( lm) and potentiated by zaleplon (ec = nm). in mock cells, real time pcr evidenced a high level of gaba a a subunit, while in s cells a significant expression of a and a was detected, whereas a mrna was downregulated by %, confirming the development of functional gaba a receptors. in the same cell lines, the presence of specific markers and the secretion of specific cytokines confirmed that ndm expression leads to a differentiation process toward a neuron-like, rather than glial-like, phenotype. was planned therefore to reconstitute a model of brain tumors in rats by orthotopic implantation of xenogenic transformed human cells. iron is important element used for chemical reaction catalysis and physiological cell functions. the reason of iron deposition is still unknown. under conditions prevailing in human brain it is expected the formation of an amorphous or minute crystalline phase. we used light, scanning (sem) and transmission electron microscopy (tem), energy-dispersive microanalysis, electron diffraction and electron paramagnetic resonance (epr) for investigation of iron deposits in globus pallidus of human brain. sem revealed iron rich particles with na, si, p, s, cl, ca and cu around glial cells. tem revealed bumpy, solid particles of platy and sometimes rounded shape with the size of lm to lm. these ones were identified as hematite. epr measurements showed the presence of fe(iii) and cu(ii), but little amount of fe(ii) can not be excluded. we consider low-temperature process of hematite formation in human globus pallidus in aqueous environment influenced by organic and inorganic factors. chemical processes leading to nanoparticles formation can be associated with neurodegenerations such as alzheimer or parkinson disease. over the past years our understanding of the basic biophysical mechanisms governing the spatio-temporal dynamics of neuronal membrane potentials and synaptic efficacies has significantly expanded and improved. much research has focussed on how ionic currents contribute to the generation and propagation of action potentials, how subthreshold signals propagate along dendritic trees, how the active properties of dendrites shape the integration of incoming signals in a neuron, and how pre-and postsynaptic activities â and potential heterosynaptic effects a determine the way synaptic efficacies change on the short-and long-term. yet, despite these advances, there have been no systematic efforts to relate the basic dynamical repertoire of neurons to the computational challenges neural circuits face, and in particular to explain systematically how the biophysical properties of neurons are adapted to process information efficiently under the constraints of noise and uncertainty in the nervous system. as an initial step in this direction, i will show how various biophysical properties of neurons, in particular short-term synaptic plasticity and dendritic non-linearities, can be seen as adaptations to resolve an important bottleneck in neuronal information processing: the loss of information entailed by the conversion of analogue membrane potentials to digital spike trains. the optogenetic toolbox has greatly expanded since the first demonstration of genetically-targeted optical manipulation of neural activity. in addition to the cation channel channelrhodopsin- (chr ), the panel of excitatory opsins now includes an array of chr variants with mutations in critical residues, in addition to other, related cation channels, and channel hybrids. the inhibitory opsin panel has similarly expanded beyond the first-described halorhodopsin (nphr), a chloride pump, to include trafficking-enhanced versions of nphr as well as the proton pumps mac and arch. while the expansion of available opsins offers researchers an increasingly powerful and diverse selection of tools, it has also made it increasingly difficult to select the optimal tool for a given experiment. one cannot extract a comparison of opsins from the current literature, since studies differ across multiple variables known to contribute to opsin performance (e.g. expression method, light power density, stimulation protocols, etc.). here, we provide the first empirical comparison of both excitatory and inhibitory opsins under standardized conditions. furthermore, we identify the set of parameters that describe the properties of an opsin in a way that is maximally relevant for biological application. o- subcellular compartment-specific distribution of voltage-gated ion channels zoltan nusser institute of experimental medicine, hungarian academy of sciences, budapest, hungary voltage-gated na + (nav) channels are essential for generating the output signal of nerve cells, the action potential (ap). in most nerve cells, aps are initiated in the axon initial segment (ais). in vitro electrophysiological and imaging studies have demonstrated that dendritic nav channels support active backpropagation of aps into the dendrites, but the subunit composition of these channels remained elusive. here, i will present evidence for the somato-dendritic location of nav channels in hippocampal pyramidal cells (pcs). using a highly sensitive electron microscopic immunogold localization technique, we revealed the presence of the nav . subunit in pc proximal and distal dendrites, where their density is -fold lower than that found in aiss. a gradual decrease in nav . density along the proximo-distal axis of the dendritic tree was also detected. we have also investigated the subcellular distribution of kv . voltage-gated k + channel subunit and found a somato-dendritic localization. in contrast to that of nav . channels, the density of kv . first increases then decreases as a function of distance from the somata of pcs. such subcellular compartment-specific distribution of voltage-gated ion channels increases the computational power of nerve cells. keywords: memory, extra cellular matrix, random walk we expose first a biological model of memory based on one hand of the mechanical oscillations of axons during action potential and on the other hand on the changes in the extra cellular matrix composition when a mechanical strain is applied on it. due to these changes, the stiffness of the extra cellular matrix along the most excited neurons will increase close to these neurons due to the growth of astrocytes around them and to the elastoplastic behavior of collagen. this will create preferential paths linked to a memory effect. in a second part, we expose a physical model based on random walk of the action potential on the array composed of dendrites and axons. this last model shows that repetition of the same event leads to long time memory of this event and that paradoxical sleep leads to the linking of different events put into memory. myelinated nerve fibres were studied with fluorescent microscopy and laser interference microscopy. ca + redistribution during prolonged stimulation, changes in morphology and rearrangement of cytoplasmic structures were compared in normal conditions and after membrane modification by lysolecithin and methyl-b-cyclodextrin. lysolecithin is a detergent known to provoke demyelination, and methyl-b-cyclodextrin extracts cholesterol from membranes. cholesterol extraction could lead to disruption of membrane caveolae-like microdomains or ''rafts'' and solubilisation of different proteins connected to them. our data suggest that methyl-b-cyclodextrin and lysolecithin lead to different changes in morphology and distribution of cytoplasmic structures. the effect was different for different regions of the nerve (node of ranvier, paranodal and internodal regions). the agents also altered the kinetics of ca + response to stimulation in myelinated fibres. extracellular carbonic anhydrase contributes to the regulation of ca + homeostasis and salivation in submandibular salivary gland nataliya fedirko, olga kopach, nana, voitenko lviv national university, human and animal physiology, lviv, ukraine the maintenance of ph in the oral cavity is important for the oral heath since even a minor drop in ph can result in dental caries and damage to the teeth. submandibular salivary gland (smg) is main source of fluid and electrolytes enriched saliva therefore its core for oral ph homeostasis. smg secretion is activated by acetylcholine (ach) in [ca + ] idependend manner and accompanied with oral ph acidic shifts. ph shifts could be due to changes in buffering capacity that is regulated by carbonic anhydrase (ca). despite the expression of different subtypes of ca in smg the role of ca in the regulation of smg function is unclear yet. we found that ca inhibition by benzolamide (bz) decreased of fluid secretion in vivo extracellular na + concentration in situ. the latter confirm the ability of ca to modify both primarily and final saliva secretion. we also found correlation between the secretion and ca + -homeostasis since bz-induced decrease of: in striated muscle ca + release from the sarcoplasmic reticulum (sr) occurs when ryanodine receptors (ryr-s) open either spontaneously or upon the stimulation from dihydropyridine receptors that are located in the adjacent transverse-tubular membrane and change their conformation when the cell is depolarized. recent observations demonstrated that muscles from animal models of ptdinsp phosphatase deficiency suffer from altered ca + homeostasis and excitation-contraction coupling, raising the possibility that ptdinsp-s could modulate voltage-activated sr ca + release in mammalian muscle. the openings of a single or a cluster of ryr-s can be detected as ca + release events on images recorded from fibres loaded with fluorescent ca + indicators. to elucidate the effects of ptdinsp-s on ca + release events, images were recorded from skeletal muscle fibers enzymatically isolated from the m. flexor digitorum breavis of mice utilizing a super-fast scanning technique. a wavelet-based detection method was used to automatically identify the events on the images. three different ptdinsp-s (ptdins p, ptdins p, and ptdins( , )p) were tested. all these ptdinsp compounds decreased the frequency of spontaneous ca + release events. supported by the hungarian national science fund (otka ), té t. calcium sparks elicited by mmol/l caffeine and by a depolarization to - mv were recorded at high time resolution on both x-y ( frames/s) and line-scan images ( lines/ ms) on intact skeletal muscle fibers of the frog. while a typical spark appeared in one frame only, . and . % of spark positions overlapped on consecutive frames following caffeine treatment or depolarization, respectively. while both caffeine and depolarization increased the frequency of sparks, as estimated from x-y images, the morphology of sparks was different under the two conditions. both the amplitude (in df/f ; . ± . vs. . ± . ; n = vs. ; mean ± sem, p \ . ) and the full width at half maximum (in lm; parallel with fiber axis: . ± . vs. . ± . ; perpendicular to fiber axis: . ± . vs. . ± . ) of sparks was significantly greater after caffeine treatment than on depolarized cells. these observations were confirmed on sparks identified in line-scan images. in addition, x-t images were used to analyze the time course of these events. calcium sparks had significantly slower rising phase under both conditions as compared to the control. on the other hand, while the rate of rise of signal mass was decreased after depolarization, it increased in the presence of caffeine. prolonged depolarisation of skeletal muscle cells induces entry of extracellular calcium into muscle cells, an event referred to as excitation-coupled calcium entry. skeletal muscle excitation-coupled calcium entry relies on the interaction between the , dihydropyridine receptor on the sarcolemma and the ryanodine receptor on the sarcoplasmic reticulum membrane. in this study we exploited tirf microscopy to monitor with high spatial resolution excitationcoupled calcium entry (ecce) in primary coltures of human skeletal muscle cells harbouring mutation in the ryr gene linked to malignant hyperthermia and central core disease. we found that excitation-coupled calcium entry is strongly enhanced in cells from patients with central core disease compared to individuals with malignant hyperthermia and controls. in addition, excitation-coupled calcium entry induces generation of reactive nitrogen species and causes the nuclear translocation of nfatc . the activation of nfatc dependent genes is consistent with an increase of the il secretion from primary coltures human myotubes from ccd patients and with fibre type predominance of skeletal muscle of ccd patients. membrane lipids, microdomains & signalling p- ftir and calorimetric investigation of the effects of trehalose and multivalent cations on lipid structure sawsan abu sharkh, jana oertel, and karim fahmy division of biophysics, institute of radiochemistry, helmholtz-zentrum dresden-rossendorf, germany e-mail: s.sharkh@hzdr.de the structure of membrane lipids is of fundamental importance for the integrity of cell and organelle membranes in living organisms. membrane lipids are typically hydrated and their headgroup charges counter-balanced by solvated ions. consequently, water loss can induce severe structural changes in lipid packing (lyotropic transitions) and can lead to the damage of lipid membranes even after rehydration. this can be one out of several factors that affect the viability of organisms undergoing desiccation. many organisms, however, are resistant to even extreme water loss. some of them synthesize trehalose which has been shown to be associated with survival of desiccation in phylogenetically diverse organisms (yeast, nematodes, brine shrimp, insect larvae, resurrection plants, and others). here we have studied hydration sensitive transitions in model lipids to determine the effect of trehalose and electrostatics on lipid order. hydration pulse-induced time-resolved fourier-transform infrared (ftir) difference spectroscopy was used to address hydration-dependent lipid structure as a function of trehalose. in combination with differential scanning calorimetry and studies of langmuir-blodget films we arrive at a structural and energetically consistent picture of how trehalose can affects lipidic phase behaviour and support a native lipid structure under water loss. experiments were performed on model lipids with different headgroups and native lipids from desiccation-tolerant organisms. controlled self-assembly and membrane organization of lipophilic nucleosides and nucleic acids: perspectives for applications martin loew , paula pescador , matthias schade , julian appelfeller , jü rgen liebscher , oliver seitz , daniel huster , andreas herrmann , and anna arbuzova humboldt universitä t zu berlin, institute of biology/ biophysics, berlin, germany, humboldt universitä t zu berlin, institute of chemistry, berlin, germany, universitä t leipzig, department of medical physics and biophysics, leipzig, germany lipophilic conjugates of nucleosides and nucleic acids such as dna, rna, and peptide nucleic acid (pna) -combining assembly properties of amphiphiles and specific molecular recognition properties of nucleic acids -allow numerous applications in medicine and biotechnology. we recently observed self-assembly of microtubes, stable cylindrical structures with outer diameters of nm and - lm and a length of - lm, from a cholesterol-modified nucleoside and phospholipids. morphology and properties of these microtubes and functionalization with lipophilic dna will be characterized. we also observed that lipophilic nucleic acids, pna and dna differing in their lipophilic moieties, partition into different lipid domains in model and biological membranes as visualized by hybridization with respective complementary fluorescently-labeled dna strands. upon heating, domains vanished and both lipophilic nucleic acid structures intermixed with each other. reformation of the lipid domains by cooling led again to separation of membrane-anchored nucleic acids. by linking specific functions to complementary strands, this approach offers a reversible tool for triggering interactions among the structures and for the arrangement of reactions and signaling cascades on biomimetic surfaces. conformational dependent trafficking of p-glycoprotein with rafts zsuzsanna gutayné tó th, orsolya bá rsony, katalin goda, gá bor szabó and zsolt bacsó university of debrecen, mhsc, department of biophysics and cell biology, debrecen, hungary p-glycoprotein (pgp), an abc-transporter playing a prominent role in multidrug resistance, demonstrate conformationdependent endocytosis on the surface of t -mdr cells. these cell surface transporters have a uic conformationsensitive-antibody-recognizable subpopulation, which is about one-third of the rest persisting long on the cell surface and perform fast internalization via rafts. we have identified that the rapid internalization is followed by quick exocytosis, in which the other subpopulation is not or only slightly involved. the exocytosis presents a cholesterol depletion dependent intensification, in contrast to the internalization, which is inhibited by cyclodextrin treatment. this continuous recycling examined by total internal reflection (tirf) microscopy increases the amount of the raft associated subpopulation of pgps in the plasma membrane, and it might have a role in restoring the cholesterol content of the membrane after cholesterol depletion. our presentation will summarize related endocytotic, exocytotic and recycling processes and that how does our data fit into our current notions regarding to the cholesterol and sphingomyelin trafficking. membrane nanodomains based on phase-segregating lipid mixtures have been emerged as a key organizing principle of the plasma membrane. they have been shown to play important roles in signal transduction and membrane trafficking. we have developed lipid-like probes carrying multivalent nitrilotriacetic acid (tris-nta) head groups for selective targeting of histagged proteins into liquid ordered or liquid disordered phases. in giant unilamellar vesicles strong partitioning of tris-nta lipids into different lipid phases was observed. for a saturated tris-nta lipid, at least -fold preference for the liquid ordered phase was found. in contrast, an unsaturated nta lipid shows a comparable preference for the liquid disordered phase. partitioning into submicroscopic membrane domains formed in solid supported membranes was confirmed by superresolution imaging. single molecule tracking of his-tagged proteins tethered to solid supported membranes revealed clear differences in the diffusion behavior of the different nta-lipids. by using bsa as a carrier, multivalent nta lipids were efficiently incorporated into the plasma membrane of live cells. based on this approach, we established versatile methods for probing and manipulating the spatiotemporal dynamics of membrane nano domains in live cells. il- is a multifunctional cytokine with pleiotropic effects on t cells. the il- r consists of the cytokine-specific a-subunit and the c c -chain shared with other cytokines, including il- and - , important regulators of t cells. we have previously shown the preassembly of the heterotrimeric il- and il- r, as well as their participation in common superclusters with mhc glycoproteins in lipid rafts of human t lymphoma cells. integrity of lipid rafts was shown to be important in il- signaling. we could hypothesize that other members of the c c cytokine receptor family, such as the il- r complex, may also fulfill their tasks in a similar environment, maybe in the same superclusters. co-localization of il- r with lipid rafts as well as with the il- r/mhc superclusters was determined by clsm. molecular scale interactions of il- ra with il- r and mhc molecules were determined by microscopic and flow cytometric fret experiments. the role of lipid rafts in il- r signaling was assessed by following the effect of cholesterol depletion on il- induced stat phosphorylation. our results suggest the possibility that preassembly of the receptor complexes in common membrane microdomains with mhc glycoproteins may be a general property of c c cytokines in t cells. to unravel the molecular processes leading to fas clustering in lipid rafts, a -mer peptide corresponding to the single transmembrane domain of the death receptor was reconstituted into model membranes that display liquid-disordered/ liquid-ordered phase coexistence, i.e. mimicking cells plasma membranes. using the intrinsic fluorescence of the peptide two tryptophans residues (trp and trp ), fas membrane lateral organization, conformation and dynamics was studied by steady-state and time-resolved fluorescence techniques. our results show that the fas has preferential localization to liquid disordered membrane regions, and that it undergoes a conformational change from a bilayer inserted state in liquid-disordered membranes to an interfacial state in liquidordered membranes. this is a result of the strong hydrophobic mismatch between the (hydrophobic) peptide length and the hydrophobic thickness of liquid-ordered membranes. in addition, we show that ceramide, a sphingolipid intimately involved in fas oligomerization and apoptosis triggering, does not affect fas membrane organization. overall, our results highlight ceramide role as an enhancer of fas oligomerization, and unravel the protective function of fas transmembrane domain against non-ligand induced fas apoptosis. organization and dynamics of membrane-bound bovine a-lactalbumin: a fluorescence approach arunima chaudhuri and amitabha chattopadhyay centre for cellular and molecular biology, hyderabad , india e-mail: amit@ccmb.res.in many soluble proteins are known to interact with membranes in partially disordered states, and the mechanism and relevance of such interactions in cellular processes are beginning to be understood. interestingly, apo-bovine alactalbumin (bla), a soluble protein, specifically interacts with negatively charged membranes and the membranebound protein exhibits a molten globule conformation. we have used the wavelength-selective fluorescence approach to monitor the molten globule conformation of bla upon binding to negatively charged membranes as compared to zwitterionic membranes. tryptophans in bla exhibit differential red edge excitation shift (rees) upon binding to negatively charged and zwitterionic membranes, implying differential rates of solvent relaxation around the tryptophan residues. our results utilizing fluorescence anisotropy, lifetime and depth analysis by the parallax approach of the tryptophans further support the differential organization and dynamics of the membrane-bound bla forms. in addition, dipole potential measurements and dye leakage assays are being used in our ongoing experiments to explore the mechanism of bla binding to membranes. these results assume significance in the light of antimicrobial and tumoricidal functions of a-lactalbumin. role of long-range effective protein-protein forces in the formation and stability of membrane protein nano-domains nicolas destainvill laboratoire de physique thé orique, université paul sabatier toulouse -cnrs, toulouse, france we discuss a realistic scenario, accounting for the existence of sub-micro-metric protein domains in plasma membranes. we propose that proteins embedded in bio-membranes can spontaneously self-organize into stable small clusters, due to the competition between short-range attractive and intermediate-range repulsive forces between proteins, specific to these systems. in addition, membrane domains are supposedly specialized, in order to perform a determined biological task, in the sense that they gather one or a few protein species out of the hundreds of different ones that a cell membrane may contain. by analyzing the balance between mixing entropy and protein affinities, we propose that protein sorting in distinct domains, leading to domain specialization, can be explained without appealing to preexisting lipidic micro-phase separations, as in the lipid raft scenario. we show that the proposed scenario is compatible with known physical interactions between membrane proteins, even if thousands of different species coexist. lipid rafts are cholesterol and sphingolipid-enriched functional microdomains present in biomembranes. rafts have been operationally defined as membrane fractions that are detergent insoluble at low temperature. here we have characterized drms from erythrocytes treated with the nonionic detergents brij and brij , at °c and °c, and compared them to drms obtained with triton x- (tx ). we have also investigated the effect of cholesterol depletion in drms formation. brij drms were enriched in cholesterol as well as tx drms. hptlc analysis showed a very similar distribution of phosphatidylcholine-pc, phosphatidylethanolamine-pe and sphingomyelin-sm in brij drms to that found in ghost membranes. sm-enriched drms were obtained only with tx while pe content was decreased in tx drms, in comparison to brij drms. immunoblot essays revealed that rafts markers (flotillin- and stomatin) were present in all drms. contrary to tx drms, analysis of electron paramagnetic resonance spectra (with -doxyl stearate spin label) revealed that brij drms are not in the liquid-ordered state, evincing the differential extraction of membrane lipids promoted by these detergents. supported by fapesp/cnpq (brazil). several biological membrane mimics have been built to investigate the topology of molecules in membranes. among them ''bicelles'', i.e., mixtures of long-chain and short-chain saturated phospholipids hydrated up to %, became very popular because they orient spontaneously in magnetic fields. disk-shaped systems of - nm diameter and - nm thickness have been measured by electron microscopy and solid state nmr and can be oriented by magnetic fields with the disc-plane normal perpendicular to the field. we have been developing recently lipids that contain in one of their chains a biphenyl group (tbbpc) affording an orientation parallel to the magnetic field, in the absence of lanthanides. a large number of hydrophobic molecules including membrane proteins have been successfully embedded and static nmr afforded finding the orientation of protein helices in membranes; mas nmr provided the d structure of peptides in bicelles. biphenyl bicelles keep their macroscopic orientation for days outside the field, thus leading to combined nmr and x-rays experiments. tbbpc allows also construction of lm vesicles showing a remarkable oblate deformation in magnetic fields (anisotropy of - ) and opens the way to applications for structural biology or drug delivery under mri. imaging membrane heterogeneities and domains by super-resolution sted nanoscopy christian eggeling, veronika mueller, alf honigmann, stefan w. hell department of nanobiophotonics, max planck institute for biophysical chemistry, am fassberg , gö ttingen, germany cholesterol-assisted lipid and protein interactions such as the integration into lipid nanodomains are considered to play a functional part in a whole range of membrane-associated processes, but their direct and non-invasive observation in living cells is impeded by the resolution limit of [ nm of a conventional far-field optical microscope. we report the detection of the membrane heterogeneities in nanosized areas in the plasma membrane of living cells using the superior spatial resolution of stimulated emission depletion (sted) far-field nanoscopy. by combining a (tunable) resolution of down to nm with tools such as fluorescence correlation spectroscopy (fcs), we obtain new details of molecular membrane dynamics. sphingolipids or other proteins are transiently (* ms) trapped on the nanoscale in cholesterol-mediated molecular complexes, while others diffuse freely or show a kind of hopping diffusion. the results are compared to sted experiments on model membranes, which highlight potential influences of the fluorescent tag. the novel observations shed new light on the role of lipid-protein interactions and nanodomains for membrane bioactivity. ca + controlled all-or-none like recruitment of synaptotagmin- c ab to membranes sune m. christensen, nicky ehrlich, dimitrios stamou bio-nanotechnology laboratory, department of neuroscience and pharmacology, nano-science center, lundbeck foundation center biomembranes in nanomedicine, university of copenhagen, copenhagen, denmark e-mail: ehrlich@nano.ku.dk & stamou@nano.ku.dk synaptotagmin- (syt) is the major ca + sensor that triggers the fast, synchronous fusion of synaptic vesicle with the presynaptic membrane upon ca +-mediated membrane recruitment of the cytosolic c ab domain. the ca +-dependent recruitment of syts c ab domain to membranes has so far been investigated by ensemble assays. here we revisited binding of wild type c ab and different c ab mutants of syt to lipid membranes using a recently developed single vesicle assay. the hallmark of the single vesicle approach is that it provides unique information on heterogeneous properties that would otherwise be hidden due to ensemble averaging. we found that c ab does not bind to all vesicles in a homogenous manner, but in an all-or-none like fashion to a fraction of the vesicles. the fraction of vesicles with bound c ab is regulated by the amount of negatively charged lipids in the membrane as well as by [ca +] . this ca + controlled all-ornone like recruitment of syt to membranes provides a possible explanation for the strongly heterogeneous behavior of the in vitro model system for neuronal membrane fusion. furthermore, heterogeneity in release probability among synaptic vesicles is a critical property in determining the output of a neuronal signaling event. new insights in the transport mechanism of ciprofloxacin revealed by fluorescence spectroscopy mariana ferreira, sílvia c. lopes, paula gameiro requimte, faculdade de ciê ncias da universidade do porto, porto, portugal keywords: fluoroquinolones; liposomes; proteoliposomes; ompf; ciprofloxacin; fluorescence; anisotropy. fluoroquinolones are antibiotics that have a large spectrum of action against gram negative and some gram positive bacteria. the interaction between these species and liposomes has been cited as a reference in the understanding of their diffusion through phospholipid bilayer and can be quantified by the determination of partition coefficients between a hydrophobic phase (liposomes) and an aqueous solution. it is also known that some porins of the bacterial membranes are involved in transport mechanism of many fluoroquinolones. ompf, a well characterized membrane protein characteristic of the outer membrane of gram negative bacteria assumes the conformation of homo-trimer, whose monomers have two tryptophan residues (one located at the interface of monomers and the other at the interface lipid/protein). thus, we proceeded to study the interaction of ciprofloxacin, a second generation fluoroquinolone, with unilamellar liposomal vesicles and ompf proteoliposomes of pope/popg, pope/popg/cardiolipin and e. coli total. partition coefficients (kp's) and the association with ompf proteoliposomes were determined by steady state fluorescence spectroscopy under physiological conditions (t= °c; ph . ). the membrane mimetic systems used were characterized by dls and fluorescence anisotropy. motivation is whether there exist differences in the patternforming capabilities of two adhesion molecules of different roles: cd , mediating ,,dynamic'' adhesion in cell rolling and icam- , mediating ,,static'' adhesion during the formation of immune-synapse. homo-and hetero-associations of cd , icam- and the mhci is investigated on the nm-and lmdistance levels on ls t colon carcinoma cells in two different conditions of lymphocyte homing: ( ) with ifnc and tnfa, both lymphokines up-regulating the expression level of mhci and icam- and down-regulating that of cd . ( ) crosslinking of cd and icam- representing receptor engagement. the observations are explained by assuming the existence of a kinase cascade-level crosstalk between the cd and icam- molecules which manifests in characteristic complementary changes in the properties of cell surface receptor patterns. for the characterisation of cluster morphology new colocalization approaches were developed: (i) ,,number of first neighbours'' distribution curves, (ii) ,,acceptor photobleaching fret-fluorescence intensity fluctuation product'' correlation diagrams, and (iii) ,,random gradient-kernel smoothing assisted decay'' of pearson-correlations, and (iv) k-function formalism. analyzing janus kinases in living cells with single-color fcs using confocal and tir-illumination thomas weidemann , hetvi gandhi , remigiusz worch , , robert weinmeister , christian bö kel , and petra schwille biophysics research group, technische universitä t dresden, germany, crtd, center for regenerative therapies dresden, technische universitä t dresden, germany, institute of physics, polish academy of science, warsaw, poland cytokine receptors of the hematopoietic superfamily transduce their signal through non-covalently bound janus kinases. there are only such kinases in humans (jak , , and tyk ), which associate to different cytokine receptor chains. here we study the dynamics of gfp-tagged jak and jak in epithelial cells with fluorescence correlation spectroscopy (fcs). jak and jak behave differently in various aspects: in the absence of receptors, jak still binds the membrane, whereas jak diffuses homogeneously in the cytoplasm. we used fcs under total internal reflection illumination (tir-fcs) and determined the membrane binding affinity of jak to be ± nm. the association of jak with the common gamma chain (c c ) is very tight as shown by fluorescence recovery after photobleaching (frap). molecular brightness analysis of single-point fcs shows that jak diffuses as a monomer in the rather small cytoplasmic pool, whereas jak diffuses as dimers, which undergo a defined oligomerization. the degree of oligomerization decays at higher concentrations, indicating that some unknown, saturable scaffold is involved. characterizing the binding and mobility schemes of the janus kinases may be important to further elucidate their specific and redundant effects in signal transduction. plasma membrane (pm)-enriched fraction obtained through subcellular fractioning protocols are commonly used in studies investigating the ability of a compound to bind to a receptor. however, the presence of mitochondria membranes (mi) in the pm-enriched fraction may compromise several experimental results because mi may also contain the interest binding proteins. aiming to analyze the subcellular fractioning quality of a standard sucrose density based protocol, we investigated (a) the na + k + -atpase (pm marker) and succinate dehydrogenase -sd (mi marker) activities; (b) the immunocontent of the adenine nucleotide translocator (ant -mi membrane marker) in both pm-and mi-enriched fractions. since several binding protocols may require long incubation period, we verified the quality of both fractions after hours of incubation in adequate buffer. our results show that pmand mi-enriched fractions exhibit contamination with mi or pm, respectively. we did not observe any effect of incubation on na + k + -atpase activity and ant content in both fractions. surprisingly, sd activity was preserved in the pm-but not in mi-enriched fraction after incubation. these data suggest the need of more careful use of pm-enriched fraction preparation in studies involving pm proteins characterization. human neutrophil peptide (hnp ) is a human cationic defensin that present microbial activity against various bacteria (both gram-positive and negative), fungi and viruses. hnp is stored in the cytoplasmic azurophilic granules of neutrophils and epithelial cells. in order to elucidate the mode of action of this antimicrobial peptide (amp), studies based on its lipid selectivity were carried out. large unilamellar vesicles (luv) with different lipid compositions were used as biomembranes model systems (mammal, fungal and bacterial models). changes on the intrinsic fluorescence of the tryptophan residues present in hnp upon membrane binding/insertion were followed, showing that hnp have quite distinct preferences for mammalian and fungal membrane model systems. hnp showed low interaction with glucosylceramide rich membranes, but high sterol selectivity: it has a high partition for ergosterol-containing membranes (as fungal membranes) and low interaction with cholesterolcontaining membranes (as in mammalian cells). these results reveal that lipid selectivity is the first step after interaction with the membrane. further insights on the hnp membrane interaction process were given by fluorescence quenching measurements using acrylamide, -doxylstearic acid ( ns) or ( ns). nanoparticles (np) are currently used in many industrial or research applications (paints, cosmetics, drug delivery materials…). recent papers demonstrate clearly their activity with biological membranes (nanoscale holes, membrane thinning, disruption). different studies of the np-membrane interaction suggest that parameters are particularly important, such as the np size, their surface properties or their aggregation state. composition of biological membranes being particularly complex, supported lipid bilayers (slb) composed of a restricted number of lipids are usually used as simplified membrane model. moreover, these two-dimensional systems are convenient for surface analysis techniques, such as atomic force microscopy (afm), giving information on the morphology of the slb and its mechanical properties. in this work, we study the behaviour of slbs made of lipids representative of the membrane fluid phase (popc) or of the raft phase (sphingomyelin). these slbs are deposited on planar surfaces (mica or glass) previously recovered with silica beads ( or nm in diameter) in order to mimick the np-membrane interaction. we will present in this work our first results obtained by afm and fluorescence microscopy. it is well known that the eukaryotic nuclei are the sphere of lipids active metabolism. the investigations demonstrated the existence of numerous enzymes in nuclei which modulate the changes of nuclear lipids during different cellular processes. although the nuclear membrane is accepted as the main place of the lipids localization, nearly % of nuclear lipids are discovered in chromatin fraction. the ability of chromatin phospolipids to regulate dna replication and transcription was already demonstrated. the chromatin phospolipids seems to play an important role in cell proliferation and differentiation as well as in apoptosis. it seems also possible that chromatin phospholipids may be participated in realization of cisplatin antitumor effects. the -hour in vivo effect of cisplatin on rat liver chromatin phospholipids was investigated. the phospholipids of rat liver chromatin were fractionated by microtlc technique. the quantitative estimation of fractionated phospholipids was carried out by computer program fugifilm science lab. image gauge v . . the alteration of total phospholipids content as well as the quantitative changes among the individual phospholipids fractions in rat liver chromatin after in vivo action of cisplatin was established. the total content of chromatin phospholipids was significantly decreased after the cisplatin action. four from five individual phospholipids fractions were markedly changed after the drug action. two cholin-content phospholipids, particularly phosphatidylcholine and sphingomyelin exhibit diversity in sensitivity to this drug: the increase of sphingomyelin content accompanied by quantitative decrease of phosphatidylcholine. the quantity of cardiolipin was markedly increased while the amount of phosphatidylinositol was decreased after the cisplatin treatment. the phosphatidylethanolamin content remained unchanged after the drug action. it seems that high sensitivity of chromatin phospholipids exhibited to cisplatin action may play an important role in antitumor effects of this drug. membrane lipids and drug resistance in staphylococci r. d. harvey institute of pharmaceutical science, king's college london, stanford street, london se nh, uk staphylococci express numerous resistance mechanisms against common antimicrobials, including peptide components of the innate immune system which have been trumpeted as being likely candidates to replace our increasingly ineffective antibiotics. the membrane phospholipid lysylphosphatidylglycerol (l-pg) appears to play a key role in staphylococcal drug resistance, since its absence in mutant bacteria renders them susceptible to a range of cationic antimicrobials. the current assumption about the role l-pg plays in drug resistance is that of facilitating charge neutralisation of the plasma membrane, leading to loss of affinity towards cationic moieties. we have investigated this phenomenon using a range of model membrane systems composed of both synthetic lipids and reconstituted natural lipid extracts, using such techniques as stopped-flow fluorescence, circular dichroism and neutron scattering. our conclusions indicate that the initial assumptions about the role of l-pg in drug resistance are over-simplistic and certainly do not tell the whole story of the physical and biological properties of this fascinating moderator or membrane behaviour. our findings show that l-pg does not inhibit antimicrobial drug action by charge dampening, hinting at a different protective mechanism. modulation of a-toxin binding by membrane composition m. schwiering, a. brack, c. beck, h. decker, n. hellmann institute for molecular biophysics, university of mainz, mainz, germany although the alpha-toxin from s. aureus was the first poreforming toxin identified, its mode of interaction with membranes is still not fully understood. the toxin forms heptameric pores on cellular and artificial membranes. the present hypothesis is that the initial binding to the membrane occurs with low affinity, and that an efficient oligomerisation, relying on clusters of binding sites, is the reason for the overall high affinity of the binding process. in order to separate the effects of increasing concentration of binding sites from this topological effect, we investigated the oligomer formation based on pyrene-fluorescence for a series of lipid compositions, where the fraction of toxin binding lipids (egg phospatidylcholine (epc) or egg sphingomyelin (esm)) was varied while their concentration remained constant. the results indicate that an increased local density of toxin binding sites occurring due to phase separation facilitates oligomer formation. furthermore, the change in local environment (number of neighboring cholesterol molecules) upon domain formation also enhances oligomer formation.we thank the dfg (sfb ) for financial support, s. bhakdi and a. valeva for production of the toxin and helpful discussions. we explored quercetin effects on lipid bilayers containing cholesterol using a spectrofluorimetric approach. we used the fluorescent probe laurdan which is able to detect changes in membrane phase properties. when incorporated in lipid bilayers, laurdan emits from two different excited states, a non-relaxed one when the bilayer packing is tight and a relaxed state when the bilayer packing is loose. this behavior is seen in recorded spectra as a shift of maximum emission fluorescence from nm at temperatures below lipids phase transition to nm at temperatures above lipids phase transition values. emission spectra of laurdan were analyzed as a sum of two gaussian bands, centered on the two emission wavelengths allowing a good evaluation of the relative presence of each population. our results show that both laurdan emission states are present with different shares in a wide temperature range for dmpc liposomes with cholesterol. quercetin leads to a decrease in the phase transition temperature of liposomes, acting in the same time as a quencher on laurdan fluorescence. this paper is supported by the sectorial operational programme human resources development, financed from the caveolins are essential membrane proteins found in caveolae. the caveolin scaffolding domain of caveolin- includes a short sequence containing a crac motif (v tkywfyr ) at its c-terminal end. to investigate the role of this motif in the caveolin-membrane interaction at the atomic level, we performed a detailed structural and dynamics characterization of a cav- (v -l ) nonapeptide encompassing this motif and including the first residue of cav- hydrophobic domain (l ), in dodecylphosphocholine (dpc) micelles and in dmpc/dhpc bicelles, as membrane mimics. nmr data revealed that this peptide folded as an amphipathic helix located in the polar head group region. the two tyrosine sidechains, flanked by arginine and lysine residues, are situated on one face of this helix, whereas the phenylalanine and tryptophan side-chains are located on the opposite face (le lan c. et al., , eur. biophys. j., , - ). to investigate the interactions between the crac motif and the lipids, we performed molecular dynamics simulations in two different environment: a dpc micelle and a popc bilayer. the results obtained are in good agreement with nmr data and the comparison between both systems provided insight into the orientation of the crac motif at the membrane interface and into its interactions with lipids. this work was partially supported by the strategic grant posdru/ / . our study suggests that conformation and positioning of hydroxyl groups significantly affects thermotropic properties of sphingolipids and sterol interaction. the polymorphism of a new series of bolaamphiphile molecules based on n-( -betainylamino-dodecane)-octyl b-d-glucofuranosiduronamide chloride is investigated. the length of the main bridging chain is varied in order to modify the hydrophilic/lipophilic balance. the other chemical modification was to introduce a diacetylenic unit in the middle of the bridging chain to study the influence of the pp stacking on the supramolecular organisation of these molecules. dry bolaamphiphiles self-organize in supramolecular structures such as lamellar crystalline structure, lamellar fluid structure and lamellar gel structure. the thermal dependence of these structures, as well as the phase transition is followed by smallangle and wide-angle x-ray scattering. once the thermal cycle is accomplished, the system remains in the kinetically stabilized undercooled high-temperature phase at temperature of °c. subsequently, the time dependence of the relaxation to the thermodynamically stable phase is followed and very slow relaxation on the order of hours or days is observed. the study of polymorphism and the stability of various phases of this new series of bolaamphiphiles is interesting for potential application in health, cosmetics or food industry, is undertaken in this work. alkylphospholipids have shown promising results in several clinical studies and among them perifosine (opp) is promising for breast cancer therapy. antitumor effect was much better in estrogen receptor negative (er-) than in estrogen receptor positive (er+) tumors in vivo. it is believed that apl do not target dna, but they insert in the plasma membrane and ultimately lead to cell death. liposomes made of opp and different amount of cholesterol (ch) showed diminished hemolytic activity as compared to micellar opp, but in most cases cytotoxic activity was lower. in order to find optimal liposomal composition and to understand better the difference in the response of er+ and er-cells the interaction opp liposomes with er+ and er-cells was studied. for liposomes with high amount of ch both cell types showed slow release of the liposome entrapped spin probe into the cytoplasm. liposmes with low amount of ch interact better with cells but the release is faster for er-as for er+ cells at °c. experiments with nitroxide-labeled opp (sl-opp) liposomes suggest that the exchange of sl-opp between liposomes and cellular membranes is fast. however, translocation of sl-opp across the plasma membrane is slow, but seems to be faster for opp resistent, er+ cells as for er-cells at °c. estimation of a membrane pore size based on the law of conservation of mass krystian kubica , artur wrona and olga hrydziuszko institute of biomedical engineering and instrumentation, wroclaw university of technology, wroclaw, poland, centre for systems biology, university of birmingham, birmingham, uk the size of biomembrane pores determines which solutes or active compounds may enter the cell. here, using a mathematical model of a lipid bilayer and the law of conservation of mass, we calculate the radius of a membrane pore created by rearranging the lipid molecules (the pore wall was formed out of the lipid heads taken from the membrane regions situated directly above and below the pore, prior its formation). assuming a constant number of lipid molecules per bilayer (with or without the pore) and based on the literature data ( % decrease in the area per chain for a fluid-to-gel transition and a matching change of one chain volume not exceeding %) we have shown that the pore radius can measure up to . nm (for a nm thick lipid bilayer) without the lipid molecules undergoing a phase transition. a further assumption of area per chain being modified as a consequence of the lipids conformational changes has resulted in an increase of the calculated radius up to . nm. finally, a comparison of the pore volume with the corresponding volume of the lipid bilayer has led to a conclusion that for the system under consideration the membrane pore can only be created with the lipids undergoing fluidto-gel conformational changes. the key signaling pathway involves tyrosine phosphorylation of signal transducers and activators of transcription (stat and stat ) by receptor-associated janus kinases. we aim to unveil of the very early events of signal activation including ligand-induced receptor assembly and the recruitment of the cytoplasmic effector proteins stat and stat in living cells. to this end, we have explored the spatiotemporal dynamics of stat recruitment at the membrane on a single molecule level. highly transient interaction of stats to membrane-proximal sites was detected by tirf-microscopy, allowing for localizing and tracking individual molecules beyond the diffraction limit. thus, we obtained a pattern of the spatio-temporal recruitment of stat molecules to the plasma membrane revealing distinct submicroscopic structures and hotspots of stat interaction with overlapping recruitment sites for stat and stat . strikingly, these stat binding sites were independent on receptor localization and expression level. simultaneous superresolution imaging of the cytoskeleton revealed the organization of stat recruitment sites within the cortical actin skeleton. characterization of molecular dynamics on living cell membranes at the nanoscale level is fundamental to unravel the mechanisms of membrane organization andcompartmentalization. we have recently demonstr ated the feasibility of fluorescence correlation spectroscopy (fcs) based on the nanometric illumination of near-field scanning optical microscopy (nsom) probes on intact living cells [ ] . nsom-fcs was applied to study the diffusion of fluorescent lipid analogs on living cho cells. the experiments allowed to reveal details of the diffusion hidden by larger illumination areas and associated with nanoscale membrane compartmentalization. the technique also offers the unique advantages of straightforward implementation of multiple color excitation, opening the possibility to study nanoscale molecular cross-correlation. furthermore, the nsom probe evanescent axial illumination allows to extend diffusion study to the membrane-proximal cytosolic region. as such, nsom-fcs represents a novel powerful tool to characterize the details of many biological processes in which molecular diffusion plays a relevant role. the growing interest in supported lipid bilayers (slbs) on conductive substrates, such as gold, is due to the possibility of designing lipid-based biosensor interfaces with electrochemical transduction. due to the hydrophobicity of gold it is still a challenge to deposit planar and continuous bilayers without previous surface modification. most studies on gold concern single-phase slbs without cholesterol or gangliosides, two vital components of biomembranes. in this work the experimental conditions suitable for the formation of complex slbs with phase-separation directly on gold are exploited. the mixtures dopc/dppc/cholesterol ( : : ) with or mol % of ganglioside gm , which should yield lipid raft-like domains according to reported phase diagrams, were studied. slb with lipid rafts were successfully formed onto bare au ( ), although surface modification with -mercapto-undecanoic acid sam stabilized the slbs due to its charge and hydrophilicity. the different experimental conditions tested had an impact on nano/microdomains organization observed by atomic force microscopy in buffer solution. surface characterization through the combined use of ellipsometry, cyclic voltammetry and afm allowed to optimize the conditions for the formation of more planar and compact slbs. it is widely accepted that the conversion of the soluble, nontoxic amyloid b-protein (ab) monomer to aggregated toxic ab rich in b-sheet structures is central to the development of alzheimer's disease. however, the mechanism of the abnormal aggregation of ab in vivo is not well understood. we have proposed that ganglioside clusters in lipid rafts mediate the formation of amyloid fibrils by ab, the toxicity and physicochemical properties of which are different from those of ab amyloids formed in solution [ , ] . in this presentation, we report a detailed mechanism by which ab-( - ) fibrillizes in raft-like lipid bilayers composed of gm /cholesterol/sphingomyelin. at lower concentrations, ab formed an a helix-rich structure, which was cooperatively converted to a b sheet-rich structure above a threshold concentration. the structure was further changed to a seed-prone b structure at higher concentrations. the seed recruited ab in solution to form amyloid fibrils. [ hepatitis c virus (hcv) has a great impact on public health, affecting more than million people worldwide since it is the cause of liver-related diseases such as chronic hepatitis, cirrhosis and hepatocarcinoma. hcv entry into the host cell is achieved by the fusion of viral and cellular membranes, replicates its genome in a membrane associated replication complex, and morphogenesis has been suggested to take place in the endoplasmic reticulum (er) or modified er membranes. the variability of the hcv proteins gives the virus the ability to escape the host immune surveillance system and notably hampers the development of an efficient vaccine. hcv has a single-stranded genome which encode a polyprotein, cleaved by a combination of cellular and viral proteases to produce the mature structural proteins (core, e , e , and p ) and non-structural ones (ns , ns , ns a, ns b, ns a and ns b), the latter associated with the membrane originated from the er in the emerging virus. the ns b protein, a fundamental player in the hcv replicative process and the least characterized hcv protein, is a highly hydrophobic protein associated with er membranes. it has recently been shown that the c-terminal is palmitoylated and the n-terminal region has potent polymerization activity. the expression of ns b induces the formation of the so called membranous web, which has been postulated to be the hcv rna replication complex. thus, a function of ns b might be to induce a specific membrane alteration that serves as a scaffold for the formation of the hcv replication complex and therefore has critical role in the hcv cycle. due to the high hydrophobic nature of ns b, a detailed structure determination of this protein is very difficult. the ns b protein is an integral membrane protein with four or more transmembrane domains. the c-terminal region of ns b is constituted by two a helices, h (approximately from amino acid to ) and h (approximately from amino acid to ), which have been studied as potential targets for inhibiting hcv replication. previous studies from our group, based on the study of the effect of ns b peptide libraries on model membrane integrity, have allowed us to propose the location of different segments in this protein that would be implicated in lipid-protein interaction. additionally, the h region could be an essential constituent in the interaction between protein and membrane. in this study we show that peptides derived from the c-terminal domain of ns b protein of hcv are able to interact with high affinity to biomembranes, significantly destabilizing them and affecting their biophysical properties. there were also differences in the interaction of the peptide depending on the lipid composition of the membranes studied. we have also applied fluorescence spectroscopy, infrared spectroscopy and differential scanning calorimetry which have given as a detailed biophysical study of the interaction of the peptide with model biomembranes. this work was supported by grant bfu - -bmc (ministerio de ciencia y tecnología, spain) to j.v. a semi-quantitative theory describing the adhesion kinetics between soft objects as living cells or vesicles was developed. the nucleation-like mechanism has been described in the framework of a non-equilibrium fokker-planck approach accounting for the adhesion patch growth and dissolution (a. raudino, m. pannuzzo, j. chem. phys. , ( )). a well known puzzling effect is the dramatic enhancement of the adhesion/fusion rate of lipid membranes by water-soluble polymers that do not specifically interact with the membrane surface. we extend the previous approach by molecular dynamics simulations in the framework of a coarse-grained picture of the system (lipid+polymer+ions embedded in an explicit water medium) in order to test and support our previous analytical results. simulations show that the osmotic pressure due to the polymer exclusion from the inter-membrane spacing is partially balanced by an electrostatic pressure. however, we also evidenced an interesting coupling between osmotic forces and electrostatic effects. indeed, when charged membranes are considered, polymers of low dielectric permittivity are partially excluded from the inter-membrane space because of the increased local salt concentration. the increased salt concentration means also a larger density of divalent ions which form a bridge at the contact region (stronger adhesion). the overall effect is a smaller membrane repulsion. this effect disappears when neutral membranes are considered. the model could explain the fusion kinetics between lipid vesicles, provided the short-range adhesion transition is the rate-limiting step to the whole fusion process. the role of ceramide acyl chain length and unsaturation on membrane structure sandra n. ceramide fatty acid composition selectively regulates distinct cell processes by a yet unknown mechanism. however, evidence suggests that biophysical processes are important in the activation of signalling pathways. indeed, ceramide strongly affects membrane order, induces gel/fluid phase separation and forms highly-ordered gel domains. the impact of ceramide n-acyl chain in the biophysical properties of a fluid membrane was studied in popc membranes mixed with distinct ceramides. our results show that: i) saturated ceramide has a stronger impact on the fluid membrane, increasing its order and promoting gel/fluid phase separation, while their unsaturated counterparts have a lower (c : ceramide) or no (c : ceramide) ability to form gel domains at physiological temperature, ii) differences between distinct saturated species are smaller and are related mainly to domain morphology, and iii) very long chain ceramide induces the formation of tubular structures probably associated with interdigitation. these results suggest that generation of different ceramide species in cell membranes has a distinct biophysical impact with acyl chain saturation dictating membrane lateral organization, and chain asymmetry governing interdigitation and membrane morphology. extra high content of cholesterol (chol) in fiber-cell membranes in the eye lens leads to chol saturation and formation of cholesterol bilayer domains (cbds). it is hypothesized that high enrichment in cholesterol helps to maintain lens transparency and protect against cataractogenesis. in model studies, the cbd is formed in a phospholipid bilayer when cholesterol content exceeding the cholesterol solubility threshold, thus, the cbd is surrounded by phospholipid bilayer saturated with cholesterol. in the present study, we carried out molecular dynamics (md) simulation of two bilayers: a palmitoyloleoylphosphatidylcholine (popc) bilayer (reference) and a : popc-chol bilayer, to investigate the smoothing effect of the saturating amount of cholesterol on the bilayer. to our knowledge, this effect has not been studied so far so this study is certainly providing new results. our results indicate that saturation with cholesterol significantly narrows the distribution of vertical positions of the center-of-mass of the popc molecules and the popc atoms in the bilayer and smoothes the bilayer surface. we hypothesize that this smoothing effect decreases light-scattering and helps to maintain lens transparency. the phospholipid content of staphylococcus aureus membranes displays a high degree of variability ( - ). the major phospholipids found in s. aureus are phosphatidylglycerol (pg), cardiolipin (cl) and lysylphosphatidylglycerol (lpg), ( ) the concentrations of which are environment dependent and see fluctuations on exposure to high concentrations of positively charged moieties ( ) . up regulation of lpg has a suspected role in neutralisation of the plasma membrane in response to cationic threats. studies have been conducted to probe biomimetic models of this theory however our focus is to look at atomic details of membrane extracts in the presence of magaininf w. s. aureus lipid extracts from cells grown at ph . and . , studies by neutron diffraction with and without peptide at two contrasts. d-spacings were assessed by vogt area fitting and bragg's law. bilayer separation at low ph was * - Å less than ph . . with peptide, bilayer separations of ph . and . extracts were reduced by * Å and * Å , respectively. reduced pg content of low ph extracts is suggested to reduce d-spacing, however presence of peptide further reduces this, possibly by an anion neutralisation effect. abnormal d-spacing on increased humidity may be due to the breakdown of lpg. activation of neutrophils releasing hocl and apoptosis of vein endothelial cells are the events documented to occur in the course of atherosclerosis. as lipid chlorohydrins, which are the key products of the reaction between hocl and unsaturated fatty acid residues, were found in atherosclerotic plaques, we decided to check their biological activity in the context of their ability to act as the mediators of hoclinduced oxidative stress and apoptosis in the culture of immortalized human umbilical vein endothelial cells (hu-vec-st). the concentration of reactive oxygen species was found to be elevated after h cell incubation with phospahtidylcholine chlorohydrins. this effect was at least partially caused by the leakage of superoxide anion from mitochondria and followed by depletion of gsh and total thiols. the significant decrease of antioxidant capacity of cell extracts was also observed. the intracellular red-ox imbalance was accompanied by the increase of the ratio between phosphorylated and dephosphorylated forms of p map kinase. after longer incubation a significant number of apoptotic cells appeared. summing up, phosphatidylcholine chlorohydrins may be regarded as signaling molecules, able to initiate signalling pathways by induction of oxidative stress. giant unilamellar vesicles (guvs) are a valuable tool in the study of lateral distribution of biological membrane components. guv dimensions are comparable to typical cell plasma membranes and lipid phase separation can be observed through fluorescence microscopy. guv studies frequently require immobilization of the vesicles, and several methods are available for that effect. one of the most common methodologies for vesicle immobilization is the use of avidin/streptavidin coated surfaces and biotin labeled lipids at very low concentration in the vesicles. here, we analyze the effect of using this methodology on lipid domain distribution for different lipid compositions. we show that as a result of non-homogeneous distribution of biotin labeled lipids between liquid disordered, liquid ordered and gel phases, distribution of lipid domains inside guvs can be dramatically affected. monitoring membrane permeability: development of a sicm approach christoph saßen , and claudia steinem institute for organic and biomolecular chemistry, university of gö ttingen, tammannstraße , gö ttingen, germany, ggnb doctoral program: imprs, physics of biological and complex systems scanning ion conductance microscopy (sicm) utilises a nanopipette containing an electrode as a probe for surface investigations with resolutions of / of the inner pipette diameter. experiments are conducted under physiological conditions, in situ and without mechanical contact of probe and sample. hence, sicm serves as a well-suited technique for the investigation of soft objects such as cells or artificial lipid membranes. using pore-suspending membranes (psm) as a model system, interactions of melittin as an example for cell penetrating peptides (cpps) and lipid membranes are investigated by means of sicm. formation of a range of solvent free psm from lipid vesicles has been achieved as confirmed by means of fluorescence microscopy and sicm. application of melittin results in rupturing of the lipid bilayer. putative insights gained from this assay are critical concentrations of membrane permeabilising ccps and answers to mechanistic questions, e.g. whether ccps translocate only or form pores within the lipid bilayer. positioning of the z-ring in escherichia coli prior to cell division is regulated by intracellular pole-to-pole oscillation and membrane binding of min proteins, allowing assembly of ftsz filaments only at the center plane of the cell. in order to investigate the influence of membrane geometry on the dynamic behavior of membrane binding of min proteins, we combined concepts of synthetic biology and microfabrication technology. glass slides were patterned by a gold coating with microscopic windows of different geometries, and supported lipid bilayers (slb) were formed on these microstructures. on slbs, min-proteins organize into parallel waves. confinement of the artificial membranes determined the direction of propagation.min-waves could be guided along curved membrane stripes, in circles and even along slalom-geometries. in elongated membrane structures, the protein waves always propagate along the longest axis. coupling of protein waves across spatially separated membrane patches was observed, dependent on gap size and viscosity of the aqueous media above the bilayer. this indicates the existence of an inhomogeneous and dynamic protein gradient above the membrane. minimal systems for membrane associated cellular processes petra schwille bio technology center biotec, technical university of dresden, germany the strive for identifying minimal biological systems, particularly of subcellular structures or modules, has in the past years been very successful, and crucial in vitro experiments with reduced complexity can nowadays be performed, e.g., on reconstituted cytoskeleton and membrane systems. in this overview talk, i will first discuss the virtues of minimal membrane systems, such as guvs and supported membranes, in quantitatively understand protein-lipid interactions, in particular lipid domain formation and its relevance on protein function. membrane transformations, such as vesicle fusion and fission, but also vesicle splitting, can be reconstituted in these simple subsystems, due to the inherent physical properties of selfassembled lipids, and it is compelling question how simple a protein machinery may be that is still able to regulate these transformations. as an exciting example of the power of minimal systems, i show how the interplay between a membrane and only two antagonistic proteins from the bacterial cell division machinery can result in emergence of protein self-organization and pattern formation, and discuss the possibility of reconstituting a minimal divisome. quantitative microscopic analysis reveals cell confluence regulated divergence of pdgfr-initiated signaling pathways with logically streamlined cellular outputs Á rpá d szö } or, lá szló ujalky-nagy, já nos szö ll} osi, gyö rgy vereb university of debrecen, department of biophysics and cell biology, debrecen, hungary platelet derived growth factor receptors (pdgfr) play an important role in proliferation and survival of tumor cells. pdgf-bb stimulation caused a redistribution of pdgf receptors towards gm rich domains, which was more prominent in confluent monolayers. pdgf-bb stimulation significantly increased relative receptor phosphorylation of the ras / mapk pathway specific tyr residues and the pi -kinase / akt pathway specific tyr residues in nonconfluent cultures. tyr residues that serve as adaptors for ras-gap which inactivates the mapk pathway and tyr residues feeding into the plc-gamma / camk-pkc pathway were the docking sites significantly hyperphosphorylation following ligand stimulation in confluent cells. we found that p-akt facilitated cell survival and pmapk dependent proliferation is more activated in dispersed cells, while phospholipase c-gamma mediated calcium release and pkc-dependent rhoa activation are the prominent output features pdgf stimulus achieves in confluent cultures. these observations suggest that the same stimulus is able to promote distinctly relevant signaling outputs, namely, cell division and survival in sparse cultures and inhibition of proliferation joined with promotion of migration in confluent monolayers that appear contact inhibited. a thermodynamic approach to phase coexistence in ternary cholesterol-phospholipid mixtures jean wolff, carlos m. marques and fabrice thalmann institut charles sadron, université de strasbourg, cnrs upr, , rue du loess, strasbourg cedex, f- , france e-mail: thalmann@ics-cnrs.unistra.fr we present a simple and predictive model for describing the phase stability of ternary cholesterol-phospholipid mixtures. assuming that competition between the liquid and gel organizations of the phospholipids is the main driving force behind lipid segregation, we derive a phenomenological gibbs free-energy of mixing, based on the calorimetric properties of the lipids main transition. gibbs phase diagrams are numerically obtained that reproduces the most important experimental features of dppc-dopc-chol membranes, such as regions of triple coexistence and liquid orderedliquid disordered segregation. based on this approach, we present a scenario for the evolution of the phase diagram with temperature. results for other phospholipid species, such as popc or psm will also be presented. interleukin- and - receptors play a central role in the activation, survival and death of t lymphocytes. they form supramolecular clusters with mhc i and ii glycoproteins in t cells. in damaged or inflamed tissues the extracellular k+ concentration increases, which can depolarize the membrane. the common signaling beta and gamma chains of il- / r are phosphorylated upon cytokine binding and get a permanent dipole moment, thus their conformation, interactions, mobility and activity may be sensitive to the membrane potential. we induced depolarization on ft . t lymphoma cells by increasing the ec. k+ level or by blocking kv . voltage gated k+ channels with margatoxin. fcs measuremens showed that the lateral mobility of fab-labeled il- / r and mhc i and ii decreased upon depolarization, while that of gpi-linked cd did not change. fret efficiency measured between some elements of the il-receptor/mhc cluster increased, which may reflect an increase of cluster size. il- -induced receptor activity, as monitored by measuring stat -phosphorylation, increased upon depolarization, whereas il- induced phosphorylation did not change. our results may reveal a novel regulatory mechanism of receptor function by the membrane potential. cytokines play an important role in t cell activation and immunological memory, whereas mhcs are known for the role in antigen presentation. we applied rnai to silence the expression of mhc i in order to study its possible role in receptor assembly and function. fret data indicated that the association of il- r and il- r with mhc i as well as between il- r and il- r weakened. fcs indicated an increase of receptor mobility also suggesting the partial disassembly of the clusters. mhc i gene silencing lead to a remarkable increase of il- /il- induced phosphorylation of stat transcription factors. in search for the molecular background of this inhibition of signaling by mhc i we checked il- binding and the formation of the receptor complex (il- r alpha -il- r beta association), but we did not find a difference as compared to the control. our results suggest that mhc i plays an organizing role in maintaining supramolecular receptor clusters and inhibits il- r signaling, revealing a nonclassic new function of mhc i beyond its classical role in antigen presentation. interleukin- (il- ) is an important cytokine involved in adaptive immunity. il- binds with high affinity the singlepass transmembrane receptor il- ra. the occupied complex, il- /il- ra then engages either il- rc or il- ra , to form an activated type i or ii receptor, respectively. this formation of heterodimers is believed to trigger cross-activation of intracellular janus kinases. here we follow a fluorescently labeled ligand through various stages of receptor activation in hek t: using fluorescence correlation spectroscopy (fcs), we see that the receptor chains diffuse as monomers within the plasma membrane. using dual-color fccs provides direct evidence for ligand induced co-diffusion of occupied il- ra and il- ra . in contrast, type i complexes containing il- rc could not be observed. however, ectopic expression of gfp-tagged il- rc/jak induced stable fluorescent speckles in or close to the plasma membrane. we identified these structures as early sorting endosomes by colocalization of surface markers like eea and rab gtpases. the il- ra chain is continuously trafficking into these compartments. these observations suggest that the formation of a type i il- r heterodimer may require internalization and that early endosomes serve as a platform for il- signaling. among the membrane associated proteins, the ras family, which is lipid-anchored g protein, plays a key role in a large range of physiological processes and, more importantly, is deregulated in a large variety of cancer. in this context, plasma membrane heterogeneity appears as a central concept since it ultimately tunes the specification and regulation of ras-dependent signaling processes. therefore, to investigate the dynamic and complex membrane lateral organization in living cells, we have developed an original approach based on molecule diffusion measurements performed by fluorescence correlation spectroscopy at different spatial scales (spot variable fcs, svfcs) ( ). we have shown in a variety of cell types that lipidbased nanodomains are instrumental for cell membrane compartmentalization. we have also observed that thesenanodomains are critically involved in the activation of signaling pathways and are essential for physiological responses ( - ). more recently, weextend the application of svfcs to characterizethe dynamics of k-rasproteinat the plasma membrane. as major result, we demonstrated that the rate of k-ras association/dissociation from the membrane is fast but vary as a functional of the activation state of the molecule as well as of specific intracellular protein interactions. we have so demonstrated that an helical lid sub-domain in the sbd is essential for monomeric as binding, but not for the anti-aggregation activity of the chaperone, suggesting that hsp is able to interact with pre-fibrillar oligomeric species formed during as aggregation and that, then, the mechanism of binding for these species is different from that of the monomeric protein. aggregation of the acylphosphatase from sulfolobus solfataricus (sso acp) into amyloid-like protofibrils is induced by the establishment of an intermolecular interaction between a -residue unfolded segment at the n-terminus and the globular unit of another molecule. we have used data from hydrogen/deuterium exchange experiments, intermolecular paramagnetic relaxation enhancements and isothermal titration calorimetry measurements on an aggregation-resistant sso acp variant lacking the -residue n-terminus to characterize the initial steps of the aggregation reaction. under solution conditions that favour aggregation of the wild-type protein, the truncated protein was found to interact with a peptide corresponding to the n-terminal residues of the full length protein. this interaction involves the fourth strand of the main b-sheet structure of the protein and the loop following this region and induces a slight decrease in protein flexibility. we suggest that the amyloidogenic state populated by sso acp prior to aggregation does not present local unfolding but is characterized by increased dynamics throughout the sequence that allow the protein to establish new interactions, leading to the aggregation reaction. amyloid-like aggregates alter the membrane mobility of gm gangliosides martino calamai and francesco pavone university of florence, lens -european laboratory for non-linear spectroscopy, sesto fiorentino, florence, italy neuronal dysfunction in neurodegenerative pathologies such as alzheimer's disease is currently attributed to the interaction of amyloid aggregates with the plasma membrane. amongst the variety of toxic mechanisms proposed, one involves the binding of amyloid species to gm gangliosides. gm takes part into the formation of membrane rafts, and exerts antineurotoxic, neuroprotective, and neurorestorative effects on various central neurotransmitter systems. in this study, we investigated the effects of amyloid-like aggregates formed by the highly amyloidogenic structural motif of the yeast prion sup (sup nm) on the mobility of gm on the plasmamembrane of living cells. preformed sup nm aggregates were incubated with cells and gm molecules were subsequently labeled with biotinylated ctx-b and streptavidin quantum dots (qds). single qds bound to gm were then tracked. the mobility of gm was found to decrease dramatically in the presence of sup nm aggregates, switching from brownian to mainly confined motion. the considerable interference of amyloid-like aggregates with the lateral diffusion of gm might imply a consequent loss of function of gm , thus contributing to explain the toxic mechanism ascribed to this particular interaction. insights into the early stages of fibrillogenesis of insulin using mass spectrometry harriet l. insulin is a vital hormone in metabolic processes as it regulates the glucose levels in the body. insulin is stored in the b cells of the pancreas as a hexamer, however its biologically active form is the monomer. the formation of fibrillar aggregates of insulin rarely occurs in the body; however localised amyloidosis at the site of injection for diabetes patients and aggregation of pharmaceutical insulin stocks present problems. in the current study oligomers formed early in the process of fibril assembly in vitro are observed by mass spectrometry (ms). ms is the only technique which allows early species to be characterised as it can identify different oligomeric orders by mass to charge ratio and show protein abundance and aggregation propensity. on mobility ms is used to examine rotationally averaged collision cross sections of oligomers in the aggregating solution. a wide array of oligomers is observed and the stability of specific species is remarked. the presence of multiple conformations for the highly charged oligomers is particularly noted and their assignment confirmed using fourier transform ion cyclotron resonance ms and collision induced dissociation. molecular modelling has been used to further explore the conformational space the oligomers inhabit. amyloid fibrils consisting of different proteins have been recognized as an accompanying feature of several neurodegenerative diseases. many proteins without known connection to any diseases have been found to form amyloid fibrils in vitro, leading to suggestion that the ability to form fibrils is the inherent property of polypeptide chain. the observed common character of protein amyloid formation enables to seek further clues of fibrillation mechanism by studying generalized sequenceless polypeptide models, e.g. polylysine. we have studied conformational transitions of polylysine, with different chain length at various ph, ionic strength and temperature by means of novel approachviscometric method. this polypeptide undergoes alfa-helix to beta-sheet transition and forms amyloid fibrils in special conditions. temperature induced a-helix to b-sheet transitions occurs at ph interval form to . and with increasing chain length is slightly shifted to the lower ph. we have found narrow ph interval, in which the thermal transition is fully reversible, suggesting the high sensitivity of polypeptide conformation on subtle changes in charge on its side chains. this work was supported within the projects vega , and , cex sas nanofluid and by esf project no. . understanding the mechanisms of the conversion from the native state of a protein to the amyloidal state represents a fundamental step in improving the purification, storage and delivery of protein-based drugs and it is also of great relevance for developing strategies to prevent in vivo protein aggregation. amyloid fibrils have a structural arrangement of cross b-sheet but they can also experience different packing into three dimensional superstructures, i.e. polymorphism. it is well known that, among others, both the geometric confinement of the molecules and shear forces can affect the final morphology of the aggregates. importantly, due to the complexity and crowding of the cellular region, such parameters also play a crucial role in in vivo processes. we present an experimental approach to study in vitro amyloid aggregation in a controlled and uniform shear force field and within microscale environments. in particular we focus on the effect of these two parameters on the formation of spherical aggregates, known as spherulites. using micro channels of different cross-sections from to lm x lm and flow rates in the range of hundreds of ll/min, the number and diameter of spherulites within the channels have been characterized using crossed polarizers optical microscopy. inhibition of insulin amyloid fibrillization by albumin magnetic fluid k. insulin amyloid aggregation causes serious problems for patient with insulin dependent diabetes undergoing long-term treatment by injection, in production and storage of this drug and in application of insulin pumps. recent studies indicate that protein amyloid aggregation causes the cell impairment and death; however, the prevention of amyloid aggregation is beneficial. we have investigated ability of albumin magnetic fluid (amf) to inhibit insulin amyloid aggregation by spectroscopic and microscopic techniques. albumin magnetic fluid consists of magnetic fe o nanoparticles sterically stabilized by sodium oleate and functionalized with bovine serum albumin (bsa) at various weight ratios bsa/fe o . we have found the positive correlation between inhibiting activity of afm and nanoparticle diameter and zeta potential. the ability of amf to inhibit formation of amyloid fibrils exhibits concentration dependence with ic values comparable to insulin concentration. the observed features make amf of potential interest as agents effective in the solving of problems associated with insulin amyloid aggregation. (this work was supported within the projects vega , and , cex sas nanofluid, apvv- - , sk-ro- - and esf project ). amyloid formation of peptides causes diseases like alzheimer's and parkinson's disease. however, the conditions for the onset of the neurotoxic beta-sheet formation are poorly understood. we focus on aggregation triggers and their interplay: interactions with hydrophobic-hydrophilic interfaces, orientation of peptides in d, metal ion complexation and lipid layers. the tailor-made model peptides exhibit defined secondary structure propensities and metal ion binding sites. the interactions of the peptide with the air-water interface and with metal ions are studied using surface sensitive methods connected to film balance measurements. x-ray diffraction, x-ray reflection, infrared reflection-absorption spectroscopy and total reflection x-ray fluorescence were applied to reveal the layer structure, peptide conformations and metal ion binding at the interface. we found that amyloid formation in d is dominated by the hydrophobic-hydrophilic interface and not comparable to the bulk behaviour. the interface can enhance or inhibit betasheet formation. the effect of metal ion complexation depends on the arrangement of the binding sites in the peptide and the preferred metal complexation geometry. the two triggers interface and metal ion complexation, can oppose each other. effect of apoe isoform and lipidation status on proteolytic clearance of the amyloid-beta peptide hubin, e. alzheimer's disease (ad) is the most common type of dementia in the elderly. the most important genetic risk factor identified for ad is the isoform, e , e or e , of apolipoprotein e (apoe), a lipid-carrying protein. one hallmark of ad is the accumulation of amyloid-beta peptide (ab) in the brain which is thought to result from an imbalance between the production of ab and its clearance. previous studies report an important role for apoe in ab degradation. we sought to determine the effect of apoe isoform and lipidation status on the degradation of soluble ab by proteinases such as insulin-degrading enzyme and neprilysin. in this study an in vitro ab clearance assay based on the competition between ab and a fluorogenic peptide substrate is developed to quantify ab degradation. to elucidate the proteolytic clearance mechanism, the fragments resulting from cleavage are identified by mass spectrometry and further analyzed to identify the interacting stretch of the ab sequence with the different apoe isoforms. the results suggest that apoe influences the rate of ab degradation. the aggregation of proteins into fibrillar nanostructures is a general form of behaviour encountered for a range of different polypeptide chains. the formation of these structures is associated with pathological processes in the context of alzheimer's and parkinson's diseases but is also involved in biologically beneficial roles which include functional coatings and catalytical scaffolds. this talk focuses on recent work directed at understanding the kinetics of this process through the development and application of experimental biophysical methods and their combination with kinetic theories for linear growth phenomena. lbs contains not only as, but also other proteins including - - proteins. - - proteins exist mainly as a dimer and its exact functions are remain unclear. however, recent work has shown that the association of - - (eta) with as in lbs. herein we show how - - (eta) can modulate as in vitro aggregation behavior, by rerouting it toward the formation of stable non-fibrillar aggregates. we also show that the resulting populations of fibrillar and pre-fibrillar aggregates exhibit a modified toxicity in vivo with respect to the unperturbed aggregates. interestingly, - - (eta) does not show any binding affinity for monomeric as, nor for the mature fibrillar aggregates. we provide evidence that it acts on the oligomeric species which form during the amyloidogenesis process of aggregation. since - - (eta) can influence the toxicity of amyloidogenesis without perturbing the functional as monomers, we are convinced that once fully understood, its mode of action could represent a promising model to mimic with synthetic drugs and peptides. what makes an amyloid toxic: morphology, structure or interaction with membranes? more than human diseases are related to amyloids. in order to understand why some amyloids may become toxic to their host and some others are not, we first developed a genetical approach in the yeast saccharomyces cerevisiae. we have chosen the amyloid/prion protein het-s prion forming domain ( - ) from the fungi podospora anserina, which is not toxic in yeast. some toxic amyloids mutants were generated by random mutagenesis. in vitro the most toxic mutant called ''m '' displays very peculiar nanofibers, which polymerized mainly in amyloid antiparallel b-sheets whereas the non-toxic wt exhibits a parallel polymerization. we further established the dynamic of assembling of the m toxic amyloid, in comparison to the wt non-toxic amyloid, and showed the presence of specific oligomeric intermediates also organized in antiparallel b-sheet structures. a more global structure/toxicity study on more than mutants clearly identified an antiparallel b-sheet signature for all the toxic mutants. therefore size, intermediates and antiparallel structures may account for amyloid toxicity in yeast but we still wonder what their cellular targets are. recently, we established the first evidences that toxic mutants may specifically bind in vitro to lipids, particularly negatively charged. interconnected mechanisms in abeta ( - ) we present an experimental study on the fibril formation of ab( - ) peptide at ph . . the kinetics of this process is characterized by the occurrence of multiple transient species that give rise to final aggregates whose morphology and molecular structure are strongly affected by the growth conditions. to observe in details the aggregation pathway as a function of solution conditions, we have used different experimental techniques as light scattering, thioflavin t fluorescence, circular dichroism and two-photon fluorescence microscopy. this approach gives information on the time evolution of conformational changes at molecular level, on the aggregates/fibrils growth and on their morphologies. the selected experimental conditions allowed us to highlight the existence of at least three different aggregation mechanisms acting in competition. a first assembly stage, which implies conformational conversion of native peptides, leads to the formation of small ordered oligomers representing an activated conformation to proceed towards fibril growth. this process constitutes the rate limiting step for two distinct fibril nucleation mechanisms that probably implicate spatially heterogeneous mechanisms. the formation of amyloid fibrils of amylin - was studied by means of molecular dynamics (md) and energy partition on three peptide ß-sheet stack systems with the same amino acid composition: wild type amylin - (amyl - ), reverse amylin - (rev-amyl - ) and scrambled amylin version scr-amyl - . the results show that for amylin - peptides, amino acid composition determines the propensity of a peptide to form amyloid fibrils independent of their sequence. the sequence of amino acids defines the shape and the strength of amyloid protofibril, which conforms with the atom force microscope (afm) data [ ] . md show that the x revamyl- - stack has looser selfassembly than the x amyl- - stack, which conforms with the results of fourier transform infrared spectroscopy (ftir) measurements for the peptides studied [ ] . the results of md show that x amyl- - could have a turn, which consists with ftir data [ ] . data on ab aggregation kinetics have been characterized by a large spread between experiments on identical samples that contain so many molecules that stochastic behaviour is difficult to explain unless caused by uncontrolled amounts of impurities or interfaces. we have therefore spent considerable effort to eliminate sources of inhomogeneity and reached a level of reproducibility between identical samples and between experiments on separate occasions that we can now collect data that can lead to mechanistic insights into the aggregation process per se, and into the mechanism of action of inhibitors. data on ab aggregation will be shown that give insight into the influence of physical parameters like peptide concentration, shear and ionic strength, as well as the effect of inhibitory proteins, model membranes and the effects of sequence variations. monte carlo simulations of amyloid formation from model peptides corroborate the finding from experiments and underscore that the very high level of predictability and reproducibility comes from multiple parallel processes. negatively-charged membranes were reported to catalyze ''amyloid-like'' fiber formation by non-amyloidogenic proteins [ ] . our study aims to elucidate the factors that govern the formation of these amyloid-like fibers. lysozyme was selected as a model of non-amyloidogenic protein and was fluorescently-labeled with alexa fluor (a -lz). first, a -lz partition towards phosphatidylserine-containing liposomes was characterized quantitatively using fluorescence correlation spectroscopy (fcs), in order to calculate the protein coverage of liposomes. secondly, the interaction between a -lz and negatively-charged lipid membranes was studied using both steady-state and time-resolved fluorescence techniques. this interaction was found to switch from a peripheral binding to the anionic headgroups, at high lipid/ protein molar ratio (l/p), to a partial insertion of protein into the hydrophobic core of the membrane, at low l/p. finally, the lipidprotein supramolecular complexes formed at low l/p were characterized by fluorescence lifetime imaging microscopy (flim). the mean lifetime of a -lz in these supramolecular structures is much lower compared to the values obtained for the free and bound a -lz at high l/p. the fiber characterization will be complemented by fcs studies. [ the conversion of normal prp c to its pathological isoform prp sc is a key event in prion diseases and is proposed to occur at the cell surface or more probably in acidic late endosomes. a convergence of evidence strongly suggests that the early events leading to the structural conversion of the prp seem to be in relation with more or less stable soluble oligomers, which could mediate neurotoxicity. as commonly shared by other amyloidogenic proteins, membrane-bound monomers undergo a series of lipid-dependent conformational changes, leading to the formation of oligomers of varying toxicity rich in b-sheet structures (annular pores, amyloid fibrils). here, we have used a combination of biophysical techniques (dynamic and static light scattering, fluorescence techniques, and quartz crystral microbalance) to elucidate the interaction of native monomeric prp and that of purified b-rich oligomeric prp on model lipid membranes. under well established conditions, three b-sheet-rich oligomers were generated from the partial unfolding of the monomer in solution, which were found to form in parallel. from single mutation and/or truncation of the full length prp, the polymerization pathway is strongly affected, revealing the high conformational diversity of prp. in our previous work, we identify the minimal region of the prp protein leading to the same polymerization pattern of the full length prp. soluble -subunits and -subunits oligomers were obtained depending on the single mutation or truncation and purified. we compare their structural properties (ftir, cd) when associated with anionic lipid bilayers and study their propensities to permeabilize the membrane. fluorescence kinetics suggest different mechanisms of membrane perturbation for the monomer and the prp oligomers. deciphering this complex network of lipid interactions and conformational diversity of the prp protein will help for understanding of how amyloidogenic proteins induce neurotoxicity. the traditional view of the lipid bilayer described as a ''sea'' of lipids where proteins may float freely, is going to be inadequate to describe the increasingly large number of complex phenomena which are known to take place in biological membranes. membrane-assisted protein-protein interactions, formation of lipid clusters, protein-induced variation of the membrane shape, abnormal membrane permeabilities and conformational transitions of membrane-embedded proteins are only a few examples of the variegated ensemble of events whose tightly regulated cross-talk is essential for cell structure and function. experimental work on the above mentionated problems is very difficult and some time not accessible, especially when the studied systems have a fast dynamics. due to the large size of the systems usually involved in this multifaceted framework, a detailed molecular description of these phenomena is beyond the possibilities of conventional amyloid aggregation, a generic behavior of proteins, is related to incurable human pathologies -amyloid-related diseases, associated with formation of amyloid deposits in the body. all types of amyloid aggregates possess rich bsheet structural motif. the recent data confirm the toxic effect of aggregates on the cells, however, it was found that reduction of amyloid aggregates plays important role in prevention as well as therapy of amyloidosis. we have investigated effect of phytoalexin derivatives on amyloid fibrillization of two proteins, human insulin and chicken egg lysozyme, by tht and ans fluorescence assays. we have found that amyloid aggregation of both studied proteins was significantly inhibited by phytoalexin derivates cyclobrassinin and benzocamalexin. for most effective phytoalexins the estimated ic values were at low micromolar concentration. the observed inhibiting activity was confirmed by transmission electron microscopy. our data suggest the potential therapeutic use of the most effective phytoalexins in the reduction of amyloid aggregation. (this work was supported within the projects vega , , cex sas nanofluid, apvv- - , sk-ro- - and esf project ). the amyloid pore hypothesis suggests that interactions of oligomeric alpha-synuclein (as) with membranes play an important role in parkinson's disease. oligomers are thought to permeabilize membranes and interfere with ca + pathways. permeabilization by as requires the presence of negatively charged phospholipids. whether as can bind and permeabilize membranes with physiologically relevant lipid compositions has not been extensively explored. here we report on the binding of as to giant unilamellar vesicles (guvs) with physiologically relevant lipid compositions. comparing different protocols of oligomer preparation, leakage assays on both large unilamellar vesicles (calcein release) and guvs (hpts efflux assay) show that as is not able to permeabilize these membranes. the presence of cholesterol has a stabilizing effect on these membrane systems. in agreement with these findings, we do not observe concentration dependent as toxicity using in vivo mts assays. however, in the calcein release assay, different as preparations show differences in kinetics and as concentrations that cause % leakage. these results motivate us to critically reassess the amyloid pore hypothesis, and suggest that membrane permeabilization may be attributable only to a very specific as species. alpha-synuclein oligomers impair membrane integrity-a mechanistic view martin t. stö ckl, mireille m. a. e. claessens, vinod subramaniam nanobiophysics, mesa+ institute for nanotechnology, university of twente, enschede, the netherlands one of the most prevalent neurodegenerative diseases is parkinson's disease (pd), which is accompanied with the loss of dopaminergic neurons. although the mechanisms leading to the death of these cells are still unclear, the protein alpha-synuclein (as) is one of the pivotal factors. previous studies indicate that especially oligomeric forms of as show a detrimental effect on membrane integrity. as an intact membrane is crucial to many cellular processes, the impairment of the membrane integrity is a likely pathway for neuronal death. we use different phospholipid bilayer model systems to investigate the mechanisms underlying this process. atomic force microscopy in combination with suspended asymmetric phospholipid bilayers, which closely mimic the plasma membrane, allows the identification of the binding sites, the measurement of penetration depths of the as oligomers into the phospholipid bilayer, and the detection of membrane thinning or creation of membrane defects. using an approach based on phospholipid vesicles we were able to observe for the first time that as oligomers cause an enhanced lipid-flip flop. suggesting that the loss of lipid asymmetry is a novel mechanism which may contribute to or trigger neuronal death in pd. amyloid protein-membrane interactions and structuretoxicity relationship study h.p. ta (toxic) has a much higher and more specific effect on negatively charged phospholipids (dops, dopi and dopg) than in the case of wt (non-toxic). therefore the insertion of protein into phospholipid monolayers, which occurred similarly for both wt and m , is not a key factor for these effects (h.p. ta et al. langmuir, in press). we are now using unilamellar vesicles as a membrane model to investigate the amyloid protein (toxic and nontoxic) -phospholipid interactions. results confirmed the high specific and strong effects of m on negatively-charged membrane. in this project, we study the chemical, physical and biological properties of fibrillar networks. the formation and the network mechanics are investigated by combining droplet-based microfluidics with optical microscopy and small angle x-ray scattering (saxs). the chosen system, fibrin network formation, plays an important role in blood coagulation processes. crosslinking of fibrinogen induced by an enzymatic reaction with thrombin leads to d fibrin network formation. the fibrillar networks are formed within picoliter droplets of aqueous solutions in an continuous oil phase. droplets containing fibrinogen and thrombin can be produced of different sizes and stored for fibrin network formation. the formation of the fibrillar networks is imaged by fluorescence microscopy. to analyze the elastic properties of the networks, the droplets flow through a microchannel device of alternating width in order to squeeze and stretch the networks. additionally, saxs experiments will give structural information about the molecular dimensions of the networks. the amyloid beta peptide (ab), implicated in alzheimer's disease (ad), is released from the amyloid precursor protein (app) by secretase-induced cleavage. this process results in the release of a range of ab peptides varying in length. the brains of ad patients often contain longer ab peptides while the total concentration of ab is unaffected. longer peptides are more hydrophobic having far-reaching consequences for their toxicity and aggregation. as ab is necessary for normal neuronal function, research activities into ad therapeutic development currently explore the possibilities of modulating c-secretase activity to produce short ab peptides. whether such an approach effectively ameliorates the toxic effect of ab has not been explored yet. to answer this question, we studied the impact of heterogeneity in ab pools in an in vitro biophysical and in cellulo context using microelectrode array to assay the synaptic activity of primary neurons. we show that various lengths of the ab peptide and mixtures thereof aggregate with distinct kinetics and notoriously affect synaptotoxic and cytotoxic response. we also show that small amounts of less abundant peptides ab and ab induce aggregation and toxicity of ab while the behavior of ab is unaffected. one of the most important irreversible oxidative modifications of proteins is carbonylation, a process of introducing the carbonyl group in reaction with reactive oxygen species. importantly, carbonylation increases with the age of cells and is associated with the formation of intracellular protein aggregates and the pathogenesis of age-related disorders such as neurodegenerative diseases and cancer. however, it is still largely unclear how carbonylation affects protein structure, dynamics and aggregability on the atomic level. here, we use classical molecular dynamics simulations to study structure and dynamics of the carbonylated headpiece domain of villin, a key actin-organizing protein. we perform an exhaustive set of molecular dynamics simulations of native villin headpiece together with every single combination containing carbonylated versions of its seven lysine, arginine and proline residues, the quantitatively most important carbonylable amino acids. surprisingly, our results suggest that high levels of carbonylation, far above those associated with cell death in vivo, may be required to destabilize and unfold protein structure through the disruption of specific stabilizing elements, such as salt bridges or proline kinks, or tampering with the hydrophobic effect. on the other hand, by using thermodynamic integration and molecular hydrophobicity potential approaches, we quantitatively show that carbonylation of hydrophilic lysine and arginine residues is equivalent to introducing hydrophobic, chargeneutral mutations in their place, and, by comparison with experimental results, demonstrate that this by itself significantly increases intrinsic aggregation propensity of both structured, native proteins and their unfolded states. finally, our results provide a foundation for a novel experimental strategy to study the effects of carbonylation on protein structure, dynamics and aggregability using site-directed mutagenesis. septins are an evolutionarily conserved family of gtp-binding proteins involved in important cellular processes, such as cytokinesis and exocytosis, and have been implicated in neurological diseases, such as alzheimer's and parkinson's diseases. the focus of this study was two septins of schistosoma mansoni, (the causative agent of schistosomiasis in south america) named smsept and smsept , which were produced in a recombinant system. our objective was to verify if these septins from a simpler organism display similar characteristics to human septins. analysis of protein structure by circular dichroism showed that both recombinant smseptins produced were folded. the gtpase activity assay showed that smsept was able to hydrolyze gtp, whereas smsept was not. aggregation studies for amyloid fibril detection by right angle light scattering and thioflavin t fluorescence assay were performed. both proteins showed a temperature dependent increase in light scattering and fluorescence emission of tht probe. this indicated that s. mansoni septins tend to aggregate into amyloid-like fibers in high temperatures, with thresholds of °c for smsept and °c for smsept . these results are in accordance to that previously reported for human septins. in our work we investigated the response to standard chemotherapy of blood lymphocytes of patients suffering with melanoma. dna single and double strand breaks were determined using comet assay; intracellular levels of marker proteins were detected using immunocytochemistry. ultimately this set of parameters allows to characterize two mechanisms of dna repair (base excision repair, ber and mismatch repair, mmr) which together with apoptosis proneness underlie response of tumor cells to chemotherapy. cell death caused by o mg adducts is promoted by mmr system by inducing unrepaired double strand breaks in dna. there was a linear correlation between the level of dsdna breaks in lymphocytes after -st cycle of chemotherapy and mmr efficiency in them. the level of double strand breaks in dna after -st cycle of chemotherapy is predictive of clinical outcome. otherwise damage at the level of ssdna (ap-sites and single strand breaks) and ber mechanism associated with it couldn't be a good prognostic factor of chemotherapy. high level of double strand breaks in dna in blood lymphocytes of melanoma patients hours after -st cycle of chemotherapy appears to be a marker of a good prognosis. self-assembly and stability of g-quadruplex: counterions, pressure and temperature effects e. baldassarri jr., p. mariani, f. spinozzi, m. g. ortore saifet dept. & cnism, marche polytechnic university, ancona, italy the important role of g-quadruplex in biological systems is based on two main features: composition and stability of telomeres, and activity of telomerase. the g-quadruplex structures are formed by supramolecular organization of basic units called g-quartets that are planar rings constituted by four guanosines linked by hoogsten hydrogen bonds. gquadruplex requires the presence of monovalent cations playing a key role in stabilizing these structures, since they give rise to coordination bonds needed for the stacking of more tetrads. we performed x-ray diffraction experiments at different pressures (ranging from to bar), and small angle x-ray scattering (saxs) changing the temperature (between - °c retinoic acid receptor (rar) is a member of the nuclear receptor superfamily. this ligand-inducible transcription factor binds to dna as a heterodimer with the retinoid x receptor (rxr) in the nucleus. the nucleus is a dynamic compartment and live-cell imaging techniques make it possible to investigate transcription factor action in real-time. we studied the diffusion of egfp-rar by fluorescence correlation spectroscopy (fcs) in order to uncover the molecular interactions determining receptor mobility. in the absence of ligand we identified two distinct species with different mobilities. the fast component has a diffusion coefficient of d = . - lm /s corresponding to small oligomeric forms, whereas the slow component with d = . - . lm /s corresponds to interactions of rar with the chromatin or other large structures. the rar ligand binding domain fragment also has a slow component probably as a result of indirect dna-binding via rxr, with lower affinity than the intact rar:rxr complex. importantly, rar-agonist treatment shifts the equilibrium towards the slow population of the wild type receptor, but without significantly changing the mobility of either the fast or the slow population. by using a series of mutant forms of the receptor with altered dna-or coregulator-binding capacity we found that the slow component is probably related to chromatin binding, and that coregulator exchange, specifically the binding of the coactivator complex, is the main determinant contributing to the redistribution of rar during ligand activation. formation of inactive nuclear with high level of dna compaction in sperm cells is accompanied by a substitution of linker histones h by a number of other proteins. among them sperm-specific histones (ssh), which are characterized by elongated arginine-rich polypeptide chain compared to the somatic h . the secondary and tertiary structure of the ssh and their interactions with dna were studied using spectroscopic and thermodynamic approaches. the histones were isolated from sperm of marine invertebrates and rat thymus. all studied ssh demonstrate no considerable compaction of dna in solutions of low ionic strength. however, at physiological conditions, ssh h from s.intermedius and a.japonica compact dna more intensively than other ssh. the somatic h from rat thymus revealed a minimal ability to compact the dna. we suggest that the ssh h are able to interact with dna not only in the major groove but also in the minor groove of the double helix inducing considerable structural changes in dna and facilitating the formation of the supercompact sperm chromatin. the authors are grateful for the financial support from the russian foundation for basic research (grants § - - and - - ) and from administration of saint-petersburg. ionizing radiation causes modification and destruction of nitrogenous bases in dna molecule. there are also local breakages of hydrogen bonds both in the lesion sites mentioned above and in other sites of the macromolecule. to reveal the amount of some of these damages we applied cd and uv absorption spectroscopy. radiation-induced changes in dna structure influence its uv absorption spectrum in different ways: partial denaturation causes hyperchromic effect, while destruction of the bases results in hypochromic shift. at the same time both of them result in the same changes in dna cd spectra: the decrease in intensity. we attempted to segregate the described damages in dna structure and studied the influence of dna ionic surroundings on the radiation effect. it is shown that the radiation efficiency of base destruction and partial denaturation increases with decreasing concentration of nacl in irradiated solution. udu (ugly duckling) has been first identified from a zebrafish mutant and shown to play an essential role during erythroid development; however, its roles in other cellular processes remain largely unexplored. facs analysis showed that the loss of udu function resulted in defective cell cycle progression and comet assay indicated the presence of increased dna damage in udu mutants. we further showed that the extensive p -dependent apoptosis found in udu mutants is a consequence of activation in the atm-chk pathway. udu appears not to be required for dna repair, because both wild-type and udu embryos similarly respond to and recover from uv treatment. yeast two-hybrid and coimmunoprecipitation data demonstrated that pah-l repeats and sant-l domain of udu interacts with mcm and mcm . furthermore, udu was colocalized with brdu and heterochromatin during dna replication, suggesting a role in maintaining genome integrity. recently, we started to work on the second zebrafish homolog, udu , and its mammalian counterpart, gon l. preliminary data showed that udu and gon l mrna injection can rescue zebrafish udu mutant phenotypes. furthermore, pah-l and sant-l domains of udu and gon l can bind to mcm and mcm and they are localized in the nucleus. these data suggest that udu and gon l are functionally equivalent to zebrafish udu. their molecular mechanism leading to udu phenotypes is currently under investigation. chromatin condensation: general polyelectrolyte association and histone-tail specific folding nikolay korolev, nikolay berezhnoy, abdollah allahverdi, renliang yang, chuan-fa liu, james p. tam, lars nordenskiö ld school of biological sciences, nanyang technological university, nanyang drive, , singapore the major component of chromatin, dna, is a densely charged polyanion. electrostatic interactions between dna and dnapackaging proteins contribute decisively to formation of its elementary unit, the nucleosome, and are also important in chromatin folding into higher-order structures. we investigate condensation of dna and chromatin and find that electrostatics and polyelectrolyte character of dna play dominant role in both dna and chromatin condensation. by comprehensive experimental studies and using novel oligocationic ligands, we suggest simple universal equation describing dna condensation as a function of oligocation, dna and monovalent salt concentrations and including the ligand-dna binding constant. we found that similar dependence was also observed in condensation of the nucleosome arrays. next, we studied how general electrostatic and specific structural alterations caused by lysine acetylations in the histone tails influence formation of -nm chromatin fibre and intermolecular nucleosome array association. for the first time, a structural model is suggested which explains critical dependence of chromatin fibre folding on acetylation of the single lysine at position of the histone h . exceptional importance of the h lys acetylation in general and gene specific transcriptional activation has been known for many years but no structural basis for this effect has yet been proposed. detection of specific dna sequences is central to modern molecular diagnostic. ultrasensitive raman measurements of nucleic acids are possible through exploiting the effect of surface-enhanced raman scattering (sers). in this work, the sers spectra of genomic dnas from leaves of different apple trees grown in the field, have been analyzed [ ] . a detailed comparative analysis of sers signatures of genomic dnas is given. sers wavenumbers (cm - ) are reported here for all types of vibrations of plant genomic dnas, including bands assigned to localized vibrations of the purine and pyrimidine residues, localized vibrations of the deoxyribosephosphate moiety, etc. proposed band assignments are given. a strong dependence of the sers spectra on dna concentration and on time have been observed. in biochemical fields, nucleic acids might be used to explore the interaction between dna and small molecules, which is important in connection with probing the accurate local structure of dna and with understanding the natural dnamediated biological mechanisms [ ] . the ph-dependent structure of dna studied by fourier transform infrared spectroscopy the region of the infrared spectrum studied covered the wave number range from cm - to cm - . ir spectra show that in ph . - . interval carbonyl (c=o) band at - cm (assigned to guanine) is reduced in intensity and slightly shifted to lower frequencies. at the ph . significantly decreases band intensity at cm - due to unbounded c =o of thymine and shifts to lower frequencies, indicating at the transition of this group in bounded form, supposedly by means of excess polarized hydroxyl ions. together this, in basic region a new intense absorption band has been observed in - cm - frequency interval, corresponding to o-h group in-plane bending vibration ( - cm - ). as for acidic conditions, it was observed that under the extreme ph (* . ) value carbonyl absorption region shifts to higher frequencies and absorption intensity significantly increased, indicated at releasing of c=o groups from h-bonding between base pairs. moreover, bands intensity at cm - and cm - corresponding to out-of-plan deformation of nh groups increased due to rupture of connections between the dna strands. during the last decade it was found that in many cases specific structural organization of multi-molecular protein and dna-protein complexes determines their functioning in living cells. although these functioning structures are usually unique, it is often possible to identify their common structural elements. one of the interesting examples of such universal elements are hmgb domains: structurally conservative functional domains of non-histone proteins hmgb / also identified in many nuclear proteins. using afm, thermodynamic approaches, circular dichroism and molecular absorption spectroscopy in far-uv and mid-ir regions we have studied structural organization of the complexes between dna and different proteins, including hmgb , hmgb-domain recombinant proteins and linker histone h . we have demonstrated, that interaction with dna leads to increasing both a-helicity of the proteins and thermal stability of dna. also, this interaction may result in formation of highly ordered supramolecular complexes facilitated by hmgb-domains. the c-terminal sequence of hmgb / regulates affinity of the proteins to dna and can be ''inactivated'' by interaction with histone h . based on the data obtained a model of the interaction of multy-domain hmgb-proteins with dna is suggested. darmstadt, germany, lmu biozentrum; munich, germany *these authors contributed equally to this work chromatin in living cells displays considerable mobility on a local scale. this movement is consistent with a constrained diffusion model, in that individual loci execute multiple, random jumps. to investigate the connection between local chromatin diffusion (lcd) and the changes in nuclear organization, we established a stable hela cell line expressing gfp-pcna. this protein, a core component of the replication machinery, serves as a cell-cycle marker and allows us to visualize sites of ongoing dna synthesis within the nucleus. to monitor lcd, we labeled discrete genomic loci through incorporation of cy -dutp. this experimental system, in conjunction with particle tracking analysis, has enabled us to quantitatively measure chromatin mobility throughout the cell cycle. our results demonstrate that lcd is significantly decreased in s-phase. to explore the connection between dna replication and reduced chromatin movement, we undertook a more detailed examination of lcd in s-phase nuclei, correlating chromatin mobility with sites of replication. our results demonstrate that labeled chromatin in close proximity to gfp-pcna foci exhibit significantly decreased mobility. we therefore conclude that presence of active replication forks constrains the movement of adjacent chromatin. single-molecule studies of dna replication antoine m. van oijen zernike institute for advanced materials, groningen university, nijenborgh . ag groningen, the netherlands e-mail: a.m.van.oijen@rug.nl advances in optical imaging and molecular manipulation techniques have made it possible to observe individual enzymes and record molecular movies that provide new insight into their dynamics and reaction mechanisms. in a biological context, most of these enzymes function in concert with other enzymes in multi-protein complexes, so an important future direction will be the utilization of single-molecule techniques to unravel the orchestration of large macromolecular assemblies. we are applying a single-molecule approach to study dna replication. i will present recent results of single-molecule studies of replication in bacterial and eukaryotic systems. by combining the stretching of individual dna molecules with the fluorescence observation of individual proteins, we visualize the dynamic interaction of replication factors with the fork. in the bacteriophage t replication system, we show that dna polymerases dynamically associate with and dissociate from the fork during replication. further, i will present new data from single-molecule replication studies in x. laevis oocyte extracts. we have developed a novel imaging scheme that permits single-molecule fluorescence experiments at concentrations of labeled protein that were hitherto inaccessible. using this method, we visualize, in real time, origin firing and fork movement. in force-extension diagrams of reference models of naked dna (freely jointed chain, wormlike chain) as well as extensionrotation diagrams of naked dna have been successfully recovered. of note, plectonemic structures are most efficiently simulated thanks to ode's collision detection code. new insights into nucleosome and chromatin fiber structure and dynamics will be presented. the study of the pkm. plasmid effect on the repair of dna j. vincze , i, francia , g. vincze-tiszay hheif, budapest, hungary, univ. debrecen, debrecen, hungary in our experiments was studied the effect of pkm. plasmid on repair of single strand breaks in dna induced by cogamma irradiation in e.coli k ab (wild type) and its different rec mutant cells. the pkm. resistant-factor in case of the control decreases the sensitivity of radiation, which as we suppose, is reached by the help of dna conformation change. it can be supposed from the well known effect of radiation biology that by the effect of pkm. , the ratio of dna radiation sensitive volumes by appearing its new conformation decreases. the pkm. r-factor in rec mutants in case of gamma irradiation shows effects in two ways. one is the ''chemical'' connection between the r-factor and dna, though the other relate to positive and negative ''induced'' radiation resistance from the local type effect of the connection of an r-factor and a rec mutant, and the two radiation resistant effects are added algebraically. as a result from the view of biology we have to categorize the radiation resistance and the connected repair processes as two different classes according to the change either in the chemical or in the induced radiation resistant effect. recent studies have indicated that two trimethylated peptides (k , k ), derived from the parental hybrid peptide ca( - )m( - ), strongly interact with a bacterial membrane model (mixture of zwitterionic and negatively charged lipids), but not with a membrane model of mammalian erythrocytes (zwitterionic lipids) [ ] . a reduction of the cytotoxicity effect and an improvement of the therapeutic index have also been reported for the derivatives when compared with the parental ca( - )m( - ) [ ] . in this work, with the aim of providing insight on the interaction phenomena of the indicated peptides with zwitterionic and negatively charged membrane models, a systematic molecular dynamics study was carried out. full hydrated bilayers of dmpc:dmpg ( : ) and pope:popg( : ) were studied in the presence of each peptide, and results analyzed in terms of peptide structure and membrane composition. lipid-water and lipid-lipid interactions at the membrane/water interface play important role in maintaining the bilayer structure, however, this region is not easily available for experimental studies. we performed molecular dynamics simulations of two bilayers composed of two different types of lipids: ( ) dioleoylphosphatidylcholine (dopc); ( ) galactolipid monogalactosyldiacylglycerol (mgdg). to investigate the properties of the membrane/water interface region, we performed analysis of lipid-lipid interactions: direct, via charge pairs (dopc) and hydrogen bonds (mgdg) as well as indirect, via water bridges. we also examined water-lipid interactions. existence of well-defined entities (lipids) linked by different types of interactions (hydrogen bonds, charge pairs, water bridges) makes the analysis of the membrane/ water interface region a suitable for a graph theoretical description. we applied a network analysis approach for comparative analysis of simulated systems. we note a marked difference between the organization as well as the dynamics of the interfacial region of the two bilayers. l-nucleoside analogues form an important class of antiviral and anticancer drug candidates. to be pharmacologically active, they need to be phosphorylated in multiple steps by cellular kinases. human phosphoglycerate kinase (hpgk) was shown to exhibit low specificity for nucleotide diphosphate analogues and its catalytic efficiency in phosphorylation was also affected. to elucidate the effect of ligand chirality on dynamics and catalytic efficiency, molecular dynamics simulations were performed on four different nucleotides (d-/l-adp and d-/l-cdp) in complex with hpgk and , -bisphospho-d-glycerate (bpg). the simulation results confirm high affinity for the natural substrate (d-adp), while l-adp shows only moderate affinity for hpgk. the observed short residence time of both cdp enantiomers at the active site suggests very weak binding affinity which may result in poor catalytic efficiency shown for hpgk with d-/l-cdp. analysis of the simulations unravels important dynamic conditions for efficient phosphorylation replacing the single requirement of a tight binding. using the van der waals density functional based on the semilocal exchange functional pw together with a longrange component of the correlation energy [ ] implemented in the siesta program code, we have calculated the band structure of the double stranded dna. the unit cell was built by taking together gc (or at) homogenous base pairs and we have considered the translational symmetry as the periodic boundary condition. the results obtained are compared with the oligomer calculations taking up to seven base pairs. the band structure obtained with this van der waals density functional is also compared with results obtained with other exchange-correlation functionals as well as with band structure obtained by the hartree-fock crystal-orbital method taking into account the helical symmetry of the double stranded dna. the role of different parts of dna (base pairs, sugar-phosphate backbone, na ions) is also presented. transmembrane (tm) proteins comprise some % to % of the proteome but owing to technical difficulties, relatively few of these structures have been determined experimentally. computational modeling techniques can be used to provide the essential structural data needed to shed light on structurefunction relationships in tm proteins. low-resolution electrondensity maps, obtained from cryo-electron microscopy (cryo-em) or preliminary x-ray diffraction studies, can be used to restrict the search in conformational space. at the right resolution, the locations of tm helices can be roughly determined even when the amino acids are not visible. when these data are combined with physicochemical characteristics of amino acids (such as their hydrophobicity) and with evolutionary conservation analysis of the protein family, the location of the amino acids can be modeled. the modelstructure may provide molecular interpretations of the effects of mutations. moreover, it can be used to suggest molecular mechanisms and to design new mutations to examine them. the overall approach will be demonstrated using two human proteins: copper transporter (ctr ), which is the main copper transporter in the human cell, and the kda translocator protein (tspo) of the outer mitochondria. modelstructures of these proteins and their functional implications in health and disease will be discussed. calcium channels play a crucial role in many physiological functions and their selectivity mechanism is still an unresolved question and a subject of debate. a physical model of selective ''ion binding'' in the l-type calcium channel is constructed, and consequences of the model are compared with experimental data. this reduced model treats only ions and the carboxylate oxygens of the eeee locus explicitly and restricts interactions to hard-core repulsion and ion-ion and ion-dielectric electrostatic forces. according to the charge/space competition mechanism, the charge of structural ions attracts cations into the filter, while excluded volume effects are trying to keep them out. this is a competition between energy and entropy, where the balance of these terms minimizes free energy and determines selectivity. experimental conditions involving binary mixtures of alkali and/or alkaline earth metal ions are computed. the model pore rejects alkali metal ions in the presence of biological concentrations of ca + and predicts the blockade of alkali metal ion currents by micromolar ca +. conductance patterns observed in varied mixtures containing na+ and li+, or ba + and ca +, are predicted. ca + is substantially more potent in blocking na+ current than ba +. in apparent contrast to experiments sing buffered ca + solutions, the predicted potency of ca + in blocking alkali metal ion currents depends on the species and concentration of the alkali metal ion, as is expected if these ions compete with ca + for the pore. these experiments depend on the problematic estimation of ca + activity in solutions buffered for ca + and ph in a varying background of ulk salt. equilibrium binding affinity (expressed as the occupancy of the selectivity filter by various ions) is computed by equilibrium grand canonical monte carlo (gcmc) simulations. the conductivity of the channel is estimated from the equilibrium concentration profiles using the integrated nernst-planck equation. our simulations show that the selectivity of l-type calcium channels can arise from an interplay of electrostatic and hard-core repulsion forces among ions and a few crucial channel atoms. the reduced system selects for the cation that delivers the largest charge in the smallest ion volume. we have also performed dynamic monte carlo (dmc) simulations for a model ca channel to simulate current directly and present our results for the dynamical selectivity (expressed as the flux carried by various ions). we show that the binding affinity of ca + versus na+ is always larger than the dynamical selectivity because ca + ions are tightly bound to the binding site of the selectivity filter of the channel and, at the same time, their mobility and drift velocity is smaller in this region. carotenoids are used in light-harvesting complexes with the twofold aim to extend the spectral range of the antenna and to avoid radiation damages. the effect of the polarity and conformation of the environment is supposed to be responsible for the tuning of the electronic, optical and vibrational properties of peridinin carotenoid both in solution and in protein matrix. we investigate the effect of vibrational properties of peridinin in different solvents by means of vibrational spectroscopies and qm/mm molecular dynamics simulations . the shift of vibrational fingerprints in the - cm - frequency region, due to the solvent polarity and proticity, is studied in three cases: cyclohexane (apolar/aprotic), deuterated acetonitrile (polar/aprotic) and methanol (polar/ protic). the frequencies and vibrational modes of the carbonyl, the allene, and the polyene chain were identified using effective normal mode analysis and compared with the present and previous experimental data . on the basis of our calculations and experiments in different solvents, we propose a classification of the four peridinins of the high-salt pcp form. the controlled self-assembly of functional molecular species on well defined surfaces is a promising approach toward the design of nanoscale architectures. by using this methodology, regular low-dimensional systems such as supramolecular clusters, chains, or nanoporous arrays can be fabricated. small biological molecules such as amino acids represent an important class of building blocks that are of interest for molecular architectonic on surfaces because they inherently qualify for molecular recognition and selfassembly [ ] . the interaction between amino acids and solid surfaces is decisive for the development of bioanalytical devices or biocompatible materials as well as for a fundamental understanding of protein-surface bonding. we investigate the adsorbtion mechanism of the cysteine on au( ) surfaces by means of the dft [ ] . our main concern is to describe the molecule-metal bonding mechanism. therefore we present a complex study, including the full determination of the density of states for the free and adsorbed molecule, the determination of molecule-surface bonding energy. the method of crystal orbital overlap populations is used in order to determine the contribution of specific atomic orbitals to the molecule-metal bond. it is now widely accepted that myoglobin (mb) is not simply an o storage/delivery system but, depending on oxygen concentration, it exerts other fundamental physiological roles. recent studies revealed a widespread expression and, in particular, an over-expression in response to hypoxia, in various non-muscle tissues, including tumor cells. in human five different mb isoforms are present. the two most expressed ([ %) differ only at the th position, k (mb-i) and e (mb-ii) respectively. since high-altitude natives from tibet are characterized by a higher mb concentration and locomotion efficiency, together with the observation that the mb overexpression is totally attributable to mb-ii, the idea that the latter might be one of the responses to high-altitude evolutionary adaptation, i.e. hypoxic environment, started to emerge. however, this is not yet supported by any structure/function investigation. we performed hundred nanoseconds md simulations on human mbs to investigate the structure and dynamics of both protein and surface water. important differences have been protein kinases play key roles in cell signaling and constitute crucial therapeutic targets. in normal cell, upon substrate binding, tyrosine kinase receptor kit undergoes extensive structural rearrangement leading to receptor dimerization and activation. this process is initiated by the departure of the juxta membrane region (jmr) from the active site, allowing the activation loop (aloop) deployment. the deregulation of kit activity is associated with various forms of cancer provoked by abnormalities in signal transduction pathways. mutations v g (jmr) and d h/v (a-loop) have been reported as oncogenic and/or drug-resistant. to contribute further in the understanding of kit activation/ deactivation mechanisms, we applied a multi-approach protocol combining molecular dynamics (md), normal modes analysis (nma) and pocket detection. disturbing structural effects, both local (a-loop) and long-range (jmr), were evidenced for kit d h/v in the inactive state. nma showed that jmr is able to depart its position more easily in the mutants than in the wild type. pockets analysis revealed that this detachment is sufficient to open an access to the atp binding site. our results provided a plausible conception of mutant dimerization and a way to explore putative allosteric binding sites. transmembrane association of leukocyte integrin heterodimer might be mediated by a polar interaction choon-peng chng and suet-mien tan biophysics group, a*star institute of high performance computing, and, school of biological sciences, nanyang technological university, republic of singapore the lateral association of transmembrane (tm) helices is important to the folding of membrane proteins as well as a means for signaling across the cell membrane. for integrin, a hetero-dimeric protein important for cell adhesion and migration, the association of its a-and b-subunits' tm helices plays a key role in mediating bi-directional mechanical signaling across the membrane. we found evidence from experiment and simulation for a polar interaction (hydrogenbond) across leukocyte integrin alb tm that is absent in the better-studied platelet integrin aiibb [ ] . our coarse-grained molecular dynamics simulations of tm helix-helix selfassembly showed more native-like packing achieved by alb within the simulation timescale as compared to its 'lossof-function' b t g mutant or aiibb [ ] . association free energy profiles also showed a deeper minimum at a smaller helix-helix separation for alb , suggestive of tighter packing. the likely conservation of this polar interaction across the b integrin family further reinforces its importance to the proper functioning of leukocyte integrins. active extrusion of drugs through efflux pumps constitutes one of the main mechanisms of multidrug resistance in cells. in recent years, large efforts have been devoted to the biochemical and structural characterization of rnd efflux pumps in gram-negative bacteria, in particular the acrb/a-tolc system of e.coli. specific attention has been addressed to the active part of the efflux system, constituted by the acrb unit. despite the presence of several data, crucial questions concerning its functioning are still open. the understanding of the structure-dynamics-function relationship of mexb, the analogous transporter in p. aeruginosa, encounters even more difficulties, because of the lack of structural data of the transporter in complex with substrates. to shade some light on the activity of mexb, we performed computational studies on mexb interacting with two compounds, meropenem and imipenem, the first known to be a good substrate, and the second a modest one. several techniques were used in the present work, ranging from flexible docking [ ] to standard and targeted molecular dynamics (md) simulations. starting from the published crystal structure [ ] we identified the most probable poses of the two compounds in both the original experimental and in the md-equilibrated structures. we used information from acrb binding pocket in order to find relevant binding sites of the two compounds in the analogous binding pocket of mexb. meropenem frequently lies with appropriate orientation in a pocket similar to the one identified for doxorubicin in acrb [ ] , while the occurrence of imipenem poses in the same pocket is very scarce. additionally, when present in the pocket, imipenem is located in a position that renders very unlikely its extrusion toward the oprm docking domain during the simulation of the functional peristalsis. the analysis of the trajectories has provided a complete inventory of the transporter and antibiotic hot spots, which is key information in terms of screening and design of antibiotics and inhibitors. clathrins are three-legged proteins with the intriguing ability to self-assemble into a wide variety of polyhedral cages. the nucleation and growth of a clathrin lattice against the cytosolic face of a cell membrane enables the endocytosis of membrane proteins and various external molecules, by wrapping the membrane around the cargo to produce a coated transport vesicle within the cell. clathrins can also self-assemble, in slightly acidic solutions devoid of auxiliary proteins, into empty cages. our simulations of this process, using a highly coarsegrained model, indicate that the key to self-assembly is neither calthrin's characteristic puckered triskelion shape, nor the alignment of four legs along all cage edges, but an asymmetric distribution of interaction sites around the leg's circumference. based on the critical assembly concentration, the binding strength in these cages is estimated at to k b t per clathrin. the simulations also answer the long-standing conundrum of how flat patches of purely hexagonal clathrin lattices, which in cryo electron microscopy are frequently seen to decorate cell membranes, can produce highly curved cages containing twelve pentagonal faces interdispersed between hexagonal faces. we present experimental evidence supporting this pathway. in eukaryotic cells, the exchange of macromolecules between the cytoplasm and the nucleus is mediated by specialized transport factors. by binding to these transporters, cargo molecules, which are otherwise excluded from entering the nucleus, can traverse the nuclear pore complex efficiently. most of the proteins mediating nuclear import and export exhibit a characteristic _-solenoid fold, which provides them with an unusual intrinsic flexibility. crm is an essential nuclear export receptor, which recognizes a very broad range of export cargoes. crm -dependent nuclear export is ran-gtpase-driven, and recognition of rangtp and cargo is highly cooperative. however, recent crystal structures show that the binding sites for export cargos and rangtp are located at distant parts of crm [ ] [ ] [ ] . we have used a combined approach of all-atom molecular dynamics simulations and small-angle x-ray scattering to study rangtp and cargo binding to crm . we have found that the allosteric effect in crm -dependent nuclear export arises from a combination of subtle structural rearrangements and changes in the dynamic properties of crm . light-induced phototactic responds in green algae chlamydomonas reinhardtii are mediated via microbial-type rhodopsins, termed channelrhodopsin- (chr ) and channelrhodopsin- (chr ) , which carry the chromophore retinal covalently linked to lysin via a schiff base and were shown to be directly light-gated ion-channels . the n-terminal putative seven-transmembrane region of chr was shown to be responsible for the generation of photocurrents and exhibits sequence similarity to the well understood proton pump bacteriorhodopsin (br) and the sensory rhodopsin anabaena sensory rhodopsin (asr) . as for the majority of membrane proteins, there is no d-structural data for chr available yet. here we present homology models of chr using two high-resolution x-ray template structures of br ( qhj ) respectively asr ( xio ) in order to get structural and functional insights into chr . with both homology models we performed molecular dynamics (md) simulations in a native membrane/solvent environment using gromacs . . . comparison of energetic and structural results revealed obvious advantages of the br-based homology model of chr . here we show that the br-based homology model is a reliable model of chr exhibiting structural features already found experimen-tally . our br-based homology model of chr allows predictions of putative crucial residues within chr . so we proposed several mutations within the chr sequence which are already accomplished. electrophysiologic and spectroscopic studies of these mutations are underway in order to confirm the functional relevance of these residues and to contribute to an optimized usage of chr as a powerful tool in optogenetics. ( neuroglobin is a recently discovered globin protein predominantly expressed in brain. its biological function is still elusive. despite the fact that neuroglobin shares very little sequence homology to the well-known globins as mioglobin and hemoglobin, they all have a characteristic globin fold with heme molecule bound to the distal pocket. the structural investigations and co binding kinetics revealed existence of cavities and tunnels within the protein matrix, where small ligands can be stored even for hundreds of microseconds [ ] . in human neuroglobin there is one internal disulfide bond possible which existence is proved to have significant effect on ligand affinity [ ] . in this study effects of temperature, ph, distal histidine mutation and presence of disulfide bond on co rebinding to neuroglobin are investigated by flash photolysis experiments. in parallel, the molecular dynamics simulations are performed in corresponding conditions in order to investigate structural change of neuroglobin and especially changes in distribution of internal tunnels and cavities able to bind diatomic ligands in response to different physical conditions listed above. the thrombospondin family, being extracelluar proteins, is known to be implicated in various physiological processes such as wound healing, inflammation, angiogenesis and neoplasia. the signature domain of thrombospondins shows high sequence identities and thus allows us to transfer results obtained, studying this complex calcium reach part of the proteins, from one member of the family to the other. the domain is known to play a key role in hereditary diseases such as psach or med. in this part of thrombospondins lies a binding site to integrins, important for cell attachment. it is further known that the lectin like globe binds to cd- , a feature known to be important in cancer research. as the theoretical unit we are trying to resolve these problems by numerical means and are constantly challenged by the size, where thrombospondin can be a huge trimeric protein as one strand can measure kda, and the large variety of subdomains found in this proteins. we are thus facing a multiscale problem which can range from solving, by means of quantum mechanics a specific ion binding site, to large scale abstraction by continuum mechanics. in our talk we will show you our newest results that we obtained by simulating calcium rich c-terminal domain which is known to be conserved across the entire family, and give you an outlook into the future of our research. the process of swift heavy ions energy deposition while penetrating a solid or scattering on its surface can result in a strong and nonequilibrium excitation of matter. an extremely localized character of this excitation, meanwhile, can make possible both selective changes in chemistry of matter and its surface nanomodifications . since possible applications have been found in bio-and it-technologies (cancer curing and nanostructuring respectively) in the last decade, the heavy ion bombardment technique has attracted a lot of scientific interest , . the processes of fast energy deposition into the solid and its further dissipation, however, are essentially perturbed with highly excited and nonequilibrium state of both lattice and electron systems. at such conditions therefore, the precision in treatment of processes of electron thermalization, fast electron heat conduction, and phase transformation of the overheated solid becomes crucial. having several physical models to handle the mentioned processes, it is nevertheless difficult to describe all of them within a scale of a single computational approach. our work is aimed on elaboration of the atomistic-continuum model of heavy ion bombardment of solids. in particular, the model will be applied to study the formation of nanohillocks in the experiments on swift heavy ion xe + scattering on srtio surface . [ ] g. aquaporins are protein channels located across the cell membrane with the role of conducting water or other small sugar alcohol molecules (aquaglyceroporins). the presence of the human aquaporin (hsaqp ) in cells proximal to airinteracting surfaces (eyes, lacrimal glands, salivary glands, lungs, stomach etc.) suggest its potentially important role in ''wetting'' these surfaces. the high-resolution x-ray structure of the hsaqp tetramer (pdb code d s) exhibits two important features: (i) lack of the four fold symmetry, common in most of the aquaporins, and (ii) occlusion of the central pore by a phosphatidylserine lipid tail. in this study we investigate the importance of these two features on the transport properties of the human aqp by means of molecular dynamics simulations. we found that the asymmetry in the tetramer leads to a distribution of monomeric channel structures characterized by different free energy landscapes felt by the water molecules passing through the channel. furthermore, the structures' distribution is influenced both by the presence/absence of the lipid tail in the central pore, and by the lipid composition of the bilayer that solvates the hsaqp tetramer. elucidating the modular structure of the protein g c fragment and human igg fc domain binding site using computer simulations hiqmet kamberaj faculty of technical sciences, international balkan university, skopje, r. of macedonia protein-protein recognition plays an important role in most biological processes. although the structures of many protein-protein complexes have been solved in molecular detail, general rules describing affinity and selectivity of proteinprotein interactions break down when applied to a larger set of protein-protein complexes with extremely diverse nature of the interfaces. in this work, we will analyze the non-linear clustering of the residues at the interface between proteins. the boundaries between clusters are defined by clustering the mutual information of the protein-protein interface. we will show that the mutations in one module do not affect residues located in a neighboring module by studying the structural and energetic consequences of the mutation. to the contrary, within their module, we will show that the mutations cause complex energetic and structural consequences. in this study, this is shown on the interaction between protein g c fragment and human igg fc domain by combining molecular dynamics simulations and mutual information theory, and computational alanine scanning technique. the modular architecture of binding sites, which resembles human engineering design, greatly simplifies the design of new protein interactions and provides a feasible view of how these interactions evolved. the results test our understanding of the dominant contributions to the free energy of protein-protein interactions, can guide experiments aimed at the design of protein interaction inhibitors, and provide a stepping-stone to important applications such as interface redesign. membrane proteins can form large multimeric assemblies in native membranes that are implicated in a wide range of biological processes, from signal transduction to organelle structure. hydrophobic mismatch and membrane curvature are involved in long range forces largely contributing to such segregation. however, the existing assembly specificity is thought to be coded in the atomic details of protein surface and topology. these are best described in high resolution structures and atomistic molecular dynamics simulations. in order to explore more systematically such forces and energetics arising at intermediate time scales and resolution, we use coarse grained molecular dynamics simulations applied to membrane systems spanning over to us. as a first glimpse we study how proteins induce lipid perturbations using a previously developed conformational entropy estimator. we show that in the model membrane where hydrophobic mismatch is present, lipid perturbations extend up to * a from the protein surface. however, significant variations in perturbation profiles are seen. parameters such as protein shape, surface topology, and amino acid physicochemical properties are studied to discover the parameters governing such perturbations. crossing energy barriers with self-guided langevin dynamics gerhard kö nig, xiongwu wu, bernard brooks national institutes of health, national heart, lung and blood institute, laboratory of computational biology, rockville, md, usa even with modern computer power, the applicability of molecular dynamics simulations is restricted by their ability to sample the conformational space of biomolecules. often high energy barriers cause normal molecular dynamics simulations to stay trapped in local energy minima, leading to biased results. to address this problem, self-guided langevin dynamics (sgld) were developed. it enhances conformational transitions by accelerating the slow systematic motions in the system. this is achieved by calculating the the local average of velocities and adding a guiding force along the direction of systematic motions. thus, the efficiency of sgld is governed by three factors: a.) the friction constant involved in the langevin dynamics b.) the local averaging time and c.) the guiding factor that determines the guiding force. however, the guiding force also causes deviations from the original ensemble that have to be corrected by reweighting the data, thus decreasing the efficiency. here, we explore the three-dimensional parameter space of sgld for several benchmark systems with particularly rough energy surfaces. based on our data, we supply guidelines for the optimal selection of sgld parameters, to allow the extension of our method to other biological problems of interest. propagation of d v/h mutation effects across kit receptor e. laine, i. c. de beauchê ne, c. auclair and l. tchertanov lbpa, cnrs -ens de cachan, france receptor tyrosine kinases (rtks) regulate critical biological processes. constitutive activation of rtks provokes cancers, inflammatory diseases and neuronal disorders. biological data evidenced that oncogenic mutations of the rtk kit, located either in the juxtamembrane region (jmr) or in the activation loop (a-loop) -as is the case of d v/h, displace the equilibrium of conformational states toward the active form. we present a multi-approach study that combines molecular dynamics (md), normal modes (nm) and pocket detection to characterize and compare the impact of d v/h on the structure, dynamics and thermodynamics of kit. we have evidenced a local structural destabilization of a-loop induced by the mutation and a long-range effect on the structure and position of jmr. we have further correlated these observations with experimental data and decipher some details about the activation mechanisms of the mutants, involving leverage of the jmr negative regulation and release of an access to the catalytic site. through the identification of ''local dynamic domains'' and the recording of interactions within the protein, we propose a model of the mutational effects propagation, which highlights the importance of both structural distortion and local conformational fluctuations. investigation of biological active azolidinones and related heterocycles refer to one of the most successful scientific projects of dh lnmu. it is based on three strategic vectors: organic synthesis, pharmacological research, rational design of ''drug-like'' molecules (including in silico approaches). while applying the research strategy we succeeded in gaining a number of interesting results that make possible to extend the field, especially in the scope of ''drug-like'' molecules design, notably it has focused on the search of new anticancer agents. anticancer activity screening was carried out for more than compounds (us nci protocol (dtp) based on obtained directed library over new compounds, among them compounds showed high activity level. for the purpose of optimization and rational design directions of highly active molecules with optimal ''drug-like'' characteristics and discovering of possible mechanism of action sar, compare analysis, molecular docking and qsa(p)r were carried out. obtained results allowed to form main directions of possible anticancer activity mechanisms, which probable are apoptosis-related. nowadays the investigation of cellular and molecular aspects of anticancer effects is in progress. regulation of (bio)chemical reactions by mechanical force has been proposed to be fundamental to cellular functions [ , , ] . atomic force microscopy experiments have identified the effect of mechanical force on the reactivity of thiol/disulfide exchange, a biologically important reaction [ ] . in order to understand the influence of the force at an atomistic level, we have performed hybrid quantum mechanicsmolecular mechanics (qm/mm) transition path sampling simulation of the reaction under external forces. the results of the simulations supported the experimental findings and demonstrated that the location of the transition state on the free energy surface was shifted due to force [ ] . in contrast to our findings, however, a recent experimental study suggests only a weak coupling between the mechanical force and the reaction rate [ ] . in this study, the reactants were covalently linked to a stilbene molecule. in this system a force can be applied by photo-isomerization from the relaxed trans to the strained cis configuration. a drawback of this system is that one cannot easily determine the forces that acting on the reaction coordinate. therefore, we have developed a force distribution analysis method for quantum mechanical molecular dynamics simulations. the results of the analysis show how isomerization of stilbene alters the forces acting on the reacting atoms. the force distribution is essential for understanding how chemistry is controlled by external forces. [ conformational space modelling (csm) is a promising method for membrane protein structure determination. it is based on the concept of the side-chain conformational space (sccs), which is formed by the allowed side-chain conformations of a given residue. each sccs can be calculated from a d structure or measured via epr-sdsl experiments. for structure determination a set of singly spinlabelled mutants is needed. the final structure is obtained by altering an initial (possibly random) d structure until the best fit between the calculated and measured sccs for the whole set is found. such optimization is computationally intensive; therefore csm includes several empirical approximations. one of them describes the effect of the lipid tails on the sccs. the implementation is not trivial as lipids diffuse in the plane of the membrane and the lipid tails behave differently at different membrane depths. to unravel this relationship adaptive biasing force md simulations were used. an alanine peptide helix was made in silico, spin-labelled at the middle and inserted perpendicularly into a dmpc membrane. the free energy of the spin-label orientation at various membrane depths was calculated. a d free energy surface describing the membrane ''depth'' effect was obtained. it is known that b-cyclodextrins (bcds) are able to modify the cholesterol content of cell or model membranes. however the molecular mechanism of this process is still not resolved. using molecular dynamics simulations, we have been able to study the bcd-mediated cholesterol extraction from cholesterol monolayers and lipid-cholesterol monolayer mixtures. we have investigated many conditions that would affect this process (e.g. lipid-cholesterol ratio, lipid chain unsaturation level) our results can be summarized as follow: i) dimerization of bcds, ii) binding of the dimers to the membrane surface assuming either a tilted (parallel to the membrane normal) or untilted ( °respect to the normal of the membrane) configuration, iii) the latter configuration is suitable to extract cholesterol at a reasonable computational time scale ( - ns), however, this process may be affected by unfavorable energy barriers (from to kj mol - ), iv) desorption of the complex brings cholesterol in solution, v) the bcd-cholesterol complex is able to transfer cholesterol to other membranes. with a clearer understanding of the basic molecular mechanism of the bcd mediated process of cholesterol extraction, we can begin to rationalize the design of more efficient bcds in numerous applications. the mechanism of the complex formation of biopolymers with ligands including the solvent molecules is an actual problem of modern biophysical and biological science. polypeptides form a secondary structure and mimic the motifs of the protein architecture. the study of complexation between polypeptides and solvent molecules leads to deeper understanding of the basic interaction of proteins with environment at atomic level. besides polypeptides are promising for the development of applications which encompass some of the following desirable features: anti-fouling, biocompatibility, biodegradability, specific biomolecular sensitivity. on this account polypeptides have a great significance for a variety modern applications ranging from the nanoscale medicine devises up to food technology and others. we compare the results of calculations of complexes between helical polypeptides (polyglutamic acid in neutral form and poly-c-benzyl-l-glutamate) and water molecules at dft pbe level and the results of ftir-spectroscopic study of the film of wetted polypeptides. vibrational spectroscopy is one of the most useful experimental tools to study non-covalent bonded complexes, and calculated spectra in comparison with experimental data are reliable test for reality of simulated complexes. platelet aggregation at the site of vascular injury is vital to prevent bleeding. excessive platelet function, however, may lead to thrombus formation after surgery. therefore, an accurate measure and control of platelet aggregation is important. in vitro platelet aggregometry monitors aggregate formation in platelet rich plasma triggered by agonists such as adp, epinephrine or collagen. the fraction of aggregated platelets is plotted versus time, and platelet function is assessed by analyzing the plot's morphology. we propose new measures of platelet function based on a compartmental kinetic model of platelet aggregation induced by adp. our model includes three compartments: single, aggregated and deaggregated platelets. it is simpler than earlier models and agrees with experimental data. the model parameters were determined by non-linear least squares fitting of published data. we associated the kinetic parameters with the activity of the adp receptors p y and p y . to this end, we studied published data obtained in the presence and in the absence of specific antagonists of these receptors. comparison of kinetic parameters of healthy subjects with those of patients with myeloproliferative disorder (mpd) shows that the function of p y is significantly reduced in mpd. coarse-grained modeling of drug-membrane interactions manuel n. melo, durba sengupta, siewert-jan marrink groningen biomolecular sciences and biotechnology institute, university of groningen, groningen, the netherlands the martini coarse-grained (cg) forcefield was used to simulate the actions of the antimicrobial peptide alamethicin and of the anti-tumoral drug doxorubicin. both drugs were shown to interact strongly with a fluid phospholipid bilayer, and aggregate there, in agreement with experimental results. because doxorubicin may establish intermolecular h-bonding, and thus lower its dipole moment, the cg representation of a doxorubicin dimer was adjusted. this less polar dimer was then tested for translocation and/or pore formation. contrary to results of atomistic simulations, alamethicin aggregates did not spontaneously open pores. they did so, however, when the size of the water beads was decreased. several small independent pores could then be observed. the magnitude of the permeability of these pores is analyzed and compared to experimental values. the occurrence of multiple small pores could indicate that the different conductance levels experimentally observed for alamethicin might simply result from the association of different numbers of these small pores. polarization refers to the asymmetric changes in cellular organization in response to external or internal signals. neuronal polarization begins with the growth of a single neurite shortly after cell division, followed by the growth of a second neurite at the opposite pole. this early bipolar shape is critical for brain function, as it defines axis of migration and consequently proper three dimensional organization and nerve circuitry. however, it is not known if a direct relationship exists between the formation of the second, opposite, neurite and the mechanisms involved in the formation of the first. we tackled this issue through mathematical modeling, based on membrane traffic (exocytosis-endocytosis), and lateral diffusion. with this approach, we demonstrated that a single pole of molecular asymmetry is sufficient to induce a second one at the opposite side, upon induction of growth from the first pole. our work gives mathematical proof that the occurrence of a single asymmetry in a round cell is sufficient to warrant morphological bipolarism. trypsin is one of the best characterized serine proteases. enzyme acylation process is required for substrate degradation. there is a lot of information about how this process undergoes. however, in order to obtain a more detailed description of the catalytic triad mechanism, a qm/mm approach was used. we used the hybrid qm/mm potential implemented in amber package. in the qm calculations a dft hamiltonian was used. we develop an approach based on the adaptively biased md in order to obtain the free energy surface of the conformational space defined by the reaction coordinates. this approach presents some characteristics of steered md and umbrella sampling procedures. our results offer information about the lowest energy trajectory, the barrier profile of the reaction, and the geometry of the transition state. this method also provides a further insight into the role of specific residues in the reaction. substituting asp , member of the catalytic triad, for ala we were able to detect an increase of the barrier profile. this was due to the loss of the interaction of carbonyl group of asp with nd of his , which make ne of this residue a worst proton acceptor. this results show our approach as a valuable method in the study of enzymatic mechanisms. the intracellular media comprise a great variety of macromolecular species that are not individually concentrated, but being preset in the same compartment they exclude each other's volume and produce crowding. crowding has a profound impact on protein structure and determines conformational transitions and macromolecular association. we investigated macromolecular association on a % w/w bovine serum albumin (bsa) solution by time-domain terahertz (thz) spectroscopy and molecular modeling. molecular crowding was simulated by including two bsa molecules in the same water box. we generated * such dimeric models, computed their thz spectra by normal modes analysis and compared them with the experimental data. the best bsa dimer model was selected based on the agreement with the experiment in the lowest frequencies domain of up to thz. symmetry constraints improve accuracy of ion channels models. application to two-pore-domain channels adina l. ion channels are important drug targets. structural information required for structure-based drug design is often filled by homology models (hm). making accurate hm is challenging because few templates are available and these often have substantial structural differences. besides, in molecular dynamics (md) simulations channels deviate from ideal symmetry and accumulate thermal defects, which complicate hm refinement using md. we evaluate the ability of symmetry-constrained md simulations to improve hm accuracy, using an approach conceptually similar to casp competition: build hm of channels with known structure and evaluate the efficiency of various symmetry-constrained md methods in improving hms accuracy (measured as deviation from x-ray structure). results indicate that unrestrained md does not improve accuracy, instantaneous symmetrization improves accuracy but not stability during subsequent md, while gradually imposing symmetry constraints improves both accuracy (by - %) and stability. moreover, accuracy and stability are strongly correlated, making stability a reliable criterion in predicting hm accuracy. we further used this method to refine hm of trek channels. we also propose a gating mechanism for mechanosensitive channels that was further experimentally confirmed. nucleotide modifications and trna anticodon-mrna codon interactions on the ribosome olof allné r and lennart nilsson karolinska institutet, stockholm, sweden molecular dynamics simulations of the trna anticodon and mrna codon have been used to study the effect of the common trna modifications cmo u and m a . in trna val these modifications allow all four nucleotides to be successfully read at the wobble position in a codon. previous data suggest entropic effects are mainly responsible for the extended reading capabilities but detailed mechanisms have remained unknown. the aim of this paper is to elucidate the details of these mechanisms on an atomic level and quantify their effects. we have applied: extensive free energy perturbation coupled with umbrella sampling, entropic calculations of trna (free and bound to the ribosome) and thorough structural analysis of the ribosomal decoding center. human neuroserpin (hns) is a serine protease inhibitor (serpin) of a tissue-type plasminogen activator (tpa). the conformational flexibility and the metastable state of this proteins underlies to misfolding and to dysfunctional mutations causing a class of rare genetic diseases which share the same molecular basis. the conformational transition of the native form, triggered upon the cleavage at reactive center loop (rlc), releases a complex of the cleaved form bound to the inactivated target protease. without rlc cleavage a stable inactive latent form can be obtained by intra/inter molecular loop insertion leading to polymerization. this work concerns the study of the three above mentioned forms of hns by md simulations to investigate the relation between their conformational stability and. the starting native and cleaved configurations are based on the x-ray structure, while the latent form is here modelled. the results of the simulation reveal a striking conformational stability along with the intrinsic flexibility of selected regions of the fold.the analysis of the essential collective modes of the native hns shows that the initial opening of the b-sheet a coincides with several changes in the local pattern of salt bridges and of hydrogen bonds. regulation of ubiquitin-conjugating enzymes: a common mechanism based on a pattern of hydrophobic and acidic residues enzyme temperature adaptation generally involves a modulation of intramolecular interactions, affecting proteins dynamics, stability and activity [ ] [ ] . in this contribution, we discuss studies of different classes of extremophilic enzymes, focusing on cold-adapted variants, as well as their mesophilic-like mutants, performed by all-atom molecular dynamics simulations with particular attention to structural communication among residues within the threedimensional architecture [ ] [ ] . common adaptation strategies turned out to be based on improved local flexibility in the proximity of the functional sites, decrease in interconnected electrostatic interactions, and modulation of correlated motions and networks of communicating residues. specific differences related to the diverse protein folds can also be detected. bneurexins and neuroligins are cell adhesion molecules and play important role in synapse junction formation, maturation and signal transduction between neurons. mutations in genes coding these proteins occurs in patients with cognitive diseases like autism disorders, asperger syndrome and mental retardation [ ] . it has been found that the complex bneurexin-neuroligin has also an important role in angiogenesis [ ] . herein we will present molecular foundations of bneurexin-neuroligin interactions obtained from all-atom molecular dynamics simulations of bneurexin, neuroligin and their complex ( b q) [ ] . ns md trajectories (charmm force field) were analyzed and roles of ca + and n-actetyl-d-glucosamine posttranslational modifications in intermolecular interactions were scrutinized. advances in hardware and software have enabled increasingly long atomistic molecular dynamics simulations of biomolecules, allowing the exploration of processes occurring on timescales of hundreds of microseconds to a few milliseconds. increasing the length of simulations beyond the microsecond time scale has exposed a number of limitations in the accuracy of commonly employed force fields. such limitations become more severe as the size of the systems investigated and the length of the simulations increase. here i will describe the force field problems that we have encountered in our studies, how we identified and addressed them, and what we have learned in the process about the biophysics of the systems we are investigating. while the quest for a ''perfect'' force field is not over (and may never be), our work has greatly improved the accuracy and range of applicability of simple physics-based force fields, to the point that reliable predictions can now be obtained from millisecond-timescale simulations of biomolecules. local anesthetics (la) are pain-relief drugs, widely used in medicine and dentistry. the relatively short duration of analgesia still restricts their clinical use for the treatment of chronic pain. nowadays, intensive research is focused on anesthetics entrapped into liposomes to enhance their activity and pharmacokinetic properties [ ] . in this work, we investigated the encapsulation of prilocaine (plc), an aminoamide local anesthetic, into a small unilamellar liposome. on the line of our previous work [ ] , we have carried out molecular dynamics (md) simulations using a coarse grain model up the microsecond time scale. in this way, we compare the effects of the concentration of la at fisiological ph. we were able to capture important features of the plc-vesicle interactions. the behavior of plcs at fisiological ph is essentially a combination of high and low ph: we found that all neutral plc molecules rapidly diffuse into the hydrophobic region of the vesicle adopting an asymmetric bimodal density distribution. protonated plc molecules (pplc) initially placed in water were instead only found on the external monolayer, with a high rate of exchange with the water phase and no access to the inner part of the liposome in a concentration dependent way. we focus on applications of molecular and mesoscale simulation methodologies to the cellular transport process of endocytosis, i.e., active transport mechanisms characterized by vesicle nucleation and budding of the cell membrane orchestrated by protein-interaction networks, and functionalized carrier adhesion to cell surfaces. we discuss theoretical and computational methodologies for quantitatively describing how cell-membrane topologies are actively mediated and manipulated by intracellular protein assemblies. we also discuss methods for computing absolute binding free energies for carrier adhesion. we present rigorous validation of our models by comparing to diverse range of experiments. the importance of delta-opioid receptors as target of a large number of drugs is well recognized, but the molecular details of interaction and action of the compounds are largely unknown. in an effort to shade some light on this important issue we performed an extensive computational study on the interaction of two compounds, clozapine and desmetilclozapine, with a delta-opioid receptor. according to experiments, the lacking of a single methyl group in desmetilclozapine with respect of clozapine makes the former more active than the latter, providing a system well suited for a comparative study. we investigated stable configurations of the two drugs inside the receptor by simulating their escape routes by metadynamics, an algorithm that allows the simulation of events that are otherwise out of range for standard molecular dynamics simulations. our results point out that the action of the compound is related to the spatial distribution of the affinity sites it visits during its permanency. desmetilclozapine has a larger distribution of residues, which is interacting with, than clozapine. however, large conformational changes of the receptor were not observed in the presence of both compounds. thus, a more dynamical picture of ligand-receptor affinity is proposed on the basis of the results obtained, involving the competition among different stable states as well as the interaction with the solvents. such information might be useful to provide hints and insights that can be exploited in more structure-and-dynamics-oriented drug design. the coupling between the mechanical properties of enzymes and their biological activity is a well-established feature that has been the object of numerous experimental and theoretical works. in particular, recent experiments show that enzymatic function can be modulated anisotropically by mechanical stress. we study such phenomena using a method or investigating local flexibility on the residue scale, which combines a reduced protein representation with brownian dynamics simulations. we performed calculations on the enzyme guanylate kinase in order to study its mechanical response when submitted to anisotropic deformations. the resulting modifications of the protein's rigidity profile can be related to the changes in substrate binding affinities that were observed experimentally. further analysis of the principal components of motion of the trajectories shows how the application of a mechanical constraint on the protein can disrupt its dynamics, thus leading to a decrease of the enzyme's catalytic rate. eventually, a systematic probe of the protein surface led to the prediction of potential hotspots where the application of an external constraint would produce a large functional response both from the mechan- hiv- protease autocatalyses its own release from gag and gagpol precursor polyproteins into mature functional proteins. as it is functional in the dimeric form, whilst initially, only a single monomer is embedded within each gagpol chain, the question arises as to what cut's the cutter. two individual monomers in different gagpol chains are known to come together to form an embedded-dimer precursor protease. mature-like protease activity is concomitant with n-terminal intramolecular cleavage of this transient embedded-dimer precursor, but how this crucial maturation-initiating step is physically achieved, has remained unknown. here, we show via all-atom explicit solvent molecular dynamics simulation runs of ns each that the n-terminal of an immature-like protease, with the n-terminal initially unbound as in the gagpol polyprotein, can self-associate to the active site and therefore be cleaved under conditions of thermodynamic equilibrium, identifying possible binding pathways at atomic resolution, in agreement with previous indirect experimental evidence [ ] . the binding pathway predominantly makes use of the open conformation of the beta-hairpin flaps characterised by us previously [ ] , and the n-terminal binds across the entire active site in good agreement with crystal structures of a cleavage-site peptidebound protease. the n-terminus serves two roles, firstly in the maturation of the protease itself by self-associating to the protein and then as a stabilizing component of the dimer interface in a mature protease. targeting the prior mechanism could be the focus of a novel therapeutic strategy involving immature protease inhibition. [ knotted proteins are the object of an increasing number of experimental and theoretical studies, because of their ability to fold reversibly in the same topologically entangled conformation. the topological constraint significantly affects their folding landscape, thus providing new insight and challenges into the funnel folding theory [ ] . recently developed experimental methods to trap and detect knots have suggested that denaturated ensembles of the knotted proteins may be knotted [ ] . we present numerical simulations of the early stage of folding of the knotted proteins belonging to protein families mtase (methyltransferase) and sotcase (succinyl-ornithine transcarbamylase), and of their unknotted homologues [ ] . our results show that native interactions are not sufficient to generate the knot in the denaturated configurations. however, when non-native interactions are included we observe formation of knots only in the chains whose native state is knotted. in addition, we find that the knots are formed through a universal mechanism. such a knot formation mechanism correctly predicts the fraction of the knotted proteins found in nature and can be used to make qualitative prediction on their folding times. shape and motility and also for numerous signaling processes. adhesion is based on non-covalent interactions between transmembrane proteins and the extracellular matrix. cells are able to create two-dimensional assemblies of integrins, so called focal adhesions, which they use to stick to the substrate and collect information about the environmental properties. the goal of this work is a deeper understanding of the formation and the stability of these adhesion clusters. bond cluster formation and disintegration is dynamically modeled with the aid of monte carlo simulations. in the model, a membrane is attached to a flat surface via a variable number of adhesion bonds. the spacial configuration of these adhesion points subjected to an inhomogeneous stress field maps into a distribution of local membrane/ surface distances. we introduce a model which explicitly accounts for the membrane elasticity and demonstrate that such models are able to explain the spontaneous formation of adhesion bond clusters. structure based models are successful at conjugating the essence of the energy landscape theory of protein folding [ ] with an easy and efficient implementation. recently their realm expanded beyond single protein structure, been used profitably to widely study large conformational transitions [ ] [ ] . still, when dealing with conformational transition between two well-defined structures an unbiased and realistic description of the local backbone and sidechain interactions is necessary. the proposed model merges a precise description of these interactions with a structure-based long range potential that takes into account different conformers. we present the results of the activation of the catalytic domain of human csrc tyrosine kinase for which we reconstructed the transition free energy and the description of the activation loop flexibility. the excellent model performances in terms of speed and the satisfactory accuracy of the description of the system and its flexibility are promising for a more systematic study of the tyrosine kinase family activation mechanisms. [ we introduce a previously undescribed technique for modelling the kinetics of stochastic chemical systems. we apply richardson extrapolation, a sequence acceleration method for ordinary differential equations, to a fixed-step tau-leaping algorithm, to produce an extrapolated tau-leaping method which has weak order of accuracy two. we prove this mathematically for the case of linear propensity functions. we use four numerical examples, two linear and two nonlinear, to show the higher accuracy of our technique in practice. we illustrate this by using plots of absolute error for a fixed-step tau-leap and the extrapolated tau-leap. in all cases, the errors for our method are lower than for a fixedstep tau-leap; in most cases they are second order of accuracy. the major tripartite efflux pump acrab-tolc is responsible for the intrinsic and acquired multidrug resistance in escherichia coli. at heart of the extrusion machinery there is the homotrimeric transporter acrb, which is in charge of the selective binding of structurally and chemically different substrates and energy transduction. the effects of conformational changes, which have been proposed as the key features of the extrusion of drugs, are investigated at molecular level using different computational methods like targeted molecular dynamics. simulations, including almost half a million atoms, have been used to assess several hypotheses concerning the structure-dynamics-function relationship of the acrb protein. the results indicate that, upon induction of conformational changes, the substrate detaches from the binding pocket and approaches the gate to the central funnel. in addition, we provide evidence for the proposed peristaltic transport involving a zipper-like closure of the binding pocket, responsible for the displacement of the drug. using these atomistic simulations the role of specific amino acids during the transitions can be identified, providing an interpretation of sitedirected mutagenesis experiments. additionally, we discuss a possible role of water molecules in the extrusion process. virus inhibitory peptide (virip), a amino acid peptide, binds to the fusion peptide (fp) of human immunodeficiency virus type (hiv- ) gp and blocks viral entry. molecular dynamics (md) and molecular mechanics/poisson-boltzmann surface area (mm/pbsa) free energy calculations were executed to explore the binding interaction between several virip derivatives and gp fp. a promising correlation between antiviral activity and simulated binding free energy was established thanks to restriction of the flexibility of the peptides, inclusion of configurational entropy calculations and the use of multiple internal dielectric constants for the mm/pbsa calculations depending on the amino acids sequence. bases on these results, a virtual screening experiment was carried out to design enhanced virip analogues. a selection of peptides was tested for inhibitory activity and several improved virip derivatives were identified. these results demonstrate that computational modelling strategies using an improved mm/pbsa methodology can be used for the simulation of peptide complexes. as such, we were able to obtain enhanced hiv- entry inhibitor peptides via an mm/pbsa based virtual screening approach. an essential step during the hiv life cycle is the integration of the viral cdna into the human genome. hiv- integrase mediates integration in a tight complex with the cellular cofactor: ledgf/p [ ] . disruption of the interaction interferes with hiv replication and therefore provides an interesting new drug target for antiretroviral therapy [ , ] . here we present the structure based discovery and optimization of a series of small molecule inhibitors that bind to hiv- integrase and block the interaction with ledgf/p . the work flow was set up according to a funnel principle in which a series of virtual screening tools were applied in such way to discard at each step molecules unlikely to be active against the desired target (including d filtering, pharmacophore modelling and molecular docking) the activity and selectivity of the selected molecules were confirmed in an alpha screen based assay, that measure protein-protein interaction in vitro, and furthermore by in vivo experiments. active compounds proceeded towards crystallographic soaking into the receptor protein crystals. these crystal structures not only validated the binding mode and activity of the hit compounds, but furthermore were used as a platform for structure based drug design which resulted in the rational discovery of new hit compounds and optimized lead compounds. in vitro and in vivo experiments validated the mechanism of action of these compounds and show that they are a novel class of antiretroviral compounds with in vivo inhibitory activity by targeting the interaction between ledgf/p and hiv- integrase. cross resistance profiling indicate that these compounds are active against current resistant viral strains. [ ] currently the most potent inhibitors show an in vivo ic of nm. these compounds are promising for future pharmaceutical optimizations to be used in the clinic as new antiretroviral agents. crystallography was used to validate the binding mode of the discovered inhibitors. insights in the interaction of the ligand-protein complex allowed for rational design of optimized inhibitors. ligand-induced structural changes are small, thermal fluctuations can play a dominant role in determining allosteric signalling. in thermodynamic terms, the entropy change for subsequent binding is influenced by global vibrational modes being either damped or activated by an initial binding event. one advantage of such a mechanism is the possibility for long range allosteric signalling. here, changes to slow internal motion can be harnessed to provide signalling across long distances. this paper considers homotropic allostery in homodimeric proteins, and presents results from a theoretical approach designed to understand the mechanisms responsible for both cooperativity and anticooperativity. theoretical results are presented for the binding of camp to the catabolite activator protein (cap) [ ] , where it is shown that coupling strength within a dimer is of key importance in determining the nature of the allosteric response. results from theory are presented along side both atomistic simulations and simple coarse-grained models, designed to show how fluctuations can play a key role in allosteric signalling in homodimeric proteins. [ reversibly switchable fluorescent proteins (rsfps) can be switched between a fluorescent (on) and a nonfluorescent (off) state which is accompanied by a cis-trans isomerization of the chromophore on the molecular level , . this unique property has already provided new aspects to various microscopy techniques, such as high resolution microscopy, fcs or monochromatic multicolor microscopy - . despite of their established potential, rsfps still have a major drawback: the wavelength for fluorescence excitation is always one of the two switching wavelengths. the imaging process thus inevitably results in the switching of a small fraction of the rsfps, which might hinder or complicate some experiments. we developed a new reversibly switchable fluorescent protein which eliminates the problem of the coupling between switching and fluorescence excitation. this fluorescent protein follows an unusual and currently unknown mechanism of switching between a fluorescent and a nonfluorescent state. it is brightly fluorescent and exhibits an excellent signal to noise ratio. in parallel studies [ ] , qd-based ligands (egf, mabs) were targeted to egfr in gliomas. cell-cultures, animal models and ex vivo biopsies of human high-grade as well as low-grade gliomas showed high probe specificity.. the aim is to define more precisely the tumor boundaries at the time of resection. we used the programmable array microscope designed for sensitive, high-speed optical sectioning, particularly of living cells. the pam is based on structured illumination and conjugate detection using a digital micromirror device (dmd) [ ] located in a primary image plane. the unique feature is the rapid, (re)programmable adjustment of local excitation intensity, dynamic, on-the-fly optimization is thus achieved, e.g. multipoint frap [ ] , light exposure minimization and object tracking [ ] , or super-resolution strategies. the features and operation of the rd generation pam will be presented. contraction of muscle cells, motility of microorganisms, neuronal activity, and other fast cellular processes require microscopic imaging of a three-dimensional ( d) volume with a video-rate scanning. we present d video-rate investigations of structural dynamics in biological samples with the multicontrast third-and second-harmonic generation as well as fluorescence microscope. the multidepth scanning is achieved by two combined laser beams with staggered femtosecond pulses. each of the beams is equipped with a pair of deformable mirrors for dynamic wavefront manipulation enabling multidepth refocusing with simultaneous corrections for optical aberrations. combined, more than frames per second lateral scanning with fast refocusing enables the d video-rate imaging of dynamically moving structures. in addition, combination of two laser beams is accomplished at two perpendicular polarizations enabling live imaging of sample anisotropy, which is important for structural studies particularly with the second harmonic generation microscopy. investigations of beating chick embryo hearts with the d video-rate scanning microscope revealed multidirectional cardiomyocyte contraction dynamics in myocardial tissue. intricate synchronization of contractions between different layers of myocytes in the tissue will be presented. the video-rate d microscopy opens new possibilities of imaging fast biological processes in living organisms. confocal fluorescence microscopy is an invaluable tool to study biological systems at cellular level also thanks to the synthesis of always new specific fluorescent probes, e.g. multiprobe labelling enables complex system characterization. however, only the recent employment of narrowband tunable filters overcomes the problems due to the use of the broadband ones. the possibility of acquiring the emission spectra in a spatially resolved way extends simple image intensity studies into characterization of complex probeenvironment relationship through the sensitivity of fluorescence spectra to the local molecular environment differences. consequently, fluorescence microspectroscopy (fms) is able to provide the spectral information in a well defined spatial region allowing the researcher to simultaneously obtain spatial and spectroscopic information. our instrument has been specially built to study live cells and their interaction with nanomaterials, drug carriers and modified cell environment. other main characteristics are reducing the bleaching effect and employing a white light source that does not limit the use to specific probes. graphical tools, such as colour coded images, have also been introduced to provide explicit and straightforward visual information. high speed fpga based multi-tau correlation for singlephoton avalanche diode arrays jan buchholz , , jan krieger we demonstrate the use of fret-imaging (forster resonance energy transfer) as an assay to directly monitor the dynamics of cross-bridge conformational changes in single fibres of skeletal muscle. we measured nm-distances of several fret pairs located at strategic positions to sense myosin head conformational changes: we focused our attention on the essential light chain, elc (specifically labelling a modified elc and exchanging it with the natural elc of the fibre) and we investigated its interaction with the sh helix, with the nucleotide binding pocket and with actin. we characterized fret in single rigor muscle fibres, determining distances in agreement with those from the crystallographic data. the results demonstrate the viability of the approach in sensing different fret efficiencies over a few nanometres, an essential requirement to follow the expected small fret variations in contracting muscle fibres. we are now performing dynamic experiments on rigor and active fibres by applying small stretch/release cycles to alter the interaction distances (estimated time resolution of nearly ms/frame). in this configuration, it will be possible to measure functional changes, shedding light on the myosin head dynamics during contraction. focal stimulation of cultured neurons is crucial since it mimics physiological molecules release. indeed, the nervous system finely tunes the activity of each synapse by regulating the secretion of molecules spatially and temporally. currently used techniques have some drawbacks such as a poor spatial resolution or a low flexibility. we propose a novel approach based on optical tweezers (ot) [ ] to overcome these limitations. ot allow an ease manipulation with sub-micrometric precision of silica beads, which can be functionalized with any protein. for a proof-of-principle study we coated , lm large beads with brain-derived neurotrophic factor (bdnf) or bovine serum album (bsa) as control. we showed that a single bead was able to activate the bdnf receptor trkb, inducing its phosphorylation. moreover bdnf beads but not control beads were able to induce c-fos translocation into the nucleus [ ] , indicating that the whole pathway was activated. finally, we positioned vectors in proximity to the growth cones of cultured hippocampal neurons [ ] . control beads didn't affect the normal development of these structures while bdnf beads significantly did. these findings support the use of the ot technology for long-term, localized stimulation of specific subcellular neuronal compartments. a key role of its photoactivity, due to the singlet oxygen production, which has a very short lifetime (ns-ls, depending of hyp environment). hyp sub-cellular localization depends on its concentration in the medium, incubation time and used delivery system. variations in activity of protein kinase c, (anti-apoptotic pkca and pro-apoptotic pkcd) in correlation with the activity of bcl- protein, cytochrome c release from mitochondria and decreasing of mitochondrial membrane potential after photodynamic action were monitored. the study was performed for two different delivery modes of hyp to u- mg glioma cells-hyp alone (membrane diffusion) vs. hyp loaded in low density lipoprotein (ldl) (endocytosis). confocal fluorescence microscopy, flow-cytometry and specific fluorescence labeling were used as main experimental techniques. our results show that hyp photoaction strongly affects apoptotic response of the cells and that the dynamics of this action significantly depends on used delivery system. correlation analysis between the monitored parameters (see above) determined for both delivery system is presented and critically discussed. surface contamination by bacteria is a natural and spontaneous process occurring in any natural environment on biotic (mucosa, tissues…) and abiotic surfaces (medical equipments, food surfaces…). whatever the bacterial nature (gram-positive or -negative), the environmental fluid (air, water, blood…) and the receptor surface (implants, medical equipments, food surfaces…), the surface contamination initiated by the first adherent bacteria can evolve to a three dimensional structure named biofilm (cohesive bacteria assembly ensured by an autoproduced extracellular organic matrix). the mechanisms by which these biofilms offer protective environment to viral particles or hypertolerance to antimicrobial action are not yet elucidated. to reach a better understanding of biofilm reactivity, we reported for the first time successful applications of correlative time-resolved optical microscopy approach by time-lapse (tl), frap, fcs, flim, for real-time analysis of molecular mobility and reaction inside biofilms. by means of non-biological or biological (virus, biocides and antibiotics) reactive compounds, significant advances to understand the roles of the extracellular matrix and bacteria physiological properties were obtained, an important step to improve pathogenic biofilm inactivation. here we present a feasibility study to develop two-photon microscopy ( pm) into a standard diagnostic tool for noninvasive, skin cancer diagnosis. the goal is defining experimental parameters to maximize image quality of optical biopsies in human skin, while avoiding tissue damage. possible diagnostic indicators will be compared for healthy tissue, benign, and malignant melanocytic lesions. we report on preliminary results of a study on pm imaging of ''ex-vivo'' biopsy samples, were autofluorescence intensity and contrast between lesion and surrounding tissues were optimised varying excitation wavelength, detection band, and dispersion pre-compensation. moreover, we determined modulation functions for laser power and detector gain to compensate losses in deep tissue imaging. as the main process of photo-damage, thermo-mechanical modifications were quantified and damage threshold powers were determined. in order to image structural changes in ordered tissue like collagen fibres, second-harmonic generation signals were recorded and optimised. in-vivo two-photon imaging of the honeybee antennal we adapted a two-photon microscope for in-vivo imaging of the honeybee olfactory system, focusing on its primary centres, the antennal lobes. the setup allowed obtaining both d-tomographic measurements of the antennal lobe morphology and time-resolved in-vivo calcium imaging of its neuronal activity. the morphological data were used to precisely measure the glomerular volume in both sides of the brain, investigating the question of volumetric lateralization. functional calcium imaging allowed to record the characteristic glomerular response maps to external odour stimuli applied to the bees' antennae. compared to previous neural imaging experiments in the honeybee, this work enhanced spatial and temporal resolution, penetration depth, and it minimized photo-damage. final goal of this study is the extension of the existing functional atlases of the antennal lobe to d and into the temporal dimension by investigating the time-resolved activity pattern. the use of voltage sensitive fluorescent dyes (vsd) for noninvasive measurement of the action potential (ap) in blood perfused heart have been hindered by low interrogation depth, high absorption and auto-fluorescence of cardiac tissue. these limitations are diminished using new near infrared (nir) vsd (di- -anbdqbs). here we validated toxicity and photo-toxicity of these dyes in guinea pig and human cardiac muscle slabs. application of nir vsd showed no effect on cardiac muscle contraction force or relaxation. optical action potentials closely tracked kinetics of microelectrode recorded aps in both field and electrode stimulated preparations. for phototoxicity assessment dye ( lm) preloaded cardiac slabs were exposed to prolonged laser radiation of various power. microelectrode ap recordings show that exposure to prolonged laser radiation ( min.; mw/mm ) of dye loaded tissue had no statistically significant effect on apd or conduction velocity, thus indicating no or weak photo-toxicity on the nir vsd. in contrast, exposure to min. laser radiation of phototoxic dyes (mitotracker deep-red) preloaded tissue caused significant reduction in apd (by %) and conduction velocity ( %). thus, due to the low photo-toxicity, nir vsd are well suited for in vivo cardiac imaging. streptomycesetes are filamentous gram-positive soil bacteria well known for their complex morphological development and secondary metabolite production. during their life cycle spores germinate to form a network of hyphae, which later develops into aerial mycelium when cross-walls are generated and spores are formed. we have examined and compared the last stage of the differentiation process in a wild-type s. coelicolor (m ) and its dcabb mutant lacking a calmodulin-like calcium binding protein. the strains were grown on four kinds of media: smms, smms with % saccharose, r and r with reduced calcium in order to study the effect of environment and osmotic stress on the sporulation of the two strains to assess the function of cabb protein. from the cultures pictures were taken at hours and after days using phase contrast, atomic force and confocal laser scanning microscope and the sizes of spores were measured. our results showed that the dcabb mutant made smaller spores, its differentiation and stress response were slower. we could conclude from it that the aberrant protein slows the metabolism, the signal transduction and has effects on sporulation, septation and air-mycelium formation. based on it we can tell that the cabb has a significant role in normal development. the mobility and reaction parameters of molecules inside living cells can be conveniently measured using fluorescent probes. typically fluorescence correlation spectroscopy (fcs) based on confocal microscopy is used for such measurements. this implies high time-resolution but only for a single spot at a time. in order to achieve a high time-resolution at multiple spots, we built a single plane illumination microscope (spim) equipped with high-speed image acquisition devices and a high-na detection optics. this allows us to do parallel fcs measurements in a thin plane (width * - lm) of the sample. our setup is equipped with a fast emccd camera (full frame time resolution ls) and a pixel array of spads. the spad array has a full frame time resolution of - ls, which is even fast enough to resolve the typical motion time-scale of small molecules (like egfp) inside living cells. the performance of the system is characterized by diffusion measurements of water-soluble fluorescent beads, as well as fcs measurements in living cells. our data acquisition system uses programmable hardware for some tasks and is fast enough to allow real-time correlation of pixels, as well as saving the complete dataset for later evaluation. electron cryo-microscopy (cryo-em) covers a larger size range than any other technique in structural biology, from atomic resolution structures of membrane proteins, to large noncrystalline single molecules, entire organelles or even cells. electron crystallography of two-dimensional ( d) crystals makes it possible to examine membrane proteins in the quasi-native environment of a lipid bilayer at high to moderately high resolution ( . - Å ). recently, we have used electron crystallography to investigate functionally important conformational changes in membrane transport proteins such as the sodium/proton antiporters nhaa and nhap, or the structure of channelrhodopsin. ''single particle'' cryo-em is well suited to study the structure of large macromolecular assemblies in the . to Å resolution range. a recent example is our Å map of a mitochondrial respiratory chain supercomplex consisting of one copy of complex , two copies of complex iii, and one of complex iv. the fit of the x-ray structures to our map indicates short pathways for efficient electron shuttling between complex i and iii by ubiquinol, and between complex iii and iv by cytochrome c. electron cryo-tomography can visualize large protein complexes in their cellular context at - Å resolution, and thus bridges the gap between protein crystallography and light microscopy. cryo-et is particularly suitable for studying biological membranes and large membrane protein complexes in situ. we found that long rows of atp synthase dimers along ridges of inner membrane cristae are an ubiquitous feature of mitochondria from all species we investigated ( mammals, fungi, plant). the proton pumps of the respiratory chain appear to be confined to the flat membrane regions on either side of the ridges. this highly conserved pattern suggests a fundamental role of the mitochondrial cristae as proton traps for efficient atp synthesis. single-particle analysis: advanced fluorescence imaging, including subdiffraction microscopy, relies on fluorophores with controllable emission properties. chief among these fluorophores are the on/off photoswitchable and green-to-red photoconvertible fluorescent proteins. irisfp was recently reported as the first fluorescent protein to combine irreversible photoconversion from a green-emitting to a red-emitting state with reversible on/off photoswitching in both the green and red states. the introduction of this protein resulted in new applications such as super-resolution pulse-chase imaging, but the properties of irisfp are far from being optimal from a spectroscopic point of view and its tetrameric organization complicates its use as a fusion tag. we have demonstrated how four-state optical highlighting can be rationally introduced into photoconvertible fluorescent proteins by developing and characterizing a new set of such enhanced optical highlighters derived from meo-sfp and dendra . one of these, which we called nijifp, was identified as a promising new multi-photoactivatable fluorescent protein with optical properties that make it ideal for advanced fluorescence-based imaging applications. introducing to medicine and biology concept of optical markers in tremendous way has changed the recent status of these two important disciplines. this was mainly due to strong development in imaging techniques which recently allow us to investigate both static as well dynamic properties of living cells, their components and their interactions with external factors. recently used molecular markers including organic dyes, fluorescent proteins or chelates containing lanthanide ions have several significant limitations. one of the alternatives for molecular markers are inorganic quantum dots (ie. cdse, cds) which are recently commonly used in many academic works. however, even if they are much better from physicochemical point of view, from the application point of view at this moment they are rather useless mainly because of their high risk of toxicity. one of the solution combining advantages of both concepts is to make nontoxic inorganic nanocrystals doped by lanthanide ions. in this work, we will present optical results obtained for nayf nanocrystals doped by different lanthanide ions. the aim of this work was to design and to synthesize these markers and to understand physical processes responsible for their emission and to optimize these processes to the physical limits. intravital microscopy has fostered a full blown of publication regarding the behavior of cells in different tissues and physiological conditions. however, a few papers describe how the motility parameters can be used to understand whether an interaction is occurring and, on balance, the distinction between interacting and non interacting cells is performed visually on the image time stack. here we describe a multi-parameter approach that allows to discern among different cell behaviors on an objective ground and we demonstrate its effectiveness valuating the mutual fate of natural killer (nk) and dendritic (dc) cells at the draining lymph-nodes in inflammatory and stationary conditions. the method is time saving and allows a wide scale characterization of the lymphocyte tracks and to build up statistics of the cell-cell interaction duration. this has allowed the development of a numerical model of the nk-dc interaction, based on a molecular-stochastic dynamic approach, whose output can be directly compared to the data. hemozoin is formed during malaria infection of red blood cells, the malaria parasite cleaves hemoglobin, leaving free heme which is then toxic to the parasite. the free heme is then bio-crystalized to form hemozoin which allows the parasite to remain viable. the hemozoin is released during the breakdown of the red blood cells, is small and can be difficult to resolve spatially. since it contains an abundance of heme protein, which has a strong absorbance at nm, it can be readily detected and tracked by using resonant raman scattering spectroscopy. here we use slit-scanning confocal raman imaging to detect the hemozoin and resolve it against the background molecules. inside a red blood cell, hemoglobin is the strongest background signal since it also contains large amounts of heme. nevertheless, the discrimination is possible, and the time-resolved observation of hemozoin is an important tool to understand effects of malaria since the hemozoin can trigger the immune response and cause inflammation in tissue. muscle performance at the molecular level is determined by the elementary displacement (working stroke) produced by the motor protein myosin ii, and its dependence on load. we developed a laser trap assay (the optical leash) capable of applying controlled loads to a single myosin head before the working stroke is initiated and probing actin-myosin interaction on the microsecond time scale. we found that the working stroke size depends both on load and on the detachment pathway followed by myosin. in a first pathway, myosin detaches very rapidly from actin (\ ms) without producing any movement. in a second pathway, myosin steps and remains bound to actin for a time inversely proportional to atp concentration; the working stroke remains constant (* nm) as the load is increased, until it suddenly vanishes as the isometric force is reached ( . ± . pn). a third dissociation pathway becomes more populated as the force is increased, due to premature unbinding of myosin from actin, resulting in a working stroke that decreases with load. taken together, these results give a new insight into the molecular mechanism of load dependence of the myosin working stroke, which is a primary determinant of skeletal muscle performance and efficiency. previously we have deleted either or both of these terminal helices genetically. surprisingly, all mutants rotated in the correct direction, showing that the shaft portion is dispensable. here we inquire if the rest of the c rotor, the globular protrusion that accounts for * % of the c residues, is also dispensable. keeping the n-and c-terminal helices that constitute the shaft, we have replaced the middle * residues with a short helix-turn-helix motif borrowed from a different protein. the protrusion-less mutant thus made retained a high atpase activity and rotated fast in the correct direction. this may not be unexpected because, in crystal structures, most of the removed residues do not contact with the a b ring. combined with the previous results, however, the present results indicate that none of the c residues are needed for rotation. the rotary mechanism of a molecular engine, the vacuolar proton-atpase, working in a biomembrane csilla ferencz , pá l petrovszki , zoltá n kó ta the rotary mechanism of vacuolar proton-atpase (v-atpase) couples atp hydrolysis and trans-membrane proton translocation. we tested the effect of oscillating electric (ac) field on v-atpase activity in yeast vacuoles. the ac technique has several advantages over direct observations: it can be applied on native membranes, there are no labels and attachments involved, and the target protein is in its natural environment. this was/is the first of its kind of experiment on v-atpase, and we got strikingly different results from previous studies on other proteins: both low and high frequency ac field reduces atpase activity in a wide frequency range. a sharp resonance is seen at . hz, where the atpase activity reaches or exceeds the control (no ac) level. we think that the resonance happens at that of the degrees rotor steps, meaning that the rotation rate of the rotor is around hz, under the given conditions. synchronisation of individual atpases by slow or matching, but not fast ac is likely via a hold-and-release mechanism. we can explain the above observations by assuming that the ac field interacts with the proton movements, and if we consider the estimated geometry of the hydrophilic proton channels and the proton binding cites on the rotor. [ ] . the ttss constitutes a continuous protein transport channel of constant length through the bacterial envelope [ ] . the needle of the type three secretion system is made of a single small protein (protomer). we analyzed the assembly and the structure of the ttss needle using different biophysical methods including fourier transform-infrared spectroscopy, nmr spectroscopy and x-ray crystallography. we show that the ttss needle protomer refolds spontaneously to extend the needle from the distal end. the protomer refolds from a-helix into b-strand conformation to form the ttss needle [ ] . regulated secretion of virulence factors requires the presence of additional protein at the ttss needle tip. x-ray crystal structure analysis of the tip complex revealed major conformational changes in both the needle and the tip proteins during assembly of the s. typhimurium ttss. our structural analysis provides the first detailed insight into both the open state of the ttss needle tip and the conformational changes occurring at the pathogen-host interface [ ] . [ the membrane-bound component f o of the atp synthase works as a rotary motor and plays the central role of driving the f component to transform chemi-osmotic energy into atp synthesis. we have conducted molecular dynamics simulations of the membrane-bound f o sector with explicit lipid bilayer, in which the particular interest was to observe the onset of helix motion in the c ring upon the change of the protonation state of asp of the c subunit, which is the essential element of the boyer's binding-change mechanism. to investigate the influence of transmembrane potential and ph gradient, i.e., the proton motive force, on the structure and dynamics of the a-c complex, different electric fields have been applied along the membrane normal. correlation map analysis indicated that the correlated motions of residues in the interface of the a-c complex were significantly reduced by external electric fields. the deuterium order parameter (s cd ) profile calculated by averaging all the lipids in the f o -bound bilayer was not very different from that of the pure bilayer system, which agrees with recent h solid-state nmr experiments. however, by delineating the lipid properties according to their vicinity to f o , we found that the s cd profiles of different lipid shells are prominently different. lipids close to f o formed a more ordered structure. similarly, the lateral diffusion of lipids on the membrane surface also followed a shell-dependent behavior. the lipids in the proximity of f o exhibited very significantly reduced diffusional motion. the numerical value of s cd was anti-correlated with that of the diffusion coefficient, i.e., the more ordered lipid structures led to slower lipid diffusion. our findings will not only help for elucidating the dynamics of f o depending on the protonation states and electric fields, but may also shed some light to the interactions between the motor f o and its surrounding lipids under physiological condition, which could help to rationalize its extraordinary energy conversion efficiency. this work has been published in march , and it was selected as one of the two featured articles of that issue. the research project is directed toward the construction of a synthetic bio-inorganic machine that consists in a single actin filament that interacts with a linear array of myosin ii motors regularly disposed on a nano-structured device. the motor array is intended to simulate the unique properties of the ensemble of motor proteins in the half-sarcomere of the muscle by providing the condition for developing steady force and shortening by cyclic interactions with the actin filament. the mechanical outputs in the range . - pn force and - , nm shortening will be measured and controlled the bacterial flagellar motor is a membrane-embedded molecular machine that rotates filaments, providing a propulsive force for bacteria to swim. the molecular mechanism of torque (turning force) generation is being investigated through the study of the properties and three-dimensional structure of the motor's stator unit. we are taking both topdown and bottom-up approaches, combining data from the molecular genetics studies, cross-linking, x-ray protein crystallography and molecular dynamics simulations. we have recently determined the first crystal structure of the protein domain that anchors the proton-motive-force-generating mechanism of the flagellar motor to the cell wall, and formulated a model of how the stator attaches to peptidoglycan. the work presented at the meeting will inform the audience on our latest work that establishes the relationship between the structure, dynamics and function of a key component of the bacterial flagellar motor, the motility protein b (motb). this work will be put in the perspective of the mechanism of rotation, stator assembly, anchoring to peptidoglycan and interaction with the rotor, and discussed in the light of the elementary events composing the cycle of electrochemical-to-mechanical energy conversion that drives flagellar rotation. members of the conserved kinesin- family fulfill essential roles in mitotic spindle morphogenesis and dynamics and were thought to be slow, processive microtubule (mt)-plusend directed motors. the mechanisms that regulate kinesin- function are still not well understood. we have examined in vitro and in vivo functions of the saccharomyces cerevisiae kinesin- cin using single-molecule motility assays and single-molecule fluorescence microscopy and found that cin motility is exceptional in the kinesin- family. in vitro, individual cin motors could be switched by ionic conditions from rapid (up to lm/min) and processive minus-end, to bidirectional, to slow plus-end motion. deletion of the uniquely large insert of amino acids in loop of cin induced bias towards minus-end motility and strongly affected the directional switching of cin both in vivo and in vitro. we further found that deletion of the functionally overlapping kinesin- kip and of the spindle-organizing protein ase affected cin velocity and processivity, but directionality was not affected. the entirely unexpected finding of switching of cin directionality in vivo and in vitro demonstrates that the ''gear box'' of kinesins is much more complex and versatile than thought. many biological motor molecules move within cells using stepsizes predictable from their structures. myosin-vi, however, has much larger and more broadly-distributed stepsizes than those predicted from its short lever arms. we explain the discrepancy by monitoring qdots and gold nano-particles attached to the myosin vi motor domains using high-sensitivity nano-imaging. the large stepsizes were attributed to an extended and relatively rigid lever arm; their variability to two stepsizes, one large ( nm) and one small ( nm). these results suggest there exist two tilt-angles during myosin-vi stepping, which correspond to the pre-and post-powerstrokes states and regulate the leading head. the large steps are consistent with the previously reported hand-over-hand mechanism, while the small steps follow an inchworm-like mechanism and increase in frequency with adp. switching between these two mechanisms in a strain sensitive, adpdependent manner allows myosin-vi to fulfill its multiple cellular tasks including vesicle transport and membrane anchoring. http://www.fbs.osaka-u.ac.jp/labs/yanagida/, http:// www.qbic.riken.jp/. ferritin deposits iron in oxyhydroxide iron core surrounded by protein shell. the iron core structure may vary in different ferritins in both normal and pathological cases. to study iron core variations the mö ssbauer spectroscopy with a high velocity resolution was applied for comparative analysis of normal and leukemia chicken liver and spleen tissues, human liver ferritin and commercial pharmaceutical products imferon, maltoferÒ and ferrum lek as ferritin models. mö ssbauer spectra of these samples measured with a high velocity resolution at room temperature were fitted using two models: homogenous iron core (one quadrupole doublet) and heterogeneous iron core (several quadrupole doublets). the results of both fits demonstrated small variations of mö ssbauer hyperfine parameters related to structural variations of the iron cores. these small structural variations may be a result of different degree of crystallinity, the way of iron package, nonequivalent iron position, etc. obtained small differences for normal and leukemia tissues may be useful for distinguishing ferritins from normal and pathological cases. this work was supported in part by the russian foundation for basic research (grant # - - -a). invasion of epithelial cells by salmonella enterica is mediated by bacterial ''effector'' proteins that are delivered into the host cell by a type iii secretion system (ttss). the collaborative action of these translocated effectors modulates a variety of cellular processes leading to bacterial uptake into mammalian cells. type iii effectors require the presence in the bacterial cytosol of specific tts chaperones. effectors are known to interact with their chaperone via a chaperone binding domain (cbd) situated at their n-terminus. this work focus on sopb, an effector with phosphoinositide phosphatase activity and particularly its interaction with the specific chaperone sige by biochemical, biophysical and structural approaches. we have co-expressed sopb with its specific chaperone sige and purified the complex, determined the limits of the cbd and purified the sopb cbd /sige complex. the structure of sige has been solved previously but no crystals could be obtained for structure determination of both complexes. we used saxs experiment combined with biophysical approach to analyse the interaction between sopb and its chaperone as well as the quaternary structure on the complex that will be described in this presentation. guanylate monophosphate kinase (gmpk) is a cytosolic enzyme involved in nucleotide metabolic pathways. one of the physiological roles of gmpks is the reversible phosphoryl group transfer from atp to gmp (its specific ligand), yielding adp and gdp. the gmpk from haemophilus influenzae is a small protein, with a number of amino acids in the primary structure. in order to determine the secondary structure changes of this enzyme, as well as some physical characteristics of its complexes with gmp and atp ligands, circular dichroism (cd) and atr -ftir studies were performed. the enzyme and its ligands were dissolved in tris -hcl buffer, at ph . and °c. from the cd spectra the content of the secondary structure elements of gmpk and gmpk/gmp, gmpk/atp (with and without mg + ) were determined. the major secondary structure elements of gmpk from haemophilus influenzae were a-helix (* %) and b-sheet (* %). atr -ftir experiments show that the amide i and amide ii bands of the gmpk are typical for a protein with great a-helix content. from the second derivative spectra, the content of the secondary structure elements were estimated. these data were in agreement with those obtained by cd. assembly of the mature human immunodeficiency virus type capsid involves the oligomerization of the capsid protein, ca. the c-terminal domain of ca, ctd, participates both in the formation of ca hexamers, and in the joining of hexamers through homodimerization. intact ca and the isolated ctd are able to homodimerize in solution with similar affinity (dissociation constant in the order of lm); ctd homodimerization involves mainly an a-helical region. in this work, we show that peptides derived from the dimerization helix (which keep residues energetically important for dimerization and with higher helical propensities than the wild-type sequence) are able to self-associate with affinities similar to that of the whole ctd. moreover, the peptides have a higher helicity than that of the wild-type sequence, although is not as high as the theoretically predicted. interesting enough, the peptides bind to ctd, but binding in all peptides, but one, does not occur at the dimerization interface of ctd (helix ). rather, binding occurs at the last helical region of ctd, even for the wild-type peptide, as shown by hsqc-nmr. as a consequence, all peptides, but one, are unable to inhibit capsid assembly of the whole ca in vitro. the peptide whose binding occurs at the ctd dimerization helix has an val?arg mutation in position , which has been involved in dimer-dimer contacts. these findings suggest that event keeping the energetically important residues to attain ctd dimerization within a more largely populated helical structure is not enough to hamper dimerization of ctd. putp is an integral membrane protein located in the cytoplasmic membrane of escherichia coli, being responsible for the coupled transport of na + and proline. it belongs to the family of sodium solute symporters (sss). structural data for putp is not available, but secondary structure predictions together with biochemical and biophysical analyses suggest a transmembrane motif. from a recent homology model based on the x-ray structure of the related na + /galactose symporter vsglt, previously published electron paramagnetic resonance (epr) studies, and recent crystallographic and epr studies on the cognate bacterial homolog of a neurotransmitter:na + symporter, leut, it has been proposed that helices viii and ix as well as the interconnecting ''loop '' region determine the accessibility of the periplasmic cavities which bind sodium and proline. we performed site-directed spin labeling of ''loop '' in combination with epr spectroscopy to investigate the structural features of this region and possible conformational changes induced by sodium and proline. analyses of spin label mobility and polarity as well as accessibility to paramagnetic quenchers allow us to refine this region in the present homology model. furthermore, our data suggest conformational changes in this region upon substrate binding including an overall motion of a helical segment. fatty acid-binding proteins (fabp) are a family of low molecular weight proteins that share structural homology and the ability to bind fatty acids. the common structural feature is a b-barrel of antiparallel b-strands forming a large inner cavity that accommodates nonpolar ligands, capped by a portal region, comprising two short a-helices. b-fabp exhibits high affinity for the docosahexaenoic (dha) and oleic acid (oa). it is also postulated that b-fabp may interact with nuclear receptors from ppar family. in the present work, we used molecular biology and spectroscopic techniques to correlate structure, dynamics and function. site-directed mutagenesis was used to produce mutants of b-fabp with a nitroxide spin probe (mtsl) selectively attached to residues located at the portal region. esr spectra of the labeled b-fabp mutants were sensitive to the location of the mutation and were able to monitor interactions in three cases: ( ) shsp are ubiquitous proteins involved in cellular resistance to various stress (oxidative, heat, osmotic…). they are able to prevent aggregation of non-native proteins through the ability of forming large soluble complexes and preventing their nonspecific and insoluble aggregation. in consequence to this molecular chaperone function, they can regulate many processes (resistance to chemotherapy, modulation of the cellular adhesion and invasion, inflammatory response in skin), and the modulation of their expression has been found to be a molecular marker in cancers, spermatogenesis, or cartilage degeneration. furthermore, they are involved in several pathologies: myopathies, neuropathies, cancers, cataracts. among the human members (hspb - ), this study focused on hspb (hsp involved in some cancers), hspb (lens specific), hspb (lens, muscle, heart, lung) and hspb -r g (responsible for a desmin-related myopathy and a cataract). as shsp form large, soluble (but polydisperse in mammals) hetero-oligomers, molecular biology, biochemistry, biophysics and bioinformatics were successfully combined to compare the functional/dysfunctional assemblies in order to understand the critical parameters between shsp members depending upon their tissue and cellular localization. ionizing radiation is a type of radiation that contains enough energy to displace electrons and break chemical bonds. this can promote the removal of at least one electron from an atom or molecule, creating radical species, namely, reactive oxygen species (ros) [ , ] . these are often associated with damages at cellular level, such as, dna mutations, cell cycle modifications and, in animal cells, cancer. to overcome this problem, organisms developed different protection/repair mechanisms that enable them to survive to these threats. dna glycosylases are enzymes that are part of base excision repair (ber) system, mainly responsible for dna repair. they can recognize a dna lesion and, in some cases, are able to remove the mutated base. here we propose to study one of those enzymes, endonuclease iii, which contains a [ fe- s] cluster [ , ] . samples were exposed to different doses of uv-c radiation and the effects were studied by electrophoretic and spectroscopic methods. [ na,k-atpase is an integral protein present in the plasma membrane of animal cells, and consists of two main subunits: the a and b. cholesterol is an essential constituent of the animal membrane cells. in order to study the interaction between na,k-atpase and cholesterol, we have used the dsc technique, and a proteoliposome system composed by the enzyme and dppc:dppe, with different percentage in mol of cholesterol. the heat capacity of purified na,k-atpase profile exhibits three transitions with , and kcal/mol at , and °c. multiple components in the unfolding transition could be attributed either to different steps in the pathway or to independent unfolding of different domains. this denaturation of na,k-atpase is an irreversible process. for the proteoliposome, we also observed three peaks, with , and kcal/mol and , and °c. this increase in dh indicates that the lipids stabilize the protein. when cholesterol was used (from to mol %), the first transition was shifted to a lower temperature value around °c. these results confirm that cholesterol has an influence on the packing and fluidity of lipid bilayer and changes in lipid microenvironment alter the thermostability as well as the activity of na,k-atpase. financial support: fapesp. we have undertaken to study the structure and function of peroxisomal multifunctional enzyme type (mfe- ) from different organisms. mfe- is a key enzyme in long-and branched-chain fatty acids breakdown in peroxisomes. it contains two enzymes in the same polypeptide and also consists of differing amount of domains depending on the species. crystal structure and enzyme kinetics of drosophila melanogaster mfe- has revealed the domain assembly and raised a question about existence of a substrate channeling mechanism. small-angle x-ray scattering studies have further resolved the assembly of domains in the human mfe- . mutations in the mfe- -coding gene in humans may cause d-bifunctional protein deficiency -a metabolic disease characterized by accumulation of fatty acyl-coa intermediates due to inactive or residually active mfe- protein. we have also studied the structure, stability and dynamics of such mutant proteins both experimentally and in silico. the latest results on all these studies will be presented. ftsz is a protein that plays a key role in bacterial division, forming a protein ring directly related to the constriction of the membrane. this process has been observed to occur without the help of molecular motors. nonetheless, the details of the self-assembly and subsequent force generation of the septal ring are still obscure. afm observations allows the study of the behaviour of ftsz solutions on a substrate with unprecedented resolution, permitting the identification of individual protein filaments. the different resulting structures can be compared to monte carlo models for a d lattice accounting for the essential interactions between monomers. these include a strong longitudinal bond that allows a limited flexibility (i.e., curvature of the filaments) and a weaker lateral interaction. the work we present follows this approach, focussing on the latest experiments with ftsz mutants. by using these mutants, it is possible to choose the specific region of the monomer that will anchor to the substrate, thus generating new structures that provide an insight into monomer-monomer interactions. in this way, we explore the anisotropy of the lateral bond in ftsz, a factor that has not been taken into account before but may prove to be of importance in fstz behaviour in vivo. modeling protein structures and their complexes with limited experimental data dominik gront university of warsaw, pasteura , - warsaw, poland conventional methods for protein structure determination require collecting huge amounts of high-quality experimental data. in many cases the data (possibly fragmentary and/or ambiguous) on itself cannot discriminate between alternative conformations and a unique structure cannot be determined. small angle xray scattering is an example of such a ''weak'' experiment. the spectrum encodes only several independent degrees of freedom that provide a global description of a molecular geometry in a very synthetic way. in this contribution we utilized both local information obtained from nmr measurements and global description of a macromolecule as given by saxs profile combined with a knowledge-based bimolecular force field to determine tertiary and quaternary structure of model protein systems. saxs curve as well as various kinds of local nmr data such as isotropic chemical shifts and their tensors, j-couplings, rdc, backbone noe and redor from nmr in solid phase are parsed with the ''experimental'' module of bioshell toolkit and utilized by rosetta modeling suite to generate plausible conformations. obtained results show the new protocol is capable to deliver very accurate models. noenet-use of noe networks for nmr resonance assignment of proteins with known d structure dirk stratmann, carine van heijenoort and eric guittet structural genomics programs today yield an increasing number of protein structures, obtained by x-ray diffraction, whose functions remain to be elucidated. nmr plays here a crucial role through its ability to readily identify binding sites in their complexes or to map dynamic features on the structure. an important nmr limiting step is the often fastidious assignment of the nmr spectra. for proteins whose d structures are already known, the matching of experimental and back-calculated data allows a straightforward assignment of the nmr spectra. we developed noenet, a structure-based assignment approach. it is based on a complete search algorithm, robust against assignment errors, even for sparse input data. it allows functional studies, like modeling of protein-complexes or protein dynamics studies for proteins as large as kd. almost any type of additional restraints can be used as filters to speed up the procedure or restrict the assignment ensemble. noenet being mainly based on nmr data (noes) orthogonal to those used in triple resonance experiments (jcouplings), its combination even with a low number of ambiguous j-coupling based sequential connectivities yields a high precision assignment ensemble. we observed, that t. thermophilus isopropylmalate dehydrogenase (ipmdh) has higher rigidity and lower enzyme activity at room temperature than its mesophilic counterpart (e. coli), while the enzymes have nearly identical flexibilities under their respective physiological working conditions, suggesting that evolutionary adaptation tends to maintain optimum activity by adjusting a ''corresponding state'' regarding conformational flexibility. in order to reveal the nature of the conformational flexibility change related to enzymatic activity, we designed a series of mutations involving non conserved prolines specific to thermophilic ipmdh. proline to glycine mutations substantially increased conformational flexibility and decreased conformational stability. the mutant enzyme variants did not show enhanced catalytic activity, but the non arrhenius temperature dependence of enzyme activity of the wild type was abolished. this phenomenon underlines the fact that the delicate balance between flexibility, stability and activity which is required for the environmental adaptation of enzymes can be easily disrupted with mutations even distant from the active site, providing further evidence that optimization of proper functional motions are also a selective force in the evolution of enzymes. the kinetoplastids trypanosoma brucei, t. cruzi and leishmania major are responsible for causing great morbidity and mortality in developing countries. the all a-helical dimeric dutpases from these organisms represent promising drug targets due to their essential nature and markedly different structural and biochemical properties compared to the trimeric human enzyme. to aid in the development of dutpase inhibitors we have been structurally characterizing the enzymes from these species. here we present the structure of the t. brucei enzyme in open and closed conformations, completing the view of the enzymes from the kinetoplastids. furthermore, we sought to probe the reaction mechanism for this family of enzymes as a mechanism has been proposed based on previous structural work but has not received any further verification. the proposed scheme is similar to that of the trimeric enzyme but differs in detail. using tryptophan fluorescence quenching in the presence of the transition state mimic alf we have been able to identify which species is the likely transition state in the reaction. the crystal structure of t. brucei in complex with this transition state analogue confirms the nature of the nucleophilic attack clearly showing how it differs from trimeric enzymes. the structure of factor h-c d complex explains regulation of immune complement alternative pathway circular dichroism (cd) spectroscopy is a widely used technique for studying the secondary structure (ss) of proteins. numerous algorithms have been developed for the estimation of the ss composition from the cd spectra. although, these methods give more or less accurate estimation for proteins rich in a-helical structure, they often fail to provide acceptable results on mixed or b-rich proteins. the problem arises from the diversity of b-structures, which is thought to be an intrinsic limitation of the technique. the worst predictions are provided for proteins of unusual b-structures and for amyloid fibrils. our aim was to develop a new algorithm for the more accurate estimation of ss contents for a broader range of protein folds with special interest to amyloid fibrils. using synchrotron radiation cd (srcd), we were able to collect high quality spectra of amyloid fibrils with good s/n ratios down to nm. the novel reference dataset with spectra that significantly differ from present reference sets, extends the information content for ss determination. our algorithm takes into account the diverse twist of the b-sheets that has a great influence on the spectral features. for the first time, we can reliably distinguish parallel and antiparallel b-structure using cd spectroscopy. monitoring the assembly of the membrane protein insertase alexej kedrov , marko sustarsic , arnold j.m. driessen groningen biomolecular sciences and bioengineering institute, university of groningen, the netherlands, university of oxford, uk molecular forces that govern membrane protein integration and folding remain a major question in current molecular biology and biophysics. each nascent polypeptide chain should acquire its unique three-dimensional folded state within a complex environment formed by the anisotropic lipid membrane and the membrane-water interface. secyeg translocase and members of a recently described yidc/oxa /alb chaperone family are recognized as primary players in the membrane protein genesis. these proteins, so called insertases, serve as membraneembedded molecular pores where the newly synthesized protein is loaded prior its release into the bilayer. here we apply fluorescence correlation spectroscopy to monitor the assembly of the insertase:ribosome:nascent polypeptide chain complexes in solution and reconstituted into nanodiscs and model membranes. results provide insights on molecular mechanisms and dynamics of the insertase functioning. conformational changes during gtpase activity induced self-assembly of human guanylate binding protein revealed by epr spectroscopy correct assembly and regulation of multi-component molecular machines is essential for many biological activities. the type iii secretion system (t ss) is a complex molecular machine that is a key virulence determinant for important gram-negative pathogens including shigella, yersinia and salmonella species [ ] [ ] . the t ss consists of multiple copies of * different proteins (totalling * mda), spans both bacterial membranes and drives insertion of a contiguous pore into the host-cell membrane. virulence factors are secreted via this apparatus directly into the host cell. in all t ss various levels of regulation occur with switching between secretion off and on states overlaid on control of which substrates are secreted. genes involved in a variety of these switches have been identified but the molecular mechanisms underlying these functions is poorly understood. we are studying the t ss of shigella flexneri, the causative agent of dysentery and will present the structure of the so-called ''gatekeeper protein'' mxia. the diacylglycerolacyltransferase (dgat ) is an integral protein from the reticulum endoplasmic membrane that plays an essential role in triacylglyceride synthesis. in cattle, this enzyme is associated to the fat content regulation on milk and meat. in this study, synthetic peptides corresponding to both dgat binding sites (sit and sit ) were designed, purified and employed to investigate the enzyme interaction with substrates and membrane models. different binding specificities in the interaction with phospholipid vesicles and micelles were noted. sit showed to bind more strongly in nonpolar membrane models, while sit was electrostatically attracted to negative phospholipid surfaces. the binding of both peptides was followed by significant conformational changes (like unordered to helix transition) in circular dichroism spectra and a nm blue shift in fluorescence emission. the binding of sit and sit peptides to negative liposome gave dissociation constants (k d ) of and . lm, respectively, and a leakage action -fold higher to sit . the difference in specificity is related to the features of the putative substrates (acyl-coas and diacylglycerol) and can be attributed to the distinct role of each dgat binding site during lipid synthesis. supported by fapesp. ftsz, the bacterial homologue of tubulin, assembles into polymers in the bacterial division ring. the interfaces between monomers include a gtp molecule, although the relationship between polymerization and gtpase activity is still controversial. a set of short ftsz polymers were modelled and the formation of active gtpase structures was monitored using molecular dynamics. only the interfaces nearest the polymer ends exhibited geometry adequate for gtp hydrolysis. conversion of a mid-polymer interface to a close-to-end interface resulted in its spontaneous rearrangement from an inactive to an active conformation. fluorescent proteins (fps) have become extremely valuable tools in the life sciences. due to the latest advances in the light microscopy, there is a steady need for fps with improved spectral properties. mirisfp is a monomeric fp that can be switched reversibly between a bright green fluorescent and a dark state by illumination with light of specific wavelengths [ ] . structurally, this photo-switching is based on a cis-trans isomerization of the chromophore. upon illumination with violet light, mirisfp can be irreversibly photoconverted from the green-emitting to a red-emitting form. the red form can again be switched reversibly between a fluorescent and a dark state. to elucidate the mechanistic details of the photoinduced reactions, we have generated mirisgfp . this variant can still undergo reversible photoswitching, but lacks the ability to photoconvert to the red state so that the photoinduced transitions of the green form can be studied without 'artifacts' due to green-to-red photoconversion. using uv/visible spectroscopy, we have characterized the on-and off-switching processes in great detail. several light-activated reaction pathways have been identified. they are highly intertwined so that the net effect achieved with light of a particular wavelength depends on the relative probabilities to photoinduce the various processes. phosducin (pd) is a g t bc-binding protein that is highly expressed in photoreceptors. pd is phosphorylated in dark-adapted retina and is dephosphorylated in response to light. dephosphorylated pd binds g t protein bc-heterodimer with high affinity and inhibits its interaction with g t a or other effectors, whereas phosphorylated pd does not. therefore pd down-regulates the light response in photoreceptors. phosphorylation of pd at s and s leads to the binding of the - - protein. the - - proteins function as scaffolds modulating the activity of their binding partners and their role in pd regulation is still unclear. the - - protein binding may serve to sequester phosphorylated pd from g t bc or to decrease the rate of pd dephosphorylation and degradation. we performed several biophysical studies of the - - :pd complex. analytical ultracentrifugation was used to determine the complex stoichiometry and dissociation constant. conformational changes of pd induced both by the phosphorylation itself and by - - binding were studied using the time-resolved fluorescence spectroscopy techniques. mö ssbauer spectroscopy with a high velocity resolution was used for comparative study of various oxyhemoglobins for analysis of the heme iron electronic structure and protein structure-function relationship. samples of pig, rabbit and normal human oxyhemoglobins and oxyhemoglobins from patients with chronic myeloleukemia and multiple myeloma were measured using mö ssbauer spectrometric system with a high velocity resolution at k. mö ssbauer spectra were fitted using two models: one quadrupole doublet (model of equivalent iron electronic structure in a-and b-subunits of hemoglobins) and superposition of two quadrupole doublets (model of non-equivalent iron electronic structure in a-and b-subunits of hemoglobins). in both models small variations of mö ssbauer hyperfine parameters (quadrupole splitting and isomer shift) were observed for normal human, rabbit and pig oxyhemoglobins and related to different heme iron stereochemistry and oxygen affinity. small variations of mö ssbauer hyperfine parameters for oxyhemoglobins from patients were related to possible variations in the heme iron stereochemistry and function. the different types of silk produced by orb-weaving spiders display various mechanical properties to fulfill diverse functions. for example, the dragline silk produced by the major ampulate glands exhibits high toughness that comes from a good trade-off between stiffness and extensibility. on the other hand, flagelliform silk of the capture spirals of the web is highly elastic due to the presence of proline and glycine residues. these properties are completely dictated by the structural organization of the fiber (crystallinity, degree of molecular orientation, secondary structure, microstructure), which in turn results from the protein primary structure and the mechanism of spinning. although the spinning process of dragline silk begins to be understood, the molecular events occurring in the secretory glands and leading to the formation of other silk fibers are unknown, mainly due to a lack of information regarding their initial and final structures. taking advantage of the efficiency of raman spectromicroscopy to investigate micrometer-sized biological samples, we have determined the conformation of proteins in the complete set of glands of the orb-weaving spider nephila clavipes as well as in the fibers that are spun from these glands. domain of rgs at . Å resolution was solved. the stoichiometry of - - f /rgs protein complex was elucidated using the analytical ultracentrifugation. to map the interaction between - - f and rgs protein we performed a wide range of biophysical measurements: h/d exchange and cross link experiments coupled to mass spectrometry, fret (fö rster resonance energy transfer) time-resolved fluorescence experiments, time-resolved tryptophan fluorescence spectroscopy and saxs (small angle x-ray scattering) measurements. based on all these results we build d model of - - f /rgs protein complex. our model revealed new details on architecture of complex formed by - - proteins. to date all known structure of - - proteins complexes suggests that the ligand is docked in the central channel of - - protein. our results indicate that the rgs domain of rgs protein is located outside the central channel of - - f protein interacting with less-conserved residues of - - f. the receptor for advanced glycation end-products (rage) is a multiligand cell surface receptor involved in various human diseases. the major alternative splice product of rage comprises its extracellular region that occurs as a soluble protein (srage). although the structures of srage domains were available, their assembly into the functional full length protein remained unknown. here we employed synchrotron small-angle x-ray scattering to characterize the solution structure of human srage. the protein revealed concentration-dependent oligomerization behaviour, which was also mediated by the presence of ca + ions. rigid body models of monomeric and dimeric srage were obtained from the scattering data recorded at different solvent conditions. the monomer displays a j-like shape while the dimer is formed through the association of the two n-terminal domains and has an elongated structure. the results provided insight into the assembly of i) the heterodimer srage:rage, which is responsible for blockage of the receptor signalling, and ii) rage homodimer, which is necessary for signal transduction, paving the way for the design of therapeutical strategies for a large number of different pathologies. clpb is a hexameric aaa? atpase that extracts unfolded polypeptides from aggregates by threading them through its central pore. the contribution of coiled-coil m domains is fundamental for the functional mechanism of this chaperone, and its location within the protein structure in previous structural models is contradictory. we present cryo-electron microscopy structural analysis of clpb from e. coli in several nucleotide states. the study reveals a novel architecture for clpb and shows that m domains form an internal scaffold located in the central chamber of clpb hexamers. this inner structure transmits local signals due to atp binding and hydrolysis by aaa? domains. surprisingly, coiled-coil m domains are seen to bend significantly around a hinge region that separates two structural motifs. our results present a new framework to understand clpb-mediated protein disaggregation. streptomyces clavuligerus isoenzymes involved in clavulanic acid biosynthesis: a structural approach clavulanic acid (ca) is a potent b-lactamase inhibitor produced by streptomyces clavuligerus. n -( -carboxyethyl) arginine synthase (ceas) and proclavaminate amidino hydrolase (pah) catalyze the initial steps in the biosynthesis of ca. recently ceas and pah genes (paralogous of ceas and pah) were related to the ca biosynthesis but their products have not been studied yet. here we present the initial structural analysis of ceas and pah using biophysical techniques. pah and ceas genes were isolated from the genomic dna of s. clavuligerus and overexpressed in e. coli. the recombinant proteins were purified by affinity chromatography and analyzed by size exclusion chromatography, non-denaturing page, dynamic light scattering, far-uv circular dichroism (cd) and fluorescence spectroscopy. our results showed that pah and ceas were obtained as hexamer and dimer respectively. both proteins showed an a/b folding, being stable up to °c. above this temperature protein unfolding was observed but the complete unfolding was not observed, even at °c. moreover ceas and pah showed to be stable over a wide ph range (ph . - . ). we are currently working on improving ceas crystals which are a promising step towards the elucidation of the ceas structure. supported by fapesp. • synchrotron radiation circular dichroism • mass spectrometry following vuv photoionisation • fluorescence imaging with lifetime and spectral measurements here we will present the srcd experiment. high photon flux of photons / sec, improved detector performances as well as user-orientated software developments have proven to be the garants for successful data collections,which considerably increased the information content obtained. the exploration into the charge transfer region of the peptide bonds is adding specifically new insights. low sample volumes of as little as ll per spectra as well as convenient sample chamber handling allow for economic and efficient data collections. typical spectra acquisition from to nm, last for min for three scans with a nm step size. prior to high resolution based techniques, srcd spectrawill answer questions about folding states of macromolecules including dna, rna and sugar macromolecules as well as their complexes with proteins and specially membrane proteins. sporulation in bacillus subtilis begins with an asymmetric cell division producing a smaller cell called the forespore, which initially lies side-by-side with the larger mother cell. in a phagocytosis-like event, the mother cell engulfs the forespore so that the latter is internalised as a cell-within-a-cell. engulfment involves the migration of the mother cell membrane around the forespore until the leading edges of this engulfing membrane meet and fuse. this releases the forespore, now surrounded by a double membrane, into the mother cell cytoplasm. membrane migration during engulfment is facilitated by the interacting proteins spoiiq and spoiiiah that are membrane-associated and expressed in the forespore and the mother cell respectively . they interact in the intercellular space and function initially as a molecular zipper and later they participate in a more elaborate complex in which spoiiq and spoiiiah are integral components of an intercellular channel. this channel is a topic of much current interest, having initially been proposed as a conduit for the passage from the mother cell to the forespore of a specific, but putative, regulator of the rna polymerase sigma factor, r g and later as a gap junction-like feeding tube through which the mother cell supplies molecules for the biosynthetic needs of the forespore. here we present data on the structure and interactions of spoiiq and spoiiiah gleaned from biophysical methods and protein crystallography. these data lead to a plausible model for the intercellular channel. the glycine receptor (glyr) is a chloride permeable ligand gated ion channel and that can mediate synaptic inhibition. due to a possible involvement in the pathophysiology of temporal lobe epilepsy, the different properties of glyrs containing alpha l and k subunit isoforms are currently investigated. previous characterizations of homomeric receptors consisting of these isoforms have shown a difference in their electrophysiological properties and their membrane distribution as observed by diffraction-limited fluorescence microscopy. we studied these isoforms, when separately expressed in transfected hek t cells, by using single molecule tracking (smt) in living cells and direct stochastic optical reconstruction microscopy (dstorm) on fixated cells. for both techniques the glyrs are stained using a primary antibody directly labeled with alexa fluor dyes. the dstorm experiments support the observation that alpha l glyr are clustered, while the alpha k glyrs are more uniformly spread. the analysis of the short range diffusion coefficients obtained by smt reveals the presence of heterogeneous motion for both isoforms. the k-isoform has a higher fraction of fast diffusion. in contrast, the l-isoform is more associated with slow diffusion and appears to undergo hindered diffusion. since nanoparticles are suitable for tumor therapy due to their passive targeting to cancer cells by enhanced permeability and retention effect [ ] , it is important to understand mechanisms of their delivery into the living cancer cells. in this respect we have developed a modular spectral imaging system based on a white light spinning disk confocal fluorescence microscope and a narrow tunable emission filter. firstly, interaction of polymer nanoparticles and cells labeled with spectrally overlapping probes was examined. the use of fluorescence microspectroscopy (fms) allowed co-localization, which showed that the size of polymer nanoparticles strongly influences their transfer across the cell plasma membrane. next, the delivery of liposomes (composed of cancerostatic alkylphospholipid (opp) and cholesterol) labeled with environment-sensitive fluorescent probe was monitored. we were able to detect a very small shift in emission spectra of cholesterol-poor opp liposomes inside and outside the cells, which would not be possible without the use of fms. this shift implies that the delivery of these liposomes into cancer cells is based on fusion with the cell membrane [ ] . [ high-resolution optical imaging techniques make now accessible the detection of nanofeatures in bio-and soft-matter by non-ionizing visible radiation. however, high-resolution imaging is critically dependent by the fluorescent probes used for reporting on the nano-environment. on account of our long-standing interest in the development of fluorescent probes, we set out to design and engineer new fluorescent systems for nanoscale imaging and sensing of biological specimens and soft-matter. these fluorophores report on fast subtle changes of their nanoscale environment at excited state and are meant to fulfill these requirements: a) optical responses (intensity, wavelength-shift, lifetime, anisotropy) predictably related to the environmental polarity, viscosity, macromolecular structure, b) high brightness allowing for single-molecule detection, c) easily conjugable to biomolecules or macromolecules of interest. notably, we aim at conjugating these properties with the capability of nanoscopy imaging based on stimulated emission depletion or stochastic reconstruction optical microscopy. in this lecture the main features and applications of the engineered probes will be reviewed and future developments in this exciting field will be discussed. foamy virus (fv) is an atypical retrovirus which shares similarities with hiv and hepatitis b viruses. despite numerous biochemical studies, its entry pathway remains unclear, namely membrane fusion or endocytosis. to tackle this issue, dual color fluorescent viruses were engineered with a gfp labeled capsid and a mcherry labeled envelope. using high resolution d imaging and d single virus tracing, we followed the entry of the fluorescent viruses in living cells with a precision of nm in the plane and nm along the optical axis. to distinguish between the two possible pathways, we developed a novel colocalization analysis method for determining the moment along every single trace where the colors separate, i.e. the fusion event. the combination of this dynamical colocalization information with the instantaneous velocity of the particle and its position within the reconstructed d cell shape allows us to determine whether the separation of capsid and envelope happens at the cell membrane or from endosomes. we then compared two types of fv and demonstrated, consistently with previous ph-dependency studies, that the prototype fv can enter the cell by endocytosis and membrane fusion, whereas the simian fv was only observed to fuse after endocytosis. phosphatidylinositol , -bisphosphate (pi( , )p ) is a minor component of the plasma membrane known to a critical agent in the regulation of synaptic transmission. clustering of pi( , )p in synaptic active zones is important for synaptic transmission. however, pi( , )p does not spontaneously segregate in fluid lipid membranes and another mechanism must be responsible for the lateral segregation of this lipid in active zones. clustering of pi( , )p is expected to be associated with lipid-protein interactions and possibly partition towards lipid rafts in the plasma membrane. here we analyze the influence of protein palmitoylation on the formation of pi( , )p clusters and on synaptic protein-pi( , )p interaction by means of fö rster resonance energy transfer measurements by fluorescence lifetime imaging (fret-flim) and fret confocal microscopy. . during sporulation an entire chromosome is transferred into the forespore. this process starts by the formation of an asymmetrically located division septum that leads to the formation of two unequally sized compartments: a large mothercell and a smaller forespore. the septum traps about % of the chromosome to be transferred into the forespore. the remaining (* mbp) are then translocated from the mother cell into the forespore by an active mechanism involving the spo-iiie dna translocase. the mechanisms of translocation, particularly the control of the directionality, still remains unknown and various models have been proposed so far. since each model predicts very different distribution of spoiiie proteins at the sporulation septum, we used palm microscopy (photoactivated localization microscopy) to investigate proteins localization in live-sporulating bacteria. using this technique, we showed that spoiiie proteins are forming a single tight focus at the septum with a characteristic size of around nm. more surprising, the focus is usually localized in the mother cell compartment and the mean distance between the spoiiie focus and the septum is nm. our data suggest that during the translocation process, spoiiie proteins are only forming stable complexes on the mother cell side, allowing then for a control of the chromosome translocation from the mother cell to the forespore. morphogenetic gradients determine cell identity in a concentration-dependent manner and do so in a way that is both incredibly precise and remarkably robust. in order to understand how they achieve this feat, one needs to establish the sequence of molecular mechanisms starting with morphogen gradient formation and leading to the expression of downstream target genes. in fruit flies, the transcription factor bicoid (bcd) is a crucial morphogen that forms an exponential concentration gradient along the embryo ap axis and turns on cascades of target genes in distinct anterior domains. we measured bcd-egfp mobility in live d. melanogaster embryos using fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. we found that bcd-egfp molecules had a diffusion coefficient on the order of * lm /s during nuclear cycles - , both in the cytoplasm and in nuclei. this value is large enough to explain the stable establishment of the bcd gradient simply by diffusion. on the other hand, in the context of the extremely precise orchestration of the transcription of the hunchback bcd target gene, it is too slow to explain how a precise reading of bicoid concentration could be achieved at each interphase without the existence of a memorization process. single molecule studies of key processes during the initiation of innate and adaptive immune response the two pillars of the vertebrate immune system are the innate and adaptive immune response, which confer resistance to pathogens and play a role in numerous diseases. here we exploit single molecule fluorescence imaging on live cells to study the key molecular processes that underpin these responses. the first project looks at the changes in the organisation of toll-like receptor (tlr ) on the cell surface of macrophages upon activation via lipopolysaccharide (lps), as it is currently not known whether a higher level of tlr organisation is required for the signalling process. macrophages natively express tlr at a low level which allows for oligomerisation to be analysed in live cells by dynamic single molecule colocalisation (dysco) using data obtained by totalinternal reflection fluorescence (tirf) microscopy. the experiments of the second project aim at determining the critical initial events in t-cell triggering by labelling key proteins like the tcr receptor and cd on the surface of live t-cells and following how their spatial distribution changes following the binding of the t-cell to a surface. this enables us to distinguish between the different models of t-cell triggering which are based on aggregation, segregation or a conformational change of the tcr. the study of cells using scanning force microscopy the motility of unicellular parasites in mammalians seems very interesting, yet very complex. in a world, were inertia cannot be used for propulsion, in a world at low reynolds numbers, most of our everyday strategies of self-propulsion do not work. one class of parasites that know their way around, the flagellate trypansome, manage not only to survive in the blood stream, which is a lot faster than its own propulsion velocity and where the trypanosome is constantly attacked by its host's immune response, but also to penetrate the bloodbrain-barrier, which actually should be to tight to enter. even though trypanosomes are known for more than years, their motility behaviour is not completely elucidated yet. now, using high-speed darkfield-microscopy in combination with optical tweezers in microfluidic devices and analyzing the recorded data, new light has been shed on the motility of these parasites. astonishing results show that trypanosomes are very well adapted to their hosts environment, they even can abuse red blood cells for their self-propulsion and use the bloodstream itself to drag antibodies bound to their surface to their cell mouth, where the antibodies are endocytosed and digested. the first part of the presentation will discuss nanoparticle (quantum dot, qd) biosensors and nanoactuators that exploit novel and unusual fret phenomena in the induction/detection of protein aggregation [ ] , reversible on-off qd photoswitching [ ] , and ph sensing [ ] . the second part of the presentation will feature the application of an integrated chemical biological fret approach for the in situ (in/on living cells) detection of conformational changes in the ectodomain of a receptor tyrosine kinase (the receptor for the growth factor egf) induced by ligand binding [ ] . the measurements were conducted with a two-photon scanning microscope equipped with tcspc detection; novel methods for lifetime analysis ad interpretation were employed to confirm the concerted domain rearrangements predicted from x-ray crystallography. the study of protein-protein interactions in vivo is often hindered by the limited acquisition speed of typical instrumentation used, for instance, for lifetime imaging microscopy. anisotropy polarization is altered by the occurrence of foerster resonance energy transfer (fret) and anisotropy imaging was shown to be comparatively fast and simple to implement. here, we present the adaptation of a spinning disc confocal microscope for fluorescence anisotropy imaging that allowed to achieve in vivo imaging at high spatial and temporal resolution. we demonstrate the capabilities of this system and in-house developed analysis software by imaging living caenorhabditis elegans expressing constitutive dimeric and monomeric proteins that are tagged with gfp. measuring intracellular viscosity: from molecular rotors to photodynamic therapy of cancer marina k. kuimova department of chemistry, imperial college london, exhibition road, sw az, uk viscosity is one of the main factors which influence diffusion in condensed media and is crucial for cell function. however, mapping viscosity on a single-cell scale is a challenge. we have imaged viscosity inside live cells using fluorescent probes, called molecular rotors, in which the speed of rotation about a sterically hindered bond is viscosity-dependent [ ] [ ] [ ] . this approach enabled us to demonstrate that viscosity distribution in a cell is highly heterogeneous and that the local microviscosity in hydrophobic cell domains can be up to higher than that of water. we demonstrated that the intracellular viscosity increases dramatically during light activated cancer treatment, called photodynamic therapy (pdt) [ ] . we also demonstrated that the ability of a fluorophore to induce apoptosis in cells during pdt [ ] , or to act as a benign molecular rotor, measuring viscosity, can be controlled by carefully selecting the excitation wavelength in viscous medium [ ] . in the field of biophysics and nanomedicine, the cellular reaction and the kinetics of gene expression after transfection of live cells with plasmid dna or gene-silencing sirna is of great interest. in a previous study on the transfection kinetics of non-viral gene-transfer [ ] we realised that the development of single-cell arrays would be a great step towards easy-to-analyse, high-throughput transfection studies. the regular arrangement of single cells would overcome the limitations in image-analysis that arise from whole populations of cells. in addition to that, the analysis of expression kinetics at the single-cell can help to identify the cell-to-cell variability within a cell population. in order to develop suitable single-cell arrays, we are currently adjusting the different parameters of such a microenvironment (e.g. size, shape, surface-functionalisation) in order to end up with a defined surrounding for single-cell transfection studies. in addition to that, we try to find the optimal uptake pathway for each of the different applications. the neurodegenerative disorder alzheimer's disease (ad) causes cognitive impairment such as loss of episodic memory with ultimately fatal consequences. accumulation and aggregation of two proteins in the brain -amyloid beta and tau -is a characteristic feature. these soluble proteins aggregate during the course of the disease and assemble into amyloid-like filaments. recently it was found that the toxicity of soluble amyloid beta oligomers must also be taken into account for the pathogenesis of cognitive failure in ad. if oligomers are the predominant toxic species it would be pertinent to determine how they disrupt and impair neuronal function. the prion protein (prp) receptor has been proposed to mediate amyloid beta binding to neuronal cells. we have characterised the interaction of the amyloid beta and the prp receptor expressed on hippocampal and neuroblastoma cells at the single-molecule level. we do not detect any colocalisation of either the or amino acids variants with the prp receptor. bacterial biofilms are of the utmost importance in the study of environmental bioremediation and the design of materials for medical applications. the understanding of the mechanisms that govern cell adhesion must be analysed from the physics point of view in order to obtain quantitative descriptors. the genus rhodococcus is widely spread in natural environments. the species are metabolically diverse and thus they can degrade a wide range of pollutants. due to their high hydrophobicity, these cells are very resistant to harsh conditions, are able to degrade hydrophobic substances (e.g. oil) and attach to high-contact angle surfaces. the hydrophobicity of several strains of rhodococcus is measured and mapped using chemical force microscopy (cfm) in the present study. cfm relies on the functionalisation of scanning force microscopy (sfm) tips using hydrophobic or hydrophilic groups. in cfm, the microscope is operated in the forcevolume mode, which combines adhesion data with topographic images. the careful control of the tip chemistry permits the study of interactions between the functional groups on the tip and the bacterial surface, thus allowing the assessment of hydrophobicity. in order to perform a cfm study, the cells need to be firmly anchored to a substrate under physiological conditions (i.e. under a nutrient media or a saline buffer). to this end, several adhesive surfaces have been tested in order to find the one that gives the best results. optical microscopy is arguably the most important technique for the study of living systems because it allows d imaging of cells and tissues under physiological conditions under minimally invasive conditions. conventional far-field microscopy is diffraction-limited; only structures larger than * nm can be resolved, which is insufficient for many applications. recently, techniques featuring image resolutions down to * nm have been introduced such as localization microscopy (palm, storm) and reversible saturable optical fluorescent transition microscopy (resolft, sted). these methods are well suited for live-cell imaging and narrow the resolution gap between light and electron microscopy significantly. we have used palm imaging to study the formation and disassembly of focal adhesions of live hela cells in a high resolution pulse-chase experiment using monomeric irisfp [ ] . mirisfp is a photoactivatable fluorescent protein that combines irreversible photoconversion from a green-to a red-emitting form with reversible photoswitching between a fluorescent and a nonfluorescent state in both forms. in our experiments a subpopulation of mirisfp molecules is photoconverted to the red form by irradiating a specified region of the cell with a pulse of violet light. migration of tagged proteins out of the conversion region can be studied by subsequently localizing the proteins in other regions of the cell by palm imaging, now using the photoswitching capability of the red species. real-time image reconstruction developed in our lab [ ] allowed instant control imaging parameters. live cell imaging of cancer cells is often used for in-vitro studies in connection with photodynamic diagnostic and therapy (pdd and pdt). especially in presence of a photosensitizer, this live cell imaging can only be performed over relatively short duration (at most hour). this restriction comes from the light-induced cell damages (photodamages) that result from rapid fluorescence photobleaching of photosensitizer. while these studies reveal exciting results, it takes several hours to discover the detailed effects of the photosensitizer on cell damage. up to our knowledge, however, there is no general guideline for modification of excitation light dose to achieve that. in this paper, the relation between excitation light doses, photobleaching of photosensitizer (pvp-hyperycin) and cell vitality are investigated using human lung epithelial carcinoma cells (a ). the strategy of this paper is to reduce the excitation light dose by using a low-power pulsed blue led such that the structures are visible in time-lapse images. fluorescence signals and image quality are improved by labelling the cells with an additional non-toxic marker called carboxyfluorescein-diacetate-succinimidyl-ester (cfse). in total we collected time-lapse images (time intervals min) of dual-marked a cells under three different light intensities ( . , . and . mw/cm ) and a variety of pulse lengths ( . , . , , . and ms) over five hours. we have found that there is a nonlinear relationship between the amount of excitation light dose and cell vitality. cells are healthy, i.e. they commence and complete mitosis, when exposed to low light intensities and brief pulses of light. light intensities higher than . mw/cm together with pulse durations longer than ms often cause cell vesiculation, blebbing and apoptosis. in all other cases, however, we found no cell death. in the future, this striking nonlinearity will be studied in more detail. progressive advances in scanning ion conductance microscopy (sicm) [ ] enabled us to convert ordinary scanning probe microscope (spm) in to versatile multifunctional technique. as an imaging tool, ion conductance microscopy is capable to deliver highest possible topographical resolution on living cell membranes among any other microscopy techniques [ ] . also, it can visualize surfaces complexity of those makes them impossible to image by other spms [ ] . ion conductance microscopy combined with a battery of powerful methods such as fluorescence resonance energy transfer (fret) [ ] , patch-clamp, force mapping, localized drug delivery, nano-deposition and nano-sensing is unique among current imaging techniques. the rich combination of ion conductance imaging with other imaging techniques such as laser confocal and electrochemical [ ] will facilitate the study of living cells and tissues at nanoscale. ) and coleoptiles of wheat (triticum aestivum l.) seedlings, which were growing in light and dark conditions, were used to determine fluorescence of whole cells. fluorescence emission spectrum was monitored by fluorescent microscopy using the spectrometer usb . fluorescence intensity f , f , f and f was determined and data was statistically analyzed in annova. we observed that bgf, rf and frf intensity increased in the first leaves with the age of the seedlings. in the coleoptiles was observed great bgf intensity increase with the age of the seedlings. in the coleoptiles decreased rf intensity of the and hours old seedlings, and bgf intensity decreased of hours old seedlings. it was found that emission spectrum and fluorescence intensity changes are induced by the lack of light and salt (nacl) stress. analysis of fluorescence spectrum can quickly and accurately indicate the outset of light and salt stress in plants. there are analogical changes in fluorescence emission spectrum of plant cells in senescence and stress conditions. it was assumed that environmental stress and senescence have common mechanisms in plants. this changes can be monitored by fluorescent microscopy. triple-colour super-resolution imaging in living cells markus staufenbiel, stephan wilmes, domenik lisse, friedrich roder, oliver beutel, christian richter and jacob piehler universitä t osnabrü ck, fachbereich biologie, barbarastraße , , germany, markus.staufenbiel@biologie.uniosnabrü ck.de super-resolution fluorescence imaging techniques based on single molecule localisation has opened tremendous insight into the sub-micrometre organisation of the cell. live cell imaging techniques such as fluorescence photoactivation localization microscopy (fpalm) are currently limited to dual-colour detection due to the restricted availability of red-fluorescent photoswitchable proteins. we employed photoswitching of the oxazine dye atto under reducing conditions for super-resolution imaging in the cytoplasm of living cells. for efficient and specific covalent labelling of target proteins, we have made use of the halotag system. atto was coupled to the halotag ligand (htl) and fast reaction of htl-atto with the halotag enyzme was confirmed in vitro by solid phase binding assays. efficient labelling of the membrane cytoskeleton using lifeact fused to the halotag was observed and super-resolution imaging was readily achieved. based on this approach, we managed to follow the nanoscale dynamics of the actin cytoskeleton as well as clathrincoated pits using clathrin light chain fused to the halotag. we combined this technique with fpalm for triplecolour super-resolution imaging of the spatial distribution of membrane receptors in context of the membrane skeleton. the erbb family of receptor tyrosine kinases consists of four transmembrane proteins that transduce signals across the membrane to control cell fate. growth factor binding results in homo-and hetero-interactions between these receptors at the membrane. erbb receptors are implicated in many cancers, making them a target for therapeutic drugs. to date, studies of erbb interactions have been limited to individual family members or specific pairs, giving an incomplete picture of the highly complex behaviour controlling positive and negative feedback loops and signalling outcomes. to investigate erbb receptor interactions, we have developed tirf-based single molecule fluorescence microscopes capable of simultaneously imaging three, and soon five, fluorescence probes in live cells. we have also developed a catalogue of extrinsic fluorescent probes for : labelling of both endogenous and transfected erbb family members in mammalian cells, plus a bayesian approach to the analysis of single molecule data. this allows us to track active and inactive erbb family members at the basal surface of a model breast cancer cell line that expresses physiological levels of all four receptors. we present here initial characterisation of the entire erbb family together in the cell membrane. the human genome contains more than g proteincoupled receptors (gpcrs); overall, - % of the mammalian genome encodes these molecules. processes controlled by gpcrs include neurotransmission, cellular metabolism, secretion, and immune responses. however it is the stoichiometry of these receptors that is the most controversial. the starting point for understanding gpcr function was the idea that these receptors are monomeric. on the other hand a lot of recent studies favour the concept that gpcr form dimers and are not capable of signalling as independent monomers. recent single molecule studies try to solve this dilemma by suggesting that gpcrs form transient dimers with a lifetime of * ms. however questions remain about the physiological relevance of the preparations necessary for these studies, since they have not been performed on endogenous receptors. here, we directly image individual endogenous receptors using an equimolar mixture of two colour fluorescent fab fragments. we can then determine the receptors stoichiometry by quantifying its dynamic single molecule colocalisation (dy-sco) recorded by total-internal reflection fluorescence (tirf) microscopy. we have recently investigated the domain dynamics of pgk ( ) . structural analysis by small angle neutron scattering revealed that the structure of the holoprotein in solution is more compact as compared to the crystal structure, but would not allow the functionally important phosphoryl transfer between the substrates, if the protein would be static. brownian large scale domain fluctuations on a timescale of ns was revealed by neutron spin echo spectroscopy. the observed dynamics shows that the protein has the flexibility to allow fluctuations and displacements that seem to enable function. [ many physiological and pathological processes involve insertion and translocation of soluble proteins into and across biological membranes. however, the molecular mechanisms of protein membrane insertion and translocation remain poorly understood. here, we describe the ph-dependent membrane insertion of the diphtheria toxin t domain in lipid bilayers by specular neutron reflectometry and solid-state nmr spectroscopy. we gained unprecedented structural resolution using contrast-variation techniques that allow us to propose a sequential model of the membrane-insertion process at angstrom resolution along the perpendicular axis of the membrane. at ph , the native tertiary structure of the t domain unfolds, allowing its binding to the membrane. the membrane-bound state is characterized by a localization of the c-terminal hydrophobic helices within the outer third of the cis fatty acyl-chain region, and these helices are oriented predominantly parallel to the plane of the membrane. in contrast, the amphiphilic n-terminal helices remain in the buffer, above the polar headgroups due to repulsive electrostatic interactions. at ph , repulsive interactions vanish; the n-terminal helices penetrate the headgroup region and are oriented parallel to the plane of the membrane. the c-terminal helices penetrate deeper into the bilayer and occupy about two thirds of the acyl-chain region. these helices do not adopt a transmembrane orientation. interestingly, the t domain induces disorder in the surrounding phospholipids and creates a continuum of water molecules spanning the membrane. we propose that this local destabilization permeabilizes the lipid bilayer and facilitates the translocation of the catalytic domain across the membrane. the limited stability in vitro of mps motivates the search of new surfactants ( ) ( ) ( ) ( ) . fss with a polymeric hydrophilic head proved to be mild towards mps ( ) .new fss were designed with chemically defined polar heads for structural applications. lac-derivative was efficient in keeping several mps water soluble and active but formed elongated rods ( ) . the glu-family was synthesized, characterized in by sans and auc and for its biochemical interest. the formation of rods is related to the low volumetric ratio between the polar head and hydrophobic tail. the surfactant bearing two glucose moieties is the most promising one, leading to both homogeneous and stable complexes for both br and the b f. it was also shown be of particular interest for the structural investigation of membrane proteins using sans ( ). by combining elastic and quasi-elastic neutron scattering data, and by applying theory originally developed to investigate dynamics in glassy polymers, we have shown that in lyophilised apoferritin above t * k the dynamic response observed in the pico-to nano-second time regime is driven by ch dynamics alone, where the methyl species exhibit a distribution of activation energies. our results suggest that over the temporal and spatial range studied the main apoferritin peptide chain remains rigid. interestingly, similar results are reported for other smaller, more flexible lyophilised bio-materials. we believe this work elucidates fundamental aspects of the dynamic landscape in apoferritin which will aid development of complex molecular dynamic model simulations of super-molecules. a detailed appreciation of the relationships between dynamics and biological function will require analysis based on such models that realize the full complexity of macromolecular material. biological systems must often be stored for extended periods of time. this is done by lyophilisation in the presence of lyoprotectants, such as sugars, which results in stable products at ambient conditions. [ ] in an effort to understand the mechanism of preservation and stabilization, the interactions between sugars and liposome vesicles, which serve as a simple membrane model, have been studied extensively. amongst the common sugars, trehalose has superior preservative effects [ ] and accumulates to high concentrations in many anhydrobiotic organisms. despite many experimental and numerical studies three mechanisms are proposed: vitrification [ ] , preferential exclusion [ ] and water replacement [ ] . to gain more insight into the stabilization mechanism we have recently investigated the effect of trehalose on the bending elasticity of fully hydrated unilamellar vesicles of , -dipalmitoyl-phosphatidylcholine (dppc) in d o at temperatures below and above the lipid melting transition (tm) using neutron spin-echo. the data was analyzed using the zilman-granek theory. at all temperatures measured, trehalose stiffens the bilayer suggesting strong interactions between trehalose and the lipid. trehalose appears to broaden the melting transition but does not change the tm. this agrees with observations using differential scanning calorimetry. influence of macromolecular crowding on protein stability sté phane longeville, clé mence le coeur laboratoire lé on brillouin, gif-sur-yvette, france cell interior is a complex environment filled with a variety of different objects with respect to shape and size. macromolecules are present at a total concentration up to several hundred grams per litre and the overall occupied volume fraction can reach ö & . - . . under crowding environment protein-protein interaction play a fundamental role. the crowding environment can affect some physical, chemical, and biological properties of biological macromolecules [ , ] . traditionally, protein folding is studied in vitro at very low concentration of proteins. under such conditions, small globular single chain proteins can unfold and refold quite rapidly depending mainly to the nature of the solvent. such processes have been very intensively studied, since folding of proteins into their native structure is the mechanism, which transforms polypeptide into its biologically active structure. protein misfolding is involved in a very high number of diseases [ ] (e.g. alzheimer, parkinson, and kreuzfeld-jacob diseases, type ii diabetes, …). theoretically, the problem was studied by the introduction of the concept of excluded volume [ ] . in recent papers [ , ] , minton uses statistical thermodynamic models to address the question. he predicted that inert cosolutes stabilize the native state of proteins against unfolding state mainly by destabilizing the unfolded state and that the dimension of the unfolded state decreases with increasing the concentration of cosolute in a measurable way. small angle neutron scattering (sans) is a technique of choice for such study because, by using appropriate mixtures of light and heavy water, it is possible to match the scattering length density of the solvent to the one of the cosolute and thus to measure the conformation of a molecule at low concentration in a presence of a high concentration of another one. we will present a complete experimental study of the mechanism that leads to protein stabilization by macromolecular crowding [ , ] . coupled dynamics of protein and hydration water studied by inelastic neutron scattering and molecular dynamics simulation hiroshi nakagawa , , mikio kataoka , japan atomic energy agency, tokai, japan, juelich centre for neutron science, forschungszentrum juelich gmbh, nara institute of science and technology, ikoma, japan proteins work in an aqueous environment at ambient temperature. it is widely accepted that the proteins are flexible and mobile. the flexibility and mobility, that is, protein dynamics are essential for protein functions. neutron incoherent scattering is one of the most powerful techniques to observe protein dynamics quantitatively. here i will talk about dynamics of protein and its hydration water. the structure of a soluble protein thermally fluctuates in the solvated environment of a living cell. understanding the effects of hydration water on protein dynamics is essential to determine the molecular basis of life. however, the precise relationship between hydration water and protein dynamics is still unknown because hydration water is ubiquitously configured on the protein surface. we found that hydration level dependence of the onset of the protein dynamical transition is correlated with the hydration water network. hydration water dynamics change above the threshold hydration level, and water dynamics control protein dynamics. these findings lead to the conclusion that the hydration water network formation is an essential property that activates the anharmonic motions of a protein, which are responsible for protein function. thermal motions and stability of hemoglobin of three endotherms (platypus -ornithorhynchus anatinus, domestic chicken -gallus gallus domesticus and human -homo sapiens) and an ectotherm (salt water crocodile -crocodylus porosus) were investigated using neutron scattering and circular dichroism. the results revealed a direct correlation between the dynamic parameters, melting -, and body temperatures. on one hand, a certain flexibility of the protein is mandatory for biological function and activity. on the other hand, intramolecular forces must be strong enough to stabilize the structure of the protein and to prevent unfolding. our study presents experimental evidence which support the hypothesis that the specific amino acid composition of hb has a significant influence on thermal fluctuations of the protein. the amino acid sequence of hb seems to have evolved to permit an optimal flexibility of the protein at body temperature. macromolecular resilience was found to increase with body and melting temperatures, thus regulating hb dynamics. where k is the trap spring constant, a is the subdiffusion exponent and e a is the mittag-leffler function. the parameters obtained by fitting this equation to the experimental msds are summarized in table . at short lag times we have not found any difference between the two cell types, contrarily to the previous results obtained by afm . for both cell lines the subdiffusion exponent, a was found close to , the value predicted by the theory of semiflexible polymers. but the crossover frequency x , was found smaller for the cancerous cells for all datasets. it corresponds to passage to the confined regime at longer times. we attribute it to the bigger impact of molecular motors. we study the spatiotemporal evolution of fibrous protein networks present in the intracellular and extracellular matrices. here, we focus on the in vitro actin network dynamics and evolution. in order to study the hierarchical self-assembly of the network formation in a confined environment and provide external stimuli affecting the system minimally, we introduce a new microfluidic design. the microfluidic setup consists of a controlling channel to which microchambers of different shapes and sizes are connected through narrow channels. this design results in having mainly convective transport in the controlling channel and diffusive transport into the microchamber. rhodamine labeled actin monomers diffuse into the chamber. after polymerization is induced, they form a confined entangled network. cross-linking proteins can then be added to increase the network complexity. moreover, we can generate gradients of reactants across the microchambers and vasodilator-stimulated phosphoprotein (vasp) is a crucial regulator of actin dynamics. it is important in cellular processes such as axon guidance and migration, promoting assembly of filopodia and lamellipodia. vasp's multiple domain structure increases the range of interactions it has with actin monomers, filaments, and other proteins and it displays multiple binding modes both in vitro and in vivo, including barbed end elongation and filament bundling. however, it is not fully understood how vasp affects the structural and mechanical properties of actin networks. we characterize vasp-mediated bundling of actin networks in a simplified in vitro system using confocal microscopy and quantify mechanical properties with rheology measurements. we show that the network properties differ from other actin bundling proteins and reflect vasp's multiple domain structure, displaying a complex bundling phase space that depends upon solution conditions. we observe the formation of large bundle aggregates accompanied by a reduction in network elasticity at high protein ratios. in addition, we change vasp's actin binding mode and eliminate bundling by introducing free actin monomers. finally, we show preliminary results from a biomimetic system that extends the range of actin-vasp interaction. cell migration or proliferation? the go or grow hypothesis in cancer cell cultures tamá s garay, É va juhá sz, jó zsef tímá r, balá zs heged} us nd department of pathology, semmelweis university, budapest, hungary background: cancer related death is constantly growing in the past decades. the mortality of solid tumors is mostly due to the metastatic potential of tumor cells which requires a fine adjustment between cell migration and cell proliferation. as the metabolic processes in the cell provide a limited amount of available energy (i.e. atp) the various biological processes like cell motility or dna synthesis compete for the atp available. the go or grow hypothesis postulates that tumor cells show either high migration or proliferation potential. in our study we investigate on a large series of tumor cell lines whether this assumption stands for malignant cells. materials and methods: twenty tumor cell lines derived from malignant mesothelioma (mesodermal origin) and malignant melanoma (neuroectodermal origin) were subjected to three-days-long time-lapse videomicroscopic recordings. cell motility and proliferation were characterized by the probability of cell division within hours and the -hour migration distance of the cells. results: we found a wide range in both the cell migratory activity and the proliferation capacity in our series. the -hour migration distance ranged from to micron and from to micron in mesothelioma and melanoma cells, respectively. the lowest -hour cell division probability was found to be . in both the melanoma and mesothelioma series while the highest proliferation activity reached . and . in melanoma and mesothelioma, respectively. interestingly, in the melanoma cell lines we found a significant positive correlation (r= , ; p= , ) between cell proliferation and cell migration. in contrast our mesothelioma cell lines displayed no correlation between these two cellular processes. conclusions: in summary our findings demonstrate that the investigated tumor cells do not defer cell proliferation for cell migration. important to note the tumor cells derived from various organ systems may differ in terms of regulation of cell migration and cell proliferation. furthermore our observation is in line with the general observation of pathologists that the highly proliferative tumors often display significant invasion of the surrounding normal tissue. many cell types are sensitive to mechanical signals. one striking example is the modulation of cell proliferation, morphology, motility, and protein expression in response to substrate stiffness. changing the elastic moduli of substrates alters the formation of focal adhesions, the formation of actin filament bundles, and the stability of intermediate filaments. the range of stiffness over which different primary cell types respond can vary over a wide range and generally reflects the elastic modulus of the tissue from which these cells were isolated. mechanosensing also depends on the type of adhesion receptor by which the cell binds, and therefore on the molecular composition of the specific extracellular matrix. the viscoelastic properties of different extracellular matrices and cytoskeletal elements also influence the response of cells to mechanical signals, and the unusual non-linear elasticity of many biopolymer gels, characterized by strain-stiffening leads to novel mechanisms by which cells alter their stiffness by engagement of molecular motors that produce internal stresses. the molecular mechanisms by which cells detect substrate stiffness are largely uncharacterized, but simultaneous control of substrate stiffness and adhesive patterns suggests that stiffness sensing occurs on a length scale much larger than single molecular linkages and that the time needed for mechanosensing is on the order of a few seconds. to explore potential role of cytoskeletal component in cardiomyocyte for adaptation to extreme conditions was carried out the comparative study of expression of cytoskeletal sarcomeric protein titin in myocardium of ground squirrels during hibernation and gerbils after spaceflight. we have revealed a two-fold increase in content of long n ba titin isoform as compared to short n b titin isoform in different heart chambers of hibernating ground squirrels. the prevalence of the long titin isoform is known to determine the larger extensibility of heart muscle that promotes, according to frank-starling law, the increase in force of heart contractility for pumping higher viscous blood during torpor and adapting the myocardium to greater mechanical loads during awakening. moreover, titin mrna level showed seasonal downregulation in which all hibernating stages differed significantly from summer active level. it is possible that the decline of mrna and protein synthesis during hibernation may be regarded as the accommodation for minimization of energetic expenditures. we have not revealed differences in titin mrna levels between control gerbils and gerbils after spaceflight. but we have also observed the two-fold growth in the amount of n ba titin isoform in left ventricle of gerbils after spaceflight that is likely to be directed to the restoration of the reduced heart contractility at zero-gravity. these results suggest that the increase of the content of the long n ba titin isoform may serve as universal adaptive mechanism for regulating of heart function in response to the extreme conditions. nuclear migration is a general term for a non-random movement of the nucleus toward specific sites in the cell. this phenomenon has been described throughout the eukaryotes from yeast to mammals. the process is however still poorly understood in mammalian cells. by using microcontact printing we are able to regulate the geometry and spreading of cultured cells. adhesive micropatterns of fibronectin provide an attachment surface for the cells whereas the passivation of the surface by pll-peg prevents protein, thus cell adhesion. live cell imaging by time-lapse microscopy has shown that under these conditions cells gain a bipolar shape, and more interestingly, the nuclei of the cells showed auto-reversed motion. our research tries to understand the molecular cues and mechanisms behind the observed cellular and nuclear movement. we have already shown that the cytoskeleton plays an important role in this phenomena but the exact players and the detailed mechanism remain to be clarified. in order to identify the most important components and their relationship have drug treatments and sirna experiments have been applied. although our research focuses mainly on the motility of the nucleus, it may also help to get a better understanding of the general theme of cell migration. cell motility involves a number of strategies that cells use to move in their environments in order to seek nutrients, escape danger and fulfil morphogenetic roles. when these processes become uncontrolled, pathological behaviours, like cancer or metastasis of cancerous cells, can occur. here we present a new method for the contextual quantification of cellular motility, membrane fluidity and intracellular redox state, by using the ratiometric, redox-sensitive protein rxyfp and the ratiometric fluidity-sensitive probe laurdan. we provide evidence that dynamic redox and fluidity changes are correlated with signaling processes involved in cellular motility. these findings may pave the way to novel approaches for the pharmacological control of cell invasiveness and metastasis. manipulation of cellular mechanics anna pietuch, andreas janshoff georg-august university, tammanstraße , gö ttingen, germany, e-mail: anna.pietuch@chemie.unigoettingen.de rheological properties of cells determined by the underlying cytoskeleton (cortex) are key features in cellular processes like cell migration, cell division, and cell morphology. today it is possible to investigate local cellular elastic properties under almost physiological conditions using the afm. by performing force indentation curves on local areas on a cell surface the use of contact mechanic models provides the young's modulus, comprising information about the elastic properties of cells. the administration of cytoskeleton modifying substances into the cell is achieved by microinjection. we are also investigating morphological changes and rearrangements of the cytoskeleton in time resolved impedance measurements. electric cell-substrate impedance sensing is a label-free and minimal invasive technique which allows monitoring morphological changes of cells in real time. readout of the impedance is sensitive to changes in cell-substrate contacts as well as density of cell-cell contacts yielding important information about the integrity of the cell layer and changes in the properties of the cell membrane. we are studying the cellular response to modification of the cytoskeleton e.g. by introducing proteins which affect directly the organization of the actin structure like ezrin. mechanical characterization of actin gels by a magnetic colloids technique thomas pujol, olivia du roure, julien heuvingh pmmh, espci-cnrs-umpc-p . paris, france the actin polymer is central in cell biology: it is a major component of cytoskeleton and it plays a fundamental role in motility, division, mechanotransduction…. its polymerization just beneath the cell membrane generates forces responsible for cell movement. actin filaments form a network whose architecture is defined by the nature of the binding proteins and depends on the location in the cell. for example, in the structure which leads cell migration, the lamellipodium, the gel is branched due to its interaction with arp / protein complex. determining the mechanical properties of such actin network is a crucial interest to understand how forces are generated and transmitted in living cells. we grow a branched actin network from the surface of colloids using the arp / machinery. the particles are super paramagnetic and they attract each other via dipole-dipole interaction to form chain. by increasing the magnetic field we apply an increasing force to the gel and we optically measure the resulting deformation. from those measurements we deduce a young modulus for a large amount of data. we are characterizing different networks by varying the concentration of the capping and branching proteins and we show how mechanics can be regulated by the different proteins. microtubules (mts) are central to the organization of the eukaryotic intracellular space and are involved in the control of cell morphology. in fission yeasts cells mts transport polarity factors to poles where growth is located, thus ensuring the establishment and maintenance of the characteristic spherocylindrical shape. for this purpose, mt polymerization dynamics is tightly regulated. using automated image analysis software, we investigated the spatial dependence of mt dynamics in interphase fission yeast cells. we evidenced that compressive forces generated by mts growing against the cell pole locally reduce mt growth velocities and enhance catastrophe frequencies. in addition, our systematic and quantitative analysis (in combination with genetic modifications) provides a tool to study the role of ?tips (plus-end tracking proteins) such as mal and tip in the spatial regulation of mt dynamics. we further use this system to decipher how the linear transport by mt interferes with the feedback circuitry that assures the correct spatial distribution of tea , the main polarity factor in fission yeast cells. the dynamics of the cytoskeleton are largely driven by cytoskeletal motor proteins. complex cellular functions, such as mitotsis, need a high degree of control of these motors. the versatility and sophistication of biological nanomachines still challenges our understanding. kinesin- motors fulfill essential roles in mitotic spindle morphogenesis and dynamics and were thought to be slow, processive microtubule (mt)-plus-end directed motors. here we have examined in vitro and in vivo functions of the saccharomyces cerevisiae kinesin- cin using single-molecule motility assays and single-molecule fluorescence microscopy. in vivo, the majority of cin motors moved slowly towards mt plus-ends, but we also observed occasional minus-end directed motility episodes. in vitro, individual cin motors could be switched by ionic conditions from rapid (up to lm/min) and processive minus-end, to bidirectional, to slow plus-end motion. deletion of the uniquely large insert in loop of cin induced bias towards minus-end motility and strongly affected the directional switching of cin both in vivo and in vitro. the entirely unexpected in vivo and in vitro switching of cin directionality and speed demonstrate that kinesins are much more complex than thought. these results will force us to rethink molecular models of motor function and will move the regulation of motors into the limelight as pivotal for understanding cytoskeleton-based machineries. morphological and dynamical changes during tgf-b induced epithelial-to-mesenchymal transition david schneider , marco tarantola , and andreas janshoff institute of physical chemistry, georg-august-university, gö ttingen, germany, max planck institute for dynamics and self-organization, laboratory for fluid dynamics, pattern formation and nanobiocomplexity (lfpn), goettingen, germany the epithelial-to-mesenchymal transition (emt) is a program of cellular development associated with loss of cell-cell contacts, a decreased cell adhesion and substantial morphological changes. besides its importance for numerous developmental processes like embryogenesis, emt has also been held responsible for the development and progression of tumors and formation of metastases. the influence of the cytokine transforming growth factor (tgf-b ) induced emt on structure, migration, cytoskeletal dynamics and long-term correlations of the mammalian epithelial cell lines nmumg, a and mda-mb was investigated by time-resolved impedance analysis and atomic force microscopy (afm) performing force-indentation measurements. the three cell lines display important differences in cellular morphology mirrored in changes of their elastic response (young modulus), as well as their dynamics upon tgf-b treatment. impedance based measurements of micromotility reveal a complex dynamic response to tgf-b exposure which leads to a transient increase in fluctuation amplitude and long-term correlation. additionally, the investigation of cellular elasticity via afm depicts the different cytoskeletal alterations depending on the metastatic potential of the used cell type. physics of cellular mechanosensitivity studied with biomimetic actin-filled liposomes bjö rn stuhrmann, feng-ching tsai, guido mul, gijsje koenderink fom institute amolf, amsterdam, the netherlands biological cells actively probe the mechanical properties of their tissue environment by exerting contractile forces on it, and use this information to decide whether to grow, migrate, or proliferate. the physical basis for cell contractility is the actin cytoskeleton, which transmits motor generated stresses to mechanosensitive adhesions sites that anchor the cell to the tissue. the origins of mechanosensing are far from understood due to the complex interplay of mechanical effects and biochemical signaling that occurs far from equilibrium. we use a quantitative biophysical approach based on biomimetic constructs to elucidate physical principles that underlie active mechanosensing in biological cells. we have built realistic in vitro models of contractile cells by encapsulating cross-linked actin networks together with myosin motors in cell-sized membranous containers (liposomes). our method has several advantages over prior methods, including high liposome yield, compatibility with physiological buffers, and chemical control over protein/lipid coupling. i will show contour fluctuation spectra of constructs and first data on mechanical response obtained by laser tweezers microrheology. our work will yield novel insights into stress generation and stiffness sensing of cells. setting up a system to reconstitute cytoskeleton-based protein delivery and patterning in vitro nú ria taberner, liedewij laan, marileen dogterom fom institute amolf, amsterdam, the netherlands keywords: microtubules, fission yeast, cell polarity, protein patterning, plus end binding proteins. many different cell types, from mobile fibroblasts [ ] to fission yeast cells [ ] , display non-homogenous protein patterns on their cell cortex. in fission yeast the cell-end marker protein tea that among others is responsible for recruiting the actin dependent cell-growth machinery, is specifically located at the growing cell ends. tea travels at the tips of growing microtubules and is delivered to the cell ends [ ] . we aim to in vitro reconstitute a minimal microtubule plus-end tracking system that leads to cortical protein patterning in functionalized microfabricated chambers. our model will allow us to perturb microtubule-based transport and diffusion independently and evaluate the resulting protein patterns. [ the tropomyosins (tm) are dimeric actin-binding proteins that form longitudinal polymers along the actin filament groove. there is a great variety of isoforms, but the division of labour between the individual tms and their significance is poorly understood. as in most cell types, also in the neurons several isoforms are present, whose spatio-temporal localisation is differentially regulated. the neuron-specific brain isoform (tmbr- ) can be found in the axon of the mature cells. we aimed to clone, express and charaterise this protein in terms of its effects on the kinetic parameters of the actin filament. using a pet a construct we purified native, tag-free protein, and examined if it influences the rate of actin polymerisation or the stability of the filaments in the presence of either gelsolin or latrunculin-a, two depolymerising agents. in cosedimentation experiments the affinity of tmbr- to actin was* lm, about six times that of skeletal muscle tropomyosin. the net rate of actin polymerisation was reduced by % in the presence of tmbr- . the depolymerisation induced by gelsolin or latrunculin-a was inhibited in a concentration-dependent manner. tmbr- seems to stabilise actin filaments against disassembly without significant effect on the net polymerisation. cell mobility and metastatic spreading: a study on human neoplastic cells using optical tweezers f. tavano the primary causes of death in cancer patients are local invasion and metastasis but their mechanisms are not yet completely understood. metastatization is accompanied by alterations of the cytoskeleton and membrane structure leading to changes in their biomechanical properties [ ] . in this study we analyzed by means of optical tweezers the mechanical properties of two different breast adenocarcinoma cell lines corresponding to different metastatic potential. ot were used to grab the plasma membrane by a , um silica bead and form a plasma membrane tether. we measured the force exerted by the cell membrane on the bead and drew the force-elongation curves. fitting data in the kelvin body model [ ] we found out the values for the viscoelastic parameters influencing the pulling of the membrane tethers. the first cell line analyzed, mcf- , associated to a low metastatic potential showed tether stiffness of pn/um in average. the second cell line, mda-mb , poorly differentiated with a high metastatic potential had a tether stiffness of pn/um in average, that is a four times lower value. these results seems to confirm the hypotesis that metastasis prone cells are softer than less aggressive cancer cells, and support the use of ot for these measurements for its sub-pn force resolution and because cells are manipulated without damage. [ tubulin polymerization promoting protein (tppp/p ) is a brain-specific protein that primary targets the microtubule network modulating its dynamics and stability. tppp/p is a disordered protein with extended unstructured segments localized at the n-and c-terminals straddling a flexible region. tppp/p is primarily expressed in oligodendrocytes where its multifunctional features such as tubulin polymerization promoting and microtubule bundling activities are crucial for the development of the projections in the course of oligodendrocyte differentiation enabling the ensheathment of axons with a myelin sheath that is indispensable for the normal function of the central nervous system. microtubule network, a major constituent of the cytoskeleton, displays multiple physiological and pathological functions in eukaryotic cells. the distinct functions of the microtubular structures are attained by static and dynamic associations of macromolecules and ligands as well as by post-translational modifications. tppp/p is actively involved in the regulation of microtubule dynamics not exclusively by its bundling activity, but also by its tubulin acetylation-promoting activity. atypical histone deacetylases, such as nad-dependent sirt and histone deacetylase- , function outside of the nucleus and control the acetylation level of cytosolic proteins, such as tubulin. acetylation-driven regulation of the microtubule network during cellular differentiation is an ambiguous issue. tppp/p has been recently identified as an interacting partner and inhibitor of these deacetylases and their interaction decreased the growth velocity of the microtubule plus ends and the motility of the cells. we have established cell models for the quantification of the acetylation degree of microtubule network in correlation with its dynamics and stability as well as in relation to aggresome formation, that mimics the pathological inclusion formation. the intracellular level of tppp/p is controlled at posttranscription level by microrna and at protein level by the proteosome machinery. under pathological circumstances this disordered protein displays additional moonlighting function that is independent of its association with microtubule system or deacetylases; it enters aberrant proteinprotein interaction with a-synuclein forming toxic aggregates within the neuronal and glial cells leading to the formation of inclusions characteristic for parkinson's disease and multiple system atrophy, respectively. the cell membrane separates the intracellular from the extracellular environment while intimately interacting with the cytoskeleton in numerous cellular functions, including cell division and motility. cell shape changes are for a large part mediated by the contractile actomyosin network forming the cortex underneath the cell membrane. to uncover molecular mechanisms of cell shape control based on actin-membrane interactions, we built a novel biomimetic model system: a cell-sized liposome encapsulating an actively contracting actin-myosin network. our fabrication method is inspired by a recent report of liposome preparation by swelling of lipid layers in agarose hydrogel films. we extensively characterize important liposomal properties, finding diameters between and lm, unilamellarity, and excellent and uniform encapsulation efficiency. we further demonstrate chemical control of actin network anchoring to the membrane. the resulting liposomes allow quantitative tests of physical models of cell shape generation and mechanics. in the cohesive structure of the cytoskeleton functionally distinct actin arrays orchestrate fundamental cell functions in a spatiotemporally controlled manner. emerging evidences emphasize that protein isoforms are essential for the functional polymorphism of the actin cytoskeleton. the generation of diverse actin networks is catalyzed by different nucleation factors, like formins and arp / complex. these actin arrays also exhibit qualitative and quantitative differences in the associated tropomyosin (tm) isoforms. how the molecular composition and the function of actin networks are coupled is not completely understood. we investigated the effects of different tm isoforms (skeletal muscle, cytoskeletal nm and br ) on the activity of mdia formin and arp / complex using fluorescence spectroscopic approaches. the results show that the studied tm isoforms have different effects on the mdia -, and arp / complexmediated actin assembly. the activity of the arp / complex is inhibited by sktm and tm nm , while tmbr does not have any effect. all three tm isoforms inhibited the activity of mdia . these results contribute to the understanding of the mechanisms by which tropomyosin isoforms regulate the functional diversity of the actin cytoskeleton. chronic thromboembolic pulmonary hypertension (cteph) is a dual pulmonary vascular disorder, which combines major vascular remodelling with small-vessel arteriopathy. the presence of fibroblasts in the clot, occluding the pulmonary arteries, and its composition create a microenvironment with increased collagen level, which might affect the local endothelial function. in this study, human pulmonary artery endothelial cells (hpaec) were exposed to collagen type i to address the effect of the thrombotic microenvironment on the vessel wall forming cells. the hpaecs, cultured under standard conditions were treated with , and lg/ml of collagen type i for h and h. the changes in the endothelial cell barrier function were investigated by performing permeability and migration test as well as ve-cadherin staining. collagen type i treatment led to a decrease in ve-cadherin signal in hpaec. the loosening of cell-cell contacts could be proven with a significant increase in permeability after h of collagen treatment with different concentrations. besides the loosening of the cell-cell contacts, the hpaec migration was also dose dependently retarded by collagen application over time. our data show that collagen-rich microenvironment leads to a disruption of the junctional proteins in hpaecs, indicating an environmental induced possible alteration in the function of endothelial cells in the clots of cteph patients. the implementation of miniaturisation and high throughput screening has quickened the pace of protein structure determination. however, for most proteins the process still requires milligram quantities of protein with purity [ %. these amounts are required as a result of unavoidable losses during purification and for the extensive screening of crystallisation space. for integral membrane proteins (imps), one of the initial steps in the structure determination procedure is still a major bottleneck -the over-expression of the target protein in the milligram quantity range. with a view of developing guidelines for over-expression of human imps, a systematic approach using the three most common laboratory expression systems (e. coli, s. cerevisiae, sf insect cells) was implemented. initial expression levels were determined by either partial purification using ni ? -nta (e. coli), green fluorescence protein (gfp) fluorescence using a c-terminal gfp tagged protein (s. cerevisiae) or flag tagged partial purification (sf cells). the results show that e. coli is suitable for the over-expression of human imps in the required quantity range however protein size and complexity is an important factor. the yeast system is fast and affordable but, for the group of human imps tested, the expression levels were borderline. finally for the insect cell system, the timelines are slower and it is in comparison costly to run, however, it can produce relatively large quantities of human imps. the cu?-atpase copb of enterococcus hirae is a bacterial p-type atpase involved in resistance to high levels of environmental copper by expelling excess copper. the membrane protein copb was purified from an over-expressing strain and solubilized in dodecyl-maltoside. by uv circular dichroism the secondary structure is predicted to contain - % a-helices and - % b-sheets in agreement with estimates based on homology with the ca atpase serca . we present cd-spectroscopic data on thermal unfolding of the protein to address the influence of the binding of the atp analogs atpcs and the fluorescent analog mant-atp on the protein stability. such analogs are used to mimic functional states of the atpase but undergo different interactions with the binding site that are not well characterized. we propose a competition-based assay for nucleotide binding using cdspectroscopy to deduce the occupancy of the nucleotidebinding site by non-fluorescent nucleotides. alternatively, the change of intrinsic fluorescence of mant-atp upon binding to the atpase is exploited in these assays. finally, we show how the simultaneous measurement of protein cd and nucleotide fluorescence in thermal denaturation experiments may help to determine the stability of several functional conformational states of copb. showing the steady-state distribution of electric potential, ionic concentrations are obtained efficiently. channel current, a summation of drift and diffusive currents, can be further computed from the flux of ionic concentrations. the influence of finite size effect will be also addressed. effect of cholesterol and cytoskeleton on k v . membrane distribution jimé nez-garduño am , , pardo la , ortega a , stü hmer w unam, mexico-city, mexico, mpi-em, gö ttingen, germany the potassium channel k v . is expressed nearly exclusively in the central nervous system. besides its function as an ion channel, k v . has also been associated with non-canonical signaling functions. various membrane proteins associated with cholesterol-sphingolipids enriched microdomains are involved in signaling pathways. in this work we studied the membrane distribution of k v . in highly purified brain-tissue plasma membranes as a function of cholesterol content versus cytoskeletal proteins. the results show that one fraction of kv . associates to cholesterol-rich domains or detergent resistant membranes (drm) and another fraction to non-drm domains. the kv . fraction inserted in drm is dependent on cholesterol as well as on cytoskeleton proteins. depletion of cholesterol leads to a doubling of k v . current density. we suggest that k v . coexists in two different populations: one where the transmembrane domain fits cholesterol enriched membranes and another able to fit into a less packed lipid bilayer. the importance of this distribution on signaling processes needs to be further investigated. we use the reduced model of an axis-symmetric water-filled channel whose protein wall has a single charged site. the channel length, radius and fixed charge are selected to match experimental data for gramicidin a. the ion current, occupancy and escape rate are simulated by the d self-consistent bd technique with account taken of the electrostatic ion-ion interaction. the bath with non-zero ion concentration on one side of the channel is modelled via the smoluchowski arrival rate. it is shown that: a) the occupancy saturates with michaelis-menten kinetics. b) the escape rate starts from the kramers value at small concentrations and then increases with concentration due to the electrostatic amplification of charge fluctuations. the resulting dynamics of the current can be described by modified reaction rate theory accounting for ionic escape over the fluctuating barrier [ ] . many membrane-protein functions are amenable to biophysical and biochemical investigation only after the protein of interest has been reconstituted from a detergent-solubilised state into artificial lipid bilayers. unfortunately, functional reconstitution has remained one of the main bottlenecks in the handling of numerous membrane proteins. in particular, gauging the success of reconstitution experiments has thus far been limited to trial-and-error approaches. to address this problem, we have established high-sensitivity isothermal titration calorimetry (itc) as a powerful method for monitoring the reconstitution of membrane proteins into liposomes. itc has previously been employed for characterising liposome solubilisation and reconstitution in the absence of protein. here we show that itc is also excellently suited for tracking the complex process of membrane-protein reconstitution in a non-invasive and fully automated manner. the approach is exemplified for the prokaryotic potassium channel kcsa, which we first purified in detergent micelles and then reconstituted into stable proteoliposomes at very high protein densities. electrophysiological experiments performed in planar lipid membranes confirmed that kcsa regained its functional activity upon itc-guided reconstitution. gating currents of low-voltage-activated t-type calcium channels family má ria karmažínová , Ľ ubica lacinová institute of molecular physiology and genetics, sav, bratislava, slovak republic t-type calcium channels are distinguished by relatively low voltage threshold for an activation and steep voltage dependence of activation and inactivation kinetics just above the activation threshold. kinetics and voltage dependence of macroscopic inward calcium current through ca v channels was described in a detail. in contrast, very little information is available on gating current of these channels. therefore we compared gating currents measured from all three ca v . , ca v . and ca v . channels. voltage dependencies of charge movement differ dramatically from those for macroscopic current. first, their slope factors are several-fold bigger that slope factors of macroscopic current activation. second, activation mid-point for ca v . channels on-gating is shifted to more positive membrane potentials by about mv compare to ca v . and ca v . channels, whose activation mid-points are similar. the same is truth for off-gating voltage dependences. kinetics of both onand off-gating is remarkably faster for ca v . and ca v . channels compare to ca v . channels. further, more charge is moved per unit of macroscopic current amplitude in ca v . channels compare to ca v . and ca v . channels. supported by apvv- - and vega / / . the local anaesthetic lidocaine (lid) is generally believed to reach its binding site in the intracellular vestibule of the voltage-gated sodium channel via the cell membrane. qx (qx) is a permanently charged, quaternary amine analogue of lid, that can access this binding site via a hydrophilic route across the channel protein. the mutation i e of the adult rat muscle-type sodium channel (rna v . ) opens such a hydrophilic pathway. when bound to the internal vestibule, lid stabilizes both fast and slow inactivated states. we wondered whether qx, once bound to the internal vestibule, exerts a similar modulatory action on inactivated states as lid. the construct i e was expressed in tsa cells and studied by means of the patch-clamp technique. when applied from the extracellular side lm qx stabilized the slow but not the fast inactivated state in i e. when applied internally, qx entered the channel, but stabilization of inactivated states could not be observed. these results suggest that binding site for use-dependent block is in the inner vestibule of the channel, fast inactivation is modulated only by the hydrophobic form of lid, and the binding site for modulation of slow inactivation by qx is only accessible from the extracellular side of the channel. we observed that lipophilicity (quantified by the logarithm of the calculated water-octanol partition coefficient, logp) is important in determining both kr and ki, but had a greater effect on ki. distribution coefficients (logd) discriminated better between kr and ki than partition coefficients (logp). the ratio of positively charged/neutral forms (quantified by the acidic dissociation constant, pka) was a significant determinant of resting affinity: predominantly charged compounds tended to be more potent against resting channels, while neutral compounds tended to be more state-dependent. aromaticity was more important for inactivated state affinity. the acidification of intracellular compartments is critical for a wide range of cellular processes. a recent candidate for ph regulation within early endosomes and tgn is the highly conserved intracellular na ? /h ? exchanger isoform (nhe ), whose mutation leads to neurological syndromes in human patients. however, due to its intracellular localization, nhe biochemical features are still poorly characterized and its biological function remains elusive. we have developed somatic cell genetic techniques that enable the selection of variant cells able to resist h ? killing through plasma membrane expression of h ? extruders. this enabled us to obtain stable cell lines with forced plasma membrane expression of nhe . we used them to measure its functional and pharmacological parameters with high accuracy, using fast transport kinetics. to summarize, this exchanger displays unique features within the nhe family, especially with respect to its affinity for its substrates, lithium, sodium and protons and for its guanidine-derived inhibitors. taken together with our results on the subcellular localization of the native nhe , these unique biochemical features provide new insights on the biological function and pathological implications of this intracellular na ? /h ? exchanger. analysis of the collective behaviour of ion channels j. miśkiewicz, z. trela, s. przestalski wrocław university of environmental and life sciences, physics and biophysics department, ul. norwida , - wrocław, poland a novel approach to the analysis of the ion current recordings is proposed. the main goal of the standard patch clamp technique is to measure single channel activity (however the whole cell configuration is also used in various researches). in the presented study the ion channels time series recordings were several (up to four) ion channels were present are analysed and the collective behaviour of ion channels is investigated. the time ion current time series are converted into dwelltime series and the channel activity is analysed. the hypothesis of collective ion channels behaviour is verified and the influence of organolead compounds (met pbcl) on collective ion channel activity is measured. the analysis is performed on the sv cation channels of vacuolar membrane of beta vulgaris. the aim of our computed study was to examine the possible binding site of primaquine (pq) using a combined homology protein modeling, automated docking and experimental approach. the target models of wild-type and mutant-types of the voltage-dependent sodium channel in rat skeletal muscle (rna v . ) were based on previous work by tikhonov and zhorov. docking was carried out on the p-loop into the structure model of rna v . channel, in the open state configuration, to identify those amino acidic residues important for primaquine binding. the threedimensional models of the p-loop segment of wild types and mutant types (w . w c, w c and w a at the outer tryptophan-rich lip, as well as d c, e c, k c and a c of the deka motif) helped us to identify residues playing a key role in aminoquinoline binding. in good agreement with experimental results, a -fold inhibition loss was observed, tryptophan is crucial for the reversible blocking effects of pq. as a result, w c abolished the blocking effect of primaquine in voltage-clamp assays. hydrogen bond formation accelerates channel opening of the bacterial mechanosensitive channel mscl yasuyuki sawada and masahiro sokabe department of physiology, nagoya university graduate school of medicine, nagoya, japan the bacterial mechanosensitive channel mscl is constituted of homopentamer of a subunit with two transmembrane inner and outer (tm , tm ) a-helices, and its d structure of the closed state has been resolved. the major issue of mscl is to understand the gating mechanism driven by tension in the membrane. to address this question, we performed molecular dynamics (md) simulations for the opening of mscl embedded in the lipid bilayer. in the closed state of mscl, neighboring tm inner helices are crossed each other near the cytoplasmic side and leu and val in the constricted part form a stable hydrophobic environment called gate. upon membrane stretch, phe in tm outer helices was dragged by lipids, leading to an opening of mscl. thus phe was concluded to be the major tension sensor. during opening, tm inner helices were also dragged and tilted, accompanied by the outward sliding of the crossings. this led to a slight expansion of the gate associated with an exposure of oxygen atoms of the backbone to the inner surface of the gate. this allows water penetration in the gate and formation of hydrogen bonds between water and the exposed oxygen, which in turn weakened the hydrophobic interaction at the crossings, causing a further opening of the gate and water permeation. mitochondrial bk ca , mitobk ca has been proposed to be cardioprotective and formed by proteins of * to * kda. thus, we investigated the molecular characteristics of this channel in isolated mitochondria from murine heart. labeling of adult mouse cardiomyocytes with plasmalemma bk ca antibodies, mitotracker, and wheat germ agglutinin yielded remarkable mitochondrial but not plasma membrane localization. nanoscale fluorescence microscopy (stimulated emission depletion) revealed to of * - nm bk ca clusters per mitochondria. further, western blot analysis of purified mitochondria showed the presence of a full length * kda protein. systematic rt-pcr exon scanning of isolated cardiomyocyte mrnas were consistent with a full length * kda alpha-subunit protein and revealed the expression of three splice inserts. insertless-bk ca robustly localized to the plasma membrane of cho cells but when a c-terminal splice insert was present bk ca was readily targeted to the mitochondria (protein proximity index was * . indicating % colocalization). hence, cardiac mitobk ca is composed by full-length bk ca protein but with splice inserts which may facilitate its targeting to mitochondria. supported by nih and aha. patch-clamp technique was used to examine effect of trimethyl-lead and -tin on the sv channel activity in the red beet (beta vulgaris l.) taproot vacuoles. it was found that the addition of both investigated compounds to the bath solution inhibit, in a concentration-dependent manner, sv currents. when single channel properties were analyzed, only little channel activity can be recorded in the presence of lm of organometal. compounds investigated decreased significantly (by about one order of magnitude) the open probability of single channels. the recordings of single channel activity obtained in the presence and in the absence of organometal showed that compounds only slightly (by ca. %) decreased the unitary conductance of single channels. it was also found that organometal diminished significantly the number of sv channel openings, whereas it did not change the opening times of the channels. taken together, these results suggest that organometal binding site is located outside the channel's selectivity filter and that the inhibitory effect of both compounds investigated on sv channel activity probably results from organometal-induced disorder in compatibility between membrane lipids and membrane proteins. the research was financed by polish ministry of science and higher education by grant no. nn . electrophysiological investigation of the hvdac ion channel in pore-spanning membranes conrad weichbrodt, claudia steinem georg-august-universitä t gö ttingen, iobc, tammannstr. , d- gö ttingen, germany, e-mail: cweichb@gwdg.de the human voltage-dependent anion channel (hvdac ) plays an important role in cell life and apoptosis since it is the main porin of the outer mitochondrial membrane. as hvdac is believed to play a pivotal role in apoptosis-related diseases such as stroke, alzheimer, parkinson and cancer, the alterations of its electrophysiological properties under different conditions are of great value. to perform different investigations, refolded hvdac is reconstituted in artificial membranes which typically consist of dphpc with a cholesterol fraction of - %. they are prepared via the mü ller-rudin-technique on a functionalized porous alumina substrate containing pores with a diameter of nm. the quality of these so-called nano-black-lipid-membranes (nano-blms) is verified via electrochemical impedance spectroscopy (eis), hvdac is reconstituted and single channel recordings are made. membranes are also prepared by spreading proteoliposomes on hydrophobized porous silicon nitride with pores of lm diameter. information about altered gating-characteristics and related conductivities is gained by application of holding potentials up to ± mv and evaluation of the resulting currents. the hvdac was a kind gift of prof. c. griesinger, mpibpc, gö ttingen. pharmacological inhibition of cardiac herg k ? channels is associated with increased risk of arrhythmias. many drugs bind directly to the channel, thereby blocking ion conduction. ala-scanning mutagenesis identified residues important for drug block. two aromatic residues y and f were found to be crucial for block of most compounds. surprisingly, some cavity blocking drugs are only weakly affected by mutation y a. in this study we provide a structural interpretation for this observation. md simulations on the y a mutant suggest side chain rearrangements of f located one helical turn below y . loss of p-p stacking induces reorientation of f from a cavity facing to a cavity lining conformation, thereby substantially altering the shape of the binding site. docking studies reveal that due to their rigid shape and compactness y insensitive drugs can still favorably interact with the reoriented f aryl groups, while molecules with more extended geometries cannot. the ankyrin transient receptor potential channel trpa is a transmembrane protein that plays a key role in the transduction of noxious chemical and thermal stimuli in nociceptors. in addition to chemical activation, trpa can be activated by highly depolarizing voltages but the molecular basis of this regulation is unclear. the transmembrane part of the tetrameric trpa is structurally related to the voltagegated k ? channels in which the conserved charged residues within the fourth transmembrane region (s ) constitute part of a voltage sensor. compared to these channels, the voltage-dependence of trpa is very weak (apparent number of gating charges * . versus in k ? channels) and its putative voltage-sensing domain most likely lies outside the s because trpa completely lacks positively charged residues in this region. in the present study we used homology modelling and molecular dynamics to create models of the transmembrane part and the proximal cytoplasmic c terminus of trpa . in combination with electrophysiological data obtained from whole cell patchclamp measurements we were able to point out several positively charged residues which mutation strongly alter the voltage sensitivity of trpa channel. these may be candidates for as yet unrecognized voltage sensor. photosynthesis p- action of double stress on photosystem aliyeva samira a., gasanov ralphreed a. institute of botany, azerbaijan national academy of sciences, baku, azerbaijan the simultaneous effect of photoinhibitory illumination and toxic action of heavy metals ions (cd ? and co ? ) on activity of ps in vitro measuring by millisecond delayed fluorescence (ms-df) of chlorophyll a was studied. during action on chloroplasts only of cd ? ( - m) the fast component of ms-df, which originates via radiative recombination of reaction center with the camn -cluster or y z on donor side of ps , is inhibited stronger than at action of only co ? . the steadystate level at cd ? treatment is remain stable, while at co ? action it is increased. simultaneous action of cd ? and photoinhibitory illumination ( lmol photons m - s - ) have shown that fast component of ms-df was inhibited faster with time than in case of action of co ? and excess light. result indicates that damage sites of action cd ? and co ? are donor and acceptor side of ps , accordingly. we assume that binding site of cd ? is y z or camn -cluster, one of the recombination partners with p ? on the donor side of ps . thereby, action of cd ions on donor side of ps leads mainly to development of mechanism of donor-side photoinhibition. field instrument for determination of the photosynthetic capacity of intact photosynthetic bacteria e. asztalos , z. gingl and p. maró ti department of medical physics and informatics, university of szeged, hungary, department of experimental physics, university of szeged, hungary a combined pump and probe fluorometer and spectrophotometer with high power laser diodes has been constructed to measure fast induction and relaxation of the bacteriochlorophyll fluorescence yield and light-induced absorption changes in intact cells of photosynthetic bacteria. the construction is the upgraded version of our previous set up with better time resolution ( ls). the compact design of the mechanics, optics, electronics and data processing makes the device easy to use as outdoor instrument or to integrate into larger measuring systems. the versatility and excellent performance of the apparatus will be demonstrated on different fields: ) organization and redox state of the photosynthetic apparatus of the whole cells under different growth conditions deduced from fluorescence characteristics including the lag phase, the amplitude and the rise time of the variable fluorescence, ) electron transfer in the reaction center, cytochrome bc complex and in between obtained from relaxation of the fluorescence and ) re-reduction kinetics of the oxidized primary donor of the reaction center and energetization and relaxation of the intracytoplasmic membrane tracked by absorption changes at and nm, respectively. previous work has established that the iron stress induced protein a (isia) synthesized by cyanobacteria under stress conditions, has at least two functions: light harvesting [ ] and photoprotection [ ] . under prolonged iron starvation isia becomes the main chlorophyll-binding protein in the cell and occurs without a photosystem association. these isia aggregates have a strong ability to dissipate light energy and there is evidence of carotenoid participation in the quenching mechanism via downhill energy transfer from chlorophyll to the s state of a carotenoid [ ] . in the present work we have measured the temperature dependence of the fluorescence of carotenoid depleted mutants (echinenone and/or zeaxanthin) and isia monomers in order to investigate the role of carotenoid and aggregation in the quenching process. pigment analysis confirms the absence of the carotenoid mutated in its biosynthesis but shows that it is mainly replaced. the monomers are lacking two carotenoids, echinenone and one of the two ß-carotenes found previously in isia aggregates. temperature dependent fluorescence shows that quenching properties are affected in the monomers and the mutants lacking zeaxanthin. soon exhausted oil resources and global climate change have stimulated research aiming at production of alternative fuels, ideally driven by solar energy. production of solar fuels needs to involve the splitting of water into protons, energized electrons and dioxygen. in photosynthetic organisms, solar-energy conversion and catalysis of water splitting (or water oxidation) proceed in an impressive cofactor-protein complex denoted as photosystem ii (psii). the heart of biological water-oxidation is a protein-bound manganese-calcium complex working at technically unmatched efficiency. in an attempt to learn from nature, the natural paragon is intensely studied using advanced biophysical methods. structural studies by x-ray spectroscopy with synchrotron radiation play a prominent role in this endeavor. time-resolved methods provide insights in the formation of intermediate states of the reaction cycle. an overview is presented focusing on (i) the efficiency of solar energy usage in psii, (ii) the interrelation between electron transfer and proton relocations, and (iii) the mechanism of water oxidation. as an outlook, new results on water oxidation by biomimetic manganese and cobalt oxides, which may become a key element in future solar-fuel systems, are presented. the peridinin-chlorophyll-protein (pcp) is a light-harvesting complex (lhc) that works as antenna in the photosynthetic process of dinoflagellates. the protein contains both chlorophylls and carotenoids molecules, the latter being responsible to extend the spectral range of captured light to regions where chlorophylls are transparent. pcp crystal structures [ ] reveal that each chlorophyll is surrounded by or molecules of the carotenoid peridinin, located in non-equivalent positions. the different protein environment of the sites might be responsible of a spectral shift of the pigments with the functional role to extend the absorption spectra of the complex and enhance its light harvesting capabilities. high resolution x-ray diffraction data on a reconstructed pcp, the refolded peridinin-chlorophyll a-protein (rfpcp) [ ] , and on the less common high salt-pcp (hspcp) opened the way to the mechanistic understanding of peridinin spectral tuning, peridinin chlorophyll energy transfer and photoprotective mechanism [ ] . the two pcp forms differ in various features: spectral properties, molar mass, amino acid sequence and, above all, pigment stoichiometry, the peridinin:chlorophyll ratio being : for the rfpcp and : for the hspcp [ ] . in the present work we perform classical molecular dynamics simulations of the rfpcp and the hspcp in explicit water solution. we analyse the structure and dynamics of the proteins and of their pigments to characterize the different peridinin sites in both pcp forms in terms of quantities that can affect the chromophore spectra, such as distorsion, fluctuations and nature of the protein environment. the comparison between the data suggests correspondences between the pigments of the two forms. quantum and mixed quantum/ classical molecular dynamics simulations are also under progress to investigate the effect of the protein environment on the electronic and optical properties of the pcp pigments. peculiar applications, like in optoelectronics, biosensors, photovoltaics. among the existing carrier matrices conductive metal oxides (e.g. indium tin oxide, ito), carbon nanotubes, graphenes, silicon (si) are the most frequently used materials because of their unique characteristics such as good conductivity, good optical properties and excellent adhesion to substrates. in our work we combined purified photosynthetic reaction center protein (rc) and porous silicon (psi) investigating the morphology and optoelectronic properties of the bio-nanocomposite material. ftir spectroscopy, scanning electron microscopy and energydispersive x-ray spectroscopy indicated the binding of the protein to the psi. specular reflectance spectra showed a red shift in the characteristic interference peak of the psi microcavity which was saturated at higher concentration of the protein. the binding was more effective if the functionalization was done by the si-specific oligopeptide compared to the classical covalent binding via aminopropyl-triethoxysilane (aptes). excitation by single saturation flashes indicated that the rc still exhibited photochemical turnover after the binding. the role of reactive oxygen species (ros) in plant stress, both as damaging agent and as potential signal molecule is often assessed in experiments using photosensitized elicitor dyes. for these studies, it is essential to know how efficiently these chemicals generate ros, whether they are specific ros sources, as well as their cellular localization and additional side effects. the present study addresses these issues using a variety of dyes known and traditionally applied as singlet oxygen ( o ) sources. rose bengal (rb), methylene violet (mvi), methylene blue (mb), neutral red (nr) and indigo carmine (ic) were studied as putative ros sources in tobacco leaves. ros products of photosensitized dyes were measured in vitro, using spin trapping epr spectroscopy. dye concentrations and irradiation concentration leading to equal absorbed excitation quanta were determined spectrophotometrically. in vivo studies were carried out using tobacco leaves infiltrated with water solutions of the putative o sources. cellular localizations were identified on the basis of the dyes' fluorescence. rb, nr and mvi reached into mesophyll cells and were used to study the effects of these dyes on photosynthesis. photochemical yields and quenching processes were compared before and after photosensitization of the elicitor dyes inside the leaf samples. chlorophyll-chlorophyll charge transfer quenching is the main mechanism of non-photochemical quenching in higher plants alfred r. holzwarth max-planck-institut fü r bioanorganischechemie, stiftstraße - , d- mü lheim a.d.ruhr, germany non-photochemical quenching (npq) in plants protects against photochemical destruction of the photosynthetic apparatus under excess light conditions.while one location of the npq process has been shown to be centered on the major light harvesting complex ii (lhcii) (q type or qequenching), an additional quenching center responsible for qi type (identical to q center) quenchinghas been suggested to be located on the minor light-harvesting complexes upon accumulation ofzeaxanthin (zx), in particularon cp and cp we have performed femtosecond transient absorption and time-resolved fluorescence measurements of npq quenching in intact leaves of higher plants, on isolated light harvesting complexes in the minor (non-aggregated)light harvesting complex cp reconstituted with violaxanthin (vx) or zx, and in the isolated major lhc ii complex in the aggregated state. in all of these situations we find the formation of chl-chl charge transfer (ct) states to be the dominant quenching mechanism. the yield of formation of carotenoid cation states and/or carotenoid s state is either extremely low or absent, thus excluding their involvement in npq quenching as a major quenching mechanism. single-molecule spectroscopy (sms) is a powerful technique that allows investigation of fluorescence properties from single fluorescing systems. this technique enabled us to investigate the dynamics of the fluorescence intensity and spectral profiles of single, isolated light harvesting complex (lhc) on timescales of milliseconds to seconds, during continuous laser illumination. we were able to observe how each complex can rapidly switch between different emission states [ , ] and to characterise the intensity and the spectral dynamics of major and minor antenna complexes from plants, in two different environments, mimicking the light harvesting and the light dissipating state, respectively. the results will be discussed with respect to the current models for nonphotochemical quenching (npq) mechanisms [ , , ] , a vital photoprotection mechanism during which the lhcs of plants switch between a state of very efficient light utilisation and one in which excess absorbed excitation energy is harmlessly dissipated as heat. phaeodactylum tricornutum is one of the most utilized model organisms in diatom photosynthesis research mainly due to availability of its genome sequence ( ). it's photosynthetic antennae are the fucoxanthin chlorophyll a/c binding proteins (fcps) which share a high degree of homology with lhcs of higher plants and green algae ( ) . for detailed investigation of the antenna system of p.tricornutum, a transgenic strain expressing recombinant his-tagged fcpa protein was created which simplified the purification of a specific stable trimeric fcp complex consisting of fcpa and fcpe proteins. excitation energy coupling between fucoxanthin and chlorophyll a was intact and the existence of a chlorophyll a/fucoxanthin excitonic dimer was demonstrated ( ). we investigated in detail the existence of specific antenna system for psi and psii in p.tricornutum as in case of higher plants. our studies indicated that at least the main light harvesting proteins fcpa and fcpe are most probably shared as a common antenna by both psi and psii. harvesting complex ii (lhciib) from spinach or in native thylakoid membranes by picosecond time-resolved fluorescence. the domain size was estimated by monitoring the efficiency of added exogeneous singlet excitation quenchers -phenyl-p-benzoquinone (ppq) and dinitrobenzene (dnb). the fluorescence decay kinetics of the systems under study were registered without quenchers and with quenchers added in a range of concentrations. stern-volmer constants, k sv and k sv -for aggregates (membranes) and detergentsolubilized complexes, respectively, were determined from the concentration dependence of the ratio of the mean fluorescence lifetimes without/with quencher (s , s). the ratio k k sv • s /s was suggested as a measure of the functional domain size. values in the range of - were found for lhcii macroaggregates and - for native thylakoid membranes, corresponding to domain sizes of - chlorophylls. although substantial, the determined functional domain size is still orders of magnitude smaller than the number of physically connected pigment-protein complexes; thus our results imply that the physical size of an antenna system beyond these numbers has little or no effect on improving the light-harvesting efficiency. the interaction between photosynthetic reaction center proteins (rcs) purified from purple bacterium rhodobacter sphaeroides r- and functionalized and non-functionalized (single (swnt) and multiple (mwnt) walled) carbon nanotubes (cnt-s) has been investigated. both structural (afm, tem and sem microscopy) and functional (flash photolysis and conductivity) techniques showed that rcs can be bound effectively to different cnts. both physical sorption and binding through -nh or -cooh groups gave similar results. however, it appeared that by physical sorption some sections of the cnts were covered by multiple layers of rcs. after the binding the rcs kept their photochemical activity for a long time (at least for three months, even in dried form) and there is a redox interaction between the cnt and rcs. the attachment of rc to cnts results in an accumulation of positive and negative charges followed by slow reorganization of the protein structure after excitation. in the absence of cnt the secondary quinone activity decays quickly as a function of time after drying the rc onto a glass surface. the special electronic properties of the swnt/protein complexes open the possibility for several applications, e.g. in microelectronics, analytics or energy conversion and storage. the decay of the high fluorescence state generated by actinic illumination of different durations was measured in whole cells of various strains and mutants of photosynthetic purple bacteria. although similar method is used in higher plants, its application in photosynthetic bacteria is novel and highly challanging. the available data are restricted and only the re-oxidation of the reduced primary quinone (q a -? q a ) is usually blamed for the decay kinetics. here, we will analyse the complexity of the kinetics over a very broad time range (from ls to s) and show that the dark relaxation of the bacteriochlorophyll fluorescence reflects the overlap of several processes attributed to the intra-and interproteinous electron transfer processes of the reaction center (rc) and cytochrome bc complex of the bacterium. in the shorter (\ ms) time scale, the dominating effect is the re-reduction of the oxidized primary donor (p ? ? p) that is followed by the re-oxidation of the acceptor complex of the rc by the cytochrome bc complex. as the life times and amplitudes of the components depend on the physiological state of the photosynthetic apparatus, the relaxation of the fluorescence can be used to monitor the photosynthetic capacity of the photosynthetic bacteria in vivo. circular dichroism (cd) spectroscopy is an indispensable tool to probe molecular architecture. at the molecular level chirality results in intrinsic cd, pigment-pigment interactions in protein complexes give rise to excitonic cd, whereas ''psitype'' cd originates from large densely packed chiral aggregates. it has been well established that the anisotropic cd (acd), measured on samples with defined orientation, carries specific information on the architecture of molecules. however, acd can easily be distorted by linear dichroism of the sample or instrumental imperfections -which might be the reason why it is rarely studied in photosynthesis research. here we present acd spectra of isolated intact and washed, unstacked thylakoid membranes, photosystem ii membranes (bby), and tightly-stacked lamellar macroaggregates of the main light-harvesting complex ii (lhcii). we show that the acd spectra of face-and edge-aligned stacked thylakoid membranes and lhcii lamellae exhibit profound differences in their psi-type cd bands. marked differences are also seen in the excitonic cd of bby and washed thylakoid membranes. thus acd provides an additional dimension to the light induced conformation changes of quinone depleted photosynthetic reaction centers (rcs) purified from the carotenoid-less rhodobacter sphaeroides r- were investigated by transient absorption (ta) and grating (tg) methods. surprisingly, the decay of the ta signal measured at nm was divided into a ns and a ls components. the latter coincides with the life time of the tg signal, which was assigned earlier [nagy et al. ( ) febs lett. , - ] to spectrally silent conformational changes. the nature of the ls phase was investigated further. although, the probability of chlorophyll triplet formation under our measuring conditions was small, possible contribution of the triplet states was also studied. the presence of carotenoid in the wild type rcs eliminated the ls component indicating the role of carotenoid in the energy transfer within the rcs. there was no significant effect of the molecular oxygen on the ta. this fact may be explained if the chlorophyll triplets inside the protein have reduced accessibility to molecular oxygen. a differential effect of osmotic potential and viscosity on conformation changes accompanying the primary charge separation was measured by the effect of ficoll, glucose and glycerol as compared the ta to the tg signals. variable chlorophyll fluorescence: in part a yield change due to light-induced conformational change gert schansker , szilvia z. tó th , lá szló ková cs , alfred r. holzwarth and gy} oz} o garab institute of plant biology, biological research center, hungarian academy of sciences, szeged, hungary, max-planck-institut fü r bioanorganische chemie, mü lheim an der ruhr, germany on a dark-to-light transition the chlorophyll fluorescence rises from a minimum intensity (f ) to a maximum intensity (f m ). conventionally, this rise is interpreted to arise from the reduction of the primary quinone acceptor, q a , of photosystem ii -although this cannot explain all presently available observations. in untreated leaves, at room temperature, the fluorescence rise follows the reduction of the electron transport chain (etc). once induced, * - % of the variable fluorescence intensity relaxes within ms in darkness and can be re-induced within ms as long as the etc remains reduced. analyzing the fluorescence relaxation kinetics, ?/-dcmu, * % of the amplitude cannot be explained by q a -re-oxidation. special properties of this phase determined on dcmu-inhibited samples: at cryogenic temperatures (below - °c), where the q a -/s recombination is blocked, it still relaxes and it exhibits a strong temperature dependence with an apparent e a & kj/mol, whereas the reduction of q a is nearly temperature insensitive. a fluorescence yield change, driven by light-induced conformational change in the reaction center complex, can explain all these observations. tuning function in bacterial light-harvesting complexes katia duquesne , edward o'reilly , cecile blanchard , alexandra olaya-castro and james n. sturgis lism, cnrs and aix-marseille university, marseille, france, department of physics, university college, london, uk purple photosynthetic bacteria are able to synthesize a variety of different light-harvesting complexes, sometimes referred to as lh , lh and lh . here we have investigated the structural origins of these different forms and the manner in which the sequence tunes the absorption spectrum of the light-harvesting system. we then consider the functional consequences of this tuning for the organization of the light harvesting system and on the ecology of the organisms. specifically by spectroscopic techniques, in particular circular dichroism and resonance raman spectroscopy, we have been able to obtain information on the organization of the bacteriochlorophyll binding sites in the unusual lh of roseobacter denitrificans. this provides a picture of how different peripheral light-harvesting complexes are able to modulate the absorption spectrum. the structure and organization of this complex is the put in the context of the the recently published variability of the light-harvesting complexes. in particular the observation of their ability to form mixed complexes containing different polypeptides. we examine quantitatively the possible reasons for maintaining such variability by considering the transport properties of membranes containing either pure or mixed complexes and show that mixed complexes can permit light-harvesting to continue during adaptation. we then consider the different constraints that may be behind this type of adaptation in different bacteria and the conditions under which different types of antenna system might be optimal finally we integrate this into the evolutionary context of adaptation to variable light intensity and the ecological niches where such organisms are found. interaction between photosynthetic reaction centers and ito t. szabo , g. bencsik , g. kozak , cs. visy , z. gingl , k. hernadi , k. nagy , gy. varo and l. nagy departments of medical physics and informatics, physical chemistry and materials science, technical informatics and of, applied and environmental chemistry, university of szeged, hungary, institute of biophysics, has biological research center, szeged, hungary photosynthetic reaction center proteins (rc) purified from rhodobacter sphaeroides purple bacterium were deposited on the surface of indium-tin-oxide (ito), a transparent conductive oxide, and the photochemical/-physical properties of the composite was investigated. the kinetics of the light induced absorption change indicated that the rc was still active in the composite and there was an interaction between the protein cofactors and the ito. the electrochromic response of the bacteriopheophytine absorption at nm showed an increased electric field perturbation around this chromophore on the surface of ito compared to the one measured in solution. this absorption change is associated with the chargecompensating relaxation events inside the protein. similar life time, but smaller magnitude of this absorption change was measured on the surface of borosilicate glass. the light induced change in the conductivity of the composite as a function of the concentration showed the typical sigmoid saturation characteristics unlike if the chlorophyll was layered on the ito which compound is photochemically inactive. in this later case the light induced change in the conductivity was oppositely proportional to the chlorophyll concentration due to the thermal dissipation of the excitation energy. the supramolecular organization of photosystem ii in vivo studied by circular dichroism spectroscopy tü nde tó th , , herbert van amerongen , , gy} oz} o garab , lá szló ková cs institute of plant biology, biological research center, hungarian academy of sciences, hungary, wageningen university, laboratory of biophysics, wageningen, the netherlands, microspectroscopy centre, wageningen university, wageningen, the netherlands the light reactions of photosynthesis in higher plants take place in granal chloroplast thylakoid membranes, which contain chirally organized macrodomains composed of photosystem ii (psii) supercomplexes associated with light harvesting antenna complexes (lhciis). the physiological relevance of this hierarchic organization, which often manifest itself in semicrystalline assemblies, has not been elucidated but the diversity of the supramolecular structures and their reorganizations under different conditions indicates its regulatory role. the present work focuses on the structural and functional roles of different components of lhcii-psii supercomplexes. we used various growth conditions, influencing the protein composition, and different arabidopsis mutants (kocp , kocp , kopsbw, kopsbx, dgd ), with altered organization of the membranes, and measured their circular dichroism (cd) spectra as well as their chlorophyll fluorescence kinetics to characterize the chiral macro-organization of the chromophores and the functional parameters of the membranes, respectively. we show that the formation of chiral macrodomains require the presence of supercomplexes. our data also reveal specific functions of some of the protein or lipid compounds in the light adaptation processes of plants. excitation energy transfer and non-photochemical quenching in photosynthesis rienk van grondelle department of physics, vu university, de boelelaan , hv, amsterdam, the netherlands the success of photosynthesis relies on two ultrafast processes: excitation energy transfer in the light-harvesting antenna followed by charge separation in the reaction center. lhcii, the peripheral light-harvesting complex of photosystem ii, plays a major role. at the same time, the same light-harvesting system can be 'switched' into a quenching state, which effectively protects the reaction center of photosystem ii from over-excitation and photodamage. in this talk i will demonstrate how lhcii collects and transfers excitation energy. using single molecule spectroscopy we have discovered how lhcii can switch between this light-harvesting state, a quenched state and a red-shifted state. we show that the switching properties between the light-harvesting state and the quenched state depend strongly on the environmental conditions, where the quenched state is favoured under 'npq-like' conditions. it is argued that this is the mechanism of non-photochemical quenching in plants. photobiology in the soil: arrested chlorophyll biosynthesis in pea epicotyl sections beá ta vitá nyi, katalin solymosi, annamá ria kó sa, bé la bö ddi eö tvö s university, institute of biology, department of plant anatomy, pá zmá ny p. s. /c, h- budapest, hungary the key regulatory step of chlorophyll (chl) biosynthesis is the nadph:protochlorophyllide oxidoreductase (por) catalyzed reduction of protochlorophyllide (pchlide)which is light activated in angiosperms. this process is usually described on artificially dark-grown plants. in this work, we studied epicotyl segments developed under the soil surface, which were dissected from pea plants grown under natural light conditions. using k fluorescence spectroscopy, pigment analyses, electron microscopy and fluorescence microscopy, we found that upper segments showed transitional developmental stages, i.e. chl appeared besides pchl(ide) and etio-chloroplasts were typical. in regions under cm depth, however, the characteristics of the segments were similar to those of plants germinated artificially in complete darkness, i.e. only pchl(ide) and etioplasts were present. the results of this work prove that these latter symptoms may occur in shaded tissues of fully developed, photosynthetically active plants grown under natural conditions. in this overview talk it will be shown how atomistic computations can complement experimental measurements in our quest to understand biological electron and proton transfer reactions. at first the molecular simulation methods for calculation of important electron transfer parameter such as reorganization free energy, electronic coupling matrix elements and reduction potentials will be explained. then three applications will be discussed where such computations help interpret experimental data on a molecular level. the first example concerns electron tunneling between heme a and heme a in the proton pump cytochrome c oxidase. this reaction is very fast, occurring on the nano-second time scale, and it is unclear if this is due to an unusually low reorganization free energy or due to high electronic coupling. carrying out large-scale all-atom molecular dynamics simulation of oxidase embedded in a membrane, we do not find evidence for unusually small values of reorganization energy as proposed previously, implying that the nanosecond tunneling rate between heme a and a is supported by very efficient electronic coupling. the second example is on electron transport in a deca-heme 'wire'-like protein, used by certain anaerobic bacteria to transport electrons from the inside of the cell to extracellular substrates. the crystal structure of such a protein has been solved recently for the first time. however, it is unclear if and in which direction the wire structure supports electron transport. here we present results of heme reduction potential calculations that help us reveal the possible electron flow in this protein. in a third example we explain how quantum mechanical/molecular mechanical methods (qm/mm) recently helped us understand why the catalase from h. pylori is prone to undergo an undesired protein radical migration reaction during catalysis. proton pumping activity of purple and brown membranes regenerated with retinal analogues k. bryl , k.yoshihara university of warmia and mazury, department of physics and biophysics, olsztyn, poland, suntory institute for bioorganic research, wakayamadai, osaka , japan the retinal protein bacteriorhodopsin (br) acts as a light-driven proton pump in the purple membrane (pm) of halobacterium salinarium (h.s.). the aim of these studies was to clarify whether the specific crystalline structure of protein and protein-substrate interactions are significant for h ? transfer into the aqueous bulk phase. two membrane systems were prepared: purple membranes (br arranged in a two-dimensional hexagonal lattice) and brown membranes (br not arranged in a crystalline lattice) were regenerated with -fuororetinals. light-induced proton release and reuptake as well as surface potential changes inherent in the regenerated systems reaction cycles were measured. signals of optical ph indicators residing in the aqueous bulk phase were compared with signals of ph indicator covalently linked to the extracellular surface of proteins and with surface potential changes detected by the potentiometric probe. the energies of activation of proton transfer have been calculated. experimental results and thermodynamic parameters (energies of activation) suggest the different mechanism of proton transfer into the aqueous bulk phase in these two systems. the implications for models of localized-delocalized energy coupling by proton gradients will be discussed. iron regulation is a vital process in organisms and in most of them it is accomplished through the metal solubilisation and storage by ferroxidase enzymes of the ferritin family, which have the ability of sequester, oxidize and mineralize ferrous ions using oxygen or hydrogen peroxide as substrate. dnabinding proteins from starved cells (dps) belong to this ferritin's family. dps belongs to the sub-type designated as miniferritins and, besides iron storage and release capability, is responsible for hydrogen peroxide resistance showing the ability to form stable complexes with dna. the preferable cosubstrate of this enzyme is h o although the reaction can occur in the presence of oxygen with lower rate [ , ] . in this work, the electrochemical behaviour of the recombinant dps from pseudomonas nautica, was assessed as a function of metal content in anaerobic environment with h o as co-substrate. the obtained electrochemical results together with spectroscopic studies allowed inferring some new hypothesis on the dps iron uptake mechanism. for myoglobin electrostatically immobilized on au-deposited mixed self-assembled monolayers (sams) of the composition: -s-(ch ) -cooh/-s-(ch ) -oh. our approach allows for a soft switching of the haem group charge state and accurate probing of the accompanying reorganizational dynamics of conformational (quasi-diffusional) and quantum (e.g. protonrelated) modes. the electron transfer rate constants were determined with h o or d o as solvent, under the variable temperature ( - k) or pressure ( - mpa) conditions, revealing the overall reorganization free energy of . ± . ev, activation volume of - . ± . cm mol - and inverse solvent kinetic isotope effect of . ± . ( °c). on the grounds of an extended charge-transfer theory, we propose specific protoncoupled et mechanism additionally coupled to the slow conformational dynamics of the protein matrix accompanied by translocation(s) of haem-adjacent water molecule(s). proton gradients across pore spanning membranes: towards on-chip energy conversion daniel frese, claudia steinem institute for organic and biomolecular chemistry, georg-august-university goettingen, germany in cell organelles, chemiosmotic potentials resulting from proton gradients across membranes are widely used to fix chemical energy in forms of atp. the high efficiency of this protein-mediated energy conversion raises interest for artificial proton gradient setups. to investigate proton transport across artificial membranes, we prepared pore spanning membranes (psms) on porous silicon substrates via painting technique. this allowed us to trap aqueous content of well-defined composition and volume inside the substrates microcavities. nigericin, a peptide that acts as an h ? -k ? -antiporter and bacteriorhodopsin, a transmembrane protein which is well known to be a light driven proton pump, were reconstituted into the pre-formed psms to achieve proton transport from one aqueous compartment to the other. changes of proton concentrations inside the pores were monitored by means of confocal laser scanning microscopy (clsm). therefore, pores were filled with pyranine, a ph-sensitive fluorescence dye and variations in intensity were measured to analyze proton translocation. we were able to show that both, nigericin and bacteriorhodopsin are capable of building up a proton gradient across psms and plan to co-reconstitute atp synthases for on-chip energy conversion by formation of atp. application of the gibbs free energy profiles to sequential biochemical reactions pé ter farkas, tamá s kiss and eugene hamori department of biological physics, eö tvö s ló rá nd tudomá ny egyetem, budapest, hungary, and department of biochemistry, tulane university, new orleans, la., usa the full understanding of the energetic details of complex metabolic reaction sequences requires a step-by-step analysis of the gibbs free energy (g) changes of the ''parasystem'' (i.e., a collection of atoms comprising all the molecules participating in a given reaction) as it gradually changes from its initialreactants state to its final-products state along the reaction pathway. knowing the respective equilibrium constants of each of the participating reaction steps and also the actual in vivo concentrations of the metabolites involved, a free-energy profile can be constructed that will reveal important information about the progress of the reaction as driven by thermodynamic forces. this approach will be illustrated on some biochemical reactions including the glycolytic/gluconeogenetic pathways. furthermore, the often misleading text-book representation of enzymatic catalysis will be reexamined and explained in thermodynamic terms using the free-energy profiles of both the non-catalyzed and the enzyme-catalyzed reactions. redox-active proteins can be diversely functionalized at metaldeposited self-assembled monolayers (sams) of a widely variable composition and thickness. the voltammetric methodology in combination with the advanced data processing procedures allow for comprehensive kinetic data within the congruent series of nano-devices and the subsequent calculation of the key physical parameters, such as the medium reorganization energy of et, the donor-acceptor electronic coupling, effective relaxation time (related to the protein's and environment's fluctuational dynamics), etc. in our studies the ''model'' redox protein, cytochrome c (cytc), was either freely diffusing to sam terminal groups (mode ), or attached to sams through the electrostatic interaction (mode ), or specific ''wiring'' (mode ). another redox-active protein, azurin, was confined at terminal sam groups through the hydrophobic interaction (mode ). diverse experimental strategies including the variation of sam thickness, solution viscosity temperature and hydrostatic pressure, allowed for a severe demonstration of the full adiabatic and nonadiabatic control (thinner and thicker sams, respectively), and the intermediary regime, in a nice agreement with the major theoretical predictions. proton transfers in a light-driven proton pump j.k. lanyi dept. physiology & biophysics, university of california, irvine, usa illumination of bacteriorhodopsin causes isomerization of alltrans retinal to -cis, -anti and a cyclic reaction ensues, in which the protein and the chromophore undergo conformational changes with an overall ten millisecond turn-over time and a proton is transported from one membrane side to the other. with crystal structures of six trapped intermediate states and plausible structural models for the remaining two intermediates, structures are now available for the initial bacteriorhodopsin state and all intermediates. they reveal the molecular events that underlie the light-induced transport: protonation of the retinal schiff base by asp , proton release to the extracellular membrane surface, a switch event that allows reprotonation of the schiff base from the cytoplasmic side, side-chain and main-chain motions initiated in the cytoplasmic region, formation of a single-file chain of hydrogen-bonded water molecules that conducts the proton of asp to the schiff base, and reprotonation of asp from the cytoplasmic surface. the observed changes can be summarized as a detailed atomic-level movie in which gradual relaxation of the distorted retinal causes a cascade of displacements of water and protein atoms results in vectorial proton transfers to and from the schiff base. electron transfer (et) processes are fundamental in photosynthesis, respiration and enzyme catalysis. the relative importance of superexchange and sequential mechanisms in biological et is still a matter of debate. the identification of any ''stepping stones'' necessary for electron hopping is a key point in the understanding of long range et. hence, the study of a single event in the sequence of reactions occurring in these phenomena is a fundamental but formidable task. muon spin relaxation (lsr) has been shown to be sensitive to charge transport on a molecular lengthscale. the muon is a very sensitive probe of electron transport, as any changes to the electronic density sampled by the muon can change its spin polarization, which can easily be measured. in this context, a very useful tool is the detection of the so-called avoided level crossing (alc) resonances [ ] . the enhancement in the loss of polarization of the muon's spin on these resonances dramatically increases sensitivity. we show that a laser pump -lsr probe technique can measure et processes at particular, and most importantly known, sites within the amino acids chain, and therefore track the time evolution of the electron over the molecule. keywords: photosynthesis, reaction center, electron transfer, proton transfer, fourier transform infrared, l dn, isotopic labeling, band assignment, histidine, mechanism in photosynthesis, the central step in transforming light energy into chemical energy is the coupling of light-induced electron transfer to proton uptake. in the photosynthetic reaction center (rc) of rhodobacter sphaeroides, fast formation of the charge separated state p ? q a is followed by a slower electron transfer from the primary quinone q a to the secondary quinone q b and the uptake of a proton from the cytoplasm to q b . previous fourier transform infrared (ftir) measurements on rc suggested an intermediate x in the q a -q b to q a q b transition. mutation of the amino acid aspl to asn (l dn mutant) slows down proton uptake and oxidation of q a -. using time-resolved ftir spectroscopy we characterized this rc mutant and proposed specific ir bands that belong to the intermediate x. to study the role of the iron-histidine complex located between q a and q b , we performed fast-scan ftir experiments on the l dn mutant marked with isotopically labelled histidine. we assigned ir bands of the intermediate x between cm - and cm - to histidine vibrations. these bands show the protonation of a histidine, most likely hisl , during the q a -q b to q a q b transition. based on these results we propose a new mechanism of the coupling of electron and proton transfer in photosynthesis. complex i of respiratory chains is an energy transducing enzyme present in most bacteria and in all mitochondria. it is the least understood complex of the aerobic respiratory chain, even though the crystallographic a-helical structures of bacterial and mitochondrial complexes have been recently determined [ ] [ ] . this complex catalyses the oxidation of nadh and the reduction of quinone, coupled to cation translocation across the membrane. rhodothermus marinus complex i, our main model system, is a nadh:menaquinone oxidoreductase and has been extensively characterized. we have made an exhaustive study in order to identify all the subunits present in the complex [ ] . the nature of the coupling charge of r. marinus complex i was investigated using inside-out membrane vesicles, which were active with respect to nadh oxidation and capable of creating and maintain an nadh-driven membrane potential (dw) positive inside. it was observed that this bacterial complex i is able of h ? and na ? transport, although to opposite directions. the coupling ion of the system was shown to be the h ? being transported to the periplasm, contributing in this way to the establishment of the electrochemical potential difference, while na ? is translocated to the cytoplasm [ ] . the sodium ion extrusion from the membrane vesicles was due to the activity of complex i, since it was sensitive to its inhibitor rotenone, and it was still observed when the complex i segment of the respiratory chain was isolated by the simultaneous presence of cyanide and external quinones. additional studies have shown that although neither the catalytic reaction nor the establishment of the dph requires the presence of na ? , the presence of this ion increased the proton transport. combining all these results, a model for the coupling mechanism of complex i was proposed, suggesting the presence of two different energy coupling sites, one that works only as a proton pump (na ? independent), and the other functioning as a na ? /h ? antiporter (na ? dependent) [ ] . this model was reinforced by further studies performed in the presence of the na ? /h ? antiporter inhibitor, -(n-ethyl-n-isopropyl)-amiloride (eipa) [ ] . a deeper inside into the coupling mechanism of this enzyme was provided by studying the influence of sodium ions on energy transduction by complexes i from escherichia coli and paracoccus denitrificans. it was observed that the na ? / h ? antiporter activity is not exclusive of r. marinus complex i, since the e. coli enzyme is also capable of such a transport, but is not a general property given that the p. denitrificans enzyme does not performed sodium translocation [ ] . due to the fact that r. marinus and e. coli enzymes reduce menaquinone while p. denitrificans complex i reduce ubiquinone, it is suggested that the na ? /h ? antiporter activity may be correlated with the type of quinone used as substrate. under anaerobic conditions some bacteria can use nitrate instead of oxygen in a process called denitrification. during denitrification, the reduction of no to n o is catalyzed by a membrane-bound enzyme nitric oxide reductase (nor). this enzyme is an important step in the evolution of a respiratory system: nor belongs to the superfamily of o -reducing heme-copper oxidases and is assumed to be the evolutionary ancestor of cytochrome c oxidase. the understanding of nor functioning was limited by the lack of structural information, but recently the first structures (cnor type from ps. aerug. and qnor type from g. stearoth.) were solved [ ] [ ] . we will present results of the first computational studies of nor (both cnor and qnor types) [ ] [ ] . the studies include: (i) large-scale all-atom md simulations of proteins in their natural environment (i.e. embedded in membrane and solvent), which were performed to describe water dynamics inside the protein and to identify potential proton transfer pathways, and (ii) free-energy calculations by the empirical valence bond (evb) method [ ] for the explicit proton translocations along the pathways established by md. among important findings are new proton pathways, which were not predicted from the x-ray structure and could be identified only by means of computer simulations. simulations also reveal that, despite a high structural similarity between cnor and qnor, these enzymes utilize strikingly different proton uptake mechanisms. our results provide insights into the functional conversion between no and o reductases, and into evolution proton transfer mechanisms and respiratory enzymes in general. the genome of the bacterium geobacter sulfurreducens (gs) encodes for c-type cytochromes ( ). genetic studies using cytochrome deficient gs strains and proteomic studies identified cytochromes that were produced under specific growth conditions ( ) ( ) ( ) . a putative outer-membrane cytochrome, omcf, is crucial for fe ? and u ? reduction and also for microbial electricity production ( ). omcf is a monoheme c-type cytochrome with sequence similarity to soluble cytochromes c of photosynthetic algae and cyanobacteria ( ). the structure of oxidized omcf was determined ( ) being the first example of a cytochrome c -like structure from a nonphotosynthetic organism. the structural features of omcf hinted a different function compared to cytochromes c from photosynthetic organisms, being an excellent example of how structurally related proteins are specifically design by nature to perform different physiological functions. in order to elucidate omcf structural-functional mechanism, isotopic labeled protein ( n and c) was produced and its structure in the reduced form determined by nmr. single point-mutations at key residues were produced by site-directed mutagenesis and their impact on the structural and functional properties of omcf will be presented. in the early s, the search for the source of nitrogen monoxide (no) production in mammals led to the discovery of three major isoforms of no-synthases (nos): the neuronal nos (nnos), the inducible nos (inos), and the endothelial nos (enos) ( ( )). years later, based on genomic analysis, numerous nos-like proteins have been identified in the genome other organism and in particular of several bacteria (bacillus anthrax, staphylococcus aureus… ( )). in spite of superimposable d structures and the ability to catalyse no production, all these enzymes are carrying different (if not opposite) physiological activities including cgmp signalling, cytotoxic activities, anti-oxidant defence, metabolism… moreover, noss become increasingly associated to oxidative-stress related pathologies ranging from neurodegenerative disorders, cardiovascular and inflammatory diseases, diabetes, cancers ( )…. this apparent paradox seems related to the belief that the strong similarity of sequence and structure of no-synthases must lead to a unique and identical functioning (no production) for all isoforms. this is blatant for bacterial nos-like proteins that are lacking the essential components required for no biosynthesis but remain considered as genuine no synthases. this approach might remain an obstacle to the understanding of nos actual biological role and could prevent the design of efficient nos-targeted therapeutic strategies. to elucidate this ''nos paradox'' our group has initiated a multidisciplinary approach that aims to relate the wide diversity of nos biological activities to variations in the catalytic mechanism of noss, to modifications of their regulation patterns and to adaptations to their physiological environment. in this context we have been investigating the mechanism of bacillus subtilis nos-like proteins with a special focus on the features that are specific to nos mechanism: i) electron and proton transfers and the role of the substrate and the pterin cofactor ii) oxygen activation and the role of the proximal ligand iii) the very molecular mechanism and the variations in the nature of reaction intermediates. for that matter we have been using a combination of radiolytical techniques (cryoreduction with co y-irradiation, pulse-radiolysis with the elyse electron accelerator), stateof-the-art spectroscopies (epr, atr-ftir and resonance raman, and picoseconds uv-visible absorption spectrosocopies), organic synthesis (synthesized substrates and cofactors analogues) biochemistry and molecular biology (site-directed mutagenesis). we will present our results on the coupling of electron and proton transfers, on the tuning of the proximal ''push effect'' and we will discuss the conditions that favour for each nos isoform no production versus other reactive nitrogen and oxygen species. photosynthetic iron oxidation (pio) is an ancient form of photosynthesis with relevant consequences in the shaping of the planet. this form of metabolism may have been involved in the deposition of geological structures known as banded iron formations, which hold key information regarding the co-evolution of photosynthesis and earth. rhodopseudomonas palustris tie- and rhodobacter ferroxidans sw both use ferrous iron as an electron donor to support photosynthetic growth (i.e. photoferrotrophy). the sw foxeyz operon can stimulate light-dependent iron oxidation by other bacteria. it codes a two-heme cytochrome, a pyrroloquinoline quinone protein and an inner membrane transporter, respectively. in tie- the pioabc operon is required for photoferrotrophy. it codes for a ten-heme cytochrome, an outer membrane beta-barrel and a high potential iron-sulfur protein (hipip) respectively. here we present functional and structural characterization of proteins involved in pio. this molecular characterization is essential for understanding this mode of bioenergetic metabolism, and may one day aid the development of biotechnological applications like microbial fuel cells and bioremediation. alongside classical, cytochrome respiratory pathway, phycomyces blakesleeanus possess alternative, cyanideresistant respiration (crr) facilitated by alternative oxidase (aox). in order to study role of oxygen in regulation of crr, the effects of cyanide on respiration of h old mycelia in aerated (control), hypoxic and anoxic conditions were measured. mycelium was incubated in these conditions for . h, h and h. after . h, aox activity was increased only in specimens incubated in anoxic conditions ( . %). after h, increase in aox activity was significant in both hypoxic and anoxic specimens ( . % and . %, respectively), with even greater increase after h, . % for hypoxic and . % for specimens in anoxic conditions. mycelia treated for h was then oxygenated for minutes. this induced decrease in aox activity of % in anoxic and even . % in hypoxic mycelia. aox is recognized as one of the mechanisms for maintaining low levels of reduced ubiquione which can function in conditions in which cytochrome chain is disabled, such as anoxia. this is in concordance with results obtained on p. blakesleeanus, where aox levels rise in hypoxic and anoxic conditions and decrease close to control level shortly after introduction of oxygen into the system. influence of escherichia coli f f -atpase on hydrogenase activity during glycerol fermentation k. trchounian , , g. sawers , a. trchounian department of biophysics, yerevan state university, yerevan, armenia, institute for microbiology, martin-luther university halle-wittenberg, haale, germany e. coli encodes four hydrogenases (hyd); only three of these, hyd- , hyd- and hyd- have been well characterized. hyd- has been shown recently to reversibly evolve hydrogen during glycerol fermentation at ph . [ ] . proton reduction was inhibited by n,n'dicyclohexylcarbodiimide suggesting a link with the proton-translocating f f -atpase. indeed, at ph . in an e. coli mutant (dk ) lacking f f overall hyd-activity was reduced to approximately % of the wild type activity; hyd- , but not hyd- , was detected in an in-gel activity assay. f f is therefore suggested to be required for hyd- activity. at ph . in glycerol medium hyd-activity in dk was * % of wild type activity and hyd- and hyd- exhibited only weak activity. this indicated a significant f f contribution towards hyd-activity as ph decreased. furthermore, at ph . hydactivity was negligible and only a very weak activity band corresponding to hyd- could be observed. these results suggest that f f -atpase is essential for hydrogenase activity during glycerol fermentation at ph . . taken together, the results suggest an interdependence between hyd- , hyd- and f f -atpase activity. [ ion channels and transporters control many facets of cancer cell biology and blocking the activity of these impairs tumor cell growth in vitro and in vivo. this new paradigm has opened new opportunities for pharmaceutical research in oncology , . we have contributed to this field showing that k v . (herg ) channels are aberrantly expressed in several human cancers where they control different aspects of the neoplastic cell biology such as proliferation and apoptosis, invasiveness and angiogenesis, the latter through the regulation of vegf secretion (reviewed in ). the herg dependent effects were shown in vitro and, more recently, in vivo. in preclinical models of both leukemia and colorectal cancer , herg overexpression confers a higher malignancy to neoplastic cells. moreover, herg blockers have therapeutic potential, since preclinical tests showed that treatment with specific herg blockers overcame chemoresistance in acute leukemias as well as reduced gi cancer growth, angiogenesis and metastatic spread . the overall message emerging from our data is that the herg protein represents a novel biomarker and drug target in oncology. up to now, herg was considered an ''antitarget'' due to the cardiac side effects that many (but not all) herg blockers produce and that result in lengthening the electrocardiographic qt interval. we report here recent studies on known and newly developed herg blockers that exhibit no cardiotoxicity and are more specific for the herg channels expressed in cancer cells. we reported previously that the increase of the cholesterol content of the cell membrane (in vitro) modified the biophysical parameters of the gating of kvl. k ? ion channels in human t-lymphocytes. in our present study we aimed to determine the effect of hypercholesterolemia on the biophysical parameters of kv . gating and the proliferation of t cells. t-lymphocytes were isolated from the peripheral blood of patients with cholesterol level considered normal (\ . mmol/l,control group) and patients with hypercholesterinaemia (hc). whole-cell k? currents were measured in patchclamped t cells and the kinetic (activation and inactivation kinetics) and equilibrium parameters (voltage-dependence of steady-state activation) of kv . gating were determined. lymphocyte proliferation was measured using cfse staining with and without anti-cd and anti-cd stimulation. our results indicate that the biophysical parameters of kv . gating are similar in the control group and in the 'hc' samples. the cfse-based assay showed that hypercholesterolemic t cells had higher spontenaous activation rate compared to control group. however, t cells from high cholesterol level patients challenged by anti-cd and anti-cd exhibited lower proliferation rate than control cells. generalized epilepsy with febrile seizures plus (gefs?, omim ) is a childhood genetic epilepsy syndrome correlated to mutations in the ancillary b-subunit of neuronal voltage-gated sodium channels (nachs). b -subunit is non-covalently associated with nach a-subunits, serving as modulator of channel activity, regulator of channel cell surface expression and as cell adhesion molecule. the first and best characterized gefs? mutation is the c w. this mutation changed a conserved cysteine residue into a tryptophan, disrupting a putative disulphide bridge that should normally maintain an extracellular immunoglobulinlike fold. in this study, we investigated the presence of this putative disulphide bond using -d-diagonal-sds-page, where the proteins were separated in the first dimension in absence of a reduction agent and in presence of it in the second dimension. this method allows to visualize the protein above the diagonal experimentally confirming that the disulphide bond is intramolecular. duchenne muscular dystrophy (dmd) is associated with severe cardiac complications. recent research suggests that impaired voltage-gated ion channels in dystrophic cardiomyocytes accompany cardiac pathology. it is, however, unknown if ion channel defects are primary effects of dystrophic gene mutations, or secondary effects of the developing cardiomyopathy. here, we studied na and ca channel impairments in dystrophic neonatal cardiomyocytes, derived from dmd mouse models, prior to cardiomyopathy development. dystrophin-deficiency reduced na current density. in addition, extra utrophin-deficiency altered na channel gating. moreover, also ca channel inactivation was reduced, suggesting that ion channel abnormalities are universal primary effects of dystrophic gene mutations. to assess developmental changes, we also studied na channel impairments in dystrophic adult cardiomyocytes, and found a stronger na current reduction than in neonatal ones. the described na channel impairments slowed the action potential upstroke in adult cardiomyocytes, and only in dystrophic adult mice, the qrs interval of the ecg was prolonged. ion channel impairments precede pathology development in the dystrophic heart, and may be considered cardiomyopathy triggers. supported by austrian fwf (p ). it has been over years since sensory neuron-specific sodium channel na v . was identified. since then na v . has been shown to play a crucial role in pain pathways, and it became a prominent drug target for novel pain killers. in contrast to myelinated neurons, mechanisms that target voltagegated sodium channels to the unmyelinated c-fibre axons are largely unknown. we investigated the localisation of na v . in unmyelinated primary sensory neurons. na v . was found to be clustered in lipid rafts on unmyelinated axons. when the lipid rafts are disrupted, remarkable reduction in both the conduction velocity and the number of cells responsive to mechanical stimuli to the unmyelinated axons were seen. using a compartment culture system, we also found that disruption of rafts in the middle region of the sensory axons caused a significant reduction in the responsiveness of the neurons to chemical stimuli to nerve endings. this is due to the failure of action potential propagation through the axons. these data suggest that clustering of na v . in lipid rafts of unmyelinated fibres is a key factor for the functional properties of the channel, which may due to a change in the voltage threshold. disruption of the na v . cluster and modifying lipid rafts in primary sensory neurons may be a useful new approach to control the excitability of nociceptive neurons. ion currents in are crucially important for activation of t-lymphocytes. our aim was to investigate how the blockage of various ion channels in isolation or in combination affects mitogen-dependent activation and proliferation of t-cells. we activated human peripheral blood lymphocytes using monoclonal antibodies against the tcr-cd complex and cd . we applied specific channel blockers inhibiting the major ion channels of the t-cell: either the kv . (tea or anuroctoxin), ikca (tram- ), or the crac channel ( -apb) alone or in combination. five days after the stimulus we measured the change in cell size and cellular granulation with flow cytometry along with the proportion of dividing cells, using cfse (carboxyfluorescein succinimidyl ester) dilution assay. our measurements indicated that the ion channel blockers suppressed the proportion of dividing cells in a dosedependent manner. increasing the strength of the stimulation reduced the potency of the blockers to inhibit cell proliferation and eventually the blockers were ineffective in decreasing lymphocyte proliferation. we experienced the greatest amount of inhibition using the combination of the blockers, which indicates synergy in the regulation pathway of the various ion channels. recently, sodium dependent phosphate transporter napi b was revealed as potential marker for breast, thyroid and ovarian cancer. in vivo, napi b is involved in maintenance of phosphate homeostasis and mutations or aberrant expressions of its gene (slc a ) are associated with several diseases including cancer. however, data about napi b mrna expression in different types of cancer and correspondent normal tissues are controversial and restricted. we investigated slc a gene expression level in normal ovarian tissues and different histomorphological types of ovarian tumors using real-time pcr analysis. it was found that slc a gene was highly expressed in well-differentiated endometrioid and papillary serous tumors, but was not expressed in low-differentiated tumors, benign tumors and in most normal tissues. mrna expression of slc a in serouse and endometrioid ovarian tumors accurately correlated with protein expression that was detected in these tumor samples by western blot analysis and immunohistochemistry in our previous investigation. upregulation of slc a gene expression in well-differentiated tumors may reflect cell differentiation processes during ovarian cancerogenesis and could serve as potential marker for ovarian cancer diagnosis and prognosis. in the present contribution a procedure for molecular motion characterization based on the evaluation of the mean square displacement (msd), through the self-distribution function (sdf), is presented. it is shown how msd, which represents an important observable for the characterization of dynamical properties, can be decomposed into different partial contributions associated to system dynamical processes within a specific spatial scale. it is shown how the sdf procedure allows us to evaluate both total msd and partial msds through total and partial sdfs. as a result, total msd is the weighed sum of partial msds in which the weights are obtained by the fitting procedure of measured elastic incoherent neutron scattering (eins) intensity. we apply sdf procedure to data collected, by in , in and in spectrometers (institute laue langevin), on aqueous mixtures of two homologous disaccharides (sucrose and trehalose) and on dry and hydrated (h o and d o) lysozyme with and without disaccharides. the nature of the dynamical transition is highlighted and it is shown that it occurs when the system relaxation time becomes shorter than the instrumental energy time. finally, the bioprotectants effect on protein dynamics and the amplitude of vibrations in lysozyme are presented. we evaluated q for genotoxicity in mcf- breast cancer cells in the presence or absence of doxorubicin (dox), docetaxel (dtx) and paclitaxel (ptx), anticancer drugs commonly used in chemotherapy of different solid tumors. damage to dna was determinated by comet assay. the cells, after treatment with investigated compounds, were washed up and cultured in fresh medium for , , , and hours. we have found that q by itself caused significant dna damage. moreover flavonol enhanced genotoxic effect of anticancer drugs. the highest amount of dna in the comet tail was observed h after treatment with combination of q with dox. similar changes were found in cells incubated with combination of q with taxanes -ptx or dtx. however, damage to dna in this case was considerably lower than damage caused by combination of q with dtx. our results confirmed anticancer and genotoxic activity of quercetin, which makes it a promising candidate for a potential use as a modulator of cytotoxicity and anticancer activity of anthracycline and taxane chemotherapeutics. although the health effects of low-frequency and intensity electromagnetic fields (lfi-emfs) are controversial, increasing evidence suggests that lfi-emfs are capable for initiating various healing processes. many (bio)physical ideas were suggested to explain the influence of lfi-emfs in living systems but the main effect of lfi-emfs on cell functions remains vague. however, some effects of lfi-emfs may be explained by redox and membrane processes. during diseases, cells not only demonstrate altered biochemical processes but also produce altered non-linear bioelectromagnetic complex patterns. thus, it is reasonable to use non-linear bioelectric and bioelectromagnetic signals from cells of the body for potential therapeutic applications that may be more effective than the artificial lfi-emfs signals. our novel emost (electromagnetic-own-signal-treatment) method is based on the utilization of the non-linear, bioelectric and bioelectromagnetic signals of the patients without any electromagnetic wave modulation and inversion of recorded output signals of subjects. here, we report our some restorative results after emost application. we also suggest that the possible effects of the emost may be achieved via redox-related processes. background or leak potassium conductances are a major determinant of resting potential and input resistance, two key components of cell excitability. these currents are not passive but finely tuned and adapted to cell specific functions. k p channels producing these currents are tightly regulated by a variety of chemical and physical stimuli including temperature, membrane stretch, free fatty acids, ph, ca ? , neurotransmitters and hormones as well as protein partners. these different stimuli converge on gating mechanisms that show remarkable conservation between intracellular k p channels (twik channels) and k p channels located at the plasma membrane (trek / channels). living at the edge: volume the conduction system of interfacial forces into the alveolar type ii cell in previous investigations, using new microscopic approaches, we found that the presence of an a li leads to a paradoxical situation: it is a potential threat that may cause cell injury, but also a important stimulus: at ii cells respond promptly, and show sustained ca ?signals that activate exocytosis. exocytosed surfactant, in turn, clearly prolonged the time to irreversible cell damage, and may be an adaptive and evolutionary defense mechanism against the harmful nature of surface forces. recently we published that at ii cells are sensing the a li but how this stimulus is conducted and converted into the cell, is still obscure. currently we are searching for potential calcium sources and it seems that the cells signal ca ? by extracellular ca ? entry probably through mechanosensitive channels. specialdesigned gene chips allowed a whole genome profiling of a li exposed single cells. these cells react with rapid changes on the transcriptional level: cellular pathways that are involved include e.g. defense response and lipid metabolism, and we identified genes associated with several lung diseases and injuries. we summarize, interface forces are strong, they are acting on the cells and triggering cellular events that are closely related with classical concepts in mechanotransduction, it is very plausible that those forces play a crucial role in the lung surfactant homeostasis. calorimetric method and instrumentation were worked out and applied for investigation aqueous solutions of proteins. thermal effects were analyzed by own construction heat-fluxtype dsc cell designed for temperature range from the boiling point of water down to k. the achieved sensitivity of heat flow rate (hf) of the instrument is better than %w tested using ll of mm aqueous solution of nacl. from the integral value of hf -the total enthalpy change dh total , the enthalpies of transitions were separated from the heat capacities. using the method, several types of proteins (bsa, erd , ubq, a-, b-casein, and wt, a t, a-synuclein mutants) were investigated in that temperature range. the results are shown in detail as an illustrative example. potential applications are outlined, which include (i) the distinction between the solvent accessible surfaces of globular and intrinsically disordered proteins, (ii) the distinction between protein mutants, and (iii) the identification of monomer and polymer protein states. this method provides a possibility to study the polymerization process (amyloid formation) and to investigate in-situ the reason and circumstances of that. morphometry due to self gravity in living organism is an integral part in understanding self gravitation bio. computational studies of biological system, especially in ab-initio embryo as self gravitating mass, floating over amniotic fluid, as if maintaining anti -gravitation (extrinsic) mechanism, would be an interesting one to explore biomechanics of intrinsic gravity. since the work would be of exploratory nature, both from the point of view of identifying appropriate stage of development of embryological morulla and available computational logics, it was contemplated to initiate functional study with a few reported ultrasound evidential works on small animals like mice, grasshopper including compact human embryo as mathematical structure that may allow the formal definition of concepts such as convergence, mapping and continuity. as many of the finite-dimensional function analysis in topological vector spaces are available, we initiated our simulation and studies on concentrating locally compact banach spaces. institute for x-ray physics, university of gö ttingen, germany we report on hard x-ray phase contrast imaging of black lipid membranes (blms), which are freely suspended over a micro machined aperture in an aqueous solution. this new way of membrane structure analysis allows investigating bio molecular and organic substances in aqueous environments by parallel and divergent beam propagation imaging, using partially coherent multi-kev x-ray radiation. the width of the thinning film is significantly smaller than the detector pixel size, but can be resolved from quantitative analysis of the intensity fringes in the fresnel diffraction regime down to its native thickness of about nm. to our knowledge this is the first time that such small features of a very weak phase object have been visualized by direct x-ray imaging techniques. we have put forward a simplified but extendable model, which enables the theoretical description of image formation and characterization of membrane thickness and its decrease during the thinning process from a bulk to a bimolecular film. on the basis of recent experiments, future investigations will be performed to study the interactions of membranes, as they are for example known from synaptic fusion, with high spatial resolution. shown that the acid incorporates mainly in the exterior part of the erythrocyte membrane, inducing creation of echinocytes. this suggests that it interacts predominantly with the outer part of the lipid layer of erythrocytes and liposomes. it was also shown that the cga decreases the packing order of the hydrophobic part of the membranes, without changing the anisotropic fluorescence of the hydrophobic part. one of the unique features of single molecule absorption/ emission is their anisotropy due to the well-defined transition dipoles for both processes allowing the determination of the molecule's d orientation. therefore, several techniques have been proposed in order to determine the full d orientation of dipole emitters on a single molecule level. we recently demonstrated a technique that combines emission distribution and polarization detection [ , , ] . as the method is an intensity distribution technique and based on single photon detection in principle, one can extend the d orientation determination to fluorescence correlation spectroscopy (fcs) as well as dynamical anisotropy measurements. this allows for the determination of the dynamics in d orientation of single molecules down to a nanosecond timescales. the d orientation is particularly interesting in non-isotropic environments. a lipid membrane is such a non-isotropic environment of enormous importance in biological systems. we therefore use giant unilamellar vesicle (guv) labeled with dyes like dio as a model system. due to the defined curvature of such vesicles all possible dipole orientations can be achieved. this allows us to show the capabilities of our method on different timescales and to quantify the error in determination of d orientation dynamics in lipid membranes. [ the aim of the studies was to determine the changes that occur in a biological membrane and the model lipid membrane as a result of interaction with strawberry leaf extract. numerous studies conducted all over the world have documented a beneficial effect of polyphenolic compounds on the human organism. however, the mechanism of the interaction on the molecular and cell level is not yet known. in the work presented, the effect of strawberry leaf extract on the erythrocyte and black lipid membranes has been investigated. the applied methods -spectroscopic, fluorimetric and electric -allowed to determine the hemolytic and antioxidant activity, and the packing order in the erythrocyte membrane as well as the electric capacity of blms. the results obtained indicate that the extract is efficient in protecting membrane lipids against oxidation, does not induce hemolysis, increases osmotic resistance and decreases packing order in the hydrophilic region of the erythrocyte membrane. moreover, it increases stability and life-time of flat lipid membranes, without altering their specific capacity. supported lipid bilayers are an abundant research platform for understanding the behavior of cell membranes as they allow for additional mechanical stability and enable characterization techniques not reachable otherwise. however, in computer simulations these systems have been studied only rarely up to now. we present systematic studies on different length scales of the changes that a support inflicts on a phospholipid bilayer using molecular modeling. we characterize the density and pressure profiles as well as the density imbalance induced by the support. it turns out that the changes in pressure profile are strong enough that protein function should be impacted leading to a previously neglected mechanism of transmembrane protein malfunction in supported bilayers. we determine the diffusion coefficients and characterize the influence of corrugation of the support. we also measure the free energy of transfer of phospholipids between leaflets using the coarsegrained martini model. it turns out that there is at equilibrium about a - % higher density in the proximal leaflet. these results are in agreement with data obtained by very large scale modeling using a water free model where flip-flop can be observed directly. we are additionally characterizing the intermediate states which determine the barrier height and therefore the rate of translocation. we also study the influence of surface roughness and curvature on the behavior. simulations in atomistic detail are performed for selected systems in order to confirm the findings. [ the inverse bar (i-bar) domain is part of the superfamily of the membrane-deforming protein is bin-amphiphysin-rvs (bar) proteins which induce either positive or negative membrane curvature both in vitro and in cells. generation of membrane curvature by these membrane deforming proteins often works together with actin dynamics. i-bar shares its function between actin bundling and membrane binding but it is still obscured what molecular mechanisms are responsible for these functions. the aim of our project is to investigate the detailed membrane binding properties of the i-bar of irsp and its relations to the actin cytoskeleton. in vitro fret experiments and fluorescence quenching studies were carried out between the i-bar and liposomes made up from different lipid constructs. we have found that the i-bar has preference to bind to the negatively charged lipids however it can also bind to the uncharged lipids. the fluorescence quenching studies reflected that the accessibility of the i-bar surface was higher toward the negatively charged lipids than for the uncharged ones. tns fluorescence assay reflected that the i-bar domain binds to the surface of the micells rather than penetrating into its core. lipid bilayers present a well-known order-disorder chain transition at ambient temperatures. this transition may become anomalous if the lipid head-group presents ionic dissociation at low ionic strength, as detected by several experimental techniques: between the gel and the liquid phases an intermediate phase appears as a shoulder in the specific heat, a deep in turbulence or a maximum in conductivity. we propose a statistical model which allows ionic dissociation of the polar group on the membrane surface and thus introduces competition between the hydrophobic interaction of hydrocarbonic chains, which favours the gel phase, at low temperatures, and the electrostatic interaction of charged head-groups, which favours the fluid phase, at higher temperatures. the model presents an intermediate fluid phase with higher dissociation and charge ordering on the membrane surface, beyond a sharp gel-fluid transition. the model presents increasing temperature of the main transition by addition of salt, as well as the shrinking of the anomalous region as chain length increases. model thermodynamic behavior is compared to results for pgs, phospholipids with a glycerol head-group. the well-programmed membrane fusion systems, operating in a weakly acidic environment, have attracted attention in the fields of biochemistry, biophysics, and pharmacy because these acidic conditions are generally observed in endosomal membranes or tumor tissues. we have reported a selective liposomal membrane fusion system toward a sugar-like cyclic cis-diol structure on the target liposome. this system consists of a lapidated phenyl boronic acid derivative as membrane -bound fusogen and phosphatidylinositol as target. here we report the preparation of a boronic acid / ph-responsive polypeptide conjugate as a novel membrane fusion device and the development of a target selective liposomal membrane fusion system with endosomal ph-responsiveness. during the course of lipid-mixing, inner-leaflet lipid-mixing, and contents-mixing assays to characterize membrane fusion behavior, we clearly observed a liposomal membrane fusion phenomenon when the ph of the experimental system was changed from . (physiological) to . (endosomal). our highly effective methods, which include a target selective liposomal membrane fusion, can be useful in the area of nanomedicine such as hybridoma technology and liposomebased drug or gene delivery. complete and reversible chemical denaturation of an a-helical membrane protein jana broecker, sebastian fiedler, sandro keller molecular biophysics, university of kaiserslautern, erwin-schrö dinger-str. , kaiserslautern, germany the question of how an unordered polypeptide chain assumes its native, biologically active conformation is one of the greatest challenges in molecular biophysics and cell biology. this is particularly true for membrane proteins. chemical denaturants such as urea have been used successfully for in vitro un-and refolding studies of soluble proteins and b-barrel membrane proteins. in stark contrast with these two protein classes, in vitro unfolding of a-helical membrane proteins by urea is often irreversible, and alternative denaturation assays using the harsh detergent sodium dodecyl sulphate suffer from a lack of a common reference state. here we present the complete and reversible chemical denaturation of the bacterial a-helical membrane protein mistic out of different micellar environments by urea. we applied multidimensional spectroscopy and techniques typically used in b-barrel membrane protein unfolding. mistic unfolds reversibly following a two-state equilibrium that exhibits the same unfolded reference state. this allows for a direct comparison of the folding energetics in different membrane-mimetic systems and contributes to our understanding of how a-helical membrane proteins fold as compared with both b-barrel membrane proteins and water-soluble proteins. in recent years, buckwheat has been of great interest in the world markets of healthy food, due to its high energy value, the content of unsaturated fatty acids, mineral constituents and vitamins. its seeds contain flavonoids which are natural, efficient antioxidants. the aim of the present studies was to investigate the effect of buckwheat extracts on the properties of biological membrane, which is the main site of the interaction between the substances buckwheat contains and the organism. the research was conducted on red blood cells and their isolated membranes, using the spectrophotometric, microscopic and fluorimetric methods. from the results obtained it follows that the compounds contained in buckwheat extracts increase the osmotic resistance of erythrocytes, making them less sensitive to the medium's osmotic pressure, induce changes in cell shape, producing increased number of echinocytes, and decrease the packing order of the polar heads of membrane lipids. it can thus be inferred that the compounds contained in the extracts penetrate the hydrophobic region of the erythrocyte membrane and alter its properties. .due to its small size, symmetric structure, amphipathicity, proteolytic stability and testable mode of activity, the gs backbone is a convenient model system to examine the structure-activity relationship of individual amino acid substitutions. we have previously reported the structure analysis of two gs analogues in which either the val or leu residues on the hydrophobic face of the molecule were substituted by the aliphatic f-labeled amino acid f-phg. using f-ssnmr in oriented lipid bilayers, we observed a re-alignment of the peptide that is compatible with the formation of a putative pore [top.curr.chem. : ]. here, we present novel analogs of gs with different f-prolines in the b-turn region, and with cf -bpg in place of leu. based on these f-ssnmr results and supported by cd, dsc and activity tests, we could demonstrate that all analogues are structurally intact and antimicrobially active. we observe, however, differences in the re-alignment propensity when comparing these gs analogues in dlpc and dmpc bilayers. these differences can be rationalized in terms of molecular shape being changed upon incorporation of unnatural amino acids at various sites of the molecule. the beta-propiolactone (bpl) is an inactivating reagent commonly used to produce viral vaccine preparations (whole virions or split-virions). although bpl has been reported to inactivate nucleic acids, its mechanism of action on proteins and the outcome on viral infection remains ill-defined. in this work, h n /victoria/ / influenza virus strain has been submitted to various bpl inactivation conditions (from lm to mm). cell infection ability was progressively reduced and entirely abolished at mm bpl. to clarify the bpl effect, we focused on membrane fusion infection steps using kinetic fluorescence molecule leakage from liposome and lipid fret assays combined with cryo electron microscopy. membrane fusion measured at ph on gm liposomes was reduced in a dose-dependent manner. interestingly the fusion activity was partially restored using the proton-ionophore monensin as confirmed by cryoem images. in addition, a decrease of molecule leakage irrespective to bpl concentration was measured suggesting that the hemagglutinin affinity for gm was slightly modified even at low bpl concentration. altogether these results strongly suggest that bpl treatment impairs m protein activity likely by preventing proton transport and bring new light on the mechanism of action of bpl. cellular membranes have a heterogeneous lipid composition, potentially forming nano-domains or membrane rafts, believed to be platforms of altered fluidity involved in protein sorting and trafficking . an alternative mechanism, potentially leading to protein sorting, has recently been proposed, suggesting that the curvature of membranes can also actively regulate protein localization . recently we showed that a variety of protein anchoring motifs are membrane-curvature sensors and thus up concentrate in regions of high membrane curvature . furthermore the curvature sensing ability of the anchoring motifs persisted independently of their structural characteristics. this leads us to speculate that curvature sensing might be an inherent property of any curved membrane and as a consequence, the lipid composition of the bilayer could potentially regulate this recruitment by membrane curvature. thus there might be an intimate, yet unrecognized, link between the way raft-like membrane domains and membrane-curvature promotes the localization of membrane-anchored proteins. we examined how changing the lipid composition of liposomes influenced the recruitment by membrane curvature of a model amphiphilic protein-anchoring motif. employing our single liposome curvature assay, we tested lipid mixtures with different ratios of dopc, sphingomyelin and cholesterol, giving rise to liposome populations of different phase-states. we found an amplified recruitment by membrane curvature for all raft-like l o phase-state mixtures when compared to the l d phase-state counterparts. based on these findings we suggest a synergetic effect when combining a raft-like lipid phase-state and high membrane curvature, resulting in a highly potent mechanism for selective localization of membrane-anchored proteins. keywords: non-lamellar lipid structure, phase transition, minerval, -hydroxylated fatty acid. minerval ( -hydroxyoleic acid), a potent antitumoral drug, is known to modulate the lipid membrane structure by decreasing the lamellar-to-non-lamellar phase transition temperature (t h ). a series of -hydroxy fatty acid derivatives, varying in acyl chain length and degree of unsaturation, have been analyzed in terms of their ability to stabilize the inverted hexagonal (h ii ) phase in palmitoyl-oleoyl-phosphatidylethanolamine membranes. differential scanning calorimetry and p-nuclear magnetic resonance showed that mono-and polyunsaturated, but not saturated, -hydroxylated fatty acid molecules were able to decrease the t h . lipid vesicles mimicking the lipid composition of a cell membrane were solubilized at °c in the presence of triton x- . the results demonstrated that the amount of detergent-resistant membranes, which are related to liquid ordered (lo) structures, decreased in the presence of -hydroxylated fatty acids. the so-called lipid membrane therapy focuses on the reversion of cell disfunction through the modulation of the membrane structure, thus altering the activity of membrane-associated proteins. the ability to modify the biophysical properties of a lipid membrane makes the studied -hydroxylated fatty acid molecules be prospective candidates for the use in the lipid membrane therapy. ]. however, a significant downward divergence occurs above mol % of probe content, which might indicate deviations to ideal mixing in fluid phase. results for b-py-c -hpc, in mixtures of popc with and mol % pops were indistinguishable from those obtained with pure popc vesicles; however excimer formation in pure pops bilayers appears to be appreciably higher. we also compared the excimer formation findings with quenching of the same probes by low concentrations of doxyl quencher groups labeled acyl phospholipid chain at the same depth of the pyrenyl group. the results are also scrutinized by the same two-dimensional kinetic formalism and good correlation was also found. derek marsh max-planck-institut fü r biophysikalische chemie, gö ttingen, germany the amassing of comprehensive data on the lipid composition of biological membranes by lipidomics initiatives provides a potent challenge to the membrane biophysicist interested in lipid structure. this resolves itself essentially into two aspects. the first systematises the dependence of membrane biophysical parameters on lipid molecular structure. lipid volumes, membrane dimensions, chain-melting temperatures and enthalpies, nonlamellar phase formation and structure, critical micelle concentrations and thermodynamics of membrane formation, membrane-membrane interactions and lipid transfer are amongst the properties of central biophysical interest. the relevant structural parameters are lipid chain length, degree of unsaturation, chain branching and headgroup configuration. the second, more complex and less well developed, aspect concerns the lipidlipid interactions that determine the membrane properties of lipid mixtures. in part, these can be obtained from binary phase diagrams, and the more limited number of ternary phase diagrams -notably with cholesterol -that are available. extrapolation to higher order mixtures lies in the future. i shall attempt to summarise some of the progress in these directions. the immediate aim is a second edition of my handbook of lipid bilayers, which, in addition to a vastly expanded database, will include interpretative features and will be available in the early part of next year. lateral diffusion dynamics in phosphatidylcholine/cholesterol bilayers has been mostly accessed by means of epr, nmr and fcs spectroscopic techniques. reliable stady-state florescence quenching analysis of diffusion-controlled processes has been ampered by the lack of a self-consistent kinetic formalism for the two-dimensional ( d) counterpart of the classical stern-volmer analysis for three-dimensional ( d) solvents. we studied the excimer formation of phospholipid-labeled pyrenyl probes (proportion of mol %) in mixed popc/cholestrol mlv liposomes by combined steady-state and timeresolved fluorescence the findings are in very good agreement with the theoretical predictions of the kinetic formalism specific for fluorescence quenching processes occurring in the small hydrophobic head group, the closely packed acyl chains, and the capability of interfacial hydrogen bonding have been suggested to govern the characteristic membrane behavior of long chain saturated ceramides: the self-segregation, and the formation of hexagonal phases and highly ordered gel phases. while it has been shown that structural alterations of the ceramide acyl chains induce position dependent effects on their behavior, we wanted to study the effect of interfacial properties, including hydrogen bonding, on ceramide membrane properties. the h-bond donor functions of nh and oh in the sphingosine backbone of palmitoylceramide were disrupted either separately or simultaneously by replacing the hydrogen with a methyl-group. when the lateral phase behavior of mixed bilayers containing cholesterol/sphingomyelin-rich domains was studied in the presence of the ceramide analogs, the o-methylated ceramide appeared to form a thermally stable, sterol-excluding gel phase with sphingomyelin, whereas the o-methylated ceramide failed in both thermal stabilization and sterol displacement. the doubly methylated analog was the poorest ceramide mimic. together with the possible steric effects induced by the methylations, the lack of nh h-bond donor function impaired ceramide membrane behavior to a greater degree than the lack of oh h-bond donor function. sugar-based surfactants are made from renewable resources using the ''green chemistry'' methods, are easily biodegradable and used in washing agents, cosmetics, and drug carriers. besides, there are attempts to use them as nonviral vectors in gene therapy. we studied the influence of new x-(alkyldimethylamonium)alkylaldonamide bromides (c n gab) with different chain lengths (n = , , , ) on the thermotropic phase behavior of dppc, and dppc/chol bilayers by means of differential scanning calorimetry. the surfactants were added either to the water phase or directly to the lipid phase (a mixed film was formed). we analyzed the changes in the temperatures, enthalpies and shapes of the main phase transitions as a function of concentration. molecular modeling methods were also used. cytotoxicicity of the c n gabs was determined in the cell line l and a . for cytotoxicity test, the cells were seeded in -well plates ml of Á cells/ml in the culture medium eagle'a or dulbecco with % calf serum, penicillin and streptomycin was deposited into each plates. the cells were treated with various doses of surfactants and incubated. the minimal concentration which was toxic to approximately % of cells was taken as tccd . this work was supported by grant n n . membrane fusion is ubiquitous in life requiring remodeling of two phospholipid bilayers. as supported by many experimental results and theoretical analyses, merging of membranes seems to proceed via similar sequential intermediates. contacting membranes form a stalk between the proximal leaflets which expand radially into a hemifusion diaphragm (hd) and subsequently open to a fusion pore. direct experimental verification of the hd is difficult due to its transient nature. using confocal fluorescence microscopy we have investigated the fusion of giant unilamellar vesicles (guvs) containing fluorescent membrane protein anchors and fluorescent lipid analogues in the presence of divalent cations. time resolved imaging revealed that fusion was preceded by displacement of peptides and lipid analogues from the guv-guv contact region being of several lm in size. a detailed analysis showed that this structure is consistent with the formation of an hd. a quantitative model of the hemifusion equilibrium and kinetics of the growing hd was developed. bilayer tension could be shown to drive hd expansion and interleaflet tension was found to act as a counterforce, because the outer leaflets are compressed upon hd growth. the model and its predictions fit nicely with observations above. concentration effects of trehalose in the equivalent polarity of fluid popc bilayers c. nobre , d. arrais , j. martins , ibb -cbme, faro, portugal, dcbb -fct, universidade do algarve, faro, portugal trehalose is an important disaccharide, formed by two units of glucose linked by a a- , glicosidic bond. it is capable of replacing water molecules in the hydration shell of the phospholipid headgroups, in cases of extreme dehydration, by establishing hydrogen bonds with their -co and -po groups, preserving this way the membrane structure. the polarity gradient is a significant feature of lipid bilayers and is influenced by the amounts of water within this medium. it is therefore important to understand the effects of different concentrations of trehalose in simple model membranes. using the pyrene empirical polarity scale, we monitorized changes in the polarity values when varying trehalose concentration in the bounding aqueous phase. for lower concentrations (until . m), we observed a decrease in polarity, comparing with popc bilayers in pure water. for higher trehalose concentrations (above . m), the polarity values are indistinguishable from those popc in water. using the freeze and thaw technique we obtained the same results, except for the lower trehalose concentrations. general anesthetics are indispensible tools of daily surgery. yet, their molecular mode of action remains elusive. while one school favors specific (direct) interactions with proteins of the central nervous system, another school adheres to a nonspecific modulation of biophysical membrane properties. one of the strongest arguments against lipid theories is the absence of stereo-specific effects in model membranes, as opposed to their detection by electrophysiological measurements on ionchannels. we have combined x-ray scattering and molecular dynamics simulations on palmitoyl-oleoyl-phosphatidylcholine bilayers with fluorescence microscopy on live cells to study the effects of the stereoisomers of ketamine on membrane properties. we find significant effects of both enantiomers on the distribution of lateral pressures at clinically relevant concentrations, being more pronounced for s-(?)-ketamine. we further calculated the effect of the lateral pressure profile changes on the opening probability of an ion-channel using crystallographic information. the observed channel inhibition compares remarkably well with clinically observed effects of the enantiomers. we thus provide first evidence for a stereo-specific, but indirect effect of general anesthetics on ion-channels. dependence of gramicidin a channel lifetime on membrane structure obtained from x-ray scattering measurements horia i. petrache department of physics, indiana university purdue university indianapolis, in , usa the activity of ion channels, in particular the lifetime of their conducting (open) state depends on the physical properties of lipid bilayers [ , ] which in turn depend on lipid headgroup and acyl chain composition. in order to investigate this dependence, we have performed measurements of gramicidin a (ga) channel lifetimes in three different lipid series. in each series, the lipid headgroups were phosphatidylcholine (pc), phosphatidylethanolamine (pe), and phosphatidylserine (ps), while the acyl chains consisted of symmetric monounsaturated di( : ), mixed ( : )( : ), and methylated di( : - me). in order to minimize the effect of headgroup electrostatics, measurements where performed in m kcl salts. we show how ga lifetimes depend on headgroup and acyl chain composition and on structural parameters determined by x-ray scattering. for the lipids considered, ga lifetimes cover a range from . seconds in the dope lipid to seconds in dphps. in this range, we find a gaussian dependence of ga lifetime on bilayer thickness, consistent with hydrophobic matching models. we discuss different aspects of channel-lipid interactions and to what extent measurements of ga lifetime in binary mixtures are consistent with measurements in pure lipid systems. [ the aim of the studies was to determine the effect of chlorogenic acid (cga), which is the main constituent of plant extracts, on properties of the model membranes. its effect was studied on temperature of the main phase transition of various lipids with and without presence of cholesterol, using the differential scanning calorimetry (dsc) method and the fluorimetric method. in particular, the degree of packing order of the hydrophilic phase of liposomes was determined using the laurdan and prodan probes, and fluorescence anisotropy of the hydrophobic phase with the probes dph and tma-dph. it had also been studied the effect of chlorogenic acid on the structure and capacity of black lipid membranes (blms), formed of egg lecithin and lipids extracted from erythrocytes. the results obtained indicate that cga lowers the main phase transition temperature slightly, without changing the fluorescence anisotropy in the hydrophobic part of the bilyer, and causes a decease in the packing order of the hydrophilic phase. by monitoring the capacity during blm formation we have found that the presence of chlorogenic acid accelerates the process of lipid self-organization into a bilayer, and increases stability and life of the blms. however, the was no effect of cga on specific capacity of the membranes, and thus on thickness of the liposome membrane hydrophobic layer. this work was sponsored by the ministry of science and education, scientific project no. n n and n n . many examples have recently been found where biological processes in the lipid bilayer are affected by the changes in the physicochemical properties of the membrane, e.g. the local curvature, the membrane tension and certainly the membrane structure. it has been shown that the activity of polyene antibiotics is strongly correlated to the phase diagram in a membrane composed of a mixture of popc and ergosterol or cholesterol (j membrane biol, : - , ( )). it is known that polyene action is quite sensitive to the type of sterol in the membrane, which enables its medical use, mainly as antifungals. it has been proposed that this selectivity of the drug to fungi is related to structure modulation by the sterols (see for example, biophys. j., , , ( )) and therefore the correlation found could be due to structural differences between popc/ergosterol and popc/ cholesterol along the corresponding phase diagrams. to investigate this, molecular dynamics simulations of the above mixtures along their phase diagrams were performed. it was found that there are indeed marked differences in structure along the phase diagrams, but for the sterol-sterol distribution function. an analysis of the behavior of this observable and the implications on polyene action is discussed. acyl transfer from lipids without enzyme catalysis: a new paradigm for membrane protein ageing? john sanderson, catherine pridmore, jackie mosely, paul yeo durham university, department of chemistry, durham, uk membrane proteins are recycled in cellulo with half-lives ranging from minutes to days. in other systems, such as enveloped viruses, proteins may equally remain membranebound for periods of days. it is therefore of interest to examine the behaviour of proteins in model membranes over extended periods in order to determine the long-term stability of the mixed systems, both in kinetic terms (attainment of equilibrium states) and chemical terms (reactivity). the reactivity of proteins towards membranes has been examined using the peptide melittin as a model for membrane proteins. acyl transfer from phospholipids to the peptide was found to occur over a period of several days, in the absence of any enzyme catalysis. transfer was detectable after days and reached % conversion in days. using tandem mass spectrometry approaches, the sites of melittin modification were localised. these sites included the side chain of lysine, opening the possibility that this residue may be modified in any membrane protein where this residue has an appropriate disposition. these observations challenge preconceptions concerning the membrane as an inert medium and highlight potential new mechanisms for membrane protein ageing. interaction of poly(l-arginine) with negatively charged bilayers studied by ft-ir spectroscopy christian schwieger, alfred blume martin-luther-university, halle-wittenberg, institute of chemistry, van-dankelman-platz , halle / saale, germany e-mail: christian.schwieger@chemie.uni-halle.de oligoarginine residues attached to macromolecules are known to facilitate the transport through lipid membranes. since the mechanism of this transport is still unclear, the effect is often called ''arginine magic''. we studied the interaction of poly(larginine) (pla) of different molecular weight with negatively charged lipid bilayers. we have shown by calorimetric and monolayer techniques that the interaction is due to a combination of electrostatic and hydrophobic forces. now we present an ft-ir spectroscopic study to reveal the effect of pla binding on membrane organisation and peptide conformation. we will show that pla binding reduces the lipid miscibility of negatively charged (pg or pa) and zwitterionic (pc) lipids within the bilayer. from the shift of the c=o stretching vibration we deduce that arginine side chains penetrate into the hydrophobic/ hydrophilic interface and replace hydration water molecules. the binding reduces the rotational freedom of the lipid molecules, as could be shown by an analysis of the ch -streching vibrations. pla binds in a b-sheet conformation to pg or pa gel phase membranes whereas its structure in bulk is random coil. the shift of the guanidyl vibration frequencies shows that also hydrogen bonds contribute to the pla -lipid interactions. neutron scattering studies of model membrane as a function of hydration and temperature federica sebastiani , , alessandra filabozzi and giovanna fragneto dipartimento di fisica, università degli studi di roma ''tor vergata'', roma, italy, institut laue-langevin, grenoble, france cell membranes carry out highly specialised functions in living materials. the composition of bacterial membranes is essential to understand the mechanism of action of antimicrobial peptides. in order to understand the role of the various components contributing to the overall behaviour, we have reproduced the membrane of bacillus subtilis and carried out neutron diffraction studies on d (small momentum-transfer diffractometer) and d (reflectometer used as a diffractometer), at ill. an ordered and homogeneous sample has been obtained by using the widely studied dmpc. the measured d-spacing of dmpc as a function of the relative humidity (rh) is related to the physical and chemical conditions affecting the sample. consequently the reliability of the humidity chamber, which has been previously upgraded, has been stated. moreover, the most suitable preparation technique has been set up. in order to investigate the component roles within bacillus subtilis membrane, three samples of phospholipids were prepared (with pope, popg and cardiolipin). neutron diffraction measurements, performed at controlled rh and temperature, suggested the presence of interesting phase transitions or coexistence of phases. the rupture of membrane vesicles near solid surfaces annamá ria taká ts-nyeste, imre deré nyi department of biological physics, eö tvö s university, h- budapest pazmany p. stny. /a, hungary the behavior of lipid membranes near solid surfaces has a great significance both in medicine and in technology. in spite of the widespread use and study of such membrane phenomena, their theoretical analysis is rather scarce. our main goal here is to understand the process during which membrane vesicles first adhere to solid surfaces, then rupture (or go through a series of transient ruptures) due to the mechanical tension induced by the adhesion, and finally spread along the surface forming a supported lipid bilayer. in our theoretical description we simultaneously consider the dynamics of spontaneous pore opening and closing; volume loss via leakage through the pores; and the advancement of the adhesion front. all these processes are supposed to follow an overdamped dynamics and coupled to each other through membrane tension. our numerical simulations reveal that the rupture process consists of three well distinguishable phases: a fast initial volume loss; followed by a slow volume loss; ending with a final burst and surface spreading. the second phase can be skipped if either the first phase advances far enough or the third phase sets in early enough. the smaller the vesicle, the further the first phase can advance. the third phase can start earlier if either the surface is smooth enough, or the adhesion energy is large enough, or the line tension is small enough. when the second phase is not skipped the time needed for the rupture process can take very long with a large variance. in the realistic range of the material properties (line tension, bending rigidity) the process is qualitatively always the same, so the most decisive parameter remains the size of the vesicle: the smaller the vesicle the faster and easier it ruptures. ( )). we chose four plant-derived polyphenols (flavonoids and stilbenes) of documented biological activity to study their influence on lipid domain number, area, shape, and borderlength. we found that resveratrol elevated the number of domains per vesicle, decreased their area and markedly increased the total length of domain border without affecting domains' circular shape. surprisingly, no such effect was observed for piceatannol differing from resveratrol by one hydroxyl group only. neither genistein nor -prenylnaringenin changed the morphology of lipid domains significantly. the possible mechanism of resveratrol-induced effect on lipid domains' morphology could be its selective accumulation in the interfacial regions between liquid ordered and liquid disordered domains. putative cholesterol recognition amino acid consensus (crac) motif in hiv coreceptors cxcr and ccr mikhail a. zhukovsky, albrecht ott biological experimental physics department, saarland university, saarbruecken, germany we identified a cholesterol recognition amino acid consensus (crac) motif in transmembrane domain (tmd ) of two g protein-coupled receptors (gpcrs), human chemokine receptors cxcr and ccr , coreceptors of human immunodeficiency virus (hiv). we suggest that residues belonging to this crac motif are involved in cholesterol binding to cxcr and ccr that is responsible for cholesterol requirement for cxcr and ccr conformation and function and for the role that cell cholesterol plays in the cell entry of cxcr -using and ccr -using hiv strains. putative crac sequences involve residues v /l -y -k in cxcr and l /v /v -y -k in ccr . in cxcr , crac motif is highly conserved across chordata species, whereas in ccr , crac motif is less conserved. t curve describe quantitatively the interfacial landscape around the protein molecules and can be used for the distinction between the globular and idp states. the behavior of the t and t data showed that there are two reorientation types present for every protein solutions below °c, irrespective for the nature of the protein or the solvent composition. local field fluctuation and the bpp models were applied, which failed for the buffered protein solutions and for the idps dissolved in water. a main cause of the failure is the changing h in the analyzed temperature range. this case is valid for the solutions of idps and for buffered solutions of both protein types. another cause can be the active relaxation channels other than dipolar when ions of quadrupolar nuclei are present. ligand-induced disorder-to-order transition plays a key role in the biological functions of many proteins that contain intrinsically disordered regions. this trait is exhibited by rtx (repeat in toxin) motifs found in more than virulence factors secreted by gram-negative pathogenic bacteria. we investigated several cyaa rtx polypeptides of different lengths ranging from to residues. we showed that the rtx proteins exhibit the hallmarks of intrinsically disordered proteins in the absence of calcium: they adopt premolten globule conformations and exhibit a strong timeaveraged apparent hydration, due in part to the internal electrostatic repulsions between negatively charged residues, as revealed by the high mean net charge. calcium binding triggers a strong reduction of the mean net charge, dehydration and compaction, folding and stabilization of secondary and tertiary structures of the rtx proteins. we propose that the intrinsically disordered character of the rtx proteins may facilitate the uptake and secretion of virulence factors through the bacterial secretion machinery. these results support the hypothesis that the folding reaction is achieved upon protein secretion and, in the case of proteins containing rtx motifs, could be finely regulated by the calcium gradient across bacterial cell wall. occupational exposure to heavy metals has been recognized to be a risk factor for parkinson's disease via metaltriggered deposition of alpha-synuclein (as) , . in the present work, al ? induced conformational change and instant oligomerization of as have been studied using fret and fcs as main techniques. donor and acceptor were labeled in the c-terminal at positions a c and a c. the average lifetime of donor in the presence of acceptor increases with the increase of al ? concentration, indicating as adopts a more extended conformation upon al ? binding. the intrinsic tyr fluorescence rises sharply within the mixing dead time, reflecting an enhanced hydrophobicity of the tyr environment and a fast conformational change of as. al ? also induces an immediate oligomerization of as as monitored by fcs. the diffusion coefficient of as changes from ± lm /s as monomer state to ± lm /s as oligomer state. the oligomerization is supposed to be induced by the ligand bridging of trivalent al ions. nearly % of human genes encode protein-kinases (pk), enzymes involved in cellular signaling and several other vital biochemical functions, which transfer phosphate groups from atp to specific target molecules, modifying their activity. [ ] deregulated pk have been linked to numerous diseases including cancer and diabetes, making them attractive targets for drug design. [ ] conformational transitions play a central role in regulating the phosphorylation activity. pk adopt an on state that is maximally active and one or more inactive states that shows minimal activity. [ ] the similarity of the relatively rigid and largely conserved atp binding site makes the design of selective inhibitors binding to the active state very difficult. indeed some of the best cancer therapies available are based on inhibitors, as imatinib, that bind to inactive states peculiar to a small subset of pk (abl, c-kit and pdgfr in the case on imatinib). thus, understanding the atomic details of the active to inactive transitions in kinases has a great importance. here we study a particular active-toinactive transition of c-src, a fundamental proto-oncogene involved in cancer and metastasis, by using multi-microsecond long fully solvated molecular dynamics simulations, metadynamics and ptmetad calculations [ , ] . the results, validated by mutagenesis, x-ray crystallography and binding kinetics, are suggestive of a functional role for the conformational transition. moreover, we were able to single out the most important residues affecting the conformational transition and to show that even a very conservative amino-acid substitution can have a dramatic effect on the conformational free energy landscape. the time-scales of protein folding events range over many orders of magnitude. in order to understand the complex folding mechanisms, peptides with well-defined secondary structure are often used as model systems as they may be regarded as smallest folding units of proteins. the formation of secondary structure elements occur on the nanosecond to low microsecond time scale. thus, stopped-flow techniques are too slow whereas pulsed laser techniques are capable to trigger folding processes in nanoseconds and to analyze faster folding events. we study ns-to-ls peptide dynamics by temperature-jump infrared spectroscopy. after initiation of a nanosecond temperature jump, the spectral response is monitored at single wavelengths in the amide i region reflecting the dynamics of the peptide backbone. relaxation rates are obtained. the helix-to-coil relaxation of polyglutamic acid is a multi-step process and requires more complex models than two-state kinetics. however, there are kinetic steps that are well described by single-exponential behavior and a two-state model. we demonstrate how equilibrium and time-resolved infrared spectroscopic data can be combined to deduce folding rates. unfolding and refolding studies using chemical denaturants have contributed tremendously to our understanding of the thermodynamics and kinetics of protein folding and stability. however, a major limitation of this approach lies in the large uncertainty inherent in the extrapolation of the free energy of unfolding in the absence of denaturant from free energy values measured at finite denaturant concentrations. here we show that this limitation can be overcome by combining multiple spectroscopic signals-including fluorescence, circular dichroism, and absorbance-recorded in a quasi-simultaneous and fully automated way at different wavelengths. we have optimised the number of wavelength values used, the integration time per data point, the increment in the denaturant concentration, and the weighting scheme applied for global data fitting. compared with the traditional approach based on the use of a single or a few wavelengths, we could thus improve the precision of the free energy value by an order of magnitude. we exemplify and validate this novel approach using representative, well-studied globular proteins and explain how it can be exploited to quantify subtle changes in membrane-protein stability which have thus far remained elusive. the rates of protein conformational changes are usually not only limited by external but also internal friction, however, the origin and significance of this latter phenomenon is poorly understood. it is often found experimentally that a linear fit to the reciprocal of the reaction rate as a function of the viscosity of the external medium has a non-zero , the physical basis of pressure unfolding is still largely unknown. we report here a specific study of cavities contributions to the volume difference between unfolded and folded states (dv u ), using four single point mutants of staphylococcus nuclease (snase). each mutation is localised in a strategic position on the protein structure and was designed to change a large buried hydrophobic side chain into alanine, thus opening tunable cavities in the snase d structure. measuring hsqcs peaks intensities up to bar monitored the equilibrium high pressure unfolding and leads us to precise estimations of dv u for more than two-thirds of the residues of each mutant. so-fast hmqc experiments were also performed to measure folding and unfolding rates from bar pressure jumps. high-pressure fluorescence experiments were done on six additional alanine mutants to complement the nmr study, allowing a more complete exploration of the local pressure sensitivity along the protein d structure. all these highly reliable measurements shed light on the real signification of the thermodynamic parameter dvu, and bring an unprecedented complex and heterogeneous picture at a residue level of the apparent two-state folding process of snase. determination of contributing factors to the volume change magnitude between unfolded and folded states (dv u ) is a longstanding question in the high-pressure field. we provide here new experimental and computational data using two wellcharacterized model proteins: notch ankyrin repeat domain (nank) and staphylococcal nuclease (snase). the repetitive nature of the nank protein was used to study influence of the protein size on dv u in a systematic way with a set of deletion mutants. high-pressure fluorescence data provided new evidences that neither peptide bonds hydration nor side chains differential hydration could be considered as major contributor to the measured dv u value. additional molecular dynamics (md) simulations rather suggested that the heterogeneous distribution of void volume in the folded states structures could explain the dv u variations among the nank deletion mutants. the specific issue of the void volume contribution to dv u values was studied using cavity mutants in snase, allowing a large structural mapping of the alanine mutations on this globular protein. combination of x-ray crystallography, highpressure fluorescence, high-pressure nmr and md simulations provided a first clear determination of the void volume contribution to the dv u values. these results also bring an unprecedented complex and heterogeneous picture at a residue level of the apparent two-state folding process of snase. we expressed an ig domain (i ) and a -residue-long fragment of the pevk domain in order to investigate the effect of temperature and pressure on their conformation. ftir spectroscopy is a useful method for investigating the secondary structure of proteins. we analyzed the amide i band to obtain information on protein structure. fluorescence labeling was also used in some experiments. to generate high pressures, a diamond anvil cell was employed. the ftir and fluorescence spectra of the protein fragments were recorded across the pressure and temperature ranges of - gpa and - °c, respectively. moderate changes were observed in the conformation of the pevk fragments in the explored range of the t-p plane, suggesting that the domain is a highly flexible, random-coil across the entire studied t-p range. by contrast, the i domain showed quite stable secondary structure. intrinsically disordered proteins participate in important regulatory functions in the cell, including regulation of transcription, translation, the cell cycle, and numerous signal transduction events. disordered proteins often undergo coupled folding and binding transitions upon interaction with their cellular targets. the lack of stable globular structure can confer numerous functional advantages, including, paradoxically, both binding promiscuity and high specificity in target interactions. nmr is unique in being able to provide detailed insights into the intrinsic conformational preferences and dynamics of unfolded and partly folded proteins, and into the mechanism of coupled folding and binding. the function of intrinsically disordered protein domains in transcriptional regulation and signaling will be described, with particular reference to the general transcriptional coactivators cbp and p , the tumor suppressor p , and the adenovirus e a oncoprotein. the globular domains of cbp/p are targets for coupled folding and binding of disordered transactivation motifs of numerous transcription factors and viral oncogenes, which compete for binding to limiting amounts of cbp/p . many intrinsically disordered proteins contain multipartite interaction motifs that perform an essential function in the integration of complex signaling networks. the role of multipartite binding motifs and post translational modifications in regulation of p -mediated signaling pathways will be discussed. the early vascular network is one of the simplest functioning organs in the embryo. its formation involves only one cell type and it can be readily observed and manipulated in avian embryos or in vitro explants. the early vascular network of warm-blooded vertebrates self-organizes by the collective motility of cell streams, or multicellular ''sprouts''. the elongation of these future vascular network segments depends on a continuous supply of cells, moving along the sprout towards its tip. to understand the observed self-organization process, we investigate computational models containing interactions between adherent, polarized and self-propelled cells. by comparing the simulations with data from in vivo or simplistic in vitro experiments, we explore the role of active migration, leader cells, invasion of the ecm, and cell guidance by micromechanical properties of adjacent cell surfaces. boron neutron capture therapy (bnct) is a promising method for treating the highly fatal brain tumor; glioblastoma multiform. it is a binary modality; in which use is made of two components simultaneously; viz. thermal neutrons and boron- . the biophysics of bnct is very complicated; primarily due to the complexity of element composition of the brain. moreover; numerous components contributes to the over all radiation dose both to normal brain and to tumor. simple algebraic summation cannot be applied to these dose components, since each component should at first be weighed by its relative biological effectiveness (rbe) value. unfortunately, there is no worldwide agreement on these rbe values. thermal neutrons were formerly employed for bnct, but they failed to prove therapeutic efficacy. later on; epithermal neutrons were suggested proposing that they would be enough thermalized while transporting in the brain tissues. however; debate aroused regarding the optimum source neutrons energy for treating brain tumors located at different depths in brain. insufficient knowledge regarding the rbe values of different bnct dose components was a major obstacle. a new concept was adopted for estimating the optimum source neutrons energy appropriate for different circumstances of bnct. four postulations on the optimum source neutrons energy were worked out, almost entirely independent of the rbe values of the different dose components. four corresponding condition on the optimum source neutrons energy were deduced. an energy escalation study was carried out investigating different source neutron energies, between . ev and . mev. mcnp b monte_carlo neutron transport code was utilized to study the behavior of these neutrons in the brain. the deduced four conditions were applied to the results. a source neutron energy range of few electron volts (ev) to about kev was estimated to be optimum for bnct of brain tumors located at different depths in brain. simulation of mutation induction by inhaled radon progenies in the bronchial epithelium balá zs g. madas, Á rpá d farkas and imre balá shá zy hungarian academy of sciences kfki atomic energy research institute, konkoly-thege mikló s ú t - ., budapest, h- , hungary radon is considered as the second most important cause of lung cancer after smoking. to understand the mechanisms leading from radon exposure to cancer formation is of crucial importance. this study focuses on the description of mutation induction by radon progenies in the bronchial epithelium. computational fluid and particle dynamics approach was applied to determine the radio-aerosol deposition distribution in the central airways. a numerical replica of a small fragment of the bronchial epithelium was prepared based on experimental data. microdosimetric computations were performed to quantify the cellular radiation burdens at the very site of deposition accumulation. a mutagenesis model was applied supposing that radiation induces dna damages and enhances the cell turnover rate. the results show that both considered mutagenic effects of densely ionising radiation contribute significantly to mutation induction and mutation rate depends non-linearly on exposure rate. furthermore, simulations suggest that the local maintenance capacity of the bronchial epithelium can be exhausted by chronic exposure to radon progenies with activity concentration characteristic of some uranium mines. the present work demonstrates possible applications of numerical modelling in radon related carcinogenesis studies. the neural crest is a group of cells found in all vertebrate embryos. it forms in the neural folds at the border of the neural plate and gives rise to a huge variety of cells, tissues and organs. one of the astonishing characteristic of neural crest cells is that they are able to migrate very long distances in the embryo. the neural crest has been called the ''explorer of the embryo'' as it is one of the embryonic cell types that migrate most during development, eventually colonizing almost every tissue. in this talk i will discuss our recent finding about neural crest migration. we have shown that neural crest cells, classically described as mesenchymal cells, migrate in large clusters cytokinesis relies on tight regulation of the mechanical properties of the cell cortex, a thin acto-myosin network lying under the plasma membrane. although most studies of cytokinetic mechanics focus on force generation at the equatorial acto-myosin ring, a contractile cortex remains at the poles of dividing cells throughout cytokinesis. whether polar forces influence cytokinetic cell shape is poorly understood. combining cell biology and biophysics, we demonstrate that the polar cortex makes cytokinesis inherently unstable and that any imbalance in contractile forces between the poles compromises furrow positioning. we show that limited asymmetric polar contractions occur during normal cytokinesis, and that perturbing the polar cortex leads to cell shape oscillations and division failure. a theoretical model based on a competition between cortex turnover and contraction dynamics accurately accounts for the oscillations. we further propose that blebs, membrane protrusions that commonly form at the poles of dividing cells, stabilise the position of the cleavage furrow by acting as valves releasing cortical contractility. taken together, our findings show that the physical properties of the entire cell are integrated into a finetuned mechanical system ensuring successful cytokinesis. collective motion of individual cells marks the onset of the transition to multicellularity in many microorganisms. this transition is often mediated by intercellular communication signals between cells. here, we show, in contrast, that the transition from single cell to collective motion in an ensemble of gliding bacterial cells can be understood as a dynamical selfassembly process of self-propelled rods. experiments were carried out with a mutant of the bacterium myxococcus xanthus moving by means of the a-motility system only and without undergoing reversals. the collective motion phase is confined to a monolayer and is characterized by the organization of cells into larger moving clusters. a transition to collective motion is detected in experiments by image analysis, that reveals a qualitative change of the cluster-size distribution at a critical cell packing fraction around %. this transition is characterized by a scale-free power-law cluster size distribution with an exponent . . we provide a theoretical model for cluster formation of self-propelled rods that reproduces the experimental findings for the cluster size distribution. our findings suggest that the interplay of selfpropulsion of bacteria and volume exclusion effects of the rodshaped cell bodies is sufficient to explain the onset of collective motion and the related changes in the cluster statistics. despite much speculation on the existence of structurally distinct oligomeric species associated with the conversion of certain monomeric proteins into amyloid fibrils, it has not previously been possible to observe them directly or to relate them to any key mechanistic steps involved in the interconversion process. we have developed a novel application of singlemolecule intermolecular fret to investigate in unprecedented detail the aggregation and disaggregation of alpha-synuclein, the protein whose pathogenic deposition as intracellular lewy bodies is a characteristic feature of parkinson's disease. our study reveals that a range of oligomers of different size and structure are formed, even at physiologically relevant concentrations. interestingly, the resistance to degradation of the aggregated state of alpha-synuclein, which is a well- we focused on the structure-dynamics interplay and showed how the fractal-like properties of proteins lead to such anomalous dynamics. we used diffusion, a method sensitive to the structural features of the protein fold and them alone, in order to probe protein structure. conducting a large scale study of diffusion on over pdb structures we found it to be anomalous, an indication of a fractal-like structure. taking advantage of known and newly derived relations between vibrational dynamics and diffusion, we demonstrated the equivalence of our findings to the existence of structurally originated anomalies in the vibrational dynamics of proteins. more specifically, the time dependent vibrational mean square displacement (msd) of an amino acid is predicted to be subdiffusive. the thermal variance in the instantaneous distance between amino acids is shown to grow as a power law of the equilibrium distance. the autocorrelation function in time of the instantaneous distance between amino acids is shown to decay anomalously. our analysis offers a practical tool that may aid in the identification of amino acid pairs involved in large conformational changes. more recently, we studied the effect of the hydrodynamic interaction between amino acids using a zimm-type model. we computed the time-dependent msd of an amino acid and the time-dependent autocorrelation function of the distance between two amino acids, and showed that these dynamic quantities evolve anomalously, similar to the rouse-type behavior, yet with modified dynamic exponents. we also studied the dynamic structure factor s(k,t) of proteins at large wavenumbers k, kr g [ [ , with r g the gyration radius, that are sensitive to the protein internal dynamics. we showed that the decay of s(k,t) is dominated by the spatially averaged msd of an amino acid. as a result, s(k,t) effectively decays as a stretched exponential. we compared our theory with recent neutron spin-echo studies of myoglobin and hemoglobin for the rouse and zimm models of hydrodynamic friction. in addition, i will mention two other projects currently underway: (i) a new elastic network model that accounts for the tensorial aspects of protein elasticity and is a combination of stretch-compress springs and bond-bending energies. (ii) the unfolding of a protein under the exertion of a large pulling force. allosteric regulation of enzymatic activity is crucial for controlling a multitude of fundamental cellular processes. yet the molecular level details underlying regulation often remain poorly understood. here we employed single molecule activity studies to dissect the mechanistic origin of enzymatic activity regulation. as a model system we employed a lipase and measured its activity as a function of accessibility to surface tethered liposomes ( ), which are known regulators of its activity. our results surprisingly revealed that the lipase oscillates between states of different activity. we accurately quantified for the first time both the interconversion rates between activity states and the inherent activity of these states. based on these we calculated the energetic landscape of the entire reaction pathway and identified that regulatory interactions redistributed the probability to reside on preexisting enzymatic activity states but did not alter the activity of these states. our findings provide the missing link between conformational and activity substates supporting and represent the first direct validation of the textbook hypothesis of conformational selection for regulation of enzymatic activity to identify the potential targets of cgmp in arabidopsis plants we adopted a proteomic approach to isolate possible cgmp-binding proteins. purification of soluble cgmp-binding proteins was performed using cgmp-agarose-based affinity chromatography procedure. next eluted proteins were analyzed by sds-page which revealed ten bands. we focused the subsequent analysis on low-molecular peptides of , and kda which were bound cgmp more intensively. after d-ief-page of the proteins isolated by cgmp-agaroseaffinity chromatography eight most abundant protein spots in the low-molecular area were visualized. these spots of interest were excised from the gel and in gel digested by trypsin. then tryptic peptides were analyzed by maldi-tof mass spectrometry and identified as isoforms of nucleoside diphosphate kinase (ndpk) from arabidopsis. thus, our data suggest that ndpk is a potential target of cgmp signaling in arabidopsis. dual-color fluorescence-burst analysis (dcfba) was applied to measure the quaternary structure and high affinity binding of the bacterial motor protein seca to the protein-conducting channel secyeg reconstituted into lipid vesicles. dcfba is an equilibrium technique that enables the direct observation and quantification of protein-protein interactions at the single molecule level. seca binds to secyeg as a dimer with a nucleotideand preprotein-dependent dissociation constant. one of the seca protomers binds secyeg in a salt-resistant manner, while binding of the second protomer is salt-sensitive. since protein translocation is salt-sensitive we conclude that the dimeric state of seca is required for protein translocation. a structural model for the dimeric assembly of seca while bound to secyeg is proposed based on the crystal structures of the thermatoga maritima seca-secyeg and the escherichia coli seca dimer. • dcfba is a flurorescence based single molecule technique that allows assessment of the stoichiometry of ligands bound to membrane receptors • dimeric seca binds asymmetrically to the protein-conducting membrane channel secyeg • monomeric seca binds secyeg but dimeric seca is required for protein translocation • protein translocation depends on receptor cycling of the dimeric seca if the dna charge is sufficiently neutralized by counter-ions, electrostatic interactions between helical charge patterns can cause attraction [ ] . helix specific interactions also cause tilt, in one direction, between two dna fragments [ ] . in braids and supercoils, this impetus to tilt breaks positive-negative supercoil symmetry. we show that these effects may cause spontaneous braiding of two molecules, lowering the dna pairing energy [ ] . the pairing is more energetically favourable for homologues (same base pair text) than for nonhomologous pairs. this might explain pairing between only homologues observed in nacl solution [ ] . also, we construct a simple model for a closed loop supercoil, including chiral electrostatic interactions. there are very interesting effects, for sufficient charge neutralization and groove localization of counter-ions. i.) positive super-coils are more energetically favourable than negative ones. ii.) a transition between loosely and tightly wound supercoils as one moves from negative to positive values of the supercoiling density. iii.) in positive super-coils the chiral interaction underwinds dna. [ von willebrand factor (vwf) is a large multimeric protein that is crucial for the force sensing cascade triggering primary hemostasis. it mediates binding of activated thrombocytes to injured epithelial tissue and serves as a transporter for coagulation factor viii. while it was shown that the hemostatic activity of vwf is affected by shear stress [ ] , the exact impact that shear forces have on the inflammatory cascade remains unclear. it is assumed that hydrodynamic forces lead to partial unfolding of vwf, which in consequence exposes more binding sites. in order to observe shear-induced changes of the protein's functionality, we measure conformational changes of vwf under flow with fluorescence correlation spectroscopy (fcs). we aim to measure the degree of uncoiling of vwf multimers under various buffer conditions, e.g. in the presence of colloids, vesicles or platelets. as only large multimers show significant hemostatic activity we intend to monitor the molecular weight distribution of vwf. shifts in this distribution indicate various pathological conditions making our multimer analysis a fast diagnostical tool for vwf-related diseases. this will serve as a basis for studies of vwf binding to collagen, fviii, gpib, vesicles and membranecoated nanoparticles under shear flow. [ data on mechanical properties of medically important proteins located in neural junctions are very limited. contactins (cntn) and paranodin proteins, located in extracellular part of ranvier nodes, are important for proper brain wiring. here we study a new series of fniii modules from human cntn- and - using a single molecule afm force spectroscopy and advanced, all-atom steered molecular dynamics (smd) computer simulations. mutations in cntns are responsible for numerous brain disorders including autism or pathological development of odor maps. perhaps mechanical properties of individual fniii mutated protein modules are compromised, thus we address this problem. a comparison of our afm force spectra with those of reference proteins will be presented [ ] [ ] , and the molecular level interpretation fniii nanomechanics, based on our smd data will be given. we believe that these data should help to understand a role of cntn in regulation of sodium ion channels in both normal and autistic subjects. supported in part by polish ministry of education and science, grant no. n , the computational center task in gdansk and license for accelrys software. recent achievements in rational dna-motors engineering demonstrate the possibility to design nano-motors and nanorobots capable of performing externally controlled or programmed tasks. a major obstacle in developing such a complex molecular machine is the difficulty in characterizing the intermediates, the final products and their activity. typically, non in-situ gel and afm and in-situ bulk fluorescence methods are used. i will present two dna-motors recently developed and studied using in-situ single-molecule fluorescence resonance energy transfer (smfret), alternating laser excitation (alex) and total internal reflection fluorescence spectroscopy (tirf), and will demonstrate that these methods can improve the way we design, construct, measure and understand highly complex dna-based machines. a motor made of bipedal dna-walker, which walks on a dna track embedded on a dna-origami, capable of long walking distance and maintaining structural stability, will be presented. the motor is non-autonomous; it receives ss-dna fuel/anti-fuel commands from outside (as in shin & pierce, jacs, ). the motors assembly stages and singlemotor's walking steps are monitored using smfret. the second motor is based on published bipedal autonomous dna-motor (seeman, science ). it is characterized by coordinated activity between the different motor domains leading to processive, linear and synchronized movement along a directionally polar track. to prove that the motor indeed walks, the authors chemically froze the motor at each step and use a complicated radioactive gel assay. i will demonstrate that using single-molecule approach, we are able to directly and in-situ measure single-motor's movements in few simple experimental steps, and measure its structural dynamics and kinetics. translation by a single eukaryotic ribosome using single molecule total internal reflection fluorescence microscopy, we observed translation of a short messenger rna (mrna) strand by single eukaryotic ribosomes. the ribosome-mrnas complexes are fixed to a microscope coverslip through the mrna, and mrnas are located through fluorescently labelled oligonucleotides hybridized to it downstream start codon. because of the ribosome helicase activity, the double strand formed by the oligonucleotide and the mrna is opened while the ribosome translates this region of the mrna. thus, the loss of the fluorescence signal allows us to measure the distribution of translation speed of single ribosomes. careful attention was given to photobleaching for the data analysis. this experiment opens the door to the study of eukaryotic translation at the single molecule level. erythrocyte hyperaggregation, a cardiovascular risk factor, has been associated to high plasma concentrations of fibrinogen. using atomic force microscopy (afm)-based force spectroscopy measurements, we have recently identified the erythrocyte membrane receptor for fibrinogen, an integrin with a a or a -like subunit [ ] . after this, we extended the study to the influence of erythrocyte aging on fibrinogen binding [ ] . force spectroscopy measurements showed that upon erythrocyte aging, there is a decrease of the binding to fibrinogen by decreasing the frequency of its occurrence (from . % to . %) but not its force. this observation is reinforced by zeta-potential and fluorescence spectroscopy measurements. knowing that younger erythrocytes bind more to fibrinogen, we could presume that this population is the main contributor to the cardiovascular diseases associated with increased fibrinogen blood content, which disturbs the blood flow. our data also show that sialic acid residues on the erythrocyte membrane contribute for the interaction with fibrinogen, possibly by facilitating the binding to its receptor. antimicrobial peptides are usually polycationic and amphiphilic with high affinity for bacterial membranes. in order to characterize their therapeutic potential it is crucial to disclose which properties of the peptide/lipids are important for target selectivity, and to examine the peptide structure and its association with lipid bilayers. in this work, first experiments have been carried out on a promising peptide called sb , which might represent the basis for developing a novel class of antibiotics. with the goal of enhancing the activity of a new semi-synthetic sequence, two identical peptides (wkkirvrlsa) were assembled via a lysine-linker, carrying also an octanoyl-lipid anchor. a highly active compound was obtained, but its structure and mode-of-action remain unexplored. this dendrimeric peptide and its linear deca-peptide counterpart are being studied in parallel to highlight the relevant properties and differences between dendrimeric structure and the sequence. monolayer intercalation is investigated with microtensiometry, fluorescence spectroscopy is applied to study thermodynamics and kinetics of the binding process. circular dichroism, nmr and md simulations are employed with the aim of elucidating the d structure in the membrane-bound state. the capability of proteins to build structures via self-organization is fascinating biophysicists since decades. with the advent of single-molecule methods, namely fluorescence correlation spectroscopy (fcs) and fluorescence resonance energy transfer (fret), the process of complex formation is becoming accessible to direct observation. coronaviruses (cov) are enveloped positive-stranded rna viruses. for sars-cov, it was shown that coronaviruses encode a rna-dependent rna-polymerase (rdrp) build from non-structural protein (nsp ) and non-structural protein (nsp ). this hexadecameric nsp -nsp complex is a hollow, cylinder-like structure assembled from eight copies of nsp and held together by eight nsp molecules [ , ] . we are aiming at understanding the assembly process and conformational changes of the complex for the related feline coronavirus. first results implicate that nsp alone forms a dimer, where interchain fret is more efficient than intrachain fret. for the complex the results indicate that nsp -nsp form a heterodimer which is different from sars-cov. our experiments highlight the potential of single-molecule fret for the study of protein complex formation. diffracted x-ray tracking (dxt) has been considered as a powerful technique for detecting subtle dynamic motion of the target protein at single molecular level. in dxt, the dynamics of a single protein can be monitored through trajectory of the laue spot from the nanocrystal which was labeled on the objective protein. in this study, dxt was applied to the group ii chaperonin, a protein machinery that captures an unfolded protein and refolds it to the correct conformation in an atp dependent manner. a mutant group ii chaperonin from thermococcus strain ks- with a cys residue at the tip of the helical protrusion was immobilized on the gold substrate surface and was labeled with a gold nanocrystal. we monitored diffracted spots from the nanocrystal as dynamic motion of the chaperonin, and found that the torsional motion of the chaperonin in the presence of atp condition was times larger than that in the absence of atp condition. and uv-light triggered dxt study using caged atp revealed that the chaperonin twisted counterclockwisely (from the top view of chaperonin) when the chaperonin closed its chamber, and the angular velocity from open to closed state was % faster than that from closed to open state. peptides or proteins may convert (under some conditions) from their soluble forms into highly ordered fibrillar aggregates. in vivo such transitions can lead to neurodegenerative disorders such as alzheimer's disease. alzheimer's disease is characterised by the extracellular deposition of abeta peptide in amyloid plaques, and the intracellular formation of neurofibrillary tangle (nft) deposits within neurons, the latter correlating well with disease severity. the major constituent of nft deposits are paired helical filaments (phf) composed of a microtubule-associated protein known as tau. studying the process by which tau forms these large aggregates may be an essential step in understanding the molecular basis of alzheimer's disease and other tauopathies. we have applied a two-colour single molecule fluorescence technique, and single molecule intermolecular fret measurements to study the soluble oligomers of tau which are formed during the aggregation and disaggregation of phf's. the neuronal protein alpha-synuclein is considered to play a critical role in the onset and progression of parkinson's disease. fibrillar aggregates of alpha-synuclein are the main constituents of the lewy bodies that are found in the brains of parkinson patients. however, there is growing evidence suggesting that oligomeric aggregates are significantly more toxic to cells than fibrillar aggregates. very little is known about the structure and composition of these oligomeric aggregates. we present results using single-molecule photobleaching approaches to determine the number of monomeric subunits constituting the oligomers. our results show that the oligomers have a narrow size distribution, consisting of * - monomers per oligomer. fluorescence correlation spectroscopy data confirm the narrow size distribution and additionally indicate a very loose packing of the oligomers. in combination with bulk fluorescence spectroscopy results of tryptophan containing mutants of alpha-synuclein, we present a structural model for the alpha-synuclein oligomer. gold colloids are widely used for in vitro and in vivo imaging. compared to the traditional optical tags sers-coded nanoparticles show a narrow emission bandwidth with structured spectra typical of the molecule used, a wider excitation bandwidth, higher emission intensity, a better photo-stability, and a lower toxicity. this is why in cancer therapy, besides being considered good tools for the delivery of anti-tumor drugs, aunp can be also good optical tags for the analyses of both np localization by laser scanning microscopy and the process of drug release inside the cells by raman. in our work we used nm diameter aunp loaded with rhodamine g, a molecule with a high raman and fluorescence efficiency, and with a chemical structure similar to doxorubicin, the antitumoral drug used in our system. the data showed that aunp are internalized by cells and sers can be performed. nm and nm diameter aunp loaded with doxorubicin were incubated at different time points with a cell line (human adenocarcinomic alveolar basal epithelial cells). only nm aunp showed intense raman emission typical of the doxorubicin phonon transitions. in recent years biomedical applications of diamond nanoparticles have become of significant interest, which rises questions of their biocompatibility and mechanisms of interactions with cells. the aim of this study was to compare the effect of nonmodified diamond nanoparticles (dnps) and dnps modified by the fenton reaction on human endothelial cells. dnps (\ nm particle size, sigma) were modified by the fenton reaction introducing surface -oh groups. immortalized human endothelial cells (huvec-st) were incubated with - lg/ml dnps in the optimem medium. diamond nanoparticles modified by the fenton reaction had smaller hydrodynamic diameter estimated by dynamic light scattering and the surface potential (zeta potential) measured using laser-doppler electrophoresis. they were more cytotoxic as evaluated by the mtt reduction assay. dnps augmented generation of reactive oxygen species in the cells, estimated by oxidation of ', '-dichlorofluorescin, the effect being higher for the fenton-modified dnps after -h incubation. cellular production of nitric oxide, estimated with daf-fm, was also affected by dnps; after h, fentonmodified oh, in contrast to non-modified diamond, decreased no production. diamond nanoparticles affected also the cellular level of glutathione and activities of main antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione s-transferase). we aim at investigating how photoreactions of proteins can be controlled by means of intense thz radiation tuned in resonance to specific vibrational modes, much in analogy to coherent control experiments conducted by fs nir laser pulses [ ] . for this we will combine a time-resolved ir difference spectroscopic setup with uniquely intense, tunable narrow bandwidth thz radiation ( - lm) at the ps beamline of the thz free electron laser felbe. these experiments will be performed on bacteriorhodopsin (br) which is the sole protein of the purple membrane of the archaebacterium halobacterium salinarum [ ] . upon illumination, the chromophore retinal isomerizes around the c -c double bond [ ] and br pumps a proton from the cytoplasmic to the extracellular side. this proton gradient is used by the bacterium to drive photosynthetic atp production under low oxygen tension [ ] . in our experiment, the photoreaction is initiated by a visible laser pulse as in standard experiments, but then the sample will be irradiated by a thz pulse from the free electron laser tuned into resonance with low-energy vibrational modes which is supposed to influence the photoreaction [ ] . such vibrational control will be monitored by time-resolved ftir spectroscopy using the step-scan technique [ ] . liposomes are increasingly studied as nanoscale drug delivery systems and biomembrane models. however the exact structure dynamics and mechanical behavior of liposomes is little known. atomic force microscopy (afm) is a powerful tool to characterize nanoscale morphology and enables the mechanical manipulation of submicron-sized vesicles. a drawback of afm, however, is that liposomes may flatten and rupture on substrates to form patches or supported planar bilayers (spb). our aim was to obtain better understanding of factors affecting liposomes on substrates and find experimental conditions at which liposomes preserve their structural integrity. in the presence of divalent cations dppc liposomes formed spb on mica. vesicles sedimented subsequently preserved their integrity and showed stronger attachment to spb. in addition to cross-bridging lipid head groups, divalent cations influence the surface charge of liposomes, thereby modulating liposome-substrate and liposome-liposome interfacial interactions. preserved vesicles stabilized by divalent cations may provide a unique experimental system for studying membrane-protein interactions. the influence of e.g. ph and ionic strength on various chromatographic bead -biomass combinations. to analyze the force curves, possible elastic contributions, e.g. from deforming cellular membranes, have to be decoupled from the interaction forces. then, bead-biomass interactions will be modeled using (extended) dlvo theory and resulting data can also be compared to real-life eba processes. the project aims for a better understanding of the interaction forces in chromatography and might help to improve the process quality of eba. long-term non-invasive in vivo monitoring of the survival, migration, homing and fate of transplanted cells is of key importance for the success of cell therapy and regenerative medicine. tools for in vivo magnetic resonance (mr) imaging of labeled cells are therefore being developed. we have prepared superparamagnetic iron oxide nanoparticles by the coprecipitation of fe(ii) and fe(iii) salts and oxidation. to stabilize the particles and to facilitate their internalization by the cells, the nanoparticles were coated with several novel low-and highmolecular weight compounds including d-mannose, poly(llysin), poly(n,n-dimethylacrylamide) and dopamine-hyaluronate conjugate. the surface-modified magnetic nanoparticles were thoroughly characterized by a range of physico-chemical methods, which proved the presence of the coating on the particles. the particles were then investigated in stem cell experiments in terms of real time cell proliferation analysis, viability, labeling efficiency and differentiation. the iron oxide concentration of the labeled cells was assessed using mr relaxometry. the advantages/disadvantages of particular iron oxide coatings will be discussed and the optimal coating suggested. excellent contrast was achieved by labeling the cells with dopamine-hyaluronate-coated nanoparticles. support of the as cr (no. kan ) is acknowledged. in the last decades the interest towards the fabrication of innovative bio-sensors with improved sensitivity and reliability for medical-diagnostics applications has been constantly risen. among the different techniques, microfluidic systems are playing a major role. in order to detect extremely low concentrations of biomolecules (pm and fm), attention should be placed on the controlled, selective functionalization of micro-and nano-channels. in this work we propose a new approach to functionalize gold patches inside fluidic channels. we start from self-assembled monolayers (sams) of thiolated molecules on a gold electrode deposited inside the channel. then, by using an electrochemical approach [ , ] we remove molecules from the sam at selected locations, by applying a negative voltage to the electrode. the newly exposed gold surface can be re-functionalized by using a thiolated biomolecule (i.e an antibody) capable to bind specific proteins flowing inside the channel. the cycle can be applied to other electrodes in the microfluidic system, creating a multiplexing device which, as we will show, can differentially measure ionic current flows in different channels. optically actuated micromanipulation and micro-probing of biological samples are increasingly important methods in the today's laboratory. microbeads as probes are the most commonly used tools in this field, although the only manipulative motion they allow is translation. we present polymerized d microstructures which can also be used for optical micromanipulation with more degree of freedom than microbeads. the two-photon polymerization (tpp), based on focused fs laser beam into appropriate photopolymers is a powerful method to build structures of arbitrary complexity with submicrometer resolution. the presented tools have the advantage of being capable of twisting and rotational manipulative motion, and also that the position of biological manipulation and optical trapping is spatially separated. different manipulative interfaces, the positioning stability and surface activation of the manipulators will be discussed. the potential application of multiplexed quantum dot labeling, mqdl, in clinical detection, prognosis and monitoring therapeutic response has attracted high interests from bioengineers, pathologists and cancer biologists. mqdl is superior comparing with conventional organic dye staining in its narrow emission bandwidths, wide signal dynamic ranges, high detection sensitivity, and low noise to signal ratios. however, the majority of the mqdl application has been limited to identification of specific cell type or cancer subtype and the improvement in labeling methodology. in this study, we focused on simultaneous detection and analysis of proteins in the c-met activation pathway, i.e. rankl, vegf, nrpln- , p-c-met and mcl- , which are known to be associated with human prostate cancer progression and metastasis. two experimental systems were analyzed: ) fixed xenograft tissues from an established ltl castration resistance human prostate cancer or crpc model; and ) clinical prostate tissue specimens from localized cancer and bone metastasis. in the presentation we will report our experience in ) the mqdl protocol optimization for the sequential reactions of individual primary antibody, the biotinylated secondary antibody and streptavidin-coated qd conjugate with nuclear dapi staining; and ) the multiplexed image catching, image unmixing, and subsequent per cell base quantification. for future multi-specimen analyses and validation, we will introduce a high throughput vectra image analysis system. carbon nanotubes (cnts) are already quite popular among many scientific and technological disciplines . in recent years they have been targeted for biotechnological and medical applications. in this work we have investigated the nanostructural self assemblies of biological lipid molecules in presence of cnts. advantage of using highly aligned cnts for this purpose being the possibility of studying the interactions of lipid molecules on the macromolecular surface as well as in the confinement of aligned cnts. we have observed various lyotropic nanostructures that are found for corresponding lipids in the bulk under dry and hydrated conditions. nanostructural studies were mainly performed using small and wide angle x-ray scattering techniques. this work is crucial for designing the nano-micro-fluidic architectures and supported model membranes where both -functionalization of cnts and nanostructural assembling of lipids, could be employed simultaneously. alternative of the commonly used measuring tools in protein research and medical diagnostics. thazolidone derivatives are novel synthetic compounds possessing various biological activities. we selected three such compounds les- , , which passed national cancer institute in vitro tests. annexin v/pi and dapi staining, dna electrophoresis in agarose gel, and western-blot analysis using specific antibodies against cellular proteins involved in apoptosis were applied to study molecular mechanisms of tumor cell death induced by these compounds. it was found that molecular targets of thiazolidones in target cells strongly depend on structure of their side groups: les- containing isatine fragment, activated caspase- involved in receptor-mediated apoptosis, while les- possessing benzthiazol residue, induced mitochondrial apoptosis mediated by caspase- , and les- which has a unique chlorine atom in side chain, also led to mitochondrial apoptosis mediated by aif (apoptosis-inducing factor). to increase anticancer potential of these molecules, in silico study was performed and most active groups of les- and les- were combined into one molecule. in vitro studies showed that such hybrid molecule called les- possessed times higher anticancer potential (ic = lm) comparing with initial compounds. we report on the synthesis and characterization of vaterite microcontainers for controlled drug release. moreover, we present experiments on possible release strategies of encapsulated substances via recrystallization, ph controlled, or by desorption methods. vaterite spherical particles were fabricated with controllable average sizes from ± nm till ± hm. we considered two ways of functionalization of the containers: encapsulation of the substances during the vaterite synthesis or their adsorption onto the prepared particles. as model experiments, vaterite containers, encapsulating rhodamine g, were imaged by two-photon microscopy, showing dye release into the aqueous medium due to recrystallization to calcite within days. differently, in ethanol only small amounts of the encapsulated markers were diffusion released after one week. the release mechanisms can be further controlled by covering the microcontainers with additional polymer layers to increase diffusion and recrystallization time. a change of the ph from neutral to acid conditions leads to the destruction of the vaterite matrix followed by a quick release of the encapsulated materials. these flexible control mechanisms make this system an interesting candidate for pharmaceutical applications. magnetic nanoparticles (np) in combination with therapeutic molecules represent one of most promising methods for targeted drug delivery. one of major current limitations of magnetic drug targeting is to achieve efficient concentration of magnetic carrier-drug complexes at the targeted sites due to poor mobility of nanoparticles in tissue structures. interstitial delivery is hindered by microscopic extracellular matrix, which represents a major barrier for nanoparticles motilities. in order to achieve efficient magnetic drug targeting it is crucial to know particle mobility in a given in vivo environment as well as to apply magnetic field having appropriate field gradient which drags magnetic nps. we used gel magnetophoresis in order to measure motilities of different magnetic nps (co-ferrite, c-fe o ) in agarose gel. numerical modeling using fem method was used to determine appropriate settings of magnets, which generate sufficient magnetic field gradient. further, we used the numerical modeling to evaluate the magnetic force on the nps for different geometries. we obtained that one of crucial factors which determines final mobility in tissue is formation of larger aggregates of nanoparticles under physiological conditions and interaction of nanoparticles with surrounding matrix. defining the forces required to gate mechanosensitive channels in mammalian sensory neurons kate poole and gary lewin department of neuroscience, max delbrueck center for molecular medicine, robert-roessle str , , berlin-buch, germany our sense of touch and mechanical pain is based on mechano-electrical transduction (met) at the terminal endings of subsets of dorsal root ganglion (drg) neurons innervating the skin. to quantify the stimulus strengths required to gate mechanosensitive channels in these subsets of neurons, we developed an approach using microstructured surfaces. the drg neurons are grown on laminin-coated pdms pillar arrays, mechanical stimuli are applied by deflecting individual pili and the deflection is monitored using light microscopy. as the pili behave as light-guides, the center of each pilus can be determined from a fit of the intensity values, allowing detection of movements of a few nanometers. the response to such stimuli is monitored using whole-cell patch-clamp. pili deflections of nm can gate the rapidly adapting-current in mechanoreceptor cells, while deflections above nm are required for gating of slowly adapting-currents in nociceptors. smaller stimuli are required to generate currents via pili deflection ( nm) vs neurite indentation ( - nm), suggesting that gating occurs at the cell-substrate interface. we have also characterized the met currents present in n a cells which we show are modulated by the substrate to which the cells are attached. enhanced stimulation of toll-like receptor via immunostimulatory nanoparticles jan rother, anna pietuch, andreas janshoff georg-august university gö ttingen, institute of physical chemistry, tammanstr. , d- gö ttingen, germany e-mail: jrother@gwdg.de among the toll-like receptor family (tlrs), the tlr has been subject of intensive research because of its predominant localization in the lysosomes of immune cells and its ligand rendering it a potential candidate for immunotherapy of autoimmune diseases and cancer. additionally, a use as an adjuvant in vaccination is aimed using synthetic cpg-oligodeoxynucleotides (cpg-odn's). albeit, immunostimulatory cpg-odns already showed promising results in animal experiments and clinical trials, several groups found that tlr is also expressed by tumor cells. first experiments show that activation of tlr displayed by cancerous cells leads to a decreased apoptosis rate and proliferation posing unpredictable threat to tumor patients exposed to cpg-odns. therefore, detailed knowledge about the impact of cpg-odns on cancer cells is inevitable for a save use in pharmaceutics. herein, we describe a sophisticated way to address tlr in cancer cells using cpg-odn functionalized ''superparamagnetic'' mno-and c-fe o -nanoparticles (nps) to stimulate tlr in a cells. analysis of impedimetric measurements revealed a cytotoxic effect of the mno-nps. cells treated with immunostimulatory fe o -nps showed an increased micromotility as well as a higher long-term correleation of the impedance signal. biomedical diagnostics like high-sensitivity, single-molecule study, easy sample preparation. furthermore, sers allows to conduct non-invasive studies of conformations of the molecules without destruction of living cells, i.e. in vivo [ ] . this work presents a sers study of cytosolic hemoglobin (hb c ) using silver nanoparticles (agnps). the hb c was isolated from cytoplasm of red blood cells taken from rat erythrocytes and diluted. agnps were prepared by developing leopold and lendl method [ ] . three types of colloids were prepared at various temperatures ( , and °c). the resulted agnps were characterized by uv-vis-, ftirspectroscopy, dls and tem. reduction of ag ions leads to the formation of predominantly spherical agnps but also silver nanorods, faceted and aggregated agnps in small quantities with a surface plasmon resonance band in the range of - nm. for agnps synthesized at °c, for example, a bimodal size distribution was observed (about and nm medium sizes, respectively). sers measurements were optimized for each type of agnps. it was demonstrated that agnps gave strong raman enhancement from hb c and types of sers spectra differ from each other. nano-zno is characterized by unique properties, low toxicity and high biocompatibility instead of a lot of others nanomaterials. for this fact nanoparticles of zno have great potential for applications in biosystems, for example biolabeling, biosensoring, delivery systems and others, which can be used in genetics, pathology, criminology, safety of food and many other industries. for these bioapplications are necessary surface modifications, which can made to the nanostructures to better suit their integration with biological systems, leading to such interesting properties as enhanced aqueous solubility, bio-recognition or applicability for biological systems. for synthesis of zno nanoparticles in aqueous solution we used -mercapto-undecanoic acid (mua) as stabilizing agent. the coating of nanoparticles with mua could allow their solubility in the water and the binding through carboxyl groups present in its structure. we defined the optimal ph for mua modificated nano-zno solubility and their ability interaction with positive charges. we studied the optical properties of pure and surface modificated nanoparticles and their conjugates with cytochrome c and also the effect of ph on the interaction between nano-mua and horse cytochrome c. the permeation of water soluble molecules across cell membranes is controlled by channel forming proteins and particularly the channel surface determines the selectivity. an adequate method to study properties of these channels is electrophysiology and in particular analyzing the ion current fluctuation in the presence of permeating solutes provides information on possible interactions with the channel surface. as the binding of antibiotic molecules in the channels of interest is significantly weaker than that of preferentially diffusing nutrients in substrate-specific pores, the resolution of conductance measurements has to be significantly increased to be able to resolve the events in all cases. due to the limited time resolution, fast permeation events are not visible. here we demonstrate that miniaturization of the lipid bilayer; varying the temperature or changing the solvent may enhance the resolution. although electrophysiology is considered as a single molecule technique, it does not provide atomic resolution. molecular details of solute permeation can be revealed by combining electrophysiology and all atom computer modeling. [ novel functionalized nanocomposites (nc) were designed and synthesized on the basis of polymeric surface-active oligoelectrolytes. the developed technology permits controlling: ) quality and quantity of structural blocks of nc, and size unimodality; ) branching at specific sites in polymer chain of nc; ) providing nc with reactive chemical groups; ) covalent conjugation of specific bio-targeting molecules. provided bioactive elements were: a) specific anticancer drugs, antibiotics, alkaloids; b) dna and sirna; c) immunoglobulins and lectins; d) lipids and amino acids; e) polyethylene glycol. fluorescent, luminescent, super-paramagnetic, or x-ray detectable compounds were also incorporated in nc to make them detectable and measurable. biocompatible nc possessing low toxicity towards mammalian cells in vitro and in vivo (mice) were created. they were effective in delivery of: ) drugs (doxorubicine and antibiotics) for chemotherapy in vitro and in vivo; ) dna for transfection of mammalian, yeast and bacterial cells; ) protein antigens for animal immunization and specific lectins for targeting apoptotic cells. these and other approaches in application of developed nc and nanobiotechnologies are considered. this work was supported by stcu grants # , # , # . in the anti-cancer drug delivery domain, nanotechnologies are a promising tool, providing a good tissue distribution and a low toxicity. drug delivery vehicles relying on solid nanoparticles have been proposed, among which diamond nanoparticle (size\ nm) is a very promising candidate [ ] . we have investigated the delivery of sirna by nanodiamonds (nd) into cells in culture, in the context of the treatment of a rare child bone cancer (ewing sarcoma), by such a gene therapy. sirna was bound to nds after nds coating by cationic polymers, so that the interaction is strong enough to pass the cell membrane without loss of the drug and does not prevent its subsequent release. the cellular studies showed a specific inhibition of the gene expression at the mrna and protein level by the nd vectorized sirna. we also uses the fluorescence of color center created in the nanodiamonds [ ] to monitor the release of fluorescently-labeled sirna in the intracellular medium. this technique brings a quantitative insight in the efficiency of sirna to stop cell proliferation. considering the success of the cell model we recently started the drug delivery in tumor xenografted on nude mice. silica nanoparticles are stable aqueous suspension of condensed siloxane nanocomposites, having an average diameter between and nm. particles containing organic functional groups on their surface are called organically modified silica nanoparticles (ormosil). due to the various chemical and physical properties of the surface groups, ormosil nanoparticles may have an enormous variety of biological applications, such as in vivo bioimaging, non-viral gene delivery or targeted drug delivery. our aim was to synthesize both void and fluorescent dye doped amino functionalized ormosil nanoparticles through the microemulsion method and use them for gene delivery. the obtained nanoparticles have been characterized by transmission electron microscopy and dynamic light scattering. furthermore, the nanoparticles have been investigated to exploit their transfection efficiency and the possible toxicity caused by surfactants used in the synthesis. the transfection efficiency was tested on various cell cultures. our further aim is the in vivo transfection of salivary glands using ormosil nanoparticles. our work has shown that the nanomedicine approach, with nanoparticles acting as a dna-delivery tool is a promising direction for targeted gene therapy. in vivo amperoetric cells for detection of fast diffusing, physiologically important small molecules lívia nagy, bernadett bareith, tü nde angyal, erika pinté r, gé za nagy university of pé cs, pé cs, hungary h s is a naturally occurring gas that is toxic in high concentration. it exists also in different tissues of living animals sometimes in concentrations as high as lm. it is generally accepted, that h s has important roles in modulating different, physiologically important biochemical processes similarly to other, fast diffusing molecules like no, co and h o . for investigation of the physiological effects of these species their local concentration in the studied biological media is important to know. this means methods needed for measuring the instantaneous concentration with high spatial resolution in living tissues without major invasion. electrometric micro, and ultramicro sensors are often gain application in experimental life sciences for measurement of local ion concentration or following neurotransmitter species in vivo measurements. in our work efforts are being carried out to improve the applicability of selective electrometric sensors in life science experiments. as a result of these work an improved h s measuring cell and improved electrode and method was developed for measurement of electroactive small molecules like no or h o . in the poster to be presented the structure, the working principles and the performances of the different sensors mentioned will be described. bacteriorhodopsin (br) is the only protein in the purple membrane of the halophilic organism halobacterium salinarium. it is a light-driven proton pump converting light into a transmembrane proton gradient through isomerization of the covently bound retinal chromophore. its stability, as well as its photoactivity in dried films, has made br an attractive material for biomolecular devices. such studies, however, have used br within the membrane, on relatively large surfaces. here, conducting-probe atomic force microscopy (c-afm) analysis was performed after isolating the protein from its native membrane environment while keeping its basic trimeric structure, and demonstrated that the molecular conductance of br can be reversibly photoswitched with predictable wavelength sensitivity. intimate and robust coupling to gold electrodes was achieved by using a strategically engineered cysteine mutant located on the intracellular side of the protein which, combined with a % delipidation, generated protein trimers homogenously orientated on the surface. c-afm proximal probe analysis showed a reproducible fold drop of br mean resistance over * cycles of interspersed illuminations at the same gold-br-gold junction when k[ nm, while no shift was observed with other wavelengths. capture of circulating tumor cells with a highly efficient nanostructured silicon substrates with integrated chaotic micromixers shutao wang ). this core technology shows significantly improved sensitivity in detecting rare ctcs from whole blood, thus provides an alternative for monitoring cancer progression. by assembling a capture-agent-coated nanostructured substrate with a microfluidic chaotic mixer, this integrated microchip can be applied to isolate ctcs from whole blood with superb efficiency. ultimately, the application of this approach will open up opportunities for early detection of cancer metastasis and for isolation of rare populations of cells that cannot feasibly be done using existing technologies. this technology helped to find a needle in a haystack and will open up the opportunity for single cell genomic and epigenetic sequencing and gene expression profiling. results from further development of this technology will assist the physicians in follow-up patients and testing vigorously the concept of personalized oncology with individualized therapy. this novel technology has recently been reviewed and highlighted by nature medicine the growing crisis in organ transplantation and the aging population have driven a search for new and alternative therapies by using advanced bioengineering methods. the formation of organized and functional tissues is a very complex task: the cellular environment requires suitable physiological conditions that, presently, can be achieved and maintained by using properly-designed bioreactors reproducing all specific functions and bioactive factors that assure viability/regeneration of cells cultured in an appropriate scaffold. the creation of biomimetic environment requires the use of biomaterials such as membranes with specific physico-chemical, morphological and transport properties on the basis of the targeted tissue or organ. tailor-made membranes (organic, functionalized with specific biomolecules, in hollow-fiber configuration), designed and operated according to well-defined engineering criteria are able to sustain specific biotransformations, to provide adequate transport of oxygen, nutrients and catabolites throughout the cellular compartment, and to supply appropriate biomechanical stimuli of the developing tissue. in this talk the author will show the development of membrane engineered constructs focusing on liver and neuronal systems. the role of membrane surface and transport properties in providing instructive signals to the cells for the guiding of proliferation and differentiation will be discussed. membrane bioreactors, which through the fluid dynamics modulation may simulate the in vivo complex physiological environment ensuring an adequate mass transfer of nutrients and metabolites and the molecular and mechanical regulatory signals, will be presented. here we present a novel but simple system for cell-based assays enabling simultaneous testing of multiple samples on a same tissue without cross-contamination between neighbouring assays, as well as sequenced or repeated assays at the same tissue location. the principle of this method lies in the spatially-controlled diffusion of test compounds through a porous matrix to the target cells. a simple microfabrication technology was used to define areas where diffusion processes are allowed or inhibited. we performed proof-of-principle experiments on madin-darby canine kidney (mdck) epithelial cells using hoechst nuclear staining and calcein-am cell viability assay. fluorescent staining superimposed properly on membrane pattern with a dose-dependent response, indicating that both compounds specifically and selectively diffused to the target cells. mdck cells similarly treated with cytochalasin b showed their actin network rapidly altered, thus demonstrating the suitability of this system for drug screening applications. such a well-less cell-based screening system enabling multiple compounds testing on a same tissue and requiring very small volumes of test samples appears interesting for studying potential combined effects of different biochemicals applied separately or sequentially. it is generally believed that all-optical data processing is the most promising direction to achieve serious improvements both in capacity and speed of internet data traffic. one of the bottlenecks of the state-of-the-art photonic integration technology is to find the proper nonlinear optical (nlo) materials that are supposed to serve as cladding media in waveguidebased integrated optical circuits performing light-controlled active functions. recently, the unique chromoprotein bacteriorhodopsin (br) has been proposed to be used as an active, programmable nlo material in all-optical integrated circuits. in integrated optical applications of br, its light-induced refractive index change is utilized. in this paper we exploit the refractive index changes of a dried br film accompanying the ultrafast transitions to intermediates i and k, which allows even sub-ps switching, leading beyond tbit/s communication rate. in the experiments direct pulses of a femtosecond laser system at nm were used along with synchronized ultrafast laser pulses at nm. we believe that the results may be the basis for the future realization of a protein-based integrated optical device, and represent the first steps to a conceptual paradigm change in optical communication technologies. last years, such autoantibodies attract an increasing attention of researchers as potential cancer biomarkers. since the sera of cancer patients typically contain a unique set of antibodies that reflect the tumor-associated antigens expressed in a particular malignant tissue, diagnosing and predicting the outcome of disease such as breast cancer based on serum autoantibody profiling is an attractive concept. to create a representative panel of antigens for detecting of breast cancer autoantibody profile we selected breast cancer associated antigens. these antigens were identified by screening of tumor cdna libraries with autologous sera using serex (serological investigation of recombinantly expressed clones) approach. all antigens were cloned, expressed, purified in bacteria and tested with sera of breast cancer patients and healthy donors in large-scale allogenic screening using elisa. the utility of selected tumor associated antigens for detecting of autoantibody profile in different types of breast cancer was evaluated. (controlled by architectural software) is carried out according to a design template, consistent with the geometry and composition of the desired organ module. structure formation occurs by the post-printing fusion of the discrete bio-ink units. when the bio-ink units contain more than one cell type, fusion is accompanied by sorting of the cells into the physiologically relevant pattern. thus structure formation takes place through self-assembly processes akin to those utilized in early embryonic morphogenesis. we demonstrate the technology by detailing the construction of vascular and nerve grafts. spherical and cylindrical bio-ink units have been employed to build fully biological linear and branching vascular tubular conduits and multiluminal nerve grafts. upon perfusion in a bioreactor the constructs achieved desirable biomechanical and biochemical properties that allowed implantation into animal models. our results show that the printing of conveniently prepared cellular units is feasible and may represent a promising tissue and organ engineering technology. femtosecond lasers have become important tools for noncontact microprocessing of biological specimens. due to the short pulse length and intensity-dependent nature of the multiphoton ionization process, fs-laser pulses affect only a small volume of a treated cell, providing a high degree of spatial localization. we employed fs-laser to address topical bioengineering and biomedical problems such as cell fusion and embryo biopsy respectively. a tightly focused laser beam (cr:f seed oscillator and a regenerative amplifier, nm, fs, hz) was used for a fusion of blastomeres of two-cell mouse embryos and for a polar body (pb) biopsy. in order to fuse blastomeres the contact border of cells was perforated by a single laser pulse. the fusion process usually completed within * min. in order to perform a noncontact laser based pb biopsy we initially drilled an opening in the zona pellucida with a set of laser pulses, and then extracted the pb out of zygote by means of optical tweezer (cw laser, nm). the energy of laser pulses was thoroughly optimized to prevent cell damage and increase the fusion and biopsy rates. the proposed techniques demonstrate high efficiency and selectivity and show a great potential for using fs lasers as a microsurgical tool. new insights into mechanisms of electric field mediated gene delivery maš a kanduš er and mojca pavlin university of ljubljana, faculty of electrical engineering, si- ljubljana, slovenia gene electrotransfer is widely used for transfer of genetic material in biological cells by local application of electric pulses and is currently the most promising non-viral delivery method for gene therapy for a series of diseases as well as for dna vaccination. current description of the process defines several steps: electropermeabilization, dna-membrane interaction, translocation, trafficking to nucleus and into nucleus. but the mechanisms of electrotransfer are still not fully understood. we present results of the systematic in vitro analysis using pegfp of all steps involved in electrotransfection from electropermeabilization, analysis of different pulsing protocols, theoretical analysis of plasmid mobility to visualization of the processes of dna-membrane interaction. we demonstrate that in order to translate in vitro results to tissue level sub-optimal plasmid concentrations have to be used. furthermore, sofar the method of dna entry into cytoplasm was only speculated. our results suggest that it is crucial that first, membrane is electropermeabilized, then sufficient electrophoretic force is crucial for insertion of dna into destabilized lipid bilayer followed by dna translocation into cytoplasm via a slow process. efficiency of electrotransfer depends also on the stage of of cell culture -cells in dividing phase are easer to electrotransfect. gentamicin interaction with b f cell membrane studied by dielectrophoresis dielectrophoresis (dep) is the translational motion of polarizable particles due to an electric field gradient. positive-dep and negative-dep correspond to particle movement forward or backward the region of high field intensity, respectively. our study reveals some of the cell membrane modifications induced by gentamicin (gt), as they are reflected in the crossover frequency f co of b f murine cells incubated with gt for different concentrations and durations. f co is the ac frequency when cells turn from positive-dep to negative-dep. gentamicin is a positively charged aminoglycosidic antibiotic, with concentration-dependent killing action; it is widely used because of its low cost and reliable bactericidal activity. gt drawbacks consist in high toxicity for renal and hearing cells; the molecular mechanisms of this toxicity are still unclear. for low external medium conductivities (& . s/m), f co of control and gt-cells was found to range from to khz. f co shifts to higher frequencies with the increase of gt concentration and incubation time. cells dielectrophoretic behavior is discussed using the cell singleshell based model. extracellular matrix (ecm) is a major obstacle for succesful delivery of genes. chitosan is a versatile and biocompatible polysaccharide derived from chitin and is a promising gene carrier. chitosan-dna interactions, and hence dna polyplexation and release can be controlled through chitosan de-acetylation degree, molecular weight and functionalization of chitosan cationic groups. grafting of poly(ethylene glycol) peg to gene delivery vectors increases circulation time of gene delivery systems in blood vessels and reduces polyplexes charge. diffusion and unpacking of pegylated and non-pegylated chitosan-dna polyplexes through articial ecms based on collagen and collagen-hyaluronic acid (ha) gels were compared using fluorescence correlation microscopy, confocal microscopy and colocalization analysis. non-pegylated polyplexes were immobilized in the gels whereas pegylated polyplexes were diffusing. the smaller charge of pegylated polyplexes seems to decrease interactions between polyplexes and ecm components. furthermore, ha might also screen collagen fibers-pegylated polyplexes interactions. pegylated polyplexes also showed a higher degree of unpacking in gels, probably due to a looser compaction of dna by pegylated chitosan compared to non-pegylated chitosan. fabrication of vesicles, a close membrane made of an amphiphile bilayer, has great potentiality for encapsulation and controlled release in chemical, food or biomedical industries but also from a more fundamental point of view for the design of biomimetic objects. methods based on lipid film hydratation , inverse emulsion techniques and more recently microfluidic techniques such as double emulsion or jetting method are limited either by a low yield, a low reproducibility, a poor control on the size, or by the presence of remaining solvent or defects. we propose a fast and robust method easy to implement: continuous droplet interface crossing encapsulation (cdice), that allows the production of defect-free vesicles at high-yield with a control in size and content. the vesicles have controlled bilayer composition with a polydispersity in size lower than %. we have shown that solutions as diverse as actin, cells, micrometric colloids, protein and high ionic strength solutions can easily be encapsulated using this process. by adjusting the parameters of our set-up, we are able to produce vesicles in the range - lm in diameter, stable for weeks. we believe this method open new perspectives for the design of biomimetic systems and even artificial tissues. under appropriate conditions. the extremely variable d domain of flagellin subunits, comprising residues - , protrudes at the outer surface of flagellar filaments. the d domain has no significant role in the construction of the filament structure. thus, replacement of d may offer a promising approach for insertion of heterologous proteins or domains without disturbing the self-assembly of flagellin subunits. our work aims at the construction of flagellinbased fusion proteins which preserve the polymerization ability of flagellin and maintain the functional properties of the fusion partner as well. in this work a fusion construct of flagellin and the superfolder mutant of green fluorescent protein (gfp) was created. the obtained gfp variant was highly fluorescent and capable of forming filamentous assemblies. our results imply that other proteins (enzymes, binding domains etc.) can also be endowed by polymerization ability in a similar way. this approach opens up the way for construction of multifunctional filamentous nanostructures. generation polyamidoamine (pamam) dendrimer has been shown to be highly efficient nonviral carriers in gene delivery. however, their toxicity limits their applications. in this study, to improve their characteristics as gene delivery carriers, g pamam dendrimer was modified with anti-tag nanobody through hetrobifunctional peg, then complexed with t-bid coding pdna, yielding pamam-peg-anti-tag nanobody/pdna nanoparticles (nps). nuclear magnetic resonance (nmr) spectroscopy, zeta sizing and gel retardation assay results provided evidence that the nanovector was successfully constructed. the transfection efficiency of vector/pdna complexes were evaluated in vitro. real time pcr results also demonstrated that anti-tag nanobody modified nps are more efficient in t-bid killer gene expressing in colon cancer cell line than the unmodified nps. in conclusion, pamam-peg-anti-tag nanobody showed great potential to be applied in designing tumour-targeting gene delivery system. dept of chemistry, faculty of science, national university of singapore, singapore, dept of biochemistry, yong loo lin school of medicine, national university of singapore, singapore, division of bioengineering, faculty of engineering, national university of singapore, singapore macromolecular crowding (mmc) is a biophysical tool which has been used extensively to enhance chemical reactions and biological processes by means of the excluded volume effect (eve). the in vivo stem cell microenvironment contains macromolecules which are crucial for stem cell selfrenewal and cell fate determination. in order to mimic this physiological microenvironment, crowders are included in cell culture medium. we have observed that the ex vivo differentiation of human mesenchymal stem cells (hmscs) into the adipogenic lineage is significantly amplified when a crowder mixture comprising ficoll and ficoll is added to the culture medium. stem cell differentiation is modulated by soluble chemical substances as well as interactions between cells and the extracellular matrix (ecm), and both these external influences may be affected by mmc. measurements we have performed by fluorescence correlation spectroscopy (fcs) show that ficoll additives cause anomalous subdiffusion within a crowder concentration range of to mg/ml. the diffusion of fluorophorelabelled molecules in artificial lipid bilayers and membranes of living cells is not changed by crowders, suggesting that these crowders do not directly alter membrane properties and cell surface signalling. however, we have data to suggest that crowders increase actin polymerization reaction rates in vitro. we have also observed that crowders are taken up by stem cells and that they localize to specific compartments. based upon our observations, we hypothesize that crowders can influence stem cell differentiation by influencing molecular kinetics. lignocellulose-based composites are becoming extremely important and perspective sustainable and renewable natural materials. fibre modification enhancing their existing properties can be obtained to broaden the application areas. in response to shortcomings of traditional chemical and physical methods, enzymes and chemo-enzymatic methods have emerged as eco-friendly catalysts working under mild conditions and enable tailoring of the material surface properties by substrate specificity and regional selectivity. recently, binding of different functional molecules to lignin-rich fibres by using an oxidative enzyme (e.g. laccase) has been reported leading to their functionalisation through free radical reactions. by the application of electron paramagnetic resonance spectroscopy (epr) laccase action was inspected. consumption of substrates was investigated and their polymerization traced. stable radical intermediates were detected with epr when substrate molecules were in contact with active enzymes. secondly, oxidation of mediators like nitroxides was determined via epr spectroscopy of stable water-soluble nitroxide radicals. finally, the generation of short-lived radicals as well as their reduction was measured via epr spin trapping using dmpo as sensitive water soluble spin trap. mammalian ovary hormone stimulation (ohs) is known to be an inalienable stage of reproductive biotechnology as well as human infertility treatment. the basic aim of the ohs is to receive a stock of valuable oocytes and early embryos for subsequent utilization in the reproductive technology, experimental work et al. however, it is known that ohs itself affects the character of ovulation and oocyte quality, which in its turn affects the development of embryos and even has distant consequences. the wideness of cell parameters and appropriate methods for investigation of gamete/embryo quality are very important. the aim of this study is determination of specific electric conductivity of mouse oocytes and early embryos which have been received after ohs in comparison with the ones that have been received in natural animal sex cycle. using techniques of electroporation the dependence of specific electric conductivity of mouse oocytes, zygotes, -cell and -cell embryos on the external electric field intensity has been studied. it is shown that the whole pool of oocytes that were obtained in the result of ohs consists of two groups of oocytes that don't differ from each other morphologically, but differ by their electric parameters and resistance to electric breakdown. at the zygote stage, dividing of embryos into two groups is preserved, but is less expressed. at the stage of -cell and -cell dividing of embryos into two groups on their electric conductivity disappeared but certain scattering of the parameters due to individual embryo peculiarities is observed. the obtained data show that ohs may lead to latent changes of oocyte state that in their turn affect embryo quality. many microbes synthesize and accumulate granules of polyhydroxyalkanoates (pha, biodegradable storage materials alternative to traditional plastics), which help them survive under stresses. in particular, the plant-growth-promoting rhizobacterium azospirillum brasilense, that is under investigation worldwide owing to its agricultural and biotechnological significance, can produce poly- hydroxybutyrate (phb) [ ] . in our work, phb synthesis in a. brasilense cells was studied under various stresses using diffuse reflectance ftir spectroscopy. phb in cells was determined from the band intensity ratio of the polyester m(c=o) at * cm - to that of cell proteins (amide ii band at * cm - ), showing a. brasilense to be able to produce phb up to over % of cells' dry weight. stresses induced phb accumulation, enhancing ir absorption in phb specific regions. analysis of a few structure-sensitive phb vibration bands revealed changes in the degree of intracellular phb crystallinity (related to its enzymatic digestion rate) at different stages of bacterial growth, reflecting a novel trait of the bacterial adaptability to an enhancing stress, which is of great importance to agricultural biotechnology. the aim of this work is to furnish enzymes with polymerization ability by creating fusion constructs with the polymerizable protein, flagellin, the main component of bacterial flagellar filaments. the d domain of flagellin, exposed on the surface of flagellar filaments, is formed by the hypervariable central portion of the polypeptide chain. d is not essential for filament formation. the concept in this project is to replace the d domain with suitable monomeric enzymes without adversely affecting polymerization ability, and to assemble these chimeric flagellins into tubular nanostructures. to test the feasibility of this approach, xylanase a (xyna) from b. subtilis was chosen as a model enzyme for insertion. with the help of genetic engineering, a fusion construct was created in which the d domain was replaced by xyna. the flic(xyna) chimera exhibited catalytic activity as well as polymerization ability. these results demonstrate that polymerization ability can be introduced into various proteins, and building blocks for rationally designed assembly of filamentous nanostructures can be created ( table ) . the support of the hungarian national office for research and technology and the hungarian scientific research fund (otka) (grants ck , nk , nanoflag) is acknowledged. cluster phases of membrane proteins an alternative scenario for the formation of specialized protein nano-domains (cluster phases) in biomembranes dna fragmentation induced in human fibroblasts by accelerated fe ions of differing energies swift heavy ion irradiation of srtio under grazing incidence'' proc. natl. acad. sci. usa forespore engulfment mediated by a ratchet-like mechanism a channel connecting the mother cell and forespore during bacterial endospore formation a feeding tube model for activation of a cell-specific transcription factor during sporulation in bacillus subtilis the scanning ion-conductance microscope imaging proteins in membranes of living cells by high-resolution scanning ion conductance microscopy nanoscale live-cell imaging using hopping probe ion conductance microscopy beta -adrenergic receptor redistribution in heart failure changes camp compartmentation simultaneous noncontact topography and electrochemical imaging by secm/sicm featuring ion current feedback regulation timasheff in protein-solvent interactions the roles of water in foods on motor and electrical oscillations in urinary tract: computer evaluation daniele martin , , viktor foltin , , erich gornik , rumen stainov , , tanya zlateva nü rnberg. icsd e.v. postfach (pob) , d- mü nchen method: parameters: motor patterns (guinea-pig) -frequency/f, amplitudes/a (% init. length, isot. & intracell. rec.) of spontaneous phasic/spc & tonic/stc contractions, also electrical spikes/s, bursts/b, burst plateaus/bp (neu et al. biophys.j. / a/jan stretch ( - mn), k-/ca-influence induced specific changes in motor/electrical parameters. special computer programme reflects exactly biophysical parameters. conclusion: acc. to earlier/recent results mechano-sensitive ca ?? -activated k ? -channels participate in electrical oscillations of detrusor/ureteral myocytes. further experiments/evaluations incl effect of hydrophobic mismatch on the light-induced structural changes in bacterial reaction centers s. s. deshmukh, h. akhavein, and lá szló ká lmá n department of physics mechanism of proton transfer in nitric oxide reductase: computational study andrei pisliakov riken advanced science institute, wako-shi proteins , . acknowledgments this work was supported by project grant ptdc/qui/ / (cas) and doctoral grants sfrh th anniversary conference aicr this work was supported by the italian association for cancer research (airc), the istituto toscano tumori and the associazione noi per voi differential hydration, void volume: which factor provides the main contribution to dv u ? inserm umr fr; roumestand@cbs.cnrs.fr introduction: globalization needs new organizational models also for biophysics. reports on necessity of int. institutes for biophysics (iib) c/o int. universities (proposed by british nobel laureate b.russell) are given conception: proposals for ebsa-discussion: . enlargement of executive committee by a. honorary & presidents (permanent - : moral support & - fixed term), b. interdisciplinary commission: scientists from biology, medicine, physics, etc. (feps/iups, iuphar, iupab, etc). . implication of interdisciplinary topics to esba/iupab congressprogrammes, . also for biophysical journals. . organization of common interdisciplinary sessions not only to biophysical, but also to other congresses. . co-operation between esba/iupab with int. interdisciplinary organisations (waas, icsd/ias, eur. academies) for creation of iib by network of national ones: successive common personnel, possibility for whole life work, etc. conclusion: realization of proposals .- . could increase scientific/political authority of ebsa/iupab, leading to model for renewal of scientific organizations collective migration of neural crest cells: a balance between repulsion and attraction roberto mayor university college london goodilin , , olga v single-molecule cut-and-paste surface assembly (smcp) has been also used to build up a biotin scaffold that streptavidin utilizing specific molecular interactions, for example between dna-binding proteins and dna or antibodies and antigens, this technique is capable of providing a scaffold for the controlled self-assembly of functional complexes. furthermore, this allows for the introduction of smcp into protein science. we aim to employ dna-binding zinc-finger variants and gfp-binding nanobodies as shuttle-tags fused to the proteins of interest. thus a fully expressible system that can be used for the step-wise assembly of individual building blocks to form single-molecule cut-and-paste surface assembly optically monitoring the mechanical assembly of single molecules nanoparticle self-assembly on a dna-scaffold written by single-molecule cut-and-paste torsional motion analysis of group ii chaperonin using diffracted x-ray tracking nanomechanical manipulation of mason-pfizer monkey retroviral rna fragment with optical tweezers melinda simon , zsolt má rtonfalvi , pasquale bianco , beá ta vé rtessy , mikló s kellermayer micro-viscosimeter generated and manipulated by light andrá s buzá s , lá szló oroszi , ló rá nd kelemen , pá l ormos temesvá ri krt proc. natl. acad. sci. usa neural signal recordings with a novel multisite silicon probe gergely má rton , anita pongrá cz , lá szló grand , , É va vá zsonyi pé ter pá zmá ny catholic university, faculty of information technology, h- , /a prá ter st multiscale pattern fabrication for life-science applications francesco valle , beatrice chelli , michele bianchi , eva bystrenova , marianna barbalinardo , arian shehu , tobias cramer , mauro murgia , giulia foschi miroslava kuricova , jana tulinska , aurelia liskova , eva neubauerova , maria dusinska , , ladislava wsolova acceleration neuronal precursors differentiation induced by substrate nanotopography gianluca grenci , jelena ban , elisabetta ruaro , massimo tormen , marco lazzarino , and vincent torre light-induced structural changes are reported near the primary electron donor of bacterial reaction centers (brc) dispersed in detergent micelles and in liposomes from lipids with different fatty acid chain lengths. in this study we present evidence for the correlation between the light-induced increase of the local dielectric constant, determined by the analysis of the electrochromic absorption changes, and the lifetime of the charge-separated state at physiologically relevant temperatures. the increase of the local dielectric constant induced a significant decrease of the oxidation potential of the primary electron donor and a slow proton release, which appears to be the rate limiting step in the overall process. systematic selection of the head group charges of detergents and lipids, as well as the thickness of the fatty acid chains of the liposome forming lipids can increase the lifetime of the charge-separated state by up to orders of magnitude. such extensions of the lifetime of the charge-separated state were reported earlier only at cryogenic temperatures and can provide new opportunities to utilize the brc in energy storage. ontogenesis of photosynthetic bacteria tracked by absorption and fluorescence kinetics m. kis, e. asztalos, p. maró ti department of medical physics and informatics, university of szeged, hungarythe development of photosynthetic membrane of rhodobacter sphaeroides was studied by absorption spectroscopy and fast induction of bacteriochlorophyll fluorescence in different phases of the growth, under various growing conditions (oxygen content, light intensity etc.) and in synchronous cell population. the results are: ) the newly synthesized components of the membranes were imbedded immediately into the proteinous scaffold independently on the age of the cell (no ,,transient'' membranes were observed). ) under aerobic conditions, the pigments were bleached and under anaerobic conditions the pigment systems showed greening. the relative variable fluorescence (f v /f max ) had small age-dependent (but not cellcycle-related) changes. the fluorescence induction kinetics was sensitive marker of the aerobiosis: the f v /f max ratio dropped from . to . and the photochemical rate constant from Á s - to Á s - with an apparent halftime of about - hours after change from anaerobic to aerobic atmosphere. ) the electrogenic signal (absorption change at nm) reflected the energetization of the membrane which showed cell-cycle dependent changes. that included periodic production and arrangement of protein-lipid components of the membrane synchronized to the cell division. interfacial water in b-casein molecular surfaces: wide-line nmr, relaxation and dsc characterization t. verebé lyi , m. bokor , p. kamasa , p. tompa , k. tompa research institute for solid state physics and optics, hungarian academy of sciences, pob. , budapest,hungary, institute of enzymology, biological research center, hungarian academy of sciences, pob. , budapest, hungarywide-line proton nmr fid, echoes, spin-lattice and spin-spin relaxation times were measured at . mhz frequency in the - °c to ? °c temperature range, in lyophilized bcasein and aqueous and buffered solutions, and dsc method were also applied. the motivation for the selection of b-casein is the uncertainty of structural order/disorder. naturally, the nmr and thermal characteristics were also evaluated. the melting of hydration water could be detected well below °c and the quantity of mobile water molecules (hydration) was measured. the hydration vs. melting temperature curve has informed us on the bonding character between the protein surfaces and water molecules. the generally used local field fluctuation model and the bpp theory were applied in the interpretation, and the limits of the models were concluded. the torsional properties of dna play an important role in cellular processes such as transcription, replication, and repair. to access these properties, a number of single-molecule techniques such as magnetic tweezers have been developed to apply torque to dna and coil it. i will briefly refer to investigations of dna-protein interactions using these techniques, and describe what has been learnt. i will then focus on the development of novel magnetic techniques that go beyond standard magnetic tweezers, such as the magnetic torque tweezers and the freely-orbiting magnetic tweezers . these approaches allow one to quantify conjugate variables such as twist and torque. for example, the magnetic torque tweezers rely on high-resolution tracking of the position and rotation angle of magnetic particles in a low stiffness angular clamp. we demonstrate the experimental implementation of this technique and the resolution of the angular tracking. subsequently, we employ this technique to measure the torsional stiffness c of both dsdna molecules and reca heteroduplex filaments. lastly, i will describe novel applications of the optical torque wrench , . the optical torque wrench is a laser trapping technique developed at cornell capable of applying and directly measuring torque on microscopic birefringent particles via spin momentum transfer. we have focused on the angular dynamics of the trapped birefringent particle , demonstrating its excitability in the vicinity of a critical point. this links the optical torque wrench to non-linear dynamical systems such as neuronal and cardiovascular tissues, non-linear optics and chemical reactions, which all display an excitable binary ('all-or-none') response to input perturbations. based on this dynamical feature, we devise a conceptually novel sensing technique capable of detecting single perturbation events with high signal-to-noise ratio and continuously adjustable sensitivity.for the first time we report a comparative approach based on surface enhanced raman spectroscopy (sers) and raman spectroscopy to study different types of haemoglobin molecules in living erythrocytes. in erythrocytes there are two fractions of haemoglobin: cytosolic (hb c ) and membranebound (hb m ). the concentration of hb m is less then , % and therefore it is impossible to study hb m with traditional optical techniques. modifications of cellular membrane can affect conformation of hb m . therefore, it can be used as a sensitive marker of pathologies. firstly, we investigated enhancement of sers signal of hb m depending on ag nanoparticles' size. we found that the intensity of sers spectra of hb m and enhancement factor increase with the decrease in ag nanoparticles' size. secondly, we investigated the dependence of haemoporphyrin conformation in both hb c and hb m ion ph values. we observed different sensitivity of hb c and hb m to the ph and found that conformational movements of haemoporphyrin (vibrations of pyrrol rings and side radicals) in hb m are sterically hampered comparably with hb c . our observation is an evidence of a benefit of application of surface enhanced raman spectroscopy to investigate properties of the hb m in erythrocytes and provide new information about conformational changes and functional properties of hb m . rna nanotechnology is an emerging field with high potential for nanomedicine applications. however, the prediction of rna three-dimensional nanostructure assembly is still a challenging task that requires a thorough understanding the rules that govern molecular folding on a rough energy landscape. in this work, we present a comprehensive analysis of the free energy landscape of the human mitochondrial trna lys , which possesses two different folded states in addition to the unfolded one. we have quantitatively analyzed the degree of rna tertiary structure stabilization, firstly, for different types of cations, and, secondly, for several naturally-occurring nucleotide modifications in the structural core of the trna lys . , thus, notable variations in the rna binding specificity was observed for the divalent ions of mg ? , ca ? and mn ? , that can be attributed to their sizes and coordination properties to specific ligands. furthermore, we observed that the presence of m g modification together with the principal stabilizing m a modification facilitates the rna folding into the biologically functional cloverleaf shape to a larger extent than the sum of individual contributions of these modifications. in order to elucidate the mechanism of the recognition, we used diffracted x-ray tracking method (dxt) that monitors real-time movements of individual proteins in solution at the single-molecule level. we found that peptides move distinctly from i-a k , and the rotational motions of peptides correlate with the type b t cell activation. in the case of diabetogenic i-a g , immediately after peptide exchange, all the peptides moves magnificently but the motion ceased in a week, then new ordered motion appears; the rotational motion of peptides correlate to t cell activation, which is analogous to the peptide in i-a k . the rotational motion of peptides may create transient conformation of peptide/mhc that recognized by a population of t cells. dxt measurement of peptide/mhc complex well correlated to other biological phenomenon too. our finding is the first observation that fluctuations at the level of brownian motion affect to the functions of proteins.mason-pfizer monkey virus (mpmv) is an excellent model for the analysis of retrovirus assembly and maturation. however, neither the structure of the viral rna, nor its modulation by capsid-protein binding are exactly known. to explore the structure of the mpmv genome, here we manipulated individual molecules of its packaging signal sequence with optical tweezers. the -base-long segment of mpmv rna corresponding to the packaging signal, extended on each side with -base-long indifferent gene segments for use as molecular handles, was cloned into a pet b vector. rna was synthesized in an in vitro transcription system. rna/ dna handles were obtained by hybridization in a pcr with complementary dna initiated with primers labeled with either digoxigenin or biotin. the complex was manipulated in repetitive stretch and relaxation cycles across a force range of - pn. during stretch, transition occurred which increased the rna chain length and likely corresponds to unfolding. the length gain associated with the unfolding steps distributed across three main peaks at * , , nm, corresponding to * , , bases, respectively. often reverse transitions were observed during mechanical relaxation, indicating that refolding against force proceeds in a quasi-equilibrium process. structural investigation of gpcr transmembrane signaling by use of nanobodies jan steyaert , structural biology brussels, vrije universiteit brussel, pleinlaan , brussel, belgium, department of structural biology, vib, pleinlaan , brussel, belgiumin , scientists at the vrije universiteit brussel discovered the occurence of bona fide antibodies devoid of light chains in camelidae. the small and rigid recombinant antigen binding fragments ( kd) of these heavy chain only antibodies -known as vhhs or nanobodies -proved to be unique research tools in structural biology. by rigidifying flexible regions and obscuring aggregative surfaces, nanobody complexes warrant conformationally uniform samples that are key to protein structure determination by x-ray crystallography:• nanobodies bind cryptic epitopes and lock proteins in unique native conformations • nbs increase the stability of soluble proteins and solubilized membrane proteins • nbs reduce the conformational complexity of soluble proteins and solubilized membrane proteins • nbs increase the polar surface enabling the growth of diffracting crystals • nbs allow to affinity-trap active protein i will focus my talk on the use of nbs for the structural investigation of gpcr transmembrane signaling to illustrate the power of the nanobody platform for generating diffracting quality crystals of the most challenging targets including gpcrs and their complexes with downstream signaling partners. dynamics of the type i interferon receptor assembly in the plasma membrane stephan wilmes, sara lö chte, oliver beutel, changjiang you, christian paolo richter and jacob piehler university of osnabrü ck, division of biophysics, barbarastrasse , osnabrü ck, germanytype i interferons (ifn) are key cytokines in the innate immune response and play a critical role in host defense. all ifns bind to a shared cell surface receptor comprised of two subunits, ifnar and ifnar . detailed structure-function analysis of ifns has established that the ifn-receptor interaction dynamics plays a critical role for signalling specificities.here we have explored the dynamics of receptor diffusion and ifn assembly in living cells. by using highly specific orthogonal posttranslational labelling approaches combined with tirf-microscopy we probed the spatio-temporal dynamics of receptor diffusion and interaction in the plasma membrane of live cells on the single molecule level. for this purpose, we employed posttranslational labelling with photostable organic fluorophores. this allowed us to map diffusion and lateral distribution of ifnar and ifnar with very high spatial and temporal resolution by using single particle tracking (spt) and single molecule localization imaging. observed events of ''transient confinement'' and co-localization with the membrane-proximal actin-meshwork suggest partitioning of ifnar / in specialized microcompartments. this will be investigated in terms influence on receptor assembly and recruitment of cytoplasmic effector proteins. cytoplasmic dynein moves through uncoordinated action of the aaa ring domains ahmet yildiz department of physics, and department of molecular and cell biology, university of california, berkeley, ca usa cytoplasmic dynein is a homodimeric aaa? motor that moves processively toward the microtubule minus end. the mechanism by which the two catalytic head domains interact and move relative to each other remains unresolved. by tracking the positions of both heads at nanometer resolution, we found that the heads remain widely separated and move independently along the microtubule, a mechanism different from that of kinesin and myosin. the direction and size of steps vary as a function of interhead separation. dynein processivity is maintained with only one active head, which drags its inactive partner head forward. these results challenge established views of motor processivity and show that dynein is a unique motor that moves without strictly coordinating the mechanochemical cycles of its two heads.o- self-controlled monofunctionalization of quantum dots and their applicaitons in studying protein-protein interaction in live cells changjiang you, stephan wilmes, sara loechte, oliver beutel, domenik lisse, christian paolo richter, and jacob piehler universitä t osnabrü ck, fachbereich biologie, barbarastrasse , osnabrü ck, germanyindividual proteins labeled with semiconductor nanocrystals (quantum dots, qd) greatly facilitate studying protein-protein interactions with ultrahigh spatial and temporal resolution. multiplex single molecule tracking and imaging require monovalent quantum dots (mvqd) capable of orthogonally labeling proteins with high yield. for this purpose, we prepared monovalance qd-trisnta by a chemical conjugation method. our results indicated that monovalent qd-trisnta was obtained in high yield by restricting the coupling by means of electrostatic repulsion. monovalent functionalization of the qd-trisnta was confirmed by assays in vitro and in vivo. two-color qd tracking of interferon receptors ifnar and ifnar based on mvqd-trisnta were realized on live cell [ ] .to broaden the multiplex toolbox of mvqds, we extended the electrostatic-repulsion induced self-control concept for mono-functionalizing quantum dots with different affinity moieties. as a first instance, we used negatively-charged biotin peptide to produce qd with biotin mono-functionalization. we confirmed our approach was a general method to rend qd monovalent by single molecule assays based on stepwise photobleaching. these mvqds facilitate obtaining spatiotemporal information of ifnars' organization in live cells. by orthogonal labeling u a cells stably expressing ifnar at low level with biotin mvqd and mvqd-trisnta-ifn, we verified colocalization and colocomotion of individual ifn and ifnar at minute scale. combined with super-resolution imaging of ifnars' cytosolic effecter stat , we observed the dynamic coming-and-going contact between the microcompartments of ifnar and stat .a micron sized viscometer was fabricated using the couette type geometry that is capable of measuring the complex viscosity of fluids. the viscometer was produced by two photon polymerization of su photopolymer using a femtosecond laser system, a high na objective and a piezo translator stage. the viscometer was manipulated by holographic optical tweezers and operated in the . - hz frequency range. video analysis algorithm was used to evaluate our measurements. we tested the viscometer with water-glycerol solutions. one of the main reasons for lack of reliability in protein analysis for disease diagnostics or monitoring is a lack of test sensitivity. this is because, for many tests, to be reliable, they need to be performed on a homogeneous, and therefore very small, sample. current in-vitro techniques fail in accurately identifying small differences in protein content, function and interactions starting from samples constituted of few or even single cells. a nanotechnology approach may overcome the current limits in low abundance protein detection. we aim at designing a microwell device for the trapping (in native environment) and the parallel characterization of rare cells (e.g. adult stem cells). such versatile device, based on soft and nanolithography, will promote cell adhesion and viability on differently functionalized bio-compatible materials, allowing for the morphological characterization of the cells, at a single cell level. in parallel, by facing our microwell device with a protein nanoarray, produced via atomic force microscopy nanolithography, we can run proteomic studies at a single/few cells level. moreover, we could foresee the possibility to deliver different stimuli to each cell, correlating the changes in chemistry/ morphology with the protein profile at a single cell level. using an electrophysiological assay the activity of nhaa was tested in a wide ph range from ph . to . . forward and reverse transport directions were investigated at zero membrane potential using preparations with inside out and right side out oriented transporters with na ? or h ? gradients as the driving force. under symmetrical ph conditions with a na ? gradient for activation, both the wt and the ph-shifted g s variant exhibit highly symmetrical transport activity with bell shaped ph dependencies, but the optimal ph was shifted . ph units to the acidic range in the variant. in both strains the ph dependence was associated with a systematic increase of the k m for na ? at acidic ph. under symmetrical na ? concentration with a ph gradient for nhaa activation an unexpected novel characteristic of the antiporter was revealed; rather than being down regulated it remained active even at ph as low as . these data allowed to advance a transport mechanism based on competing na ? and h ? binding to a common transport site and to develop a kinetic model quantitatively explaining the experimental results. in support of these results both alkaline ph and na ? induce the conformational change of nhaa associated with nhaa cation translocation as demonstrated here by trypsin digestion. furthermore, na ? translocation was found to be associated with the displacement of a negative charge. in conclusion, the electrophysiological assay allowed to reveal the mechanism of nhaa antiport and sheds new light on the concept of nhaa ph regulation. swimming motility is widespread among bacteria. however, in confined or structured habitats bacteria often come in contact with solid surfaces which has an effect on the swimming characteristics. we used microfabrication technology to quantitatively study the interaction of swimming cells with solid boundaries. we tracked bacteria near surfaces with various engineered topologies, including flat and curved shapes. we were able to study several surface related phenomena such as hydrodynamic trapping and correlated motion. we think that our results may help to understand how physical effects play a role in surface related biological processes involving bacteria such as biofilm formation. cell labeling efficiency of oppositely charged magnetic iron oxide nanoparticles-a comparative study raimo hartmann , christoph schweiger , feng zhang , wolfgang. j. parak , thomas kissel ,# , pilar rivera_gil ,# biophotonics, institute of physics, philipps university of marburg, pharmaceutical technology, institute of pharmacy, philipps university of marburg e-mail: kissel@staff.uni-marburg.de; pilar.riveragil@physik.uni-marburg.dethe interaction of nanomaterials with cells is a key factor when considering their translocation into clinical applications. especially an effective accumulation of nanoparticles inside certain tissues is beneficial for a great number of applications. predominantly size, shape and surface charge of nanoparticles influence their cellular internalization and distribution. to investigate this, two series of maghemite (c-fe o ) nanoparticles were synthesized either via aqueous coprecipitation or via thermal decomposition of organometallic precursor molecules. size and the spherical shape of both nanoparticle types were kept constant whereas the charge was changed by modifying the surface of the nanoparticles with polymers of opposite charge, in detail poly(ethylene imine) (pei) and a polymaleic anhydride derivative (pma). the positively and negatively charged c-fe o nanoparticles were characterized with respect to size, zeta potential, colloidal stability and magnetic properties. furthermore, the uptake rate and localization of both formulations into a carcinoma cells after fluorescent labeling of the carriers as well as the resulting alteration in mr-relaxation times were evaluated. membrane proteins are the target of more than % of all drugs and are encoded by about % of the human genome. electrophysiological techniques, like patch-clamp, unravelled many functional aspects of membrane proteins but usually suffer from poor structural sensitivity. we have developed surface enhanced infrared difference absorption spectroscopy (seidas) , to probe potential-induced structural changes of a protein on the level of a monolayer. a novel concept is introduced to incorporate membrane proteins into solid supported lipid bilayers in an orientated manner via the affinity of the his-tag to the ni-nta terminated gold surface . full functionality of surface-tethered cytochrome c oxidase is demonstrated by cyclic voltammetry after binding of the natural electron donor cytochrome c. general applicability of the methodological approach is shown by tethering photosystem ii to the gold surface . in conjunction with hydrogenase, the basis is set towards a biomimetic system for h -production. recently, we succeeded to record ir difference spectra of a monolayer of sensory rhodopsin ii under voltage-clamp conditions . this approach opens an avenue towards mechanistic studies of voltage-gated ion channels with unprecedented structural and temporal sensitivity. initial vibrational studies on the novel light-gated channelrhodopsin- will be presented . probing biomass-chromatographic bead interactions by afm force spectroscopy gesa helms, marcelo ferná ndez-lahore, rami reddy vennapusa, and jü rgen fritz school of engineering and science, jacobs university bremen, bremen, germany e-mail: g.helms@jacobs-university.dein expanded bed adsorption (eba), bioproducts are purified from an unclarified fermentation broth by their adsorption on chromatographic beads in a fluidized bed. the unspecific deposition of biomass onto the adsorbent matrix can severely affect the process performance, leading to a poor system hydrodynamics which then decreases the success of this unit operation. to quantify the bead-biomass interactions different chromatographic beads are attached to afm cantilevers, and force spectroscopy experiments are performed with these colloidal probes on model surfaces and cells in solution. the experiments are conducted under varying conditions to study uncovering physiological processes at the cellular level is essential in order to study complex brain mechanisms. using multisite signal recording techniques in the extracellular space, functional connectivity between different brain areas can be revealed. a novel microfabrication process flow, based on the combination of wet chemical etching methods was developed, which yields highly reproducible and mechanically robust silicon-based multielectrode devices. the fabricated shaft of the probe is lm wide, lm thick, has rounded edges and ends in a yacht-bow like, sharp tip. its unique shape provides decreased invasivity. the sensor contains platinum recording sites at precisely defined locations. murine in vivo experiments showed that the probes could easily penetrate the meninges. high quality signals, providing local field potential, multi-and single unit activities, were recorded. the interfaces between the tissue and the platinum contacts were further improved by electrochemical etching and carbon nanotube coating of the metal sites. the integrated optical mach-zehnder interferometer is a highly sensitive device, considered a powerful lab-on-a-chip tool for specific detection of various chemical and biochemical reactions. despite its advantages, there is no commercially available biosensor based on this technique. the main reason is the inherent instability of the device due to slight changes of environmental parameters. in this paper we offer a solution to this problem that enables the optimal adjustment of the working point of the sensor prior to the measurement. the key feature is a control unit made of a thin film of the lightsensitive chromoprotein bacteriorhodopsin deposited on the reference arm of the interferometer. after showing the transfer characteristics of such a device, we demonstrate its applicability to sensing of specific protein-protein interactions. we expect our method to become a rapid and cost-efficientthe combination of unconventional fabrication technology and biomaterials allows both to realize state-of-the-art devices with highly controlled lateral features and performances and to study the main properties of the biomolecules themselves by operating at a scale level comparable with the one crucial for their activity. soft lithography and microfluidic devices offer a tool-box both to study biomolecules under highly confined environments [ ] and to fabricate in an easy way topographic features with locally controlled mechanical and chemical surface properties, thus leading to a finer control of the interplay of mechanics and chemistry. i will present an application of this technology to the control of cell fate that is becoming a key issue in regenerative medicine in the perspective of generating novel artificial tissues. patterns of extracellular matrix (ecm) proteins have been fabricated, by a modified lithographically controlled wetting (lcw), on the highly antifouling surface of teflon-af to guide the adhesion, growth and differentiation of neural cells (shsy y, n , ne- c) achieving an extremely accurate guidance [ ] . local surface topography is also known to influence the cell fate [ ] , thus, integrating this parameter in the substrate fabrication could increase the complexity of the signals supplied to the cells. in this perspective we have developed a novel fabrication technique, named lithographically controlled etching (lce), allowing, in one step, to engrave and to functionalize the substrate surface over different lengthscales and with different functionalities. i will conclude showing how we have been developing ultrathin film organic field effect transistors (ofets) as label-free biological transducers and sensors of biological systems. ofets are low dimensional devices where ordered conjugated molecules act as charge transport material. unconventional patterning techniques and microfluidics have been adapted to proteins and nucleic acids to dose the molecules on the ofet channel with a high control of the concentration. in another set of experiments, we have also been addressing the signalling from neural cells and networks grown on pentacene ultra-thin film transistosr [ , ] .advances in nanotechnology are beginning to exert a significant impact in medicine. increasing use of nanomaterials in treatment of diseases has raised concerns about their potential risks to human health. in our study, the effect of poly(lactic-co-glycolic acid) (plga) and titanium dioxide (tio ) nanoparticles (nps) on function of b-and t-lymphocytes was investigated in vitro. human blood cultures were treated with plga and tio nps in concentrations: . ; and lg/cm for h. lymphocyte transformation assay was used to assess the effect of nps on lymphocyte function. lymphocytes were stimulated with mitogens: concanavalin a, phytohaemmagglutinin (t-cell response) and pokeweed mitogen (b-cell response). our findings indicate immunomodulatory effect of plga nps. proliferative response of t-and b-lymphocytes exposed in vitro to the highest dose of plga for h was suppressed significantly (p. , p. ). on the other hand, we observed stimulative effect of exposure to middle dose of plga nps on b-lymphocyte proliferation (p. ). no alteration was found in lymphocyte proliferation treated in vitro with tio nps for h. in conclusion, proliferation of lymphocytes in vitro might be one of the relevant tests for evaluation of nps immunotoxicity.embryonic stem (es) cell differentiation in specific cell lineage is still a major challenge in regenerative medicine. differentiation is usually achieved by using biochemical factors (bf) which concentration and sides effects are not completely understood. therefore, we produced patterns in polydimethylsiloxane (pdms) consisting of groove and pillar arrays of sub-micrometric lateral resolution as substrates for cell cultures. we analyzed the effect of different nanostructures on differentiation of es-derived neuronal precursors into neuronal lineage without adding biochemical factors. neuronal precursors adhere on pdms more effectively than on glass coverslips but the elastomeric material itself doesn't enhance neuronal differentiation. nano-pillars increase both precursors differentiation and survival with respect to grooves. we demonstrated that neuronal yield was enhanced by increasing pillars height from to nm. on higher pillar neuronal differentiation reaches * % hours after plating and the largest differentiation enhancement of pillars over flat pdms was observed during the first hours of culture. we conclude that pdms nanopillars accelerate and increase neuronal differentiation. key: cord- -un ysc v authors: nan title: poster presentations date: - - journal: j pept sci doi: . /psc. sha: doc_id: cord_uid: un ysc v nan the boron neutron capture therapy (bnct) based on the interaction between b isotope and thermal neutron has been highly noted in recent years as one of promising techniques for treatment of cancers. p-( b)borono-l-phenylalanine (l- bpa), in which boron atom is enriched with b isotope, is now using clinically as an effi cient b carrier for treatment of patients in particular with malignant brain tumor and melanoma ( ) . in order to develop a practical method for the synthesis of l- bpa, we have recently examined the enantioselective method utilizing the negishi reaction based on the coupling of -( b)boronoiodobenzene derivative with -iodo-l-alanine derivative ( ) . from the standpoint of diagnosis of cancers, the magnetic resonance imaging (mri) is noted as one of common techniques. in particular, mri based on the measurement of f atom is becoming a remarkable one. to create practical materials utilizing as not only the b carrier but also mri probe, we had already synthesized the compounds containing both b and f atoms in a single molecule such as - [ -( b)borono- , -difl uorophenyl]-dl-alanine [dl- bpa( , f )] ., - [ -( l-proline is conformationally unique among coded amino acids in that its torsion angle is blocked (- ± ) by its characteristic fi vemembered pyrrolidine ring structure and the preceding torsion angle (tertiary amide) can undergo cis ( ) <-> trans ( ) isomerization much easier than the secondary amides of the usual peptide bonds. in addition, its torsion angle is commonly found either in the right-handed -/helical region (- ÷ - , or cis' conformation) or in the left-handed, semiextended, region [- ± , or trans', or poly-(l-pro) n conformation]. methylation at the c -position of a pro residue was suggested to block the preceding tertiary amide ( ) torsion angle of the resulting ( me)pro to the trans disposition and to restrict the , surface to the single region where the / -helices are found. we have synthesized a large set of n -blocked, ( me)pro-containing, dipeptide n'alkylamides having the general formulas p-d-( me)pro-xxx-nhipr and p-xxx-d-( me)pro-nhipr, where p is ac or boc and xxx is d-ala, l-ala, aib, gly, d-( me)pro, or l-( me)pro. the results of the present x-ray diffraction analysis clearly show that the region of the conformational map overwhelmingly preferred by ( me)pro is indeed that typical of / -helices, but the semi-extended, type-ii poly(pro) n helical region can exceptionally be explored by this extremely sterically demanding c -tetrasubstituted -amino acid. in addition, the known high propensity for -turn formation of the pro residue is even enhanced in peptides based on its c -methylated derivative. to complete the picture of the preferred conformation of ( me)pro, the synthesis and crystal-state investigation of a series of terminallyprotected homo-peptides from d-( me)pro are currently in progress in our laboratory the interest in guanidine-containing compounds is due to their important role in biological recognition processes. five-member cyclic n-amidinoamino acids represent hybrid structures, combining both proline rigidity and high positive charge of arginine guanidine group. structurally similar n-amidino-proline, n-amidino-pyroglutamic acid and cyclocreatine ( imino- -imidazolidine acetic acid) were chosen as objects of our study. the problem of their incorporation into peptide structure requires use of substituted derivatives or effective guanidylation technique. recently we have presented the synthesis of mts-protected n-amidinoproline. nevertheless, its application in n-termini modifi cation of peptides shows slow condensation rate in classical as well as solid phase synthesis for short peptides ( - residues) or completely fails in case of longer ones ( residues). on the other hand, polymer supported guanidylation of proline by bis-boc-protected carboxamidine- hbenzotriazole seems to be preferred route to n-amidino-proline containing peptides. three methods of n-amidino-pyroglutamic acid synthesis have been investigated and model dipeptide has been acquired on wang polymer by the cyclization of guanidine-glutamic acid. for selective boc-deprotection on wang resin the literature technique has been utilized. however, in this case esi-ms and nmr analyses evidence for side-reaction of triethylamine alkylation by polymer linker fragment. hplc analysis shows n-amidino-pyroglutamyl-phenylalanine stability at acidic and physiological ph but fast ring opening in water solution at ph . for cyclocreatine application in peptide synthesis we suggest the use of its p-toluenesulfonate having good solubility in dmf and showing effective coupling in cl-hobt/dic condensation. the examples of namidino-amino acids practical application in peptide synthesis will be presented. tryptophan is often a key pharmacophore which determines the affi nity of peptide ligands for their receptors. cyclic analogues of tryptophan which introduce local constraints and reduce the fl exibility of the indol moiety are very valuable tools to probe the bioactive conformation of the peptide ligands. one of the possibilities to freeze indol moiety of tryptophan is the synthesis of the additional -member ring by the formation of , , , -tetrahydro--carbolines. we report the synthesis of , -disubstituted , , , -tetrahydro--carbolines via the pictet-spengler reaction. methyl ester of tryptophan or dipeptides with n-terminal trp (trp-ala-ome, trp-leu-ome) were used as arylethylamine substrates and -amino aldehydes derived from l and d-amino acids were used as carbonyl components. we determined that there were no differences of the stereoselectivity of the pictet-spengler reactions in the case of trp-ome or trp-dipeptides. we also investigated the conformation of the newly created -membered ring in cis and trans diastereomers. the roesy spectra were used to assign the more stable conformation for each isomer. the conformations of cis isomers derived from tryptophan and dipeptides were the same and substituents on c- and c- were in both cases equatorial. the conformation of trans isomers were depended on the carboxyl part of trp. for methyl ester of tryptophan the ester group was axial, whereas for dipeptides we observed the opposite conformation with equatorial substituent on c- . our results show that the pictet-spengler reaction can be successfully performed for amino acids and peptides and the conformation of the newly created -membered ring depends on the substrates and the size of c- and c- substituents. helical-screw handedness of peptides composed of diastereoisomeric cyclic amino acids nagano helical-screw handedness in proteins is believed to result from thecarbon chiral center of l--amino acids. recently we have reported that the helical-screw sense of oligopeptides can be controlled without a chiral center on the peptide-backbone but by chiral centers at the side chain. that is to say, we designed and synthesized an optically active cyclic , -disubstituted amino acid (s,s)-ac( )c(dom), in which the -carbon atom is not a chiral center but chiral centers exist at the side chain. conformational analysis of the (s,s)-ac( )c(dom) peptides revealed that the hexapetide formed left-handed -helices both in solution and in the solid state, and the octapeptide assumed a left-handed -helix. herein we designed new two diastereoisomeric cyclic , -disubstituted amino acids; ( s, s)-and ( r, s)- -amino- -(methoxy)cyclopentanecarboxylic acid (ac( )c(om)) having chiral centers both at the -carbon atom and at the side chain. the amino acids ( s, s)-and ( r, s)-ac( )c(om) were synthesized starting from l-(-)-malic acid. that is to say, at fi rst, the malic acid was converted to diiodide compound, and bisalkylation of dimethyl malonate with the diiodide gave a cyclic diester. monohydrolysis of the diester, followed by curtius rearrangement produced separable mixtures of ( s, s)-and ( r, s)-ac( )c(om). we prepared both diastereoisomeric ( s, s)-and ( r, s)-homooligomers by solution-phase methods, respectively, and studied their preferred secondary structures using h nmr, ft-ir, cd and x-ray crystallographic analysis. zobel, ansgar; gaus, katharina; wollschläger, katrin; nieß, anke; juodaityte, jovita; sewald, norbert organic and bioorganic chemistry, bielefeld university, germany novel highly active and specifi c dna binding molecules have a considerable potential particularly with regard to applications in medical science. the major and minor grooves of the dna serve as recognition areas. it is of high interest for chemical biology to control dna-binding abilities of synthetic molecules. a possible modulator is light in combination with photoswitchable molecules. triostin a is a natural bisintercalator which belongs to the quinoxaline antibiotics originally isolated from streptomyces s- - . it consists of a bicyclic depsipeptide with n-methylated amino acids and a cystine bridge. its heteroaromatic quinoxaline moieties are able to intercalate gc-specifi cally into the dna inducing a change in conformation and therefore inhibit the transcription by blocking spezifi c enzymes. tandem is the n-unmethylated derivative of triostin a which binds at selectively to the dna due to the change in the hydrogen bonding pattern. the exchange of the cystine bridge of tandem with substituted azobenzene units leads to photoswitchable triostin a analogs. the synthesis of the depsipeptidic basic structure which contains the quinoxaline moieties is introduced as well as the coupling with azobenzene amino acids. the double cyclization step is accomplished under pseudo high dilution conditions. in order to differ between cisand trans-confi gurations of the molecule, nmr-and ir-spectroscopy was carried out as well as the photoswitchability of different analogues was tested by irradiation and rp-hplc analysis. for synthetic peptide vaccine prototype development: (pc) , which has an heteroatom in its structure and their bioconjugates obtained by microwave and carbodiimide methods were explained. for pc obtaining, the synthesis of the monomer, azabicyclo[ . . ]octane, (c), was carried out by organic methods which then will be used in polymer synthesis [ ] [ ] . consequently, polymers having different molecular weights and their water soluble bioconjugates were synthesized and the characterization of these polymers and conjugates were done by different methods such as uv, atr ft-ir, sec with four detectors and zeta sizer. for the chemical modifi cation of pc, bromoacetic acid was used and a water soluble polyampholyte synthesis was achieved with the quaternization of polymer. atr ft-ir spectra of pc was recorded and molecular weights, polidispersity values of polymers, mark-houwink constants and molecular diameters of the polymers were measured with size-exclusion chromatography with four-detectors (light-scattering, refractive index, viscosity and uv). with zeta sizer, size-analysis of polymer chains were done and their zeta potentials were measured. it is determined that molecular weights of the polymers were affected by the change in the amount and the type of initiators used and also from the polymerization media. analysing of having biodegradable characteristics of the synthesized polymers has been being reviewed. after synthesis and modifi cation of pc, its conjugates with antigenic peptides like avian infl uenza hemagglutinin (ha - ) ypydvpdya and rgdsggc cell receptor peptide were obtained. their characterization was also performed. japan; in alzheimer fs disease research, sparing water-solubility and uncontrolled self-assembly of amyloid peptide (a ) - are signifi cant obstacles to establish an experimental system that clarifi es a pathological mechanism of a - . to solve these problems, we herein disclose water-soluble "click peptides" based on an o-acyl isopeptide method. these peptides contain an o-acyl instead of n-acyl residue at the gly -ser of a - , and are converted to a - via an o-n intramolecular acyl migration triggered by phchange (ph-click) or photo-irradiation (photo-click). these peptides had remarkably higher water-solubility than a - . in addition, these peptides clearly adopted monomer state with a random coil structure, which were verifi ed by various physicochemical assays. importantly, we could establish an in situ system predominantly comprised of monomer a - as a result that the monomer click peptide was converted to a - quickly and quantitatively in accordance with ph-change. both selfassembly and conformational change of the produced a - in situ were observed with time. similarly, the photo-triggered click peptide afforded a - by uv-irradiation. because the in situ production of intact a - from the click peptides could overcome the handling problems of a - , this strategy would provide a reliable experimental system for investigating a pathological function of a - in alzheimer fs disease. the antioxidant effect of introducing phenylpropenoyl moiety in either in n-terminal group of analgesic oligopeptides or in c-terminal end of opioide active amino acid, modifi cated with polyamines have been evaluated. the series of hydroxycinnamoyl peptide and amino acid amides were synthesized by standard method used in peptide chemistry. obesity is a major public health problem associated with morbidity and mortality and continues to increase worldwide. the gastrointestinal tract and the pancreas release hormones regulating appetite and body weight. obestatin is a recently described amino-acids peptide derived from preproghrelin. it was identifi ed by bioinformatic prediction ( ) . obestatin decreases food intake and body weight gain, decelerates gastric emptying, promotes sleep in rat, inhibits water drinking. it has been described that obestatin effects on feeding behavior and an u-shaped dose-response relationship was found: low doses ( . - nmol/kg) and high doses ( - nmol/kg) were ineffective. the amino-acid carboxy-amidated human obestatin nalapropheaspvalglyilelysleuserglyvalglntyrglnglnhissergln alaleunh and corresponding rat obestatin hpheasnalapropheasp valglyilelysleuserglyalaglntyrglnglnhisglyargalaleunh were synthesized by different schemes. synthesis was developed on the basis of solid phase synthesis of protected peptide fragments followed by their assembly into the fi nal product. fragments - , - , - , - , - were built on the acid-labile -chlorotrityl chloride resin using the orthogonal fmoc/tbu strategy. fragment - (hcl•hargalaleunh ) was obtained in solution. the assembly of the full-length peptide was carried out by fragments coupling in solution or using solid phase method. the free peptide was obtained by treating the protected peptide with mixture of tfa: h o:tis ( : . : . ). the peptide was purifi ed with hplc and isolated by lyophilization with %+ purity according to analytical reversed-phase hplc. the mass of molecular ion determined by maldi-tof spectrometry was in fi ne agreement with calculated value. the inverstigation of biological actions of obestatins is in progress. straightforward synthesis of enantiopure tfm-amino acids from chiral cf brigaud, thierry; chaume, grégory; huguenot, florent; caupène, caroline university of cergy-pontoise, france trifl uoromethylated amino acids (tfm aas) are current synthetic targets due to their unique properties and their synthesis in enantiopure form remains a challenge. we will report that chiral -trifl uoromethyl- , oxazolidines (fox) are highly versatile synthons for the stereoselective synthesis of various functionalized -trifl uoromethylamino compounds such as -amino nitriles , -and -amino acids, diamines and amino alcohols. , moreover, trifl uoropyruvate-based oxazolidines proved to be very valuable building blocks for the stereoselective synthesis of both enantiomers of -trifl uoromethyl proline and -tfm-pyroglutamic acid, in enantiopure form. moreover, the synthesis of the (s)--tfm--allylglycine and the novel (s)--tfm norvaline were achieved in a few steps from this starting material. we will also report a recent straightforward synthetic route to tfm-dihydroxyprolines in enantiopure form from trifl uoropyruvate-based chiral oxazolidines. chitinases catalyse the hydrolysis of chitin, the natural homopolymer of ( , )-linked n-acetyl-d-glucosamine. chitin is a key structural component of the cell walls, exoskeletons, and eggshells of pathogenic fungi, insects, and nematodes, respectively, which all rely on the ability to hydrolyse chitin at specifi c points in their life cycles. chitinase inhibitors are now attracting considerable interest as novel fungicides and insecticides, as well as chemical tools to study human diseases as diverse as asthma and malaria. in this context, the cyclic pentapeptide natural products, argifi n and argadin, are two exciting inhibitors which pose some interesting synthetic challenges. the argifi n structure includes two sensitive -linked asp residues, as well as an unusual carbamoylated arg side chain, while argadin contains a unique aspsemialdehyde residue, that is cyclised to the peptide backbone to generate a potentially labile hemiaminal. we will describe improved routes to both compounds that allow us to avoid signifi cant side reactions such as aspartimide formation and homoserine-mediated backbone cleavage that are observed in our previously reported syntheses. [ , ] this is achieved by carrying out the assembly and cyclisation of both peptides, including key side chain derivatisation steps, entirely on solid phase. the application of the strategies devised to the automated synthesis of argifi n and argadin and related natural products will be described. during the last two decades combinatorial chemistry has become an important tool for the quick synthesis of large numbers of small molecules used, for example, in the generation of the new lead structures for medicinal applications. additionally, it allows rapid access to diverse chemical libraries with novel structures and properties. small molecules libraries are generally prepared on solid support simplifying tedious purifi cation steps of the intermediates and facilitating the entire synthesis. piperazines and keto-piperazines are amongst the important backbones in today's drug discovery. the piperazine framework has been defi ned in medicinal chemistry as a "privileged scaffold". it is a molecular backbone with versatile affi nity properties representing a frequently-occurring binding motif, and providing potent and selective ligands for a wide range of biological targets. the high number of positive hits revealed in biological screens with the piperazine scaffold urged chemists to develop plenty of different synthetic methods that allow fast and effi cient building of these heterocyclic systems on solid support as well as homogenous chemistry. however, the majority of these methodologies is not stereospecifi c and the complex mixtures of the stereoisomers are generated. in order to preserve important chiral centers in potential hits we propose to use pipearzine backbone, initially prepared in optically pure form, bearing various tethers with orthogonally protected groups applicable via solid phase organic chemistry (spoc). we will describe a novel synthesis of keto and diketopiperazine building blocks. these chiral building blocks are applied in "around-the-scaffold" modifi cation strategy by spoc, introducing valuable physico-chemical properties in independent diversity points. solid-phase strategies for constraining peptide structures can serve for elucidating the relationships between conformation and activity. for example, the systematic substitution of natural amino acids by their aza-amino acid counterparts has been accomplished using a fmoc strategy featuring couplings with aza-amino acid chlorides to provide insight into the biologically active conformers of the melanocortin receptor agonist ac-his-d-phe-arg-trp-nh as well as the calcitonin gene-related peptide antagonist [d , p , f ]cgrp - ( ) ( ) ( ) . employing cyclic sulfamidates, we have now developed fmoc strategies for the effective solid-phase introduction of alpha-and beta-amino gamma-lactam constraints into peptides. since their pioneering use in a somatostatin analog with about fold greater activity ( ), lactam restraints have been used to study a variety of relevant targets ( , ) . our strategies now provide effective means for performing lactam scans of biologically active peptides as demonstrated using the growth hormone secretagogue ghrp- , and an allosteric modulator peptide antagonist of the il- receptor. our presentation will focus on recent developments in the solid-phase chemistry for synthesizing such constrained peptide analogs and the relationships between peptide conformation and biology, elucidated by these novel methods. references: barcelona science park, university of barcelona, -barcelona, spain; linaclotide is a -residue peptide currently undergoing phase ii clinical trials for the treatment of gastrointestinal diseases such as chronic constipation (cc). linaclotide, which can be administered orally, is an agonist of the guanylate cyclase type-c receptor found in the intestine. from a structural point of view, this small peptide presents a constrained structure with the presence of three disulfi de bridges between cys -cys , cys -cys , and cys -cys . h-cys( )-cys( )-glu-tyr-cys( )-cys( )-asn-pro-ala-cys( )-thr-gly-cys( )-tyr-oh in order to reach the large amounts required for a marketed peptide, an effi cient synthesis needs to be attained. to optimize the synthesis, its fundamental limitations need to be determined and addressed. in the case of linaclotide, the key points are related to the numerous cys (some of them consecutive) present in the peptide, for two reasons: the potential risk of racemization upon assembling the linear chain, and the misfolding of the three disulfi de bridges. for that, the concourse of different protecting groups and folding conditions as well as the analysis of the disulfi de bridges in the fi nal folded peptide has been studied and will be discussed in this presentation. balalaie, saeed ; arabanian, armin ; mohammadnejad, mahdieh ; gross, juergen h. university, iran (islamic rep.); university, germany gnrh analogues have been used for the treatment of steroid-dependent tumors, such as prostate and breast cancers. the development of more potent gnrh analogues depended largely on the important made in the science of peptide chemistry. ugi- mcr reaction is known for the synthesis of amide bond. we wish to report herein an effi cient method for the synthesis of some gnrh analogues based on ugi reaction using fourcomponent reaction of n and c-terminus peptides, aromatic aldehydes and isocyanides. this ugi- mcr could describe to build up novel gnrh analogues deriving from triptorelin and gonadorelin. all of the products were purifi ed using preparative hplc and the structures were assigned according to maldi mass spectrometry data. peptide synthesis is based in a proper combination of protecting groups and in the right choice of the right coupling method. nowadays, almost all peptide bond formed are carried out in the presence of hydroxybenzotriazole (hobt) or its derivatives (hoat, cl-hobt). thus, hobt derivatives are used in combination with a carbodiimide or another coupling agent or built into a standalone reagent such as immonium ( thus, a replacement of hobt should be found for preparation of peptides for research purposes and, more important, for the production of peptide based apis. herein, several alternatives to hobt will be discussed taking into account the explosivity properties. furthermore, a new family of immonium salts, which incorporates an proton acceptor in its carbocation skeleton. the novel proton acceptor coupling reagent has shown superiority to the described previously. an oxygen in the carbocation moiety confers to the reagent more solubility, enhances coupling yields and decreases racemization, allowing the use of just one equivalent of base. examples on the use of the non-explosive replacement for hobt together with carbodiimides or built into phosphonium and immonium salts, with the proton acceptor, will be discussed. thiocarbamate-linked peptides by chemoselective peptide ligation using phenylthiocarbamate chemistry peptide chemical ligation chemistries, which allow the chemoselective coupling of unprotected peptide fragments, are useful tools for synthesizing native polypeptides or unnatural peptide-based macromolecules. native chemical ligation (ncl) ( ), staudinger ligation ( ) lead to the formation of a native peptide bond at the ligation site. other methods result in the formation of unnatural covalent bonds such as oxime ( ), thioester ( ) linkages. these methods are of great interest when native peptide bonds are not absolutely required. in this context, we examined the potential use of the phenylthiocarbamate group in ligation chemistry. the phenylthiocarbonyl group can be easily introduced into peptides on alpha or epsilon amino group using phenylthiochloroformate and standard solid-phase method ( ) . it reacts chemoselectively with cysteinyl peptides to give an alkylthiocarbamate bond. s,n-shift of the alkylaminocarbonyl group from the cys side chain to the alpha-amino group did not occur. the method was used for linking two peptides chain through their n-termini, for the synthesis of a cyclic peptide or for the synthesis of di-or tetravalent multiple antigenic peptides. synthetic peptide derivatives which contain secondary amide moiety at c-terminus are inhibitors of serine and cysteine proteinases and thus could be effi ciently used as ligands in affi nity chromatography in order to isolate these enzymes. there is a number of chemical syntheses of amidous peptide derivatives in the literature, but the reaction yields are usually low and reaction conditions and purifi cation procedures are too complicated. it is also known that the application of enzymatic catalysis allows to obtain high reaction yields with simultaneous simplifi cation of synthetic and purifi cation procedures, and at the same time preserves the optical purity of target compound. subtilisin sorbed on silochrome could be effi ciently used as catalyst of acylation of secondary amides by the esters of acylpeptides. subtilisin's wide substrate specifi city allows to carry out the syntheses with peptides which contain hydrophilic, hydrophobic, dicarbonic-and diamino-acids at c-terminus. piperidine, morpholine, indole, diethylamine and other secondary amines are used in the syntheses as the amino-components. the reaction yield equals about - % in dependence of the nature of c-terminal amino acid of acylating peptide, pka of amine and the hydrophobicity of the fi nal compound. the distribution of the reaction product in liquid and solid reaction phases is investigated, the optimal reaction conditions for enzymatic acylation of secondary amines are established. all the compounds obtained are characterized by its nmr and mass spectra. the inhibition constants for some proteolytic enzymes with the compounds of research are defi ned. unnatural amino acids are becoming increasingly important substrates in modern drug design, synthesis and discovery research. in particular, arylhistidines naturally occur in the active site of the heme-copper oxidases as well as in cytotoxic and antifungal marine peptides ( ) . a method of choice for the preparation of unsymmetrical biaryl systems is the suzuki-miyaura cross-coupling of an aryl halide with an arylboronic acid. it has been shown that microwaves signifi cantly enhance this reaction leading to higher overall yields and purities as well as shorter reaction times. although a great variety of biarylic compounds have been prepared following this approach, so far, it has not been applied to the arylation of the histidine imidazole ring. initially, we studied a methodology for the synthesis of -arylhistidines via a microwave-assisted suzuki-miyaura cross-coupling reaction in solution ( ) . taking into account the advantages of the synthesis on solid support, such as the avoidance of tedious work-up which are particularly valuable for palladium-catalyzed reactions, we studied the application of the above methodology on solid-phase. here, we report the suzuki-miyaura reaction between a -bromohistidine and an arylboronic acid on solid support. the reaction conditions were optimized by varying several parameters such as the solvent, the reagent concentrations and the reaction time. the optimized conditions were subsequently applied to the synthesis of peptides containing a -arylhistidine residue. this work constitutes the fi rst suzuki-miyaura coupling involving the imidazole ring of a histidine on solid support. microwave-assisted attachment of fmoc-amino acids to resins via triazine "superactive esters". the substitution level of the new functionalized resin was calculated following a standard protocol based on the spectroscopic measurement of the soluble chromophore piperidine-dibenzofulvene. coupling reactions to the resin proceeds at room temperature in hours. moreover we carried out the time-consuming attachment of fmoc-amino acids to the resin by an automatic monomode microwave (mw) instrument (liberty, cem), in fact microwave is proposed as a valid alternative to enhance effi ciency of coupling reactions and it has been widely applied to spps. novel peptide-hetorocycle conjugates: derivatives of -(benzimidazol- -yl)alanine ( ) . considering the biological activity and complexing abilities of benzimidazoles we developed a direct solid-phase synthesis of benzimidazole-peptide conjugates, expecting these new compounds to express novel biological properties ( ) . the peptide-heterocycle conjugates are obtained by on-resin reaction between aldehydes and peptides containing a specially designed -( , -diaminophenyl)alanine residue ( ) . the stoichiometric amount of aldehyde leads to '-substituted -( h-benzimidazol- -yl)alaninecontaining peptides, the increase in aldehyde content results in `, `disubstituted derivatives. the reaction with dialdehydes gives novel amino acid residues containing tricyclic systems, in the case of ophthalic aldehyde -the pyrido-[ , -a] benzimidazole. the compatibility of our method with the fmoc solid phase peptide synthesis protocols was proven by the synthesis of analogues of immunosuppressory fragments of ubiquitin and hla-dq [ , ] . the biological activity of the modifi ed oligopeptides was compared to that of original fragments . as well as to similar quinoxaline-peptide hybrids. the collision-induced dissociation of substituted benzimidazole-peptide conjugates leads to characteristic fragmentation ions, which may be used as a diagnostic tool in the fragmentation of the benzimidazole-peptide hybrids. cardona, valérie; oswald, benoit genzyme pharmaceuticals, switzerland dehydroamino acids are an important class of compounds due to their presence in many biologically active natural products including the antrimycins, tentoxin, phomopsin a, or the phosphatase inhibitors like microcystin and nodularin. in the last decade an increasing interest in -branched dehydroamino acids has been developed based on their importance as commodity chemicals and value as tools in structure relationship studies. the incorporation into a peptide of such unsaturated amino acids introduces an element of conformational rigidity, as well as changes in reactivity, allowing for the development of high affi nity ligands for receptors. a variety of methods exist for the synthesis of dehydroamino acids. some of these approaches rely on thermodynamic control to dictate the alkene geometry. if there is no strong thermodynamic preference, or if the desired product is not the thermodynamically favoured isomer, the existing synthetic methods are often ineffective. we describe here an effi cient stereoselective method for the synthesis of , -branched dehydroamino acids (iii) from -aryldehydroamino acids (i). the -aryldehydroamino acids (i) were prepared from commercially available z/boc-phosphonoglycine trimethylester and aldehydes using the schmidt protocol. the transformation of (i) into their -bromodehydroamino acid derivatives (ii) is performed by a hoerrner reaction in a very good yield giving the trans derivatives. suzuki cross-coupling on (ii) with a boronic derivative generated the high value building blocks (iii). a variety of side chains can be incorporated using this type of reaction. the stereochemistry of these compounds has been determined using noe enhancement experiments. we report here a scalable and high yielding synthesis of stereospecifi c , -aryldehydroamino acid derivatives. versatile methods for synthesizing acyl-tetramic acids peptide analogs. davidov, gali; mozes, tamar; khandadash, raz; byk, gerardo bar ilan university, israel acyl-tetramic acid derivatives have been identifi ed as potential antiviral and antibacterial compounds. in this work we have improved their synthesis starting from tetramic acid lactams using peptide coupling reagents such as bop under microwave heating for generating the carbon-carbon bond of the acyl-tetramate. using this procedure we have synthesized a number of new tetramic acid derivatives in solution and generated a series of acyl-tetramic acid building blocks that were introduced into peptides using conventional spps methods. additionally, we present here a new solid phase microwave assisted synthesis of acyltetramates on pre-synthesized peptides and amino acids. antibacterial and antiviral activity of the products will be presented. peptide sequence and include all side-chain groups. in practice this is diffi cult to achieve. the task of making a turn mimetic can be regarded from the perspective of the backbone dihedral angles -to make a beta turn mimetic the phi and psi angles of two consecutive residues have to be controlled. limiting the dihedral angle space is most effectively accomplished with a cyclic constraint -one or more may be used. in this case a single medium ring cyclisation from the (i) carbonyl to the (i+ ) amine -in place of the classical hydrogen bond -forces the four backbone dihedral angles into a turn conformation. due to the polyfunctional nature of peptide systems the introduction of unnatural constraints can be synthetically challenging. in the present case a number of side reactions were encountered and had to be overcome. some were well known such as diketopiperazine formation and others involving transannular interactions were unexpected and gave rise to interesting byproducts. ultimately the side reactions were overcome by choice of cyclisation position and synthetic modifi cations. auto-assembling antimicrobial cyclic pseudopeptides including aza- -amino acids antibiotic resistance of pathogens against conventional antibiotics is increasing at a rate that far exceeds the pace of new development of drugs. so, antimicrobial peptides, both synthetic and from natural sources, have raised interest as potential useful drugs in the future. however, due to proteolytic degradation, peptides are not ideal candidates for pharmaceutical development. that is why numerous researches try to develop non natural peptidic analogues for enhancing metabolic stability, bioavailability, and biological absorption. in this class of peptidomimetics, pseudopeptides consisting exclusively or including aza -amino acids have emerged as a promising new class of compounds that favour hydrogen bond formation and can enhance biological activities when compared to natural parent peptides ( ) . we have designed "mixed" cylic pseudopeptides composed of -and aza- -amino acids that target bacterial cell wall and induce the death of the pathogens. particularly, some of these cyclic pseudopeptides have broad spectrum antibactericidal activities on gram positive and gram negative bacteria with low minimum inhibitory concentrations (mic). on the other hand, this type of molecules is not haemolytic and cytotoxic at antimicrobial activity levels( ) now, we try to explain the mechanism of action of our pseudopeptides that act on the microbial membranes. with nmr studies we have demonstrate that in solution cycles auto-associate at high concentrations and we investigate their behaviour in presence of small unilamellar lipidic vesicules (suv) ( ) . this phenomenon of auto association seems to be facilitated at the lipid interface. liu, hongqiang ; pattabiraman, vijaya r. ; vederas, john c. university of alberta, canada; lantibiotics are a class of antimicrobial peptides containing lanthionine ( ) and/or -methyllanthionine ( ) residues in cyclic moieties, and are currently used for food preservation. they also have potential as human therapeutics. lacticin is a two-peptide lantibiotic produced by l. lactis subsp. lactis dpc , and its components (a and a ) are active against a wide range of gram-positive bacteria by a synergistic mechanism in sub nm concentrations. however, the oxidation of the thioether of lanthionine ( ) and -methyllanthionine ( ) is a major source of lantibiotic instability and loss of activity. to investigate structure-activity relationships and determine whether sulfur can be replaced, an analogue of lacticin a in which oxygen atoms replace sulfur was synthesized by solid phase peptide synthesis. utilizing the stereochemicaly pure oxa-lanthionine ( ) and oxa--methyllanthionine ( ) with orthogonal protecting groups, the conformationally constrained tricyclic moiety in oxa-lacticin a was constructed. the results of biological evaluation of the oxidatively stable analogue will also be described. controlling -helical secondary structure of oligopeptides and its use as a chiral catalyst replacement of -hydrogen atom of -amino acids results in ,disubstituted amino acids. as an , -disubstituted amino acid, achiralaminoisobutyric acid (aib) is well-known, and widely used to construct helical secondary structures of oligopeptides. the helical secondary structures constructed by using aib usually show -helices, but nothelices in the case of short oligopeptides. recently we designed and synthesized a chiral cylic , -disubstituted amino acid (s,s)-ac c dom , in which the -carbon atom is not a chiral center but chiral centers existing at the side chain. homooctapeptide composed of (s,s)-ac c dom formed a left-handed -helix both in solution and in the solid state. furthermore, in the case that the (s,s)-ac c dom was incorporated into l-leuhexapeptide, the hexapeptide cbz-{l-leu-l-leu-(s,s)-ac c dom } -ome preferentially formed a right-handed -helix in the crystal state, whereas the hexapeptide cbz-{l-leu-l-leu-aib} -ome formed a right-handed -helix. the fi nding that the propensity of cyclic amino acid ac c dom is to form -helix over -helix, stimulated us to use the -helical oligomer containing the cyclic amino acid as an asymmetric catalyst. we prepared several l-leu-oligopeptides containing , -disubstituted amino acids, analyzed their preferred secondary structures, and studied a enantioselective reaction of prochiral substrate using -helical oligopeptides as a chiral catalyst. fbp domains: spps using pseudoproline and depsipeptide approaches, stability and structure of glutamine-rich analogs (nature biotech., ) . one peptide will soon be tested in clinical studies. the success depends on effi cient synthetic access to large amounts of the peptide. systematic modifi cations of the lead structure hbvpres/ - have to be performed resulting in a panel of peptide variants to be studied with respect to their applicability, pharmacokinetics and serum stability. we selected a series of peptides carrying deletions, point mutations, d-amino acid exchanges and sequence permutations. the syntheses of derivates were performed by fmoc-solid-phase synthesis on a rink amide am resin using hbtu/dipea activation on an applied biosystems a peptide synthesizer. after completion of the peptide sequence, stearic acid was attached to the n-terminus. the products were deprotected and detached from the resin by tfa treatment and subsequently purifi ed by hplc. the stability of the peptides was determined in human serum. we were able to synthesize all hbvpres lipopeptides in acceptable yields ( - %). preparative hplc was used to obtain intended compounds in high purity as determined by hplc and esi mass spectrometry. the serum stability studies revealed exceptionally long biological half-lives (t / > h for all lipopeptides) mainly due to the fatty acid residue. the peptides belong to a class of intrinsically unfolded peptides and are therefore not prone to aggregations within the synthesis. consequently solid phase peptide synthesis provides a suitable access to the peptides described. it can be speculated that the peptides are protected against degradation due to an association via their lipophilic end to serum proteins. sugar derivatives for self-assembling -cyclic peptides brea last years, numerous inorganic and organic nanotubes have been developed. self-assembling peptide nanotubes (spn) made from cyclic peptides have structural and functional properties that may be suitable for various applications in biology and material science. recently, our group have reported , -cyclic peptide ( , -cp) that forms highly stable homo-and/or heterodimers with partial hydrophobic cavities. in the present communication we will describe the design, synthesis and applications of a new class of self-assembling -cyclic peptides containing sugar derivatives that modify both the inner and the outer surfaces of the resulting supramolecular entities. peptido .rotaxanes: can a tetramide macrocycle travel to a station by wrapping up around a helical peptide thread? we are currently expanding this fi eld by synthesizing and studying the properties of new sets of peptido .rotaxanes. the initial set examined includes symmetrical, achiral compounds with a fmoc stopper at each terminus, threads built up with a central fumaric diamide station and two helical aib ( -aminoisobutyric acid) homo-peptides of different lengths (from to residues), and an aromatic tetramide macrocycle. these supramolecular systems have been characterized spectroscopically and two of them by x-ray diffraction as well. the fundamental interactions between macrocycle and thread (the same interactions offering the major contribution to the template-directed preparation of this family of molecules) are intercomponent h-bonds comprising the four amide groups in the ring and the two amide bonds in the fumaric derivative. the second, more complex, set of peptido .rotaxanes examined is represented by non-symmetrical compounds involving a thread based on a central helical -(aib) -peptide linker and two stations of opposite chirality (a -d-leu-gly-gly-tripeptide at the n-terminus and a fumaric diamide-l-leu moiety at the c-terminus). as stoppers and the macrocycle, we selected two diphenylacetyl groups and the usual aromatic tetramide, respectively. as spectroscopically assessed, the macrocycle initially positions on the fumaric diamide-l-leu station. subsequently, by using photons as stimuli to induce the fumaric<->maleic equilibrium, we were able to switch partially the relative macrocyclebinding affi nity in favor of the -d-leu-gly-gly-tripeptide station. this is the fi rst example of a rotaxane where the ring makes a journey to one of the stations by wrapping up around a helical peptide thread. synthetic novel n phenyl and bi-phenyl tetracarboxamides bis peptides. an approach to dna threading intercalators as expected potential pharmaceutical carriers for catatonic agents. naphthalene diimides were reported to bind to dna opposite grooves via the threading intercalation mode ( , ) . on the other hand, peptides constitute an excellent class of molecules for rapid drug discovery and lead optimization. the title bis dipeptides may, consequently, offer signifi cant dna biological probes. potential anticancer drugs or drug carriers could thus be presumed. herein, the title bis peptides a and b were suggested and synthesized as new prototype candidates of such class compounds. pre-assembled and purifi ed peptides via the conventional methods of peptide synthesis are, subsequently, coupled to either , , , -benzene tetra-carboxylic dianhydride or , , , -naphthalene tetra-carboxylic dianhydride. the expected structures of obtained a and b as preliminary candidates were confi rmed via the chemical, chromatographic and spectroscopic methodologies. the chemistry of both a and b will be discussed. synthesis of other candidates, the corresponding biological, pharmacological and physicochemical investigations are under realization. x = phenyl or bi-phenyl ring [compound a and b respectivel the fragment pth( - ) is suffi cient to bind and activate the pth type i receptor (pth r). the molecular mechanisms by which pth binds to and activates the pth r have been extensively investigated. recent investigations focusing on the interaction of n-terminal modifi ed fragments pth( - )nh with pth r showed that certain modifi cations can increase signalling potency and that enhancement of the -helicity in the pth( - ) sequence yielded potent analogues of pth( - )nh . the design of cyclic analogues represents a widely used strategy to increase peptide stability and potency. the structural constraint induced by cyclization reduces conformational fl exibility and may enhance potency, selectivity, stability and bioavailability as well as membrane barrier permeability. initially, the work was concentrated on conformational constrains in n-terminal such as the introduction of ctetra-substituted amino acids. global restrictions in the conformation of a peptide are possible by limiting the fl exibility of the peptide strand through cyclization. to this purpose, the amino acid side chains that are not involved in receptor recognition are connected together or with the peptide backbone. previous works on the role of side chains of aa determined through d-scan, analogues which maintained better -helical structure, contained d-gln in position and . recently gardella and co-workers reported that an analogues of pth( - ) cyclized between and residues exhibited almost the same activity of linear analogues. so, in this work two new potent cyclic analogues in position and were synthesized directly on spps, using side chains of lys and glu or ser. then they were biologically tested and analyzed by cd, according to our previous work. boudreau, marc; vederas, john university of alberta, canada the neopetrosiamides a and b are two diastereomeric tricyclic peptides that inhibit amoeboid invasion of human tumor cells. they were isolated by anderson and co-workers from the marine sponge neopetrosia sp. collected in papua new guinea, with their subsequent structure elucidation by the same group in . the peptides are residues in length and contain three disulfi de bonds, as well as the unusual amino acid methionine sulfoxide at position . the two peptides differ only by being epimeric at this sulfoxide functionality. we report the total synthesis of neopetrosiamides a and b as well as an analog wherein the methionine sulfoxide has been replaced by norleucine, the carbon analog of methionine. the strategy involved solid phase peptide synthesis to generate the linear species, and selective formation of the disulfi de bonds from orthogonally protected cysteine residues. synthesis of mimetics of the antibody d a : a new class of antitrombotics. in the lab for tromboses research of the kulak (belgium) an antitrombotic antibody (ab) called d a has been characterized ( ) . the ab inhibits the interaction between the plasma peptide von willebrand factor (vwf) and by injury exposed collagen, a prerequisite interaction in thrombusformation in arteries (high blood shear). it has been shown in vivo that the ab d a has an antitrombotic effect without showing the bleeding complications that known antitrombotics exhibit. this makes the ab an attractive lead for antithrombotic drug design. by x-ray and mutagenesis studies, the paratope (active site) of the antibody has been determined. amino acids, discontinuous in the primary structure of the antibody but a quasi linear unit in space, play an important role in the interaction. incorporation of the functional groups of the amino acids and fi xation of the secondary structure of the paratope will be the aim in the design of the paratope mimetics. with this rational design we will try to develop an orally available, synthetic paratope peptidomimetic with the same antitrombotic effect as the antibody. the development (solid phase and solution peptide synthesis) of the mimetics is a stepwise process. in each step conformational restrictions are introduced. in this we hope to divert away from the peptide and go to a drug like molecule. peptide ligands for sh -and ptp domains containing phosphotyrosine are of great interest to infl uence the activity of kinases, phosphatases and other functional proteins. backbone cyclization can help to stabilize these ligands against proteolytic degradation and to form their bioactive conformation. till now backbone cyclization was performed with bifunctional and in few cases with trifunctional amino acids but not with phosphotyrosine. the assembly of such peptides requires preformed building units. because the necessary reductive alkylation of phosphotyrosine derivatives leads only to very low yields we used n-functionalized pseudodipeptides with the unprotected phenolic hydroxyl group. for fragment condensation we synthesized building units of the common structures: fmoc-aaØ[co-n(x)-(ch )n-nh-alloc)tyr(oh)-oh and fmoc-aaØ[co-n(ch )m -cooall)tyr(oh)-oh. depending on the steric hindrancy of the n-terminal amino acid these pseudodipeptides were synthesized in solution or at sasrinresin. they were purifi ed by fl ash chromatography and analytically characterized by hplc, esi-ms and nmr. we tested our strategy on the synthesis of an octapeptide-ligand for the n-terminal sh -domain of the phosphatase shp- : glu-gly-leu-asn/abu-ptyr-nle-asp-leu-nh . couplings of the dipeptide units were performed with pybop, stepwise coupling to the peptide fragments with unprotected phenolic hydroxyl group with pentafl uoro phenylesters. after fi nishing the assembly the obtained polymer bound octapeptides were consecutively cyclized, phosphorylated, removed from the resin and purifi ed by hplc. based on elucidated side reactions the synthetic strategy was optimized. the obtained backbone cyclic and phosphorylated ligands were tested for their infl uence on the phosphatase activity of shp- . the found enzymatic activities are correlated to size, direction, hydrophobicity and conformational fl exibility of the lactam bridges. biology and medicine. in this context, we have devised a new family of compounds named « polyamide amino acids » (paas), constituted by a pna (peptide nucleic acids) backbone mimicking rna sugarphosphate backbone, onto which aminoacid residues are linked. therefore, these paas could constitute a new type of rna ligands, liable to specifi cally interact with an rna target through an original interaction mode. to assess the ability of paas to be potential rna binders, we fi rst prepared tetra-paas (t -t ) via solid-phase synthesis, starting from paa monomers deriving from alanine, phenylalanine, lysine and arginine residues. interactions of the tetramers with a hiv- tar rna fragment, taken as a target model, was investigated by fl uorescence spectroscopy and circular dichroism. to give insights about specifi city, binding affi nities to tar were also assessed using an excess of a trna mixture. results showed kd values varying from . to m, indicating the importance of the aminoacid side chains in the interaction. thermodynamic analyses revealed that even if electrostatic interactions play a part in the complex formation, the binding is enthalpy-driven, highligthing the importance of non-electrostatic interactions in the recognition process. these results are of special interest since rna/ ligand association specifi city is typically assumed to be due to shortrange non-electrostatic interactions. moreover, t -t were shown to be specifi c to tar in the presence of an excess of trna. all together, these results are encouraging and they highlight the potential of paas as rna ligands. indeed, the use of only four paa monomers as building blocks leads to tetra-paas displaying both affi nity and specifi city for their rna target. studies on the solid phase synthesis and selective detection of peptide derived amadori products by mass spectrometry stefanowicz mass spectrometric analysis of glycation products of proteins and peptides attracts increasing attention ( ) . peptide-based amadori products could be used as markers of diabetes mellitus, which makes them the subject of interest in clinical chemistry. recently, several procedures of siteselective synthesis of amadori-modifi ed peptides has been published [ , ] . in this communication we will present the synthesis of a new, fully protected derivative of glycated lysine which was successfully applied as a building block for incorporation of the glycated lysine moiety into peptide chain according to the standard fmoc-solid phase synthesis protocol. we will also present a new and straightforward method of selective detection of peptide-derived amadori products by esi-ms basing on characteristic neutral losses in the sugar moiety. the proposed approach in contrast to the neutral loss scanning, which requires triple quadrupol mass spectrometer, can be performed on the instruments with higher resolution and sensitivity, like q-tof . new apa inhibitors interacting with the s subsite bring new insights in the apa substrate specifi city mediated by the calcium ion. aminopeptidase (apa, ec . . . ) is a membrane-bound zinc metallopeptidase involved in the maturation of brain angiotensin iii, a peptide which exerts a tonic stimulatory action on blood pressure in hypertensive animals ( ) . therefore, developing inhibitors of this enzyme should result in new antihypertensive agents with possible new application in the treatment of certain forms of hypertension ( ) ( ) . we and others have previously reported the design of such inhibitors ( ) ( ) . nevertheless, the improvement of the affi nities of the inhibitors is relatively impaired since the three dimensional ( d) structure of the enzyme is not available. with the aim of getting insight into inhibitors optimization, a d model of apa was constructed in our group as an alternative ( ) . furthermore, it is well established that apa substrate specifi city and enzymatic activity are calcium dependent. in order to render this model more accurate and so on to identify the amino acids residues involved in calcium binding, we have designed new inhibitors able to explore the apa s subsite to better understand its specifi city. we will report the synthesis of these new inhibitors, as well as an inversion of the specifi city of apa s subsite in absence of calcium. these results and the identifi cation of the amino acids implicated in calcium binding will be discussed on the basis of site-directed mutagenesis studies. one of the designed inhibitors, ni (ki = nm) is to date the more potent inhibitor of apa activity measured without calcium. altogether, these data should allow the improvement of apa inhibitors by structure aided design. synthesis, resolution, and absolute confi guration of bpaib, a benzophenone-containing c -tetrasubstituted -amino acid photoreactive amino acids with benzophenone side chains, the prototype of which is bpa ( -benzoyl phenylalanine), have found numerous applications as photo-probes for covalent modifi cation of enzymes and receptors, as well as in intramolecular quenching by a nitroxide free radical in trichogin peptide analogs. however, the remarkable fl exibility of the bpa side chain may question the extrapolation of results of photo cross-linking experiments and photophysical data to protein mapping and intramolecular distances, respectively [m. saviano et al., chembiochem, , , - and references cited therein]. to overcome this problem, we designed a new "constrained bpa" amino acid, bpaib, belonging to the sub-class of the ci <->ci cyclized, c -tetrasubstituted -amino acids (strong -turn and helix inducers in peptides). racemic boc-bpaib-oh was prepared by bis(alkylation) of ethyl isocyanoacetate under phasetransfer conditions with -benzoyl- , -(bis)bromomethyl benzene as alkylating agent, followed by acidic hydrolysis, n -boc protection, and saponifi cation of the ester function. resolution was achieved through poster abstracts the terminally-blocked dipeptide bz-bpaib-l-phe-nhchx with chromatographic separation of the diastereomers and acidic hydrolysis. x-ray diffraction analysis of a crystal of a z-bpaib-l-phe-nhchx diastereomer allowed the assignment of the absolute confi guration of the bpaib enantiomers. photo-crosslinking studies with this residue are currently in progress. n-methylation of n -acetylated, c -ethylated, fullyextended homo-peptides: synthetic and conformational aspects moretto peptides characterized by single or multiple n-methylated, ctrisubstituted (protein) -amino acids are one of the subject of increasing interest in medicinal chemistry. several naturally occurring peptides, remarkably stable to proteolytic attacks, are based on n-methylated peptides. n methylation of the -conh-function is a useful tool for discriminating solvent exposed from intramolecularly h-bonded secondary amide groups in peptides. we are currently extending this reaction to linear peptides based on c -tetrasusbtituted -amino acids. after having investigated synthesis and conformation of the n-methylated homo-peptides from the c -methylated, helicogenicaminoisobutyric acid and c -methylnorvaline residues [a. moretto et al., biopolymers (pept. sci.) , , - ], in this work we examined the n-methylation reaction on homo-peptides from c , -diethylglycine (deg), known to overwhelmingly adopt the fully-extended, multiple c , conformation. we studied the following peptide series: ac-(deg) n -n(et) , with n = - . under the experimental conditions used, only monomethylation (on the n-terminal, acetylated residue) takes place. our ft-ir absorption, nmr, and x-ray diffraction analyses support the view that the fully-extended conformation preferred by the original peptides is dramatically perturbed in all of the derivatives mono-methylated at position . computational study on secondary structure of oligopeptides containing chiral , -disubstituted -amino acids we have shown mcmm conformational search method and amber* force fi eld using macromodel is useful to predict secondary helical structures ( -helix, -helix) of oligopeptides prepared from ,disubstituted -amino acids. moreover, we have studied conformational analysis of oligopeptides containing chiral , -disubstituted -amino acids to predict the helical screw sense of helical structures. we calculated , -disubstituted peptide using mcmm conformational search with various force fi elds (amber*, mmff, opls) . in the case of using amber* force fi eld the results were in agreement with those of x-ray and were most stable conformation evaluated by - g level molecular orbital calculation. these results indicated that computational simulation using conformational search calculations with amber* force fi eld is most useful for conformational analysis of oligopeptides containing , -disubstituted -amino acids. a new colorimetric test for solid-phase amines and thiols. simple colorimetric tests still remain the most convenient tests for providing information on the course of solid-phase reactions. a colour test, which indicates the presence or absence of a functional group by visual detection, is a simple, practical tool to monitor the completeness of the reaction. although the variety of colour tests for amines, there is still a need for a reliable and sensitive test in some synthetic approaches. here, we wish to report a new simple, fast and sensitive colorimetric assay for the visual detection of solid-phase bound primary, secondary amines and solid-phase bound thiol groups using -alkyl- -aryl-imidazo[ , -a]pyrimidinium salts. in the search for a new synthetic approach to polysubstituted -aminoimidazoles ( ), we have reported a new procedure, employing substituted -aminopyrimidines and -bromocarbonyl compounds. the intermediate imidazo [ , a] pyrimidinium salts in the synthesis of substituted -aminoimidazoles appeared to give a strong colour when reacted with primary and secondary amines. due to the strong colour change of amino functionalised resins after reaction with the "desc" reagent ( ), a protocol for the detection of resin bound primary and secondary amines has been developed. the "desc" test is a new, practically, simple, quick and reliable test for the visual detection of resin bound primary and secondary amines in a sensitive way. the "desc" reagent is accessible from the cheap starting materials and can be prepared in two steps. tetrafl uoroborates of chiral n-triazinylammonium salts were found stable and useful in enantioselective (ee up to %) peptide synthesis from racemic amino acids in solution. several chiral n-triazinylammonium tetrafl uoroborates were obtained as stable l and/or d selective coupling reagents ( ) . broad range of common amines (strychnine, brucine, sparteine etc.) were found useful as a chiral auxiliary, opening access to amino acids of both required confi gurations. we have attempted to expand the scope of application of this family of reagents expecting their effi ciency in enantiodifferentiating reactions in solid phase peptide synthesis (spps). tetrafl uoroborates of chiral n-triazinylammonium salts were obtained by treatment -chloro- , -dimethoxy- , , -triazine with tetrafl uoroborates of appropriate tertiary amines in the presence of sodium bicarbonate ( ) . enantiodifferentiating reagents were used in synthesis on wang resin under classic condition for triazine based coupling reagents ( ) . the most important advantage of application of chiral n-triazinylammonium tetrafl uoroborates is the predictability of confi guration and repeatability of enantiomeric purity of incorporated amino acid. even if only threefold excess of racemic carboxylic acid, enantiomeric purity of coupling products in spps reached ee - . the demand for diversity of non-proteinogenic amino acids, willingly used as new building blocks, is severely restricted by laborious procedures leading to enantiomerically homogeneous individuals. typical sources such as isolation from natural products, biotechnological methodology, even the asymmetric syntheses posses a limited value because complex or tedious synthetic procedures or limited access to the pool of chiral auxiliaries. herein we present the novel, general approach allowing enantioselective incorporation of any enantiomer of amino acids directly from racemic substrate. according to our procedure, the chiral auxiliary is used only at the stage of enantioselective activation. the departure of chiral auxiliary yields in the traceless way, the activated derivative of n-protected amino acid identical with those which is obtained with the well known, classic, achiral triazine coupling reagent ( ) . therefore, the traceless chiral coupling reagents can be successfully applied directly in the coupling stage without additional studies of their reactivity. several chiral n-triazinylammonium tetrafl uoroborates were obtained as stable l and/or d selective coupling reagents. broad range of common amines (strychnine, brucine, sparteine etc.) were found useful as a chiral auxiliary, opening access to amino acids of both required confi gurations. the most important advantage of application of chiral n-triazinylammonium tetrafl uoroborates is the repeatability and predictability of enantiomeric purity and confi guration of incorporated amino acid. freeman, noam s.; hurevich, mattan; gilon, chaim hebrew university jerusalem, israel azapeptides are peptide analogues in which one or more of thecarbons, bearing the side chain residues, has been replaced by a nitrogen atom. azaamino acid residues conserve the pharmacophores necessary for biological activity while inducing conformational changes and increased resistance to proteolitic degradation. these properties make azapeptides an attractive tool for structure-activity relationship studies and drug design. a general approach for solid phase synthesis of azapeptides has been developed based on the in-situ activation of n- -( , dimethoxyphenyl)propan- -yloxycarbonyl (ddz), n €™-substituted hydrazines, with phosgene, followed by introduction to n-terminus of resin-bound peptide. the ddz-aza-amino building units include aliphatic, aromatic and functionalized side chains, protected for synthesis by the fmoc strategy. solid-phase azapeptide synthesis is demonstrated including selective mild deprotection of ddz with mg(clo ) and coupling of the next amino acid with triphosgene. the mild ddz deprotection is also orthogonal with boc chemistry. we describe the synthesis of n €™-substituted ddz protected hydrazines which have wide applications in the synthesis of azapeptides as well as in general synthesis of substituted hydrazines and aza containing peptidomimetics. ddz protected hydrazines offer the possibility to construct novel and unique drug-candidate structures such as branched and cyclic azapeptides. fast improved synthesis of insulin-like peptides barlos the a-and b-chains of insulin-like peptides were synthesized by various methods. the best results were achieved by dividing the sequences of the a-chain into three protected fragments and that of the b-chain into two fragments. the fragments were prepared on -chlorotrityl resin and/ or -methoxybenzhydryl resin using fmoc-amino acids. condensation of the fragments was carried out either by solution phase or solid phase techniques. the joining of the a and b chains was also studied using either biomimetic or chemoselective oxidative folding methods. a new approach for amides and peptides chemical synthesis by means of phosphonic acid/alkylene oxide chemistry few different derivatives of l-phe were synthesized, using originally discovered method of phosphonic acid/alkylene oxide chemistry. this method provides the successful synthesis of variety amides and dipeptides without preliminary protection of alfa-nh group of lamino acids. the supposed mechanism displays the formation of nphospholane carboxyanhydride ( -oxy- -aza- -phospholane- -one), or an o-hydroxypropyl h-phosphonate salt of an amide of l-phe. the nphospholane carboxyanhydride is protected at the n-terminus, as well as is activated at the c-terminus. this pathway favors the desired reaction with a variety of nucleophiles from the n-to c-terminus. this method also allows the synthesis of different esters of amino acids employing alcohols as nucleophiles previously described by us ( ). formation of disulfi de bonds in synthetic peptides is one of the more challenging transformations to achieve in peptide chemistry, in view of possible formation of oligomeric by-products and other side reactions, as well as occasional solubility problems in aqueous oxidizing media. the recently introduced polymer-supported oxidant, clear-ox™, has proven to be a very valuable tool in the preparation of disulfi de-bridged peptides. [ , ] oxidations using clear-ox were carried out at ph ranging from to in water/acetonitrile solutions, with concentrations - times higher than comparable solution oxidations. the latest progress in the preparation of various monocyclic and bicyclic peptides will be presented. the amyloid -peptide (a ) is believed to play a causal role in alzheimer's disease (ad). one of the hypotheses of a neurotoxicity is that it induces the generation of reactive oxygen species (ros) and hydrogen peroxide (h o ) formation by binding to metals such as copper and iron. it is hypothesized that the sole tyrosine residue plays an important role in a -mediated toxicity, due to its ability to form a dityrosine cross-link from tyrosyl radicals generated in the highly oxidative environment in the brain. hence, studies of the dityrosine cross-linked a peptide dimers would increase our understanding of the oxidative alteration and dimerisation of a in amyloid formation and ad related neurodegeneration. we are investigating the synthesis of the a peptide dimers containing the dityrosine cross-link. dityrosine, suitably protected for solid-phase peptide synthesis (spps), has been prepared from iodotyrosine using a miyaura borylation-suzuki coupling method. studies on the synthesis of dityrosine-linked peptide dimers through incorporation of dityrosine in spps are underway. initial model studies employing , -diaminopimelic acid (dap) have validated this approach. preparation of the model daplinked a peptide dimers will be discussed, as well as progress towards the dityrosine-linked a peptide dimers. nepomniaschiy, natalia; brik, ashraf ben-gurion university of the negev, israel the staudinger reaction, discovered nearly a century ago, occurs between a phosphine and an azide to form an aza-ylide. this transformation was further explored and developed, leading to several reactions of highly synthetic importance. traceless staudinger ligation is one example of such modifi cations, in which an ester moiety is placed within the phosphine structure to capture the nucleophilic aza-ylide, by intramolecular cyclization, leading to a stable amide bond. while the aza-ylide intermediate is known to be stable in organic solvents, it tends to hydrolyze rapidly under aqueous media to furnish the primary amine product. in the traceless staudinger ligation, however, the reduction of the azide to amine is a competing side reaction, as it would reverse the capture step leading to two peptide fragments. we have found that such reduction step would be benefi cial if an electrophile and an azide (rather than the phosphine) are placed within the switch peptide system. investigating various reducing reagents led us to the discovery that tris( -carboxyethyl) phosphine hydrochloride (tcep) is excellent azide reducing reagent. both the reduction and the acyl transfer steps are rapid and occur in a few minutes. applying the tcep-mediated triggering in switch peptides, derived from the a to understand the currently unclear processes of pathological folding, self-assembly, and aggregation of amyloid peptide will also be reported. fast fmoc-deprotection reagent for peptide synthesis diffi cult sequences have long been known in solid phase peptide synthesis, theses sequences can experience sever aggregation both during synthesis and under purifi cation. the aggregation is believed to depend mainly on -sheet formation or/ and hydrophobic properties of the peptide. now a days there are several strategies to circumvent theses problems, one of the most successful is to incorporate a backbone protective group as the hmb group, develop by johnson and co-worker( ), and thereby prevent aggregation due to -sheet formation. an additional charge will increase the solubility of peptides towards aqueous solutions and thereby facilitate the purifi cation by hplc (using acetonitrile and water system) and analysis (by maldi-tof mass spectrometer) of the peptide. here we present the nmec (n-methyl-n-[ -(methylamino)ethyl] carbamoyl) group( ) that is a orthogonal protective group for tyrosine side chain and the hydroxyl moiety of the hmb group. the nmec group is fmoc compatible and is protected during the synthesis with a boc group. the fi nal cleavage from the resin with tfa renders the amine of the nmec group protonated and will increase the solubility of the peptide during purifi cation and analysis. the nmec group can be easily removed with a mild alkaline treatment by a cyclization elimination reaction. the nmec group has been employed in the synthesis of diffi cult and hydrophobic peptides as the membrane spanning sequence of the calciton receptor, amyloid ( - ) and amyloid ( - ) with success. [ , ] . for example, infl uenza a virus-related peptide (h-gilgfvftl-h) with a diffi cult sequence was synthesized using o-acyl isodipeptide unit. analysis of the crude peptide revealed high purity of the product with no by-product derived from the diffi cult sequence or epimerization. using ch cl as solvent in coupling the isodipeptide unit, a - was also synthesized with almost no major side reaction .. racemization-free segment condensation method was developed by employing n-segments possessing a c-terminal urethane-protected o-acyl ser/thr residues .. the synthesis of long peptides/proteins by racemization-free segment condensation has thus become possible at ser/thr residues and not only c-terminal gly/pro residues. growing interest to peptide substrates, inhibitors and other peptidomimetics required new highly effective synthetic techniques. the important goal of enzymatic peptide bond formation is the optical purity of the peptide, which facilitates the product isolation. thus using enzymatic condensation as a last step in peptidomimetics production is preferable. fixing of enzymes on/in suitable insoluble supports has many advantages: high operational stability, ease of separation, possibility of recycling and improved activity in low water media. chitosan, natural hydrophilic polysaccharide, was used as a matrix as it has distinct advantages over the other supports due to (a) its renewable nature -it is available in large quantities as a waste product from fi shing industry and (b) its excellent fi lm-forming ability, allowing attachment to reactor walls for advanced processing. serine proteases subtilisin and chymotrypsin, and cysteine protease papain were immobilized onto chitosan. the fi lms of biocatalysts were prepared by drying the mixed solution of chitosan and enzyme in acetate buffer ph . . treatment with glutaraldehyde was found to give material with high stability and good mechanical properties. the obtained biocomposites showed high amidase activity against specifi c chromogenic peptide substrates and protein substrate azacasein. immobilized subtilisin and chymotrypsin also possess esterase activity against p-nitrophenyl acetate. the longterm storage in aqueous buffer and acetonitrile had a little effect on the hydrolytic activity of subtilisin-based biocomposite. the dependence of subtilisin/chitosan hydrolytic activity on temperature and ph was studied. obtained samples possessed high synthetic activity and were capable to catalyze peptide bond formation in dmfa/mecn mixture in reaction zaalome+fpna zaalfpna for subtilisin, zaaome+lpna zaalpna for papain. the product yield reached - % in h. acknowledgement: this work was supported by rfbr - - synthetic chemistry or on solid phase and b) the chemical ligation of the [cys (acm)]-( - )-thioester with the [cys (acm)]-( - ) segment, both deprotected and hplc purifi ed. the required intermediate peptides were prepared by optimized fmoc-based methods either stepwise or by convergent synthesis. for the formation of the two disulfi de bridges a two step procedure was investigated, involving a dmso oxidation step to form the cys -cys linkage, followed by iodine oxidation to form the cys -cys bond. native chemical ligation at valine haase, christian; rohde, heike; seitz, oliver humboldt-universität zu berlin, germany native chemical ligation is perhaps the most useful technique for peptide segment coupling in water. ( ) a c-terminal peptide thioester reacts with an n-terminal cysteine. the requirement for this rare amino acid represents the major bottle neck to the synthesis of native proteins. several strategies have been developed to overcome this limitation. in the extended ligation, n-terminally attached auxiliaries allow access to other ligation junctions by mimicking the cysteine-thiol moiety. the ligation-desulfurization approach employs -thiol amino acids for fragment coupling followed by sulphur removal. herein, cysteine acts as precursor of the abundant alanine( ) and phenylalanine can be obtained by using its -mercapto derivative. ( ) . we demonstrate the application of penicillamine in the generation of valine junctions employing the ligation-desulfurization approach. the , -dimethylcysteine building block is commercially available with various protecting group patterns suitable for routine solid phase synthesis of peptides. the ligation at penicillamine proceeded surprisingly fast despite the steric demand at the thiol group. even leu-val ligation sites, which appear in hydrophobic peptide segments, are accessible. we also present an improved method for achieving metal-free desulfurization and show applications in the synthesis of valine-containing peptides.( ) references: . p. e. dawson the application of n-alkyl cysteine (nac)-assisted thioesterifi cation reaction to the synthesis of polypeptides by the thioester method azide as a protecting group for lysine side chains on the solid phase peptide synthesis oriented toward the peptide condensation by the thioester method peptide ligation chemistry has been developed based on the use of peptide thioester as a building block in the thioester method ( ) and native chemical ligation ( ) . we found that a cysteine-containing peptide is transformed into the corresponding s-peptide (peptide thioester) by the n to s acyl shift reaction ( ). on the other hand, in , zanotti et al. reported that a diketopiperazine thioester, cyclo(-cys(coch ph)-pro-) ( ) was formed when a dipeptide p-nitrophenyl ester, phch co-cys(st-bu)-pro-onp ( ), was treated under reductive aqueous conditions ( ). thioester would be formed via intramolecular n-s acyl shift reaction followed by diketopiperazine formation. based on these observations, we designed a cysteinyl prolyl ester (cpe) autoactivating unit for preparation of the peptide thioester and for the peptide ligation [ , ] . a peptide containing the cpe unit, peptide-cpe , was transformed into a peptide thioester of diketopiperazine, cyclo(-cys(peptide)-pro-) , then thiol-thioester exchange reaction with an external thiol produced the peptide thioester and a diketopiperazine, cyclo(-cys-pro-) ( ) . the poster abstracts peptide-cpe was also able to ligate with a cysteinyl peptide , via the thioester in one-pot, to give a polypeptide . ( ) has enabled the synthesis of entire proteins including those carrying posttranslational modifi cations. one of the most frequently encountered modifi cation of eukaryotic secretory and cell surface proteins is the attachment of oligosaccharides to asparagine residues (n-glycosylation). despite many efforts in this fi eld the function of nglycosyation is poorly understood, which is mainly caused by the lack of pure glycoproteins. since purifi cation of natural glycoproteins is quite tedious due to heterogeneity in the sugar part, the total synthesis of homogeneous glycoproteins has become an attractive target ( ) . we have addressed this topic by choosing rnase c as a model glycoprotein. instead of the oligomannosidic type rnase c is containing a complex type n-glycan. for synthetic reasons ligations had to be conducted in a sequential manner ( ) by fi rst reacting the recombinant - cys-segment ( ) with an n-terminally protected and glycosylated thioester - .. selective deprotection of the ligation product - and ligation with synthetic thioester - in a one pot manner was accompanied by unexpected side reactions. these drawbacks were fi nally overcome leading to full-length glycosylated rnase c displaying enzymatic activity. a new pseudo-native ligation: multiple, successive azidealkyne cycloadditions. ( ) the reaction has become a classics. as triazoles are stable to acid and basic hydrolysis, and reductive and oxidative conditions, the reaction has been widely used in chemistry and biochemistry, proving to be an useful tool in combinatorial, bioconjugate or medicinal chemistry. the reaction has been applied in the peptide fi eld as an easy way to synthesize cyclic, labelled and side chain modifi ed peptides including effi cient synthesis of pseudo-glycopeptides. conformational and structural studies prove that the triazole ring is an excellent trans-amide surrogate and thus, can be considered as a new pseudo-native linkage. ( ) in order to explore the potential of the reaction as a way to successively assemble several peptide chains to generate mimics of proteins, we have elaborated a new strategy for consecutive triazole formation based on a semi-orthogonal alkyne protection scheme. ( ) so far, we have improved our fi rst one-pot successive cycloaddition approach performing the fi rst three successive cuaac in mild conditions thanks to a highly selective deprotection of two different alkyne groups. the application of this strategy represents a new promising method for the chemoselective pseudo-native ligation dedicated to the synthesis of protein chimera or for the decoration of molecular templates. solid phase synthesis using lightly crosslinked polystyrene supports has proved to be a successful route to the manufacture of peptides. the methodology introduced by merrifi eld nearly fi fty years ago has remained largely unchanged. during the last twenty years or so, it has been argued that incorporating a hydrophilic polymer within the conventional hydrophobic polystyrene matrix is benefi cial. others have suggested that polyamide or polyether-based resins may prove to be superior to polystyrene. despite this, large-scale peptide manufacture has generally relied on traditional polystyrene supports. this is due in part to the diffi culty in producing composite supports economically in large volumes, but also due to the handling diffi culties that are often associated with very high swelling polar polymers. other problems arise from the fact that many of these types of resin are relatively low loading and so yields are greatly diminished. in this poster we offer a solution to these problems: a polystyrene derived support which is modifi ed to create economical, amphipathic resins suitable for large scale peptide synthesis. attachment of appropriate handles or linkers enables both peptide acids and peptide amides to be produced. the synthesis of a variety of peptides is demonstrated. peptide synthesis in water using boc-amino acids nanoparticles reactants. here, we studied in-water solution-phase method using water-dispersible nanoparticulate boc-amino acids, which are the most common building blocks but are diffi cult to use for peptide synthesis in water. water-dispersible boc-amino acid nanoparticles were prepared by pulverization using a planetary ball mill in the presence of peg, and the size of the resulting water-dispersible nanoparticles was determined by dynamic light scattering analysis. the scanning electron microscopy image of water-dispersible boc-phe-oh nanoparticles also revealed nanosize particles. we studied in-water coupling reaction using waterdispersible boc-amino acid nanoparticles, and leu-enkephalinamide was successfully synthesized in water according to the boc chemistry. an alternative way for conopeptide formation kádár, kinga ; panyi, györgy ; tóth, gábor k. university of szeged, hungary; university of debrecen, hungary the chemistry used to oxidize the free thiol bonds to the corresponding disulfi de bond in a controlled fashion remains a signifi cant challenge in spite of many advances in peptide chemistry. the primary reason of this lies in the diffi culties involved in the formation of multiple regioselective disulfi de bonds. in this work we focused on the elucidation of synthetic strategies for the preparation of multiple disulfi de containing peptide venoms regulating the ion channels of immune cells-anuroctoxin and tc -, and a neuropeptide with regulatory functions-orexin a. for the synthesis of these naturally occuring cys-rich peptides we had chosen the oxidative folding being the most simple of the methods available. in the case of anuroctoxin we isolated the native form of the peptide toxin with high selectivity and we optimized the folding conditions, so within an hour the majority of the linear peptide folds in the cyclic form with all four disulfi de bonds in the correct form. the regioselectivity of the isolated isomer was verifi ed with biological measurements. for the synthesis of orexin a we optimized the folding conditions and, using a polymer-supported oxidant-the clear-ox resin-, within two hours we could isolate the correctly folded isomer as the major reaction product. the correct structure was proven by coelution of the isolated isomer and the commercially available orexin a. but oxidative folding does not always give the correctly folded isomer. the best proof for this is the tc scorpion toxin which albeit our tryings always gave the misfolded isomers if we used the linear, unprotected peptide. therefore a new chemical synthesis using different side-chain protection needs to be taken into consideration. the synthesis of orthogonally protected tc and its use for the preparation of the correctly folded peptide to be underway. poster abstracts have fundamental importance in biological recognition processes. one of the most challenging task among of them the rational preparation of the glycosylated peptides especially having oligosaccharide moieties. there are two main strategies for the synthesis of glycopeptides: the synthon and global (convergent) method. both of them can be implemented in liquid or solid-phase. since the glycosylation could appear on o and n atoms of the amino acid side-chain, due to the different reactivity of the glycosidic linkage different chemical strategies will necessitate. in this presentation we compare several chemical strategies for the preparation of two model peptides (leu-lys-asn*-gly-gly-pro, gly-val-glu-asp-ile-ser*-gly-leu-pro-ser-gly,*site of glycosylation). as glyco-part several mono, di and trisaccharide including chitobiose, galactosilxilose, mannosil-n-acetyl-glycosil-n-acetyl-glucosamine were used and several of the used strategies led to successful preparation of these glycoconjugates. site-specifi c pegylation of human igg -fab using a rationally designed trypsin variant in the present contribution we report on a novel, highly selective biocatalytic method enabling c-terminal modifi cations of proteins with artifi cial functionalities under native state conditions. the approach is based on the fourfold trypsin variant k e/n h/e h/d k which was generated by site directed mutagenesis and initially specifi ed in terms of selectivity and activity using a peptide library of the general structure bz aax aa x bb x cc aag-oh. the trypsin variant was found to bear a restricted proteolytic activity towards the rare recognition sequence yrh with an occurance of less than . % in native proteins. the specifi city is zinc ion dependent due to an artifi cial metal binding site in the s ´-subsite ( ). placing of the recognition sequence at the cterminal region of respective proteins via standard mutagenesis protocols provides suitable precursor targets for site-specifi c modifi cation via a transamidation reaction. due to the great demand for polymer-modifi ed antibody fragments for pharmaceutical purposes we evaluated the function of the approach on example of the c-terminal pegylation (peg...polyethylene glycol) of the human igg fab fragment. the fragment itself was expressed with the recognition sequence and an additional strep-fusion enabling effi cient purifi cation. the derivatization of the antibody fragment with kda peg via the trypsin variant (mw ~ kda) proceeds effi ciently with a total yield of isolated modifi ed protein of %. interestingly, no undesired cleavage reactions could be detected leading to fully active modifi ed antibody fragment. references: . willett, w.s., brinen, l.s. et al. ( ) . "delocalizing trypsin specifi city with metal activation." biochemistry ( ): - . -chloro- , -bis-( , , -trifl uoroethoxy)- , , -triazine and n- , -( , , -trifl uoroethoxy)- , , -triazin- yl)ammonium tetrafl uoroborates as highly effi cient coupling reagents jastrzabek, konrad; kolesinska, beata; kaminski, zbigniew, j. we expected that modifi cation of substituents in the triazine ring improve activity, stability and solubility of new triazine based coupling reagents. in order to increase activity we prepared -chloro- , -( , , trifl uoroethoxy)- , , -triazine by treatment of cyanuric chloride with , , -trifl uoroethanol. taking advantage of modular structure of triazine coupling reagents the entire family of n-( , -( , , -trifl uoroethoxy- , , -triazinyl- )ammonium tetrafl uoroborates have been obtained. effi cient coupling reagents should be useful for amide bond formation between broad range of substrates, work in stoichiometric quantities, be soluble and stable in most of the solvents. it should function effi ciently in solution as well as in spps, to get high purity crude products and minimize racemization of the products. we found n-( , -( , , trifl uoroethoxy- , , -triazinyl- )ammonium tetrafl uoroborates useful for activation of carboxylic components. the participation of triazine "superactive ester" as intermediate in the condensation has been proved in the model experiments. utility of reagents n-( , -( , , trifl uoroethoxy- , , -triazinyl- )ammonium tetrafl uoroborates were confi rmed by peptide synthesis in solution in high yield. in our study we focused our attention on the performance (in terms of purity of the crude and extent of racemization) testing the solid phase synthesis of acp( - ) and model peptides with aibaib fragment, which are a good example of diffi cult peptide sequence. purifi cation of chemically synthesised proteins by use of cleavable imac-based n -terminal protein protecting groups (tags) ( ), there remains a challenge in terms of time and cost, for the separation of impurities from the chemically synthesised product in spps. this is particularly acute in the case of protein synthesis where the crude product mixture contains capped (acetylated) truncates of comparable size and structure to the protein product. such a common situation results in extensive, and expensive, purifi cation techniques such as large scale hplc. to this end we designed a hydrophobic tag, tbfmoc ( ) , which incorporated the base-labile characteristics of the fmoc ( ) group. the tbfmoc group causes retention on hydrophobic columns and hence separation from untagged impurities. although this was effective, we were of the opinion that a complementary tag was required which would have the characteristics of metal-complexation and cleavage by -elimination for subsequent removal of the tag from the protein product after imac. considerations implicit in the design of such an n -terminal protecting group, or tag, will be discussed , and applications to the chemical synthesis of chemokines will be dealt with in the presentation. in last few decades, acridines which are known as anticancer, antimicrobal and antiviral agents have been in the center of interest of scientists. the heterocyclic molecules demonstrate a noteworthy group of compounds which interact with biological targets e.g. topoisomerase i, ii [ , ] . we wish to pay our attention to a new group of tuftsin-acridine conjugates. tuftsin offers a wide range of biological activities such as supporting of immune system, bactericidal, tumoricidal activity. however tuftsin is unstable in plasma which reduces its effi cacy. that is the reason for search of new analogues that were more resistance to proteolysis degradation ( ). tuftsin-acridine conjugates were synthesized using a fmoc solid phase strategy. the elongation of peptide chain was based on twostep procedure: deprotection and coupling reaction with dic and the additive of hobt in dmf/dcm/nmp mixture. tuftsin analogues were modifi ed at -amino group of lysine via introduction of the simple amino acids to obtain isopeptide bond. tuftsin analogues were conjugated to the acridine molecule via fl exible linkers. the carboxylic group of linker was connected to n-terminal group of peptide-resin using standard method for amide bond formation. the coupling reaction was performed with peptide-resin and the -fold excess of -nitro-acridine derivative using tbtu, hobt in the presence of diea in dmf for h. simultaneous deprotection of peptide side chain and cleavage from resin was done with the tfa cocktail. final products were purifi ed by spe and characterized by elemental analysis, ms and h-nmr spectroscopy. the effect of microwave irradiation under controlled temperature conditions on the solid-phase synthesis of peptides was investigated. for optimization studies a model peptide (h-gly-ile-leu-thr-val-ser-val-ala-val-oh) was selected which suffers from poor synthetic effi ciency under standard spps conditions. synthesis of the nonapeptide was performed using various combinations of solid supports (polysterene, tentagel, chemmatrix) and solvents employing fmoc/but orthogonal protection strategy. applying controlled microwave heating, the reaction times were signifi cantly reduced while maintaining a high purity of crude product with no racemization being observed. the optimized microwave synthesis method was succesfully applied for longer, aggregated peptides. microwave coupling and cleveage were accomplished in a dedicated reactor setup that allowed accurate internal reaction temperature measurment using a fi ber-optic probe system. comparison studies between microwave-and conventionally heated reactions will be presented. during the last few years microwave assisted peptide synthesis has became popular and it was reported to be useful in some cases. there is also an ongoing discussion about an additional "microwave effect". based on this background we have synthesized several model peptides on a microwave synthesizer and compared with the synthesis on conventional batch and continuous fl ow synthesizers with and without microwave irradiation. during this investigation we have also compared reaction rates and purity of the peptides synthesized under the infl uence of microwave irradiation or conventional heating on this different synthesizer systems by various conditions. in no case we can fi nd any additional postulated "microwave effect". the results of this investigation will be presented and discussed. conotoxins form a large family of peptide toxins from cone snail venoms that act on a broad spectrum of ion channels and receptors. the subgroup -conotoxins specifi cally and selectively bind to subtypes of nicotinic acetylcholine receptors (nachrs), which are targets for treatment of several neurological disorders. the aim of this work is to develop an improved method able to generate conotoxins in high yield and purity. this will overcome a key barrier currently preventing the effi cient synthesis of small focused libraries in order to investigate the structureactivity relationship (sars) of those peptides. the development of general synthetic strategies for the preparation of conotoxins and analogues are essential to effi ciently approach important questions within the area of neurobiology and for the development of novel drugs for treatment of various neurological diseases. a new and highly effi cient synthetic strategy for the synthesis of alpha-conotoxin-mii has been developed. this strategy combine solid phase synthesis with microwave assisted heating to produce the two disulfi de bonds peptide in high yield and purity. we fi rst report here the use of microwave assistance in order to form a disulfi de loop. this technique demonstrates the advantage of preparing the fi rst disulfi de bridge while the peptides are resin bound. this step is critical to provide the dicyclic native peptide, that was performed by a followed classical in solution oxidation by iodine strategy. an enhanced total procedure for the synthesis of ctxmii is recommended, which can be effi cient applied at several small disulfi de rich peptides of biological importance. solid phase peptide synthesis in aqueous environment using microwave assistance heating galanis, athanassios s. since the fi rst reports on the use of microwave heating more than years ago, microwave-assisted organic synthesis (maos) has become an important tool for rapid and effi cient synthesis of organic molecules. microwave assisted heating has been further applied to peptide synthesis in order to accelerate the rate of synthesis and to improve the yields for the synthesis of diffi cult sequences. as far as water as solvent is concerned, numerous recent publications report the combination of water as an environmentally benign solvent for chemical transformations with the use of microwave irradiation as an effi cient heating method. we report herein, the combination of the microwave-assisted heating and the use of water as solvent for solid phase peptide synthesis. a variety of common amino acids derivatives and coupling reagents have been studied in order to optimize coupling reactions in water by microwave-assisted heating. we also describe the total synthesis of a small peptide using water as solvent. the solid-phase synthesis using the environmental friendly aqueous medium dramatically reduces the cost of the synthesis and could be broadly applied in research or in industrial production of peptides. vanier, grace; collins, jr., michael; singh, sandeep; collins, jonathan cem corporation, united states the application of microwave energy for solid phase peptide synthesis (spps) represents a major breakthrough for overcoming incomplete and slow reactions typical of conventional spps. microwave energy has been applied successfully in a manual and automated approach for enhancing synthesis of peptides and peptidomimetics. common side reactions such as racemization and aspartamide formation have been studied and shown to be easily controllable with optimized methods that can be applied routinely. we will present the latest research on improving the synthesis of diffi cult peptides with microwave energy. synthetic antifreeze glycopeptides and analogues: synthesis, structural analysis, and functional studies norgren, anna s. glycosylated peptides are involved in various biological processes such cell adhesion and differentiation. in contrast to mucin-type oglycan peptides which are present on the membrane of mammalian cells antifreeze glycopeptides (afgps) are a little investigated example of glycosylated peptides containing similar components. afgps usually consist of a varying number of repeating units of (ala-ala-thr) with minor sequence variations and the threonine hydroxyl oxygen glycosylated with the disaccharide -d-galactosyl-( - )--n-acetyl-d-galactosamine. antifreeze activity has been proven by different experimental observations like suppression of recrystallisation and ice nucleation, thermal hysteresis and change of the crystal habitus. although it is known that the n-acetyl group at the c position of the galactosamine, the -confi gured glycosidic bond to the threonine hydroxyl group and the -methyl group are essential, the adsorption mechanism is not yet understood. ( ) . in contrast to fragment condensation, solid phase peptide synthesis gives the possibility to prepare one defi ned product and to introduce structure inducing amino acids such as proline. the disadvantage of spps with the bulky glycosylated amino acids is the low coupling effi ciency. this problem was overcome by using more active coupling reagents and microwave-enhanced methods during coupling leading to suffi cient coupling effi ciency without having to apply great excess of the glycosylated amino acid. after purifi cation the peptide structure was examined by cd and nmr in water and dmso at different temperatures. additionally, the substances were microphysically analysed according to their recrystalisation inhibition activity and their infl uence on the crystal habitus. microwave technology applied to spps has been recently proposed as valid support to the enhancement of coupling rates. we also used microwave energy in conjugation process between polyelectrolytepeptide bioconjugation with edc, hbtu. the use of peptide epitops of viruses particles in the composition of polymeric conjugates as vaccine has several potential advantages over whole viral or bacterial preparation. recently, our group report a novel approach to a totally synthetic vaccine which consists of fmdv (foot and mouth disease virus) vp peptides, prepared by covalent conjugation of peptide biomolecules with membrane active carbochain polyelectrolytes in the present study, peptide epitops of vp protein both - (p ) amino acid residues (ser-lys-tyr-ser-thr-thr-gly-glu-arg-thr-arg-thr-arg-gly-asp-leu-gly-ala-leu-ala-ala-arg-val-ala-thr-gln-leu-pro-ala) and triptophan (trp) containing on the n terminus - amino acid residues (trp- - ) (p ) were synthesized by using the microwave assisted solid-phase methods. synthesis of peptides were performed by microwave assisted spps. peptides characterized by lc-ms and purifi ed by rp-hplc. bioconjugation between polyelectrolytespeptide were synthesized by two different microwave assisted method. the fi rst one is classical carbodiimid activating method. the second one is hbtu activating method. the second method is a novel and effective method for bioconjugation process. peptides and polyelectrolytespeptide bioconjugates analysed and comparised by gpc system with four dedector (uv. refractive index, light scattering, viscosity). microwave-assisted solid-phase synthesis of peptide probes to detect specifi c biomarkers: shifting off limitations affecting conventional synthetic strategies biomarkers play a key role in development of diagnostic/prognostic tools. in fact, since their involvement in several diseases such as autoimmune diseases (i.e., multiple sclerosis, rheumatoid arthritis) and neurodegenerative diseases as alzheimer's disease, biomarkers represent a great promise for the development and set up of tuned therapies. for autoimmune diseases, we proposed the development of synthetic posttranslational modifi ed peptides as antigenic probes for characterization of specifi c and high affi nity autoantibodies as biomarkers. by an innovative "chemical reverse approach" we propose optimisation of antigenic probes by a statistically signifi cant screening of sera guided by autoantibodies circulating in patients' biological fl uids ( ) . spps via the building block approach is the principal strategy leading to modifi ed peptides. high purity is a condicio sine qua non for effi cient biomarker detection. these syntheses can present several diffi culties as result of internal aggregation of resin-bound peptides during elongation steps, reducing reagents penetration, and signifi cantly decreasing reaction rates in both acylation and deprotection steps. such events strongly affect purity of the crude peptides and therefore, fi nal yield. we demonstrated that application of microwave (mw) energy in spps of modifi ed complex peptide probes exhibits several advantages improving coupling rates, possibly because of the decrease of chain aggregation during the synthesis ( ) . we report the mw-assisted synthetic conditions of diffi cult peptides (i.e. glycosylated, citrullinated, multiple antigenic, amyloidogenic peptides, etc.) by which we strongly improved preparation of diagnostic/prognostic probes in terms of crude purity, fi nal yield, and time-consuming compared to conventional protocols. fidan, zerrin; volkmer, rudolf charité berlin, institute for med. immunology, germany the alpha-helical coiled-coil structure is a versatile protein-interactiondomain, in which the coiled-coil is composed of at least two righthanded amphipathic alpha-helices, which are coiled up around each other into a left handed supercoil. this widespread structural motif is involved in many biological procedures like transcription, scaffolding or signalling. in addition, the most characteristic and important identifying feature of a coiled-coil is the recurrence of a periodic heptad repeat sequence. coiled-coils are able to form complexes up to heptamers of different orientation. based on this signifi cant occurrence and also their structure coiled-coil peptides have the ability to function as molecular recognition molecules. our goal is to use self-associating coiled-coil sequences as molecular building blocks (lego brick). the ligation of desired functionalities on coiled-coil sequences opens up the opportunity to add different functionalities on artifi cial complex peptide molecules that stick together via coiled-coil moieties. this makes coiled-coil sequences to useful tools during complex oligopeptide assembly. we want to present novel chimeric oligopeptides which are build up by two segments, one responsible for association the other for functionality. as a model for the association segment several gcn -leucine zipper mutants ( ) and as functionality segments amongst others ww domains were used. both segments were linked by the native chemical ligation approach. we will present in detail the synthesis of a gcn -leucine zipper mutant and also the following c-terminal thioester formation for the subsequent ligation step. furthermore, we will present the synthesis of the functional device, a ww-domain with a n-terminal cystein residue and also the native chemical ligation of both synthesized peptide fragments supported by hplc and mass-chromatography. dialysis-related amyloidosis, a disease arising from long-term dialysis is characterised by gradual accumulation of -microglobulin ( m) amyloid fi brils in bones and ligaments. although -m is known to form amyloid fi brils in vitro under acidic ph conditions, seeding of preformed amyloid fi brils or as an effect of ionic strength, the mechanism underlying aggregation of soluble -m into insoluble fi brils under physiological conditions is largely unknown. interestingly, eakin and co. recently showed that the chemical basis of the amyloidogenesis process is a backbone isomerisation of the conserved pro . our aim is to understand the molecular mechanism associated with -m amyloidogenesis using fourier transformed infrared (ftir) spectroscopy and site-directed-isotope labelling, a method in which the vibration of the labelled residue is shifted from its original position in the spectrum. we already demonstrated that ftir spectroscopy along with site directed-isotope-labelling is a promising approach to obtain information on the microenvironment of tyrosine side chain residues. to use this approach in the case of -m amyloidogenesis, we decided to synthesise an isotopically labelled -m at crucial residues such as pro that should undergo drastic variations in their microenvironments during the misfolding. with its residues, -m is over the limits of a reasonable synthesis using spps but the two suitably positioned cysteines in its sequence offer the possibility of a chemical synthesis using native chemical ligation. thus, we were able to realise the chemical synthesis of an isotopically labelled -m in a good yield using a three segments strategy with disconnections at the two cysteines and thiazolidine as a masked cysteine for the middle segment. the synthetic -m obtained was in full agreement with the characteristic -m ftir spectra. a novel cyanophycin synthetase from thermosynechococcus elongatus bp- catalyzes non-primer-dependent cyanophycin synthesis. cyanophycin (multi-l-arginyl-poly[l-aspartic acid]) is synthesized by cyanophycin synthetase (cpha). it was believed that cyanophycin synthetase requires asp, arg, atp, mg + and primer (low-molecular mass cyanophycin) for cyanophycin synthesis and catalyzes the elongation of low-molecular mass cyanophycin. despite extensive studies of cyanophycin, the mechanism of primer supply is still unclear. in the present study, we searched for a cyanophycin synthetase that synthesizes cyanophycin from asp and arg without added primer in vitro. cyanophycin synthetase from thermosynechococcus elongatus bp- (tlr protein) was produced by an escherichia coli geneexpression system as a c-terminal his-tagged protein. we found that tlr protein synthesized cyanophycin without added primer. the tlr protein had strict substrate specifi city and used only asp and arg as substrates. the optimal ph was . , and mg + or mn + was essential for cyanophycin synthesis. atp could not be substituted by gtp, ctp, or ttp. the molecular mass of the tlr protein as estimated by gelfi ltration chromatography was } kda. thus, the tlr protein appeared to be a homo-tetramer of -kda subunits including the his-tag sequence. the tlr protein had thermal stability and fully retained its activity after a -min incubation at ‹c. additionally, we examined cyanophycin synthesis at ‹c, ‹c, ‹c, and ‹c. sdspolyacrylamide gel electrophoresis showed that the molecular mass of cyanophycin increased with increased reaction temperatures. synthesis of peptide thioester by fmoc chemistry through hydroxyl side chain anchoring barta , ) . azapeptides are usually synthesized on solid phase. a method, permitting the convergent synthesis of azapeptides starting from unprotected fragments, would offer the possibility to study aza amino acids effect in complex polypeptides or proteins. we have focussed our study on ligation reactions leading to the formation of an azagly residue at the ligation point, as gly is a frequent amino acid in peptides or proteins. the fi rst synthetic strategy relies on the reaction between a peptide thioester and a n-terminal azaglycine peptide. experimental conditions were found which permitted the chemoselective formation of azapeptide but with racemisation of the c-terminal amino acid of the thioester fragment. alternately, a synthetic strategy based on the chemistry of the phenylthiocarbonyl group, permitted the successful synthesis of azapeptide without racemisation. ( ), native glycoforms are of therapeutic interest. our retrosynthetic strategy implies sequential native chemical ligation ( ) and the split of il- into three fragments. an n-terminal fragment il- ( - ) thioester and the cysteine fragment il- ( - ) were expressed in e. coli. the carbohydrate carrying fragment il- ( - ) was synthesized via spps. to obtain the recombinant fragments specifi c termini were required. the n-terminal il- ( - ) was fused between two inteins and after expression and refolding from inclusion bodies the target peptide was released as a c-terminal thioester after dual intein cleavage. the cysteine fragment il- ( - ) was expressed as a single intein fusion also leading to inclusion bodies. this enabled the release of il- ( - ) with an n-terminal cysteine after intein cleavage of the refolded fusion protein. the central segment il- ( - ) contains a sugar residue attached to the n-glycosylation-site at asn . this fragment features both a c-terminal thioester and an n-terminal cysteine protected as a thiazolidine. sequential ligations leading to the full length il- glycoprotein will be presented. synthetic chemistry syntheses of shorter fragments which are fi nally linked together ( ) . this "small building block approach" should also simplify synthesis of modifi ed prion protein. in our plan of mouse prion (moprp) synthesis, we have employed consecutive chemical ligations. we have started with moprp( - ) in which it is possible to study native chemical ligation between cysteine in the position of and methionine in the position of ( ) several moprp( - ) peptide thioesters with aryl and alkyl thiols were prepared for further study of the native chemical ligation of moprp( - ) and moprp( - ). the optimal ligation conditions were found by a kinetic study which was carried out in a range of ph and with various thiols. infl uence of electron withdrawing and electron donating groups was studied in both aromatic and aliphatic thiols and it was found that it is possible to affect the ligation by choosing an appropriate thiol. ligation optimal conditions, kinetic study results, and suitability of various thiols for the ligation are discussed. low-cost industrial chemo-enzymatic synthesis of pharmaceutical, nutraceutical and diagnostic oligopeptides the production costs for oligopeptides and derivatives thereof by chemical synthesis are extremely high. therefore dsm pharmaceutical products embarked on a research programme on chemo-enzymatic peptide synthesis. important advantages of this technology are that no expensive stoichiometric coupling reagents nor side-chain protection are required and that no racemisation occurs. focus is on elongation in the n ¨c terminal direction using novel enzymatic c-protecting group interconversion methods. one of the preferred building blocks are amino acid amides, but selective deprotection of peptidic c-terminal amides is a challenge. we discovered that using a peptide amidase c-terminal peptide amides can be directly converted to methyl esters in almost pure methanol. thus, separate enzymatic hydrolysis and reactivation steps are no longer required. other versatile building blocks are amino acid t-butyl esters. we discovered that peptide c-terminal t-butyl esters can be transformed, using commercially available proteases, to activated alkyl esters such as methyl-and benzylesters which can be directly used in another protease-mediated coupling reaction. furthermore, we found that using commercial enzymes side chain unprotected peptides with a free c-carboxyl terminus can be directly transformed to cterminal thioesters and c-terminal aryl amides in the presence of the corresponding thiol and aniline, respectively. finally, a real-life example of large-scale industrial chemo-enzymatic peptide production will be shown. it is known that chiral amino acids, as well as their dipeptides, may catalyze the asymmetric condensation of glycolaldehyde in water. on the basis of the particularly large erythrose enantiomeric excesses (ee) obtained when utilizing the chiral l-val-l-val catalyst and given the possibility of an abundant delivery of other types of amino acids to the early earth, we have studied the catalytic effect on this synthesis of the peptides based on c -methylated -amino acids, such as iva (isovaline or c -methyl, c -aminobutyric acid) and c -methylvaline, ( me)val, that are abundant in meteorites. results of the catalysis experiments showed the all c -methylated peptides to the tetramer level exhibit signifi cant chiral infl uence on the synthesis of tetroses and mimic the effect of the l-val-l-val catalyst in having a larger erythrose ee than threose ee, as well as in their confi guration relationship with the sugars (the product erythrose acquires ee of confi guration opposite to that of the catalyst in case of peptides, while it is the same for amino acids). interestingly, the largest ee ( % for erythrose) was obtained with the iva homo-tetrapeptide under mild conditions. the homo-dipeptides of both iva and ( me)val also produced a signifi cant ee ( % for erythrose) that appears to increase with time. because c -methylated amino acids are non-racemic in meteorites, do poster abstracts not racemize in aqueous environments, and are known to be ( )-helix formers in peptides with as few as four residues, these results suggest that meteoritic, c -methylated, -amino acids may have contributed to molecular evolution upon delivery to the early earth by catalytically transferring their asymmetry to other prebiotic molecules. synthesis' and application of peptide adhesives for wound healing natural polymers with repeating peptide sequences, like spider silk and mussel glue have interesting properties that can be used for biomedical applications. however, the isolation from their natural sources is rather diffi cult and the desire to introduce modifi cations necessitates the development of novel methods to synthesize such polymers with repeating peptide sequences. recently, we have shown that the microwave-assisted copper(i)-catalyzed , -dipolar cycloaddition can be used in the polymerization of the model dipeptide azido-phe-alapropargyl amide. by varying the reaction conditions we could either obtain small cyclic oligomers ( - aas) or linear polymers which consisted up to aas residues ( ). to broaden the scope of this polymerization reaction, two novel biodegradable monomers, azido-phe-ala-lys-propargyl amide and azido-phe-ala-glyc-lys-propargyl amide were polymerized. both monomers are water-soluble and contain recognition sites for the proteases trypsin and chymotrypsin; moreover, the tetrapeptide can be chemically hydrolyzed depending on the ph of the solution. the molecular weight of the polymers could be tailored between - kda ( to aas residues). the enzymatic degradability of the polypeptides was monitored by a ninhydrin-based colorimetric assay and by maldi-tof. the results showed that the peptidic polytriazoles were smoothly degraded by trypsin and chymotrypsin. from these data we can conclude that the microwave-assisted copper(i)-catalyzed , -dipolar cycloaddition reaction is an effective tool to synthesize biodegradable functional polymers which provides new opportunities to design (novel) biomedical materials. details of monomer synthesis, polymerization reactions and degradation studies will be presented. peptide synthesis on cellulose membranes, known as spot synthesis, is an ideal tool to determine biological interactions and/or key positions in proteins. in general we can differ in two kinds of synthetic peptides with different possibilities: over the past years synthetic peptide library techniques have emerged as powerful approaches to determine t-cell epitopes and to specify peptide binding to mhc-i and/or mhc-ii molecules. the sequence-based approach is a straightforward method to directly identify t-cell antigens belonging to a specifi c target (e.g. a virus of interest) without the need of any further deconvolution steps. the entire protein sequence is presented as a set of overlapping peptides (peptide scan), which are subsequently screened for t-cell stimulation. several cd- t-cell epitopes in selected cmv proteins have been identifi ed using standard spps techniques. however, the peptide sequences synthesized by standard spot synthesis can only be cleaved without authentic c-termini (ß-alanine or glycine as c-terminal amino acid). here, we summarized three different established methods to generate cleavable peptides with authentic c-termini in adequate amounts, with suffi cient purity and within a justifi able time-scale. the standard spot synthesis to generate membrane bound peptides use glycine or ß-alanine membranes to map epitopes of b-cells, allergenic proteins, antibodies etc. furthermore, we have also established the "method of inverted peptides" to screen protein domains requiring peptidic ligands with free c-termini such as pdz-or . . domains. another highlight of peptide libraries is the screen of posttranslational modifi cation in proteins by phosphorylation at serine, threonine, and tyrosine residues which plays a role in eukaryotic cell cycle regulation, cell differentiation, apoptosis, or cytoskeletal regulation. labeled peptide libraries, consisting of -helices, -loops and -sheets peptides, have been successfully prepared for use in peptide arrays that act as a protein-detection system and have been applied for proteinrecognition studies [ ] [ ] [ ] . it is known that more than half the proteins in a mammalian cell are post-translationally modifi ed by such processes as phosphorylation, glycosylation, and acetylation. such modifi cations play an important role in various bio-recognition, for instance glyco- proteins are involved in cell-adhesion, infection, and biological protection. hence the designed peptide library used for arrays has been expanded to include fatty acid attached peptides and glycopeptides. fatty acids were easily introduced by conventional fmoc-spps, while the synthesis of glyco-peptides was not easy. in the present paper we describe the effi cient construction of o-glycoside structured and labeled peptides by fmoc-spps using a building block strategy. glycosylated threonine has been used as a building block and was prepared in large amounts. glucose, mannose, galactose and lactose were acetylated and coupled to the hydroxyl group of fmoc-thr in the presence of bf oet . the resulting protected sugar-amino acids were manually assembled on to the peptidyl resin containing a fl uorescent dye using a peti-syzer® (hipep laboratories). after construction of the glycol-peptides the acetyl groups on the sugar were removed by hydrazine hydrate, and the peptides were then cleaved and purifi ed. the resulting glycopeptides were characterized by lcms. the present protocol has been used to prepare ca. one hundred glycopeptides that have been tested against various carbohydrate-binding proteins. parallel small scale peptide synthesis meets a fast, lowcost purifi cation method for the production of high quality peptide microarrays to analyze dna/protein interactions herein, a parallel synthesis in a small scale ( , mol) which takes place in -micro well plates is demonstrated. with regard to a fast but yet effi cient, low-cost purifi cation in a parallel manner a ''fmoc-on'' purifi cation method, which is integrated into the synthesis process, was developed. peptides were cleaved from the resin with their fmoc-group still on. the crude products were transferred by extraction into another -micro well plate fi lled with purifi cation material. purifi cation takes place due to the high affi nity of the terminal fmoc-group to this material. hplc measurement shows a high recovery ( %) and purity ( %). cleavage of the fmoc-group during the purifi cation process is also possible. here, hplc measurement demonstrated a recovery of % and a purity of %. a fully automated synthesis of more than peptides in a -micro well plate combined with a parallel ''fmocon'' purifi cation is shown. the peptides were used for the production of microarrays to analyze dna/protein interactions. the "fmoc-on" purifi cation allows easy and cost-effi cient purifi cation of peptides for all kind of biological applications. is an antibiotic-macrolide related to a group of tylosin (tyl) which structure is based on -member lactone with carbohydrate substitutes attached. macrolides are well known translation inhibitors, they bind to ribosomal tunnel (rt) in a way that their lactone ring is located orthogonally to the long axis of the rt, covering most of its cleft; hence, the mechanism of protein synthesis inhibition by macrolides relies on the mechanical obstruction they provide to the passage of nascent polypeptide chain through the rt. recently we have designed and synthesized a number of peptide derivatives of macrolides where the peptide part modeled the growing chain, while the antibiotic served as an "anchor" for positioning the peptide at the specifi c site of rt. these derivatives are of interest both as antibacterial agents and as potential probes for investigation of the interactions of nascent peptide chain with the specifi c sites of the rt and their infl uence on the translation. now we report a new type of peptide -macrolide conjugates in which peptide binds by its -amino function through a spacer to the '-hydroxyl group of the mycaminose residue of des. two proline-containing peptides are chosen: gp and ggp. proline residues of these peptides are supposed to interact with the peptidyl transferase center region when the complexes of these '-peptidyldesmycosins with bacterial ribosomes are formed. the fi rst step of the synthesis was acetylation of '-, "-, "and "'-oh-groups of tyl by ac o in pyridine following by hydrolysis in n sulphuric acid resulted in des with all protected oh-groups except '-oh-group of mycaminose. this free hydroxyl group was used for reaction with succinic anhydride leading to formation of reactive carboxyl group. the next step was a reaction between this carboxyl function with -amino groups of peptide methyl esters. the structures of new '-peptide derivatives of des were proved by ms maldi and nmr-spectroscopy. synthesis . ]octane, (c), was carried out by organic methods which then will be used in polymer synthesis [ ] [ ] . consequently, polymers having different molecular weights and their water soluble bioconjugates were synthesized and the characterization of these polymers and conjugates were done by different methods such as esi lc-ms, uv and atr ft-ir spectroscopy. for the chemical modifi cation of pc, bromoacetic acid was used and a water soluble polyampholyte synthesis was achieved with the quaternization of polymer. it is determined that molecular weights of the polymers were affected by the change in the amount and the type of initiators used and also from the polymerization media. analysing of having biodegradable characteristics of the synthesized polymers has been being reviewed. atr ft-ir spectra of pc and pc-hemagglutinine peptide was recorded. for peptide synthesis we have used microwave assisted solid phase peptide synthesis method. lc-ms was used to determinate of molecular weight of hemagglutinin. after we have obtained m/z values of peptide we used preparative hplc for purifi cation of hemagglutinin. after synthesis and modifi cation of pc, its covalent conjugates with hemagglutinin (ha - ) were obtained by carbodiimide activation. its characterization was also performed. the peptide coupling reagent fi eld has clearly evolved in the last decade from carbodiimides to onium (phosphonium and uronium) salts. the era of industrial coupling reagents began in with the introduction of dicyclohexylcarbodiimide (dcc), which at that time was already known and well studied, as a reagent for the formation of amide bond. unfortunately, carbodiimides did not comply with the concept of ultimate coupling reagents because its high reactivity provokes racemization and side reactions during the coupling reaction. at the beginning of the 's, -hydroxybenzotriazole (hobt) was proposed as an additive to dcc to reduce racemization and from then on other benzotriazole derivatives such as -hydroxy- -chlorobenzotriazole (cl-hobt) or -hydroxy- azabenzotriazole (hoat) have also been used. the obt active esters are less reactive than the o-acylisourea, but are more stable and less prone to racemize. the use of the most reactive aminium salt, hatu , is inconvenient because of the price, which makes its use detrimental for industry. hctu/tctu, based on cl-hobt, are a good alternative to hbtu/tbtu, because of the presence of the chlorine atom that stabilizes the structure, hence, making these reagents less hazardous. herein, pyclock, the phosphonium salt of the cl-hobt is introduced p - n-methylation of biologically active peptides is of interest because it results in increased conformational integrity and stability against enzymatic degradation. furthermore, n-methylated peptides have a decreased ability to form h-bonds to water molecules and, consequently, a better ability to cross biological barriers. this is exemplifi ed by the naturally occurring peptide cyclosporine which is orally active. in an effort to improve the blood-brain barrier permeability of the cyclic enkephalin analogues h-dmt-c[d-cys-gly-phe-d(or l)-cys]nh (dmt = ', '-dimethyltyrosine), we prepared analogues that were n-methylated at phe and/or cys . n-methylated cys derivatives were prepared either by direct methylation (ch i)/nah) or by using oxazolidinone chemistry. single n-methylation of the two cyclic peptides at phe or d(or l)-cys produced four analogues that all showed very high mu and delta opioid agonist potencies in the guinea pig ileum and mouse vas deferens assays. the two analogues n-methylated at both phe and d(or l)-cys also retained high agonist activity at both receptors. results from molecular mechanics and molecular dynamics studies on the conformational behavior of these peptides will be presented. during the last years the number of deaths due to hemostatic impairments such as coronary angioplasts, coronary thromboembolisms, myocard heart attack, pulmonary embolism etc. has become equal to those caused by cancer formations. haemostasis is a key process whose correct functioning is an important defence mechanism of the human organism. it is a blood coagulation process activated in case of injury of the blood system. if it is functioning correctly vascular-motor and cell reactions are triggered and the blood coagulation cascade is activated. one of the most important enzymes in the blood coagulation cascade is factor xa. that's why its inhibitors are promising alternative against thrombotic disorders. in our previous work, we reported the synthesis of hybrid structure between isoform and of antistasin and the active sequences d-phe-pro-arg; d-arg-gly-arg; phe-ile-arg and tyr-ile-arg ( ). beside the analogues with c-terminal cooh group, the peptide d-phe-pro-arg-pro-lys-arg-nh was synthesized. the biological activity of the last one was times bigger than natural isoform of ats and some times more active than all other synthesized analogues. in the current work we described synthesis and biological activity of c-terminal amide analogues of all early synthesized peptides in order to deduce the structure-activity relationship. the anticoagulant activity according to the aptt and ic was determined. the neo-angiogenesis is the formation of new blood vessels from a pre-existing vasculature which enables the supply of the nutrients and oxygen necessary for tumor growth. consequently, inhibiting the blood vessels sprouting in order to starve the tumour constitutes an attractive anti-tumoral therapy. the vascular endothelial growth factor or their receptors (vegfr- and ) are key mediators of neo-angiogenesis and constitute nowadays validated targets for anti-angiogenic therapies ( ) . despite this fact, strategies aiming to develop pure receptors antagonists and not inhibitors of the tyrosine kinase activity remain scarce. we have previously designed antagonists that interact with the extra-cellular domain of vegf receptor and thus prevent vegf binding ( ) . these antagonists were proved to be potent and able to interfere with the vegf signalling pathway. because these compounds were rationally designed by using the co-crystallized structure of vegf with the immunoglobulin like fragment of vegfr- , we raise the following question: do these antagonists really target the immunoglobulin like domain of vegfr- ? in order to respond to this question but also with the future aim of optimizing our antagonist in a rational way we need to co-crystallize at least one of our antagonists with the d fragment of the vegfr- . furthermore, since human vegfr- d is a potential target in the development of angiogenesis modulators, the availability of this protein domain, and mutants, should be useful in the screening and development of novel specifi c ligands. up to now, the -amino acid polypeptide chain of vegfr- d has been only produced by gene expression ( ) . here, we report the fi rst solid phase peptide synthesis of the vegfbinding domain of the vegf receptor and the in vitro experiments which permitted to verify its biological activity. chung, nga n. different types of turns are important elements of secondary structure in peptides and proteins. the most common ones are different kinds of -turns involving four consecutive amino acid residues. the dipeptide unit containing the second and the third residues of such turns exists in the cis-conformation. our cispeptide bond motif -aminopyroglutamic acid promotes the -turn type vi/vi', and can be treated as a hybrid of glycine and alanine. this feature prohibits its utilization as a replacement for any possible dipeptide unit. in order to convert our compound into a more valuable tool for probing the existence of a particular peptide bond in the cis-conformation, we have elaborated the synthesis of nmonoalkylated derivatives of -aminopyroglutamic residue through a reductive alkylation reaction performed on solid support. following this idea, we have obtained analogues with n-benzylated and n-(phydroxy)benzylated -aminopyroglutamic residues as scaffolds for the phe-ala and tyr-ala dipeptides units, respectively. only one of enkephalin analogues, [n(bzl)-(r,r)-apy - ]-enkephalin amide, possesses similar agonist potency as leu-enkephalin in the gpi assay, indicating that the c-terminal part of this peptide may assume the cisconformation upon binding to the receptor. this is the fi rst active opiod analogue containing a -aminopyroglutamic acid residue. supported by kbn (poland) grant p f and nih (u.s.) grant da- mif- (l-prolyl-l-leucyl-glycine amide, plg) is a brain peptide, presented in hypothalamic tissue, as well as the c-terminal tripeptide of the neurohypophiseal hormone oxytocin and inhibits the release of melanocyte-stimulating hormone (msh) in some systems. recently, some chemical modifi cations of mif- to enhance opiate agonist/ antagonist actions as well as binding activity of analogues have been reported. on the other hand, the concept of structural modifi cation in peptide fragments to confer them specifi c properties is of current interest in the study and design of new bioactive targets. a well known example is the incorporation of unnatural amino acids into the molecule of natural biologically active peptides leading to analogues with signifi cant theoretical and practical importance. with this idea in mind we studied possibilities of introducing unnatural amino acids canaline (can), norcanaline (ncan), canavanine (cav), nor-canavanine (ncav) and slys into mif-moiety in order to achieve a better analgesic effect. to obtain the peptide mimetics, both fmoc-and boc-based spps approach were used. analgesic activity was determined by the paw-pressure (pp) and hp tests. the experiments were carried on male wistar rats. the changes in the mechanical nociceptive threshold of the rats were measured by the randall-selito paw pressure test using analgesimeter (ugo basile). it was found that substitution of leu in position of mif-molecule by unnatural amino acids increased the pain threshold. the analgesic effect with cav-substitution was highest, whereas parent mif- showed only a minute increase of pain-threshold. the compound m had the strongest effect on learning and memory and the compound p showed the strongest and fastest analgesic effect. the compound m and p were also able to modify the effect of the model cns-drugs (hexobarbital and pentylenetetrazole). theoretically and experimentally determined octanol/water logp values of the compounds correlate with their cns-effects. m and p had higher logp than m and p and showed better antinociceptive and anticonvulsant activity in vivo. compounds possessed also chelating activity towards fe ions. the stronger chelating activity of the compounds with the -spacer clearly correlated with their better neuropharmacological activity in vivo (in comparison to the compounds with the -spacer). the position isomery may also contribute for the variations in their pharmacological activity. the limited number of the compounds does not allow derivation of well-defi ned structure-activity relationships, however, their d models show possibility for many low energy conformers with different atoms involved in formation of fe chelating complexes. a major challenge in opioid peptide chemistry is the synthesis of novel compounds mimicking the endogenous peptide ligands. these new peptidomimetics should be biologically active and more stable against enzymatic degradation than their parent ligands. one of the possibilities is the introduction of -amino acids into the peptides sequence. monosubstituted -amino acids ( -or -) due to their similarity in structure to -amino acids, moreover their tendency to give folded structures even in short peptides and to the stability towards mammalian peptidases may be very useful in the creation of the new potentially active compounds. we have developed simple and effi cient two step conversion of the cyanoacetate into fully protected -amino acids. the procedure involves knoevenagel condensation of the methyl cyanoacetate and aromatic aldehydes (for aromatic path) or alkylation of the methyl cyanoacetate with various alkyl halides (for aliphatic path) at fi rst then reduction and boc-protection (performed in one pot) of the resulting fi rst stepproducts. we focused our attention on applying above method to the synthesis of different -amino acids (as homologues of -amino acids) as elements for the synthesis and structure -activity relationship study of endomorphin analogues. small library of analogues has been created in which -amino acids in every position with exception of pro were substituted by their respective -analogues. as templates, endomorphin- (tyr-pro-trp-phe-nh ), endomorphin- (tyr-pro-phe-phe-nh ) and its d-ala -analogue (tapp) have been used. in this communication the pharmacological consequences of such modifi cation in endomorphins will be discussed. solid-phase synthesis and effects of amino acid and peptide analogues of non-protein amino acid canavanine on nociception non-protein amino acids have been widely used as components of peptides to enhance biological activity, proteolytic stability, and bioavailability. it is well known that unnatural amino acids with guanidine functionality exhibit diverse pharmacological effects when introduced in biologically active systems. our previous efforts were focused on the preparation and the characterization of unnatural amino acids, particularly those containing a basic functionality in the side chain. we have synthesized numerous unnatural amino acids, structural analogues of arginine and lysine, which demonstrated certain biological effects. recently, as part of our ongoing research focused on the search of novel arginine mimetics, we developed an effi cient approach for solid-phase synthesis of unnatural amino acids nor-canaline (ncan) and nor-canavanine (ncav). next we studied the possibilities of introducing ncan and ncav into the molecule of biologically active peptides. we also studied their antinociceptive effects using the paw pressure (pp) and hp tests. in the last few decades, considerable attention has been devoted on the potential role and activity of certain environmental pollutants (edcs) in increasing anomalies that involve the endocrine system of wild species and man. ( )the development of a fast high-throughput detection system for the quantitative analysis of edcs requires the development of a novel sensitive solid phase edcs extraction method. because of the high affi nity of edcs with the estrogen receptor, this has been greatly investigated. this study has leaded the recognition of the amino acids located in the ligand binding domain of the receptor which interact with edcs. use of a dipodal scaffold molecule and different combination of the crucial amino acids, will allow the generation of a library of small to medium size biomimetic receptors. in this communication, we will disclose our fi rst results in the preparation of a small library of biomimetic receptors, with and without the dipodal scaffold, to evaluate their affi nity towards edcs and the role played by the scaffold structure. romeralo tapia, rosa ; van der eycken, johan ghnet university, belgium; ghent university, belgium endocrine disrupting chemicals (edc's) are an important class of pollutants, which have in common that they show affi nity for the hormone binding domain of the estrogen receptor, thus disturbing the endocrinal system. detection in waste water has not been succesful due to their low concentration. therefore, new sensitive screening methods are highly demanded. a possible solution is the use of estrogen receptor mimics for affi nity chromatography. to this end, scaffold will be synthesized and used for building a tetrapodal peptidomimetic library. amino acids known to be important for the estrogen-receptor interaction will be preferably incorporated. first of all, we set out to prepare one dipodal peptide library member . the orthogonally protected scaffold was synthesized in one single step starting from the commercially available amino- -nitrobenzoic acid. using fmoc solid phase chemistry on wang resin as the solid support, this dipodal scaffold allowed the attachment of two different oligopeptide chains.employing this methodology the synthesis of a small library will be performed, and the affi nity of the different peptidomimetics for edc's will be investigated. on-resin microwaves-assisted ring closing metathesis for the synthesis of octreotide dicarba-analogues di . octreotide is an antitumoral agent used mainly as a carrier of radionuclides for cancer diagnosis and therapy. it shows the same disulphide bridge of the parent srif that is prone to be opened by oxidizing and reducing agents. this prompted us to search a more stable tether bridging the active motif of the cognate molecule. the premier reaction of rcm was performed in an oil bath under severe experimental conditions i. e. anhydrous argon atmosphere and long reaction times [ ; ] . the microwaves assisted version was, instead, effi cient for the cyclopeptides yield and required very short reaction times. we also evaluated the effi cacy of different grubbs catalysts in the microwaves assisted rcm, operating in different conditions of temperature and time. in the search for potential antimicrobial drugs, we have been dealing with two microbial enzymatic systems: n á -succinyl-l-diaminopimelic acid desuccinylase (dape , ) and n á -acetyl-l-ornithine deacetylase (arge , ), which might be promising targets for potent and selective enzyme inhibitors based on the modifi cation of n á -succinyl-diaminopimelic acid (dap) and n á -acetyl-ornithine (orn). the inhibition of both the enzymes would possibly interrupt the pathways leading to development of bacteria due to a role of meso-dap as essential component of peptidoglycan based bacterial cell walls and orn, as well, being a component of biosynthetic pathway for arginine that can serve as a source of both the carbon and nitrogen in microorganisms. our effort was focused on the synthesis and characterization of two series of n á -substituted derivatives of dap and orn, potentially interfering with the hydrolytic action of dape and arge in the processes of bacterial growth. in this introductory study, the compounds prepared were also assayed against bacterial strains escherichia coli and bacillus subtilis and inhibitory activity of orn derivatives was found with regard to differences in the structure of n á -substituent. the fi rst report on practically effective bradykinin (bk) antagonists for b receptors was published in . the key to conversion of bradykinin into an antagonist was replacement of pro with an aromatic d-amino acid; d-phe was fi rst used. however, our studies demonstrated that the d-amino acid residue at position of the bradykinin antagonist, until recently considered to be necessary for b antagonism, can be replaced by suitable l-amino acid or achiral residue or, together with the amino acid occupying position , by a sterically restricted dipeptide unit. having all this in mind we synthesized and bioassayed two new analogues of bradykinin. the peptides were designed by substitution of position of bradykinin b receptor antagonist ([d-arg ,hyp ,thi , ,d-phe ]bk), previously described by stewart's group, with structural isomer of proline: -iso-pro or its homologue: -homo-pro. it's worth emphasizing that position in bradykinin molecule is occupied by proline residue. our previous results demonstrated the importance of the position in the peptide chain into which the sterically restricted -aminocyclohexane- -carboxylic acid residue (acc) was inserted. these fi ndings prompted us to investigate how introduction of l-pipecolic acid residue (l-pip) in position or of stewart's antagonist will affect pharmacological properties of resulting compounds. in comparison to the acc residue, the ring of l-pipecolic acid also consists of six atoms, but includes the nitrogen atom. bearing in mind that acylation of the n-terminus of several known b blockers with a variety of bulky groups has consistently improved their antagonistic potency in the rat blood pressure assay, the aforementioned four analogues were also synthesized in the n-acylated form with -adamantaneacetic acid (aaa). the activity of eight new analogues was assessed in isolates rat uterus and in rat blood pressure test modifi cations of the myelin basic protein epitope mbp - divert th to th : immune responses in peripheral blood mononuclear cells (pbmc) from multiple sclerosis patients. postranslational modifi cations (citrullination, phosphorylation, deamidation, methylation, glycosylation) are common biological processes that alter specifi c parts of a protein after synthesis. nearly all known proteins undergo some form of postranslational modifi cation and almost all amino acids can be altered by one or more of these processes. the modifi ed protein thus, contains new or rare amino acids or new specifi c side groups that can have critical infl uence on the structure and function of the protein molecule. conversion of arginine to citrulline, an important postranslational modifi cation, was fi rst described by fearon. arginine residues in proteins can undergo this modifi cation and the resulting citrulline remains part of the protein in the position of arginine. citrulline is not a natural amino acid in proteins, and may induce immune responses. such responses have been recently implicated in the pathogenesis of autoimmune/infl ammatory diseases such as ms and rheumatoid arthritis . herein we investigated cytokine secretion in peripheral blood mononuclear cells (pbmc) of ms patients and controls, and attempted to correlate cytokine polarization with the nature of the antigenic stimulus. we synthesized peptide analogs that map to the myelin basic protein ( . analogs p and p resulted from the citrullination of the and arginine residues in epitopes p and p . we then tested ms and control pbmc with various concentrations of the peptides and investigated cell proliferation by the brdu proliferation assay and cytokine secretion by elisa. we suggest that citrullination of self-antigens maybe an important step in triggering disease in susceptible individuals. incorporation of aza- -amino acid into rfa( - ), the endogenous ligand of gpr : structural analysis. aza- -peptides, mixing -and aza- -amino acids (the aza analogs of -amino acids), represent a novel and exciting type of peptidomimetics. in particular, we have shown that aza- -amino acid induces a n-n or hydrazino turn, stabilized by an eight-membered-ring intramolecular hydrogen bond between the carbonyl acceptor group of the residue i- and the amide proton of the residue i+ . interestingly, this n-n turn promotes a well-defi ned -turn formation (hydrogen bond between the co of the residue i- and the hydrazidic proton of the aza- -moeity) when an -amino acid is foregoing. rfa, a novel neuropeptide of the rfamide superfamily, exhibits high affi nity for gpr and induces a potent orexigenic effect in mice. in biomimetic environment, rfa encompasses an -helix between pro and arg residues and a canonical -turn centered on ser . rfa( - ), whose sequence is strictly conserved across species, is about times less potent than rfa. this heptapeptide shows important distortions of the -turn that may be responsible for its weak potency. the aim of this study was to restore the -turn formation in rfa( - ) (ggfsfrf-nh ) by the presence of an aza- -amino acid. for this purpose, we have (i) synthesized the aza- -counterpart of each residue of rfa( - ), (ii) individually incorporate the surrogate into the heptapeptide and (iii) investigated the d structure under nmr restraints of the hybrid peptides. the results will be presented with a particular attention on the serine position. the analysis of the structure-antithrombotic activity correlation for peptide receptors of adhesive glycoproteins with the general formula arg-xaa-asp gribovskaya, olga; martinovich, vera; golubovich, vladimir institute of bioorganic chemistry, belarus structural analogues of the arg-gly-asp sequence with the general formula arg-xaa-asp, where xaa -ala, d-ala, -ala, -abu, -ak, pro, d-pro, asn-trp, were synthesized using classical methods of peptide chemistry in solution, the levels of their antithrombotic activity were determined and stable conformations were calculated using a pairwiseadditive approximation method. interatomic distances in the obtained conformers were calculated between various atoms in the functional groups, including carbons in the carboxylic groups, nitrogens in the -aminogroups, amino and imino nitrogens in the guanidine group, distances in total. the analysis of correlation of the calculated interatomic distances and measured values of antithrombotic activity was carried out using statistical methods. we show that the distance between the amino nitrogen of the guanidine group of arginine and the -carboxyl carbon of aspartic acid determines the antithrombotic activity. it has the optimum value in the arg--ala-asp peptide, which was the most active among the synthesized analogues of the arg-gly-asp sequence (ic - . mkm, adp, . mkm). microbial proteases with narrow specifi city as an instrument of peptide chemistry kotlova the problem of selective removal of short n-terminal peptides from the gene-engineered proteins is actual by many reasons. at fi rst it is connected with gene-engineering method of protein preparation in form of its precursors. the problem of n-terminal formyl-met removal from gene-engineered proteins, which are produced by bacillacae may be solved by introduction of specifi cally-cleaved insert after formyl-met. subsequent specifi c removal of such short n-terminal peptide results in mature protein. moreover, the analysis of cleaved short peptides could characterize the process of mature protein formation. we suggest microbial enzymes with narrow specifi city to solve the mentioned problem. for this aim we have isolated the trypsin-like enzyme and postproline-specifi c endopeptidase from aspergillus sp. and glutamyl-specifi c protease from b. intermedius. these enzymes have high specifi city approved by hydrolysis of short synthetic peptides and long polypeptides. for example, specifi city of postproline protease was established by hydrolysis of mellitin. the fi nal peptide mixture was analyzed by rp-hplc and mass-spectra. the use of enzymes with narrow specifi city is demonstrated in the analytic method of detection of formyl-met presence at the n-terminus of gene-engineered -interferon. trypsine hydrolysis of -interferon leads to more than peptides are formed including the -membered n-terminal peptide. being hydrolyzed with glutamyl-specifi c enzyme -interferon is converted to peptides, including -membered n-terminal peptide. application of postprolinespecifi c enzyme for -interferon hydrolysis leads to formation of short -membered peptide and long fragments. experimental data show that in case of analytic control of -interferon maturity the utilization of postproline-specifi c protease is preferable. in other cases the enzyme selection is ruled by the features of protein primary structure. bimodal action of cystatin related epididymal spermatogenic (cres) protein and its reactive site loop derived s-s bridge bicyclic peptides on proprotein convertase- (pc ) activity several precursor proteins found on sperm surface and reproductive tissues were proposed or confi rmed as substrates of pc . these include adam proteins, growth factors proigf and and hormonal protein pacap. lack of pc leads to impaired fertility in mice, suggesting its important role in reproduction. pc is thus considered an important target for development of nonhormonal contraceptive agents. pc -inhibitors are expected to fi nd therapeutic, clinical and biochemical applications. a lot of interest has grown to develop specifi c pc inhibitors. recently natural inhibitor of serpin family have been described for pc , , and furin, but not for pc . however, a new serpin, cres, has been reported from epididymis fl uid, where pc may be found. so far, cres has only been shown to inhibit pc , which is not present in reproductive tissues. we propose that cres may represent a natural regulator of pc , based on localization and other studies. in this study we generated recombinant human cres protein ( aa), and its reactive-site loop (rsl) derived acyclic and cyclic -mer peptides with various s-s linkage combinations. cres inhibited pc with ic ~ um, while rsl-peptides were found to be more potent with ic - nm, depending on the s-s linkage locations. furthermore, we noted that at lower concentrations, cres and its peptides fi rst enhanced pc activity before any inhibition occurred. this bimodal behavior may be due to cleavage. molecular modeling showed strong interactions between cres and pc via some of their key residues. overall, we noted that "rsl" is the most crucial element required for pc inhibition by cres. funds were from from cihr (ab). the de novo design of peptides and proteins has assumed considerable interest didehydroresidues, in particular alpha, in the recent years. alpha, beta-beta-didehydrophenylalanine (deltaphe) are being considered important conformational constraints inducing tools in de novo peptide design. deltaphe is a noncoded, achiral residue, an analog of the naturally occurring phenylalanine amino acid with a double bond between calpha and cbeta atoms. introduction of deltaphe in peptide sequences is known to induce conformational constraint, both in the peptide backbone as well as the side chain, and to provide the peptide with increased resistance to enzymatic degradation. in small peptides containing eta turn structure, and in peptides containing a single deltaphe, a type ii b more than one deltaphe residues, helical structures are stabilized. a number of deltaphe peptides varying in length, content and position of deltaphe residues have been found to contain helices of both screw senses. following these design principles, we were able to design, synthesize and characterize super secondary structural motifs like helix-turn-helix, helical bundle and glycine zipper. we have extended this work to the de novo design of peptides with antibiotic and anti-fi brillization activity. a series of cationic peptides containing deltaphe residues have shown remarkable antibiotic activity and are being developed further. deltaphe containing peptides may have longer in vivo half life owing to their ability to resist enzymatic degradation. more recently, we have observed that small peptides containing deltaphe self-assemble in nanotubular and nanovesicular structures. these nanostructures have also been used to entrap small drug like molecules. we have fully characterized these systems and are exploring their potential as delivery agents in biological systems. the synthesis and design of peptidomimetic oligomers that adopt designed, compact conformations (foldamers) have become a challenging task in recent years. among them, -peptide foldamers exhibit rich diversity of secondary structures. [ , ] a relationship has been established between the backbone chirality pattern and the prevailing secondary structure, which underlines the role of stereochemical control in the -peptide foldamer design.( ) an important challenge is to introduce proteinogenic side-chains to generate diverse anchor points on the molecular surface promoting their application in drug discovery. it has been proved that stable helices can be obtained when sequences were coupled with -amino acid enantiomeric pair motifs in a lego approach.( ) in this work, -amino acids and open chain functionalized -amino acids were inserted between the enantiomeric pair design element for and , respectively. confi gurations in the backbone were designed to promote helix formation. nmr and ecd measurements augmented with ab initio calculations revealed that forms a h - helix and forms a h / helix. these helices are unprecedented in the literature. to prove that the stereochemical patterning is crucial in the folding, we perturbed the confi guration motifs of the backbones by swapping the sequence of the central -amino acids. the peptides with exchanged residue order could not fold into helices. these novel structures can be useful scaffolds for biomedical applications. introduction small length synthetic peptides, based on sp c-terminal fragment, increase the secretion of tnf-and prevent the proliferation of several cancer cell lines. we have already shown the antiproliferative activity of tri-and tetra-peptoids in breast and prostate cancer cells. the aim of this study was the synthesis of hexa-peptoids, analogs of sp c-terminal region, containing the residues d-trp and tic and the peptoid ones nhn(r)ch co, nphe and nala in their sequence and their evaluation against cancer cells proliferation. methods and results all the syntheses were carried out stepwise using the fmoc/ but methodology on the solid support -cltr resin and dic/hobt as coupling reagent. all analogs were purifi ed (hplc) and identifi ed ( cyclotheonamides constitute a family of structurally related cyclic pentapeptides of marine origin that inhibit trypsin-like serine proteases. their binding mode has been elucidated by the x-ray structure of cyclotheonamide a in complex with trypsin. an extended peptide conformation which is stabilized by macrolactamization allows to address in a substrate-like manner beside the s pocket also the s ' and s pocket. the s ligand, (s)- -amino- -guanidino- -oxo-hexanoic acid, interacts via its guanido function with asp at the bottom of the s pocket. in addition, the ketone covalently modifi es the gammaoxygen of ser by hemiketal formation ( ) . recently, two novel cyclotheonamides have been isolated from a marine sponge of the genus ircinia. one of them, cyclotheonamide e , is a potent inhibitor of human beta-tryptase ( ) . in this study, cyclotheonamide e was modifi ed at two positions: (i) the s ligand was replaced by beta-homolysine or as betahomoarginine to obtain reversible acting tryptase inhibitors, and (ii) the alpha amino function of (s)- , -diamino propionic acid, which is not part of the cyclic backbone, was used as anchoring point for basic p residues to exploit interactions with the negatively charged glu of tryptase. these analogs were synthesized by a combination of solid phase and solution phase chemistry .. synthetic details as well as the inhibitory profi le of these novel cyclotheonamide e analogs will be discussed. oostatic peptides containing d-amino acids: activity and degradation in the fl esh fl y neobellieria bullata bennettová deteriorating effect of c-terminus truncated p and p analogues [ , ] of decapeptide h-tyr-asp-pro-ala-pro -oh ( ) on ovarian development (i.e. oostatic effect) of insect species diptera, orthoptera and hemiptera has stimulated an analysis of metabolic degradation of corresponding peptides after application to the insect body [ ] [ ] [ ] [ ] . radiolabeling in different positions of the peptide chain allowed determination of the degradation decisive steps -the fast splitting off the c-terminal pro from the p followed by successive cleavage of tyr and asp from the nterminus. after introduction of corresponding d-amino acids into peptide chain of p, we could see the same or even stronger oostatic effect of the analogues in comparison with parent peptide after application on the fl esh fl y neobellieria bullata. in the degradation assay, an elimination of enzymatic cleavage of peptide bonds pointing from the central labeled pro residue to either d-ala or d-asp residues in the neighbourhood was observed, resulting in total increase of the analogs stability. structure-activity relationships( sar) studies of camk ii inhibitors. in fact, in vitro it can phosphorylate up to proteins, including enzymes, ion channels, transcription factors and a number of these proteins appear to be physiological substrates. for example, cam-kii is highly concentrated in the postsynaptic density of glutamatergic synapses where it phosphorylates and potentiates current through the a-amino- -hydroxy- -methyl- -isoxazolepropionic acid-type glutamate receptor ion channel (ampa-rs). in fact, this family is encoded by four genes ( , , and ), whereas the and isoforms are expressed in diverse tissues and , isoforms are most prominent in neural tissues. the identifi cation of camk ii inhibitors is important to better defi ne its physiological. in literature is reported a -aa brain-specifi c protein that and potently inhibited kinase and bound the catalytic domain of cam-kii activity with an ic of nm. the inhibitory protein (cam-kiin), and a -residue peptide derived from it (camkiintide, krppklgqigrakrvvieddriddvlk), was highly selective for inhibition of cam-kii with little effect on cam-ki, cam-kiv, cam-kk, protein kinase a, or protein kinase c. cam-kiin interacted only with activated cam-kii (i.e., in the presence of ca +/cam or after autophosphorylation). here we report a structure-activity relationships study using as template the camkiintide. we synthesised a peptide contains all amino acids present in camkiin-tide, however, in random sequence (camkiin-tide scramble). then we operated a progressive deletion of amino acids from c-terminal and dall'n-terminal to detect minimum active sequence. moreover camkiintide and cankiintide scrumble was made cell-permeable by n-terminal addiotion of an antennapedia (rqikiwfqnrrmkwk). here we report the biological results of our study. ptprj: a receptor-type protein tyrosine phosphatase as a target of new peptides with antitumoral activity to be signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. it has been demonstrated that ptprj (also known as hptpeta, dep- , cd ) is able to inhibit cell growth promoted by specifi c ptk some of which are over-expressed in certain types of cancer. moreover a role for ptprj was also assessed in vasculogenesis; infact its reduced activity in enhanced vegf-induced vegfr activity leads to increased cellular responses, supporting a ptprj-vegfr interaction. on these basis it is important the identifi cation of molecules with agonist activity which are able to stimulate the function of residual ptprj in malignant cells and stopping the proliferation and possibly trigger programmed cell death. using as a template a peptide, already identifi ed, with agonist activity against ptprj(h-[cys-his-his-asn-leu-thr-his-ala-cys]-oh), here we report a structure-activity study carried out through endocyclic modifi cations (ala-scan, d-substitutions, single residue deletions, substitutions of the disulfi de bridge) and the preliminary biological results of this set of compounds. faulty of chemistry, university of gdansk, poland; faculty of chemistry, university of gdansk, poland isolated in from the sunfl ower seeds trypsin inhibitor sfti- is up to date the smallest naturally occurring peptidic proteinase inhibitor and therefore is an excellent starting structure to design peptidomimetic serine proteinase inhibitors. peptomeric library consisting of monocyclic analogues of sfti- was designed and synthesized by the solid phase method with the intension of select chymotrypsin and cathepsin g inhibitors. all peptomers contained in positions and nbenzylglycine (nphe) that mimics proteinogenic phe. in the synthesized library different peptoid monomers were introduced in the segment - . this is a turn region that makes an important contribution to structural integrity and rigidity of sfti- . deconvolution of the library against both proteinases by the iterative method in solution revealed that the highest chymotrypsin inhibitory activity displayed analogue with n-[ -( -aminoethyl)morpholyl]-glycine (naem) in position . this analogue was even more active than the one with pro in this position which is absolutely conserved in bownan-birk inhibitors. while, deconvolution carried out against cathepsin g indicated that analogue with npiperonylglycine (npip) in position and and with n-butylglycine (norleu) in position presents the highest inhibitory activity. acknowledgements: this work was supported by ministry of science and higher education (grant no. /h / / ). a platform for the design and optimization of new antimicrobial peptides effective against specifi c pathogens has been set up. the main features expected for these peptides are low environmental impact, broad spectrum of activity, reasonable bacterial selectivity, and low eukaryotic cytotoxicity. our approach also includes the use of a design of experiments protocol in order to fi nd peptide sequences that fi t these features. the obtained peptides would represent an alternative to currently used antibiotics or pesticides. this work focused on fi nding new control agents against economically important plant pathogenic bacteria such as erwinia amylovora, pseudomonas syringae and xanthomonas vesicatoria for which the available methods are not suffi ciently effective. nowadays, their control is mainly based on copper compounds and antibiotics. although antibiotics are highly effi cient, they are not authorized in several countries and resistance has been developed on plant pathogens. we have synthesized combinatorial libraries of cyclic decapeptides and linear undecapeptides. these libraries have been screened for antibacterial activity and eukaryotic cytotoxicity, and have led to the identifi cation of peptides with mic values of . - . microm. notably, cyclic peptides active against e. amylovora have been found, constituting the fi rst report of this type of peptides with activity towards this bacteria. the best peptides are bactericidal, display a low eukaryotic cytotoxicity at concentrations - times higher than the mics, and show a low susceptibility towards protease degradation. best peptides have been tested in vivo by evaluating their preventive effect of inhibition of p. syringae, x. vesicatoria and e. amylovora infections. the most active peptide is slightly less effective than streptomycin, currently used in fi eld. therefore, the best analogues can be considered as good candidates for the development of antibacterial agents for use in plant protection. selection of chromogenic and fl uorogenic substrates of neutrophil serine proteases using combinatorial chemistry approach human serine neutrophil and mastocytes proteases such as cathepsin g, neutrophil elastase, proteinase and -tryptase are involved in several physiological processes. unwanted activity of those enzymes yields to severe pathological states like infl ammation, wegener granulomatosis or various types of cancer. therefore monitoring of the activity of these proteases is crucial for the proper therapeutical treatment. the simplest method for determination of protease activity is the use of synthetic substrates. they are also useful for characterization of the enzyme specifi city. in this work we report selection of chromogenic and fl uorogenic substrates of human serine neutrophil and mastocytes proteases applying combinatorial chemistry approach. peptide libraries were synthesized by the portioning-mixing method. deconvolution of synthesized libraries was performed using iterative approach in solution. -amino- -nitro benzoic acid attached to the c-termini of synthesized peptides served as a chromophore released upon the interaction of peptide with enzyme. additional introduction of -methoxy- -coumaryl acetic acid or -amino benzoic acid on á-amino groups of the chromogenic substrates converted them into fret displaying compounds. the most active substrates were subjected to further modifi cation applying non-proteinogenic amino acids. as a result, one of the most selective substrates of these proteases was obtained. we also attempted to construct a simple tools to detect activity of human serine neutrophil and mastocytes proteases by immobilization, through amide and peptoid based linkers, of selected substrates on solid phase. angiogenesis modulation: identifi cation of tetrameric tripeptide as inhibitor of vegfr- by the screening of peptide combinatiorial libraries vascular endothelial growth factor receptor- (vegfr- , flt- ) and their ligands are involved in complex biological processes associated to severe pathological conditions, like angiogenesis, infl ammation and metastasis formation ( ). thus, the search for antagonists of flt- has recently gained a growing therapeutic interest. in order to identify new molecules able to selectively bind flt- and neutralize its activity, a screening of a combinatorial tetrameric tripeptide library built with nonnatural amino acids has been carried out by a competitive elisa-based assay. the library has been designed on a branched tetrameric structure in order to obtain molecules with a high recognition surface and, using building blocks, a complexity of . different peptides has been achieved. peptide mixtures composing the library have been utilized as competitors of the flt- /plgf (placental growth factor) interaction and the most active components have been isolated following an iterative deconvolution procedure. the selected most active hit shows a selective binding to flt- over kdr and inhibits in vitro its interaction with both plgf and vegf-a. the peptide is fully stable in biological environments, prevents flt- phosphorylation and blocks huvec capillary-like tube formation stimulated by plgf or vegf-a. in vivo the peptide inhibits the vegf-induced neoangiogenesis in chicken embryo chorioallantoic membrane assays and also stimulates cornea neovascularization (cnv) by displacing vegf from a complex with sflt- . all data suggest that the compound has potential applications in diseases characterized by pathological angiogenesis, such as tumor growth and ischemic retinopathy. heinlein, christian; unverzagt, carlo bioorganische chemie, universität bayreuth, gebäude nw i, bayreuth, germany since the purifi cation of homogeneously glycosylated glycoproteins remains a diffi cult task the synthesis of entire glycoproteins is a fi eld of emerging interest ( ) . especially the synthesis of the required protein fragments in high purity and good yields demands for effi cient methods. fragment condensation of protected peptides can reduce deletions in the crude product and thus facilitates purifi cation. condensation of peptide fragments in cspps ( ) is however subject to epimerization upon cterminal carboxyl activation. to overcome this problem the use of cterminal gly or pro is usually performed. peptides containing c-terminal pseudoprolines can also be coupled without stereomutation because the serine or threonine residue has been reversibly protected as a prolinelike oxazolidine ( ). this concept facilitates the synthesis of long peptides by epimerization-free fragment condensation at c-terminal ser/ thr residues in addition to gly/pro residues thus increasing the number of safe condensation sites. after solving several problems associated with the generation of peptide acids with c-terminal pseudoprolines the synthesis of the glycosylated rnase fragment was carried out as a racemization-free fragment condensation. tuftsin is liberated from fc-domain of the heavy chain of igg by two specifi c enzymes. being the tetrapeptide of biological origin is extremely important product because it can activate a few elements of immune system such as granulocyte and macrophage. tuftsin indicates not only immunological stimulating factor but also antibacterial, antivirial and antitumor properties. in spite of its wide range of activity, the peptide is unstable in plasma and it has become the aim of the formation of novel analogues more resistant to proteolysis degradation. the introduction of the additional residue at -amino group of lysine caused that new bond became stronger than peptide bond in central chain [ ] [ ] [ ] . we synthesized linear tuftsin derivatives that were prepared on the solid phase using a fmoc/tbu procedure. the method of elongation of peptide chain was based on two-step procedure: deprotection and coupling step. segment coupling reaction was carried out with tbtu, hobt and in the presence of diea in dmf/dcm/nmp mixture. the introduction of the simple amino acid (ala, -ala, val, ile or gly) at -amino group of lysine let us obtain the isopeptide bond. the modifi cation was achived by introducing lysine residue, protected at -amino group with mtt. the selective removal of mtt group was caused by % tfa treatment. peptides were cleavage from resin and purifi ed. tuftsin derivatives were confi rmed by ms, amino acid analysis, elemental analysis and rp-hplc analysis. peptides were sent to assay their microbiological properties and the results will be described as a structure-activity relationship. the synthesis and structural study of iso-aß( - ) the aß( - ) peptide is diffi cult to synthesize, because of its high tendency for aggregation during the synthesis. both the couplings and the fmocremoval can be troublesome, due to the steric hindrance. the incorporation of the ester-bond disturbs the structure, thus ease the synthesis after the th residue. if the peptide would be synthesized using boc-chemistry the removal of the -amino protecting group would be less problematic, because the % tfa/dcm mixture solubilizes well the peptide chain. thus a boc-strategy was devised for the synthesis of iso-aß( - ). the purifi ed peptide was studied with cd-spectroscopy and dynamic light scattering. these structural studies revealed that the isopeptide has some ß-sheet content even in a ph solution. it was realized also that small aggregates are present in the precursor peptide. both techniques mentioned above showed, that after altering the ph to . , the ß-sheet content of the peptide increases and aggregation takes place. with the use of the isopeptide, aß oligomers and -with prolonged incubation -fi brillar structures can be formed for biological studies. continuing our program of syntheses of muramyl dipeptide (mdp) and nor-muramyl dipeptide (nor-mdp) conjugates as potential immunomodulators, we designed novel conjugates of mdp or nor-mdp with tuftsin derivatives containing isopeptide bond between -amino group of lysine and carboxylic group of simple amino acids such as alanine, glycine and valine. the synthesis of a greater number of conjugates will enable structure-activity relationship studies. tuftsin analogues containing isopeptide bond showed increased chemical resistance and activity in relation to tuftsin. the introduction of the additional residue at -amino group of lysine by nhco-formation caused that isopeptide bond became stronger than peptide bond in central chain. the protected pentapeptides (h-thr-lys(y)-pro-arg(no )-obn, y= ala,gly,val) were synthesized by the conventional chemical procedure using mixed anhydride method. acylation of the thr amino group of partially protected pentapeptides by -benzyl-mdp or -benzyl-nor-mdp was performed using the mixed anhydride method with isobutyl chloroformate and n-methyl-morpholine (nmm) in dry dmf. the protected conjugates were isolated and purifi ed with a preparative tlc. the identities of the protected products were confi rmed by high resolution h-nmr ( mhz, cosy, tocsy, roesy, ghsqc, ghmbc) spectroscopy. the fi nal products were hydrogenated with h / pd/c in % methanol-acetic acid and purifi ed with preparative tlc. the identities of the conjugates were confi rmed by tlc qualitative amino acid analysis, and elemental analyses. finally, the combined use of muramyl peptides with other immunomodulators, e.g. such as tuftsin, retro-tuftsin other chemotherapeutics is promising in the therapy of different infections, autoimmunological diseases and anticancer therapy. effi cient microwave-assisted synthesis of myelin epitopes mog - and mog - using cltr-cl resin fmoc/tbu methodology. unlike conventional heating, microwave energy directly activates any molecule with a dipole moment and allows for rapid heating at the molecular level. the protected peptides were synthesized using the cem liberty automated microwave peptide synthesizer in and . hours respectively, with a -minute fmoc deprotection ( % piperidine solution in dmf) and -minute coupling reactions using dic/hobt in dmf solution. the maximum temperature reached during both the deprotection and coupling reactions was °c except for the coupling of fmochis(trt)oh ( °c). the fi nal crude products were of high purity as identifi ed by analytical rp-hplc. siah up-regulates the hif- alpha hypoxic response pathway: siah binding peptides coupled to cpps inhibit siah activity and demonstrate proof of concept that siah is a viable anti-tumor drug target vegf, a secreted downstream component crucial for angiogenesis, has been successfully targeted clinically using antibody therapy (avastin). the hypoxic response, however, activates other pathways that stimulate tumor growth, suggesting that inhibitors of upstream components in the pathway may be useful to give a broader spectrum of inhibition. we have focused on the siah proteins that regulate hif- ƒÑ levels by ubiquitylation and degradation of the prolyl hydroxylases (phds) immediately upstream of hif- ƒÑ in the hypoxic response pathway. in a proof-of-principle study, we have shown that siah inhibition by expressed protein fragments (from a drosophila high affi nity interacting protein, phyllopod) can inhibit the stabilization of mammalian hif- ƒÑ during hypoxia and limit tumor growth in a mouse tumor model. a amino acid peptide sequence (phyl) from the phyllopod protein has been identifi ed as the interaction site with siah. the aim of this work was to show that a small peptide could mirror the activity of the transfected recombinant phyllopod protein. to achieve this, phyl was covalently attached to cell penetrating peptides (cpps) and tested for its ability to inhibit hif- ƒÑ stabilization and hypoxic response in human u os cells. both the tat sequence and penetratin were utilized as cpps and attachment was via disulfi de, maleimide or through a pro spacer sequence. the cpp¡vphyl constructs were found to have varying activities but a penetratin-pro -phyl was found to be inhibitory in the u os cell-line hif- ƒÑ stabilization assay. this result demonstrated proof of concept that siah inhibition could be attained by targeting a restricted and specifi c protein-protein interaction site. ( ) . the principal event in the development of prion disease is the transformation of the normal cellular prion protein (prpc), which is highly helical in nature, into the pathogenic isoform prpsc which is insoluble and has an extensive ƒÒ sheet structure. the events surrounding this transformation are very poorly understood, but the mechanism of the detailed steps involved in this process is fundamental to the understanding of prion disease pathogenesis. in an endeavour to study the structure of polypeptide component of the n-terminal section of prpc, synthesis of a range of prp polypeptides were assembled on a cem liberty microwave synthesiser. these fragments ranged in length from to amino acids and span the protein sequence from position to . standard cem synthetic coupling cycles were used, except when peptides were greater than amino acids in length, whereby longer coupling cycles were employed. a number of purifi cation strategies were employed, such as c and c rp-hplc at either c or c and size exclusion chromatography. the purifi ed peptides were characterised by rp-hplc and esi-ms. in addition, they were analysed for secondary structure by cd spectrometry, and evaluated for cell toxicity and fi bril forming ability with tht. the synthesis of a mer peptide for investigating the mechanism of action of the hiv fusion inhibitor t however, the mechanism of action of t is still in debate. it is believed that peptides derived from gp chr region may share a common mechanism, by binding to gp nhr coiled coil and preventing formation of the fusogenic gp core-six helix bundle ( hb), thereby inhibiting fusion between the virus and target cell membrane. we have synthesized a -mer nhr peptide-n -covering all the binding sites for any length of the chr-peptide. synthesis of the polypeptide was carried out using conventional as well as microwave assisted solid phase synthetic methods. details, including comparison of the synthetic approaches will be presented. using c , another anti-hiv chr peptide containing the pocket-binding domain, as a control, we analyzed the activity of t to interact with n- to form hb and to inhibit the -hb formation between n- and c by cd spectroscopy and elisa. we found that t- , unlike c could neither form a stable hb with n , nor inhibit the -hb formation of the fusogenic hb core. our results thus suggest that t- and c- peptides inhibit hiv fusion by different mechanisms of action. the oligomerisation equilibrium of ts is modulated by a fi ne tuning; shifting this equilibrium towards the monomeric form would cause ts inactivity and low translation, overcoming resistance mechanisms encountered for (co)substrate-like inhibitors as the inactive monomer regulates its own expression. our group designed some small ligands to interfere with thymidylate synthase dimerisation, including short peptides taken from the interface sequence that represent the natural ligand for this region, mimicking the other subunit without forming a functional dimer. interesting biological data are arising from the activity tests but a rapid screening assay is necessary to prove they are really interfering with ts oligomerisation. fret is a spectroscopic phenomenon whose intensity depends on the distance between two fl uorophores; when this value varies due to conformational changes of the protein the probes are linked to, consequent fret variation can be used to sense the state of the protein(s) ( stromal derived factor- (cxcl ) is a amino acid cxcchemokine with critical role in homing, migration and guiding of different cell types including hematopoietic progenitor cells (hpc), stem cells, tumour cells and neuronal cells during embryogenesis and in adults ( ). these various functions in physiological as well as pathophysiological processes make this small protein interesting for regenerative medicine. to apply this chemoattractant in medicine, it is needed to form spatially and temporally controllable concentration gradients of active sdf- in response to a non-tissue damaging trigger like visible or near uv-light. after irradiation of an inactive prodrug dramatic change in conformation or lack of sterical hindrance should then lead to fully biological activity under physiological conditions within a few minutes. to prove the principle and assess its potential in photodynamic therapy of neuronal injuries a water-soluble, photosensitive sdf- analogue has been developed. for this the expressed protein ligation (epl) approach has been used, in which one segment has been recombinantly expressed and purifi ed using the impact ® -system to yield the corresponding peptide thioester ( ) . the modifi ed peptide fragment has been chemically synthesized on solid phase using fmoc-strategy. the photocleavable moiety, the -nitroveratryloxycarbonyl (nvoc) protecting group, has been introduced at a side chain amino group. furthermore, the analogue has been characterised by physico-chemical methods and has also been tested in vitro on transfected cos- cells in order to determine its biological activity after activation. sdf- is a chemokine that plays a major role in traffi cking of hematopoietic stem cells (hsc). thus it enables the formation of bone marrow during embriogenesis and later in adult life it supports retention and homing of these cells in the bone marrow. furthermore it is involved in organogenesis and regeneration, respectively. due to these promising features the subform sdf- could serve as a therapeutic target. for studies on the small protein concerning its molecular properties as well as its therapeutic potentials, it needs to be modifi ed chemically. in order to reach these goals, the n-terminus sdf- - has been cloned and expressed recombinantly in e. coli er as a thioester, while the c-terminus sdf- - has been synthesized via solid phase peptide synthesis. modifi cations are thereby introduced at the c-terminus at lys . up to now carboxyfl uorescein has been coupled to the -amino group of the lysine residue. the two fragments then have been ligated via expressed protein ligation (epl), a subform of the native chemical ligation (ncl). activity studies and fl uorescence microscopy on hek cells transfected with the sdf -specifi c g-protein coupled receptor cxcr have been conducted. stueber, werner; lewandrowski, peter; frisch, juergen; weinschenk, toni; singh, harpreet immatics biotechnologies gmbh, germany ima is a multiple peptide vaccine for the treatment of renal cancer (rcc). the tumor-associated peptides (tumaps) contained in ima were identifi ed by immatics directly from primary renal cells (= primary rcc tumor tissue samples), selected regarding their over-expression in rcc and proven to be immunogenic using in vitro t-cell assays. ima consists of individual peptides ( tumaps) and nonactive ingredients which are used as excipients of the pharmaceutical presentation of ima . all peptides are synthesized by conventional fmoc chemistry. the sequences of the peptides will be presented and technical issues will be discussed. in the fi nal formulation of ima g of each peptide plus excipients are fi lled into glass vials and lyophilized. the challenges of the production of multi peptide drugs will be discussed. such challenges comprise the synthesis, the production of the formulation as well as the analyses of the fi nal presentation of ima . results of the phase trial in vaccinated rcc patients showed that ( ) ima was safe, ( ) multiple t-cell responses to vaccinated peptides correlated with favourable clinical outcome and ( ) patients with a lower percentage of regulatory t cells (tregs) were more likely to develop a vaccine-induced multiple t-cell response. novel peptides -msh analogs with high candidacidal activity in an attempt to improve the candidacidal activity of -msh and to better understand the peptide structure-antifungal activity relations, we designed and synthesized novel peptide analogs. because previous data suggested that the peptide [dnal- , phe- ]--msh( - ) has greater candidacidal activity than -msh and is the most potent of the analogs tested in the past, this compound has became our lead ( , ) . from this lead compound we have synthesized a new library of peptides where we have replaced the glycine in position with unconventional amino acids. here, we report new analogs with a strong antimicrobial and candidacidal activity. the obtained results are very encouraging in that they show the great potential of these peptides as a truly novel class of candidacidal compounds. derivative were modifi ed by the same amino acid to establish infl uence of the adjacent amino acid on antimicrobial activity of the compounds studied. the activity of all obtained peptides was screened against model gram-positive (bacillus subtilis) and gram-negative (escherichia coli) bacteria whereas antifungal activity was tested against yeast pichia pastoris. all tests were performed using antibiogram method whereas the minimal inhibitory concentrations were determined using two-fold serial dilution technique. huang, yen-hua ; colgrave, michelle l. the cyclotides are a family of naturally occurring macrocyclic peptides that combine the unique features of a head-to-tail cyclic backbone and a cystine knot motif, which impart extraordinary stability to this peptide family. a recent study demonstrated that the prototypic cyclotide kalata b possesses signifi cant activity against two economically important sheep nematodes haemonchus contortus and trichostrongylus colubriformis. an alanine scan of the molecule highlighted the residues critical for activity. in this work, we explore the relative importance of positively charged residues in different regions of the molecule to aid the understanding of the structural and biological basis of its nematocidal activity. a lysine scan has been conducted, in which each of the non-cys residues in this amino acid peptide has been successively replaced with lysine and the suite of peptides have been assayed against the two sheep nematodes. substitution of residues in loop , v , g , n , t , w , v , l , p , and v decreased or completely abolished the activity, suggesting that these residues are critical to the nematocidal activity of kalata b . on the other hand, incorporation of a positive charge in positions g , t , t , n , and g signifi cantly enhanced the anthelmintic activity of the grafted peptides, up to fourfold, compared to native kalata b . these increases in activity after lysine incorporation into the kb scaffold raise the possibility of being able to engineer greater anthelmintic activity into the peptides, further highlighting their potential as anthelmintic agents. ivanova, v.p. interaction of cells with extracellular matrix (ecm) affects many aspects of cell behavior including growth, morphology, migration and differentiation. integrins are known to mediate cell adhesion to proteins of ecm. ligand-binding properties of integrins depend not only on composition of ecm ligands and level of integrin expression in cell, but also on spectrum and activity of soluble factors indirectly infl uencing cell-matrix contacts. interaction of cells with substratum may be divided into cell adhesion and spreading. following an initial cell attachment event, cell may or not spread depending on cell type and the nature of the molecular signals they receive. oligopeptides released from different proteins during their proteolysis in or out of cells may act as the such short-time existing signals. in the present study we investigated the effect of multiply repeated peptide fragment in different collagen types on cell spreading. murine embryonic fi broblasts ( /ml) were allowed to adhere for min at ° c with or without peptide (in various concentrations) to plastic surface pre-coated or not with gelatin. it was shown that the synthetic peptide increased the number of spread cells and caused shape changes in cells spread on different substrata. a -min pretreatment of cells with the peptide (before conducting of cell spreading assay) resulted in inhibiting of cell spreading on gelatin. our results suggest that the peptide regulation of cell spreading could be related to its effect on re-distribution of integrin receptors in sites of cell contacts with substratum. bioactive peptides from cyanobacteria cyanobacteria are versatile source of small peptides from which vast majority are cyclic. cyanobacterial culture collection in university of helsinki contains over strains and this collection is used for screening of new bioactive compounds. cyclic heptapeptides, microcystins are the best known cyanobacterial peptide family. over structural variants have been described from which most are strong hepatotoxins. our research group have participated to the determination of the structure of many novel microcystins and other cyclic and linear cyanobacterial peptides. in recent years we have found highly toxic and novel microcystins from lichen associated cyanobacteria ( ) and from anabaena strains of baltic sea ( ). in the structural analysis of new peptides from the known peptide families we have used liquid chromatography ion trap mass spectrometry which have proven to be very effective method and is in many cases the only method needed in the verifi cation of a new structure. with this lc-itms method we have found many new structural variants from anabaenopeptin, anabaenopeptilide and spumigin peptide families. in collaboration with many research groups we have found cyanobacterial compounds/extracts which inhibit protein kinase c activity, inhibit/activate boar sperm motility, disintegrate cell membranes or are antidotes for microcystin toxicity. structures of the compounds are under study except the microcystin antidote which structural analysis showed that it belongs to a rare peptide family containing imino bond in cyclic skeleton. the results of n-terminal modifi cation of arginine vasopressin with cis- -amino- -phenyl-cyclohexane carboxylic acid. the highly potent oxytocin receptor antagonists arginine vasopressin (avp) is a cyclic nonapeptide with multiple functions. the main peripheral physiological roles of avp are the regulation of water balance, the control of blood pressure, and the release of adrenocorticotropin hormone (acth). moreover, avp also exhibits to some extent typical oxytocin (ot, a closely related neurohypophyseal peptide) activities such as the galactogogic and the uterotonic effects. all peptides were tested for the pressor, antidiuretic and uterotonic in vitro activities in the rat. cis-apc modifi cation at position of avp is suffi cient to change the pharmacological profi le of the peptides. analogues i -iv were moderately potent antidiuretic agonists with prolonged action. in regard to the uterotonic activity, all peptides with cis-apc were highly potent antagonists, except compound i. it supports our earlier hypothesis that an amino acid residue in position has signifi cant impact on pharmacological activities. the incorporation of unnatural non-proteinogenic -amino acids into peptides has emerged as a novel and promising approach in peptide modifi cation. the conformationally restricted amino acid derivatives are of particular interest. this thesis are also supported by our already -years research focused on the effects of steric restriction and the presence bulky substituent in the n-terminal part of avp molecule on biological properties of the resulting analogues. all peptides were tested for the pressor, antidiuretic and uterotonic in vitro activities in the rat. all the analogues were devoid of the pressor potency and exhibited only negligible antidiuretic activity. interestingly, in regard to the uterotonic activity, the new compounds exhibited moderate (i) or high (ii -iv) antioxytocic potency. it should be point out that single substitution e.g. replacement of tyr in avp molecule with igl results in moderately potent and highly selective antagonists of oxytocin (i). our new igl substituted peptides proved that the presence of the sterically restricted amino acid residue may result in high active and selective antiuterotonic agents. these in our opinion interesting fi nding demonstrates the usefulness of our approach in the design of new highly active and selective analogues with desired pharmacological properties. arginine vasopressin (avp), a neurohypophyseal hormone and neuromodulator, is a cyclic nonapeptide with a disulfi de bridge between cys residues at positions and . as a hormone avp exerts its biological effects upon binding to three receptor subtypes termed: v a , v b (v ), and v . furthermore, avp to some extent can interact with the oxytocin receptor (ot). biological activity of peptides is determined by their structure and conformation. conformational restriction of bioactive peptides is therefore a well-established strategy to change their pharmacological profi le. peptide fl exibility can be restricted by a local constraint imposed, e.g. by introducing amino acids with limited conformational freedom, that has an impact on specifi c orientations of the peptide backbone and the side chains. in this work, we decide to check the infl uence of the bulky ( design, synthesis and biological activities of temporin a and temporin l analogues. temporins a (ta) and l (tl) are antimicrobial peptides isolated from the skin of red european frog "rana temporaria". temporins are active against a broad spectrum of microrganism: ta (flpligrvlsgil-nh ) is preferentially active against gram-positive bacterial strains; tl (fvqwfskflgril-nh ) has the highest activity against fungi, and bacteria, including resistent gram-negative strains, but it shows haemolytic activity too. ta exerts its antimicrobial activity by its ability to form a transmembrane pore via a 'barrel-stave' mechanism or to form a 'carpet' on the membrane surface via the 'carpet-like' model. recently we investigated the preferential conformation of tl and ta in sds and dpc solutions which mimic bacterial and mammalian membranes, respectively. in sds, the peptides prefer a location at the micelle-water interface; in dpc, they prefer a perpendicular location to the micelle surface, with the n-terminus imbedded in the hydrophobic core. tl shows higher propensity, with respect to ta, in forminghelical structures in both membrane mimetic systems and the highest propensity to penetrate the micelles ( ). on these results we designed and synthesized new ta and tl analogues and found interesting differences in their effi cacy against microbial species, and fi nding a new potent antimicrobial agent without haemolytic activity. arginine vasopressin (avp), a neurohypophyseal nonapeptide hormone [cycle - (h-cys -tyr -phe -gln -asn -cys -pro -arg -gly -nh )], elicits a variety of responses both centrally and peripherally by acting on three distict g-protein coupled receptors: v a (vascular), v b (pituitary) and v (renal). it also binds to the oxytocin (ot) receptor. in addition to its well-known antidiuretic activity, avp has also complex cardiovascular actions and adrenocorticotropic hormone (acth) releasing activity. binding of avp to the v a receptor subtype also stimulates glycogenolysis in the liver and promotes platelet aggregation. it is generally accepted that the conformation of the n-terminal part of neurohypophyseal hormones analogues is important for their pharmacological activity. in continuing our work aimed at the design of selective avp analogues, we synthesized twelve new analogues of avp containing mercapto propionic acid ( we also studied the effect of modifi ed c-terminal amide on biological potency of the new avp analogues. the analogues were synthesized by fmoc/bu t solid phase methodology and were tested for their rat uterotonic in vitro activity, rat pressor activity and antidiuretic activity using conscious rats. the modifi cations performed had a signifi cant impact on pharmacological activities of the analogues. acknowledgements: we thank the european social fund (esf), operational program for educational and vocational training ii (epeaek ii), and particularly the program pythagoras i, for funding the above work. it was also supported by the research project no. z of the academy of sciences of the czech republic. three-dimensional structure and mechanism of action of an antifungal peptide generated from hemocyanin cleavage in a penaeid shrimp an antifungal peptide, pvhct, which corresponds to the amino acid c-terminal sequence of the shrimp respiratory protein hemocyanin, has been previously identifi ed in the plasma of the penaeid shrimp litopenaeus vannamei ( ) . it is generated by proteolytic cleavage in response to a microbial challenge. similarly, a c-terminal fragment of hemocyanin displaying antimicrobial activity has been isolated from crayfi sh plasma ( ) . these peptides are believed to contribute to the crustacean defence. the phenomenon of in vitro antimicrobial peptide generation from a respiratory pigment already observed with hemoglobin thus appears not to be restricted to mammals ( ). pvhct displays a broad spectrum of antifungal activity with minimum inhibitory concentrations (mics) in the range - m ( . m against the shrimp pathogen fusarium oxysporum). its activity would be based on the inhibition of spore germination ( ) . to contribute to the elucidation of the mechanism of pvhct antifungal activity, we determined its three-dimensional structure by circular dichroism (cd), nmr and molecular modelling and examined its effects on the f. oxysporum spore ultrastructure by transmission electron microscopy (tem). cd and nmr data indicate that pvhct is unfolded in an aqueous environment but adopts a similar helical structure in methanol solution and in dodecylphosphocholine (dpc) micelles used to mimic biological membranes. the structure consists of an amphipathic -helix spanning residues to . tem shows that pvhct induces structural changes of the plasma membrane accompanied by a disorganization of the cytoplasm and a signifi cant decrease of lipid bodies. glucagon-like peptide- (glp- ), glucagon (gcg), and oxyntomodulin (oxm) are highly homologous peptide hormones derived from posttranslational processing of the preproglucagon gene. the sequence of oxm in particular, is identical to the sequence of gcg, with an amino acid extension at the c-terminus. pharmacological doses of oxm activate both the glp- receptor (glp r) and the glucagon receptor (gcgr) albeit with lower affi nity compared to glp- and glucagon, respectively. in order to understand the origin of this dual specifi city, we synthesized a number of oxm analogs in which one or more of the native amino acids were replaced. first, a chimera was produced in which all the residues differing between glp- and gcg were grafted into the oxm native sequence, yielding an analog with the same pharmacologic profi le as glp- . further studies showed that surprisingly, the switch from glp r/gcgr co-agonism to glp r-selective agonism could be obtained by a single amino acid substitutions at position of oxm. in particular, the analog with gln substituted by glu (oxm-q e) showed the same activity as native oxm on glp r, but complete loss of activity on gcgr. oxm and oxm-q e were compared in a hyperglycemic clamp study performed in diet-induced obese (dio) mice. due to the short half-life of the peptides, both were infused intravenously at ~ g/kg/ min in chronically catheterized mice. the amount of exogenous glucose required to maintain the hyperglycemic level was -fold greater with the selective glp r agonist oxm-q e than with the glp r/gcgr coagonist oxm, showing that abolishment of gcgr activity signifi cantly improves the glucose-lowering effect. we believe that these fi ndings could be useful for the development of a peptide therapeutic for the treatment of type diabetes. hospitals across europe and north america have lately been plagued with infections caused by clostridium diffi cile, a diffi cult to treat bacterium due to its resistance to many commercially available antibiotics. recently, we isolated a two-component bacteriocin, thuricin, produced by bacillus thuringiensis that exhibits activity against c. diffi cile. we found that these peptides, called trn and trn , operate together in a synergistic fashion to inhibit bacterial growth. the peptide combination was found to be highly active against a wide range of c. diffi cile strains, including the virulent epidemic strain of the o ribotype, as well as most other clostridia and some bacilli and listeria species. maldi mass spectrometry revealed that both peptides have molecular weights that are lower than those predicted from their genetic sequences, indicating that they are post-translationally modifi ed. sequencing of the mature peptides through tandem mass spectrometry showed that each peptide has three modifi ed amino acid residues near its c-terminus. these residues were found to be two units lighter than their expected natural amino acid masses, suggesting a loss of two hydrogen atoms through dehydrogenation or oxidation. in order to confi rm these proposed modifi cations, we are investigating the production of c-and n-labeled trn and trn for structure elucidation by nmr. isolation of labeled peptides will be achieved by growing b. thuringiensis on defi ned media containing [u- c] glucose and ( nh ) so . subsequent nmr experiments will enable us to determine the three-dimensional solution structures of trn and trn , individually and bound together. by elucidating thuricin's structure, we aim to better understand its mechanism of action against c. diffi cile. the most potent peptidic human bradykinin (bk) b receptor antagonist is hoe- (icatibant). in this study we present the synthesis of nine new analogues of hoe- substituted at position , or with chosen d-amino acid residues and/or nonproteinogenic amino acid residues, e.g. n-cyclohexylglycine (nchg), -aminocyclohexane- -carboxylic acid (acc), octahydroindole- -carboxylic acid (oic), piperidine- -carboxylic acid (nip) and -phenylpiperidine- -carboxylic acid (ppc). in the next nine peptides we combined the above mentioned modifi cations with the placement of -adamantane acetic acid (aaa) at position . all new analogues were tested on human umbilical vein for their antagonistic potency. only three compounds containing nchg , nchg or ppc exhibited noticeable antagonistic activities on human bradykinin receptors thus still less potent then original hoe- sequence. each of them with aaa showed lower activity then parent analogues. peptides: d-arg-arg-pro-hyp-gly-thi-ser-d-phe-nip-arg and aaa-d-arg-arg-pro-hyp-gly-thi-ser-d-phe-nip-arg, although strongly potent antagonists against bk-induced blood pressure lowering responses in rats, did not show noticeable antagonistic activity on bk-induced contraction of the isolated human umbilical vein, a well-established b r bioassay system, suggesting a species dependent activity of the compounds. due to advances made in the peptide fi eld during the last few years, peptides as therapeutics have continued to gain more interest. the interest in peptide therapeutics generally comes from their high specifi city to targeted sites, their diversity and their usually low toxicity. pt- , also known as bremelanotide, and mt-ii are potential future drugs and both are heptapeptides with a -membered cyclic monomeric lactam bridge. they are melanocortin receptor agonist and an analog of alpha-melanocyte stimulating hormone (a-msh). the pt- molecule has a c-terminal acid group while mt-ii has a c-terminal amide function. both neuropeptides have been tested in treating male sexual and erectile dysfunction as well as female sexual arousal disorder. pt- , patented by palatin technologies, new jersey, usa, is the only known synthetic aphrodisiac and unlike other erectile enhancers like viagra, it does not act upon the vascular system. instead, it directly increases sexual desire and is used nasally as a spray. a scalable method for the synthesis of both peptides using fmoc-chemistry and the problems involved during the synthesis process will be discussed in details. tateaki, wakamiya; takahiro, nishimaru; kiyoe, mori; yoshihiro, yamaguchi kinki university, japan l-glutamate (glu) is known to be major excitatory neurotransmitter not only in the mammalian central nervous system but also in the ganglia of arthropods. binding of spider toxins to glutamate receptors (glurs) results in the inhibition of glu-mediated neurotransmission. in order to elucidate the mode of binding between glu and glurs, we focused on the visualization of glurs by complex formation with fl uorescentlabeled analogs of nptx- ( ), a spider toxin with the structure of n -( , -dihydroxyphenylacetyl-l-asparaginyl)-n -l-lysyl- , -diaza- , -dodecanediamine [dhpa-asn-dada( lys)]. in the present study, the modifi ed nptx- , i.e., dhpa-asn-dada( abg) ( ) in which the lys residue of was replaced with the n-( -aminobutyl)glycine (abg) residue, was employed as a template compound to create the fl uorescentlabeled analogs of nptx- , since the biological activity of is three times higher than that of . we thus carried out the modifi cation of dhpa in the analog , i.e., ) dhpa was replaced with the coumarintype acyl residues (type- ); ) the phenylacetyl residues having alkyne side chains that can be converted into suitable fl uorophores based on the click chemistry (type- ); and ) the coumarin-type acyl residues having the mercapto group to form disulfi de bond with the cys residue in glurs (type- ). this paper presents the synthesis and biological activity of various nptx- analogs to adopt as probes for visualization of glutamate receptors. an investigation of the functional requirements of apidaecin ib c-terminal fragment by means of peptoidpeptide hybrids by acting on one or more intracellular targets, without damaging the cytoplasmatic membrane. their particular killing mechanism and their low toxicity against mammalian cells make them attractive for the development of new antibiotics. structure-activity relationships studies have been carried out on short pro-arg rich antimicrobial peptides isolated from insects and the characterization of various natural isoforms of the -residues peptide apidaecin ib allowed to identify an evolutionary conserved region in the c-terminal part of the molecule. even a single point mutation in this region results in reduction or loss of antimicrobial activity. we recently described the synthesis of some apidaecin ib peptoid-peptide hybrids in which each arginine was replaced by the corresponding n-alkyl glycine residue ( ). the afforded modifi cation made the resulting peptoid-peptide hybrids more resistant to proteolysis but moving the [narg]residue from the n-to the c-terminal end of the molecule progressively reduced the antibacterial activity. here we report the synthesis of a series of novel analogues containing a n-homoarginine or n-norarginine residue in position , or . the effect of the size of the side-chain of the peptoid residue on the antimicrobial activity is also reported. in order to strengthen the peptide resistance to proteolysis and by considering that enzymic cleavage of the arg -leu peptide bond yields a fully inactive compound, we also prepared the [nleu ]-apidaecin analogue. the conformational properties of the resulting peptoid-peptide hybrid, which is devoid of any antimicrobial activity, will be compared to those of apidaecin ib and the other [narg]peptoid-peptide hybrids. a wide variety of organisms produce antimicrobial peptides as part of their fi rst line of defense. however, antimicrobial activity is not the main function of some of these peptides. in the cuttlefi sh sepia offi cinalis we observed an antibacterial activity for the neuropeptide : h-alsgdaflrf-nh ( ). this decapeptide belonging to the fmrfamide family involved in regulation of reproduction and chromatophore function and is able to inhibit the growth of marine bacteria. circular dichroism studies have revealed for this amphiphilic peptide a helical structuration in sds micelles and in % tfe. to improve this antimicrobial activity, we fi rst introduce a lysine residue instead of aspartic residue, the antimicrobial activity of this new peptide has been improved by augmentation of the positive net charge (+ to + ). moreover preliminary results have shown that the incorporation of aza- -amino acids analogues could create original hybrid pseudopeptide with superior activity . the natural peptide is not effi cient against staphylococcus aureus whereas the pseudopeptidic analogue revealed a minimum inhibiting concentration (mic) between - m. therefore some -amino acids were replaced by aza- -amino acids. we will show in this communication that depending on the substituted residue and on the structuration, these modifi cations could lead either to no activity or to a drastic enhancement of the antimicrobial activity, demonstrating that aza- -amino acids can facilitate the burying in lipidic bilayer of antimicrobial peptides that acts on bacterial membranes( ) references: isolation and structural characterization of capistruin, a lasso peptide predicted from the genome sequence of burkholderia thailandensis e the poor overall fragmentation behavior in ms n studies suggested a branched cyclic peptide with a rigid lasso structure. optimization of the fermentation conditions increased the production by -fold and subsequent nmr structural studies proved the lasso structure of the peptide that was named capistruin. heterologous production of the lasso peptide in e. coli showed that the identifi ed genes are suffi cient for the biosynthesis of capistruin, which exhibits antimicrobial activity against closely related burkholderia and pseudomonas strains. to our knowledge, this is the fi rst rational based identifi cation of a novel lasso peptide and the presented approach should be advantageous for the isolation of further lasso peptides in the future. endogenous antimicrobial peptides (amps) are the earliest molecular factors in the evolution of innate immunity. marine invertebrate animals have no acquired immunity with a system of antibodies diversifi cation. they are presumed to use an amps-based system as principal defense against potential pathogens. we have discovered a new family of small ( -residue) amps, termed arenicins, in coelomocytes of marine polychaeta lugworm arenicola marina. these amps exhibited activity against gram-positive, gram-negative bacteria and fungi. complete amino acid sequences were determined for each isoform. arenicins have one disulfi de bond (cys -cys ). arenicins have no structure similarity to any previously identifi ed antimicrobial peptides. a novel -residue antimicrobial peptide, aurelin, exhibiting activity against gram-positive and gram-negative bacteria, was purifi ed from mesoglea of a scyphoid jellyfi sh aurelia aurita. complete amino acid sequence of aurelin was determined. aurelin has cysteines forming three disulfi de bonds. the total rna was isolated from the lugworm coelomocytes and from the jellyfi sh mesoglea, rt-pcr and cloning were performed, and cdnas were sequenced. a -residue preproarenicin contains a putative signal peptide ( amino acids) and a long prodomain. a residue preproaurelin contains a putative signal peptide ( amino acids) and a propiece of the same size ( amino acids). aurelin reveals partial similarity both with defensins and k+ channel blocking toxins of sea anemones and belongs to shkt domain family. overlapping of biological properties of marine animal amps and toxins along with their sequence homology might be a consequence of divergent evolution from a common ancestor. antimicrobial peptides from marine organisms could afford design of new antibiotics manifesting broad-spectrum antibacterial activity. cyclic enkephalin analogs containing two alkylurea units we shall present some structure-activity results for enkephalin analogs, derived from the exhaustive combinations of d-lys and d-orn in position with lys, orn, dab and dap in position , both positions coupled ¦Ø-¦Ø¡¯ by means of the urea bridge. accordingly, they all are restrained by - -membered rings. in addition, we introduced the -nh-ethylurea unit instead of -nh of amide group in previously published analogs [ ] [ ] [ ] . their in vitro activities were determined in the gpi and mvd assays. the peptides are more active than enkephalin in the gpi while have similar activities to the latter in the mvd assay. the effect of the introduction of ethylurea unit at the c-terminus on the activities is also discussed. chemical shifts of the peptides in water were fully assigned and their sequences confi rmed. several cross-peaks between the protons of amidoalkylurea unit and preceding residues have been observed in each case, suggesting that the unit may be involved in specifi c interactions between residues and . understanding the structure/activity relationships of hepcidin iron is an essential element for nearly all living organisms and plays a key role in a range of processes including oxygen transport and storage, catalysis of redox reactions, production of metabolic intermediates and host-defence ( ). until recently, little was known about the regulatory elements involved in the control of iron uptake and distribution within the body. the recently discovered peptide hepcidin has been shown to be a key regulator of iron metabolism within the body in response to a range of conditions, including infl ammation, hypoxia and anaemia ( ) . this talk will focus on work towards elucidating structure/activity data for hepcidin with the aim of gaining a better undertstanding of the interaction between hepcidin and its receptor, ferroportin. we hope that this information will facilitate the design of synthetic agonists or antagonists of ferroportin to be used as potential drug leads. three-dimensional structures of investigated analogues were determined using two-dimensional nmr spectroscopy and molecular dynamics simulations with time-averaged restraints. the analysis of structural differences exhibited by different modifi cations provides the basis for understanding conformation -activity relationships and thereby the mechanism of interactions of the analogues with receptors. nmr structure of the micelle-bound rfa and rfa, two peptide ligands of the gpr receptor a novel rfamide peptide, named rfa and with no meaningful similarity with other members of this family, has been recently characterized. its precursor encompasses several potential cleavage sites and thus may generate various mature peptides including an nterminally extended form of rfa, termed rfa. both peptides act as endogenous ligands of the g-protein coupled receptor gpr . this receptor has been recently implicated in the bone metabolism regulation the determination of the d structure of such peptides is essential for the elucidation of their structure/function relationships and for the design of potent agonists or antagonists. although structure elucidation of a ligand in the absence of the target receptor can deliver limited insight into the bioactive conformation, there is emerging evidence that interactions with the cell membrane is a key step required for receptor recognition. in this context, we have investigated the solution conformation of rfa and rfa by cd, nmr and molecular modelling in different media, in particular in one miming the "free" form of the molecule (methanol or mixture tfe/water) and in a cell membrane mimetic medium (dpc micelles). in an organic solvent, both peptides adopt the same conformation, i.e. an amphipathic alpha-helical structure, fl anked by two n-and c-terminal disordered regions. when bound to dpc micelles, the n-terminus remains fl exible and an helix is present at the same position as in the "free" form for both molecules. in contrast, the cterminal extremity becomes structured adopting an inverse gamma-turn conformation. these data represent the fi rst step for the rational design of new molecules that might be used in the treatment of osteoporosis. acknowledgements: supports were obtained from inserm and ifrmp . the nmr spectrometers are supported by grants of the conseil régional de haute-normandie. nmr and molecular modelling facilities are provided by the centre de ressources informatiques de haute-normandie. antimicrobial activity of analogues of a peptide isolated from venom glands of social wasps polistes major major inhabiting the dominican republic recently we have described isolation and biological activities of several new peptides from the venom glands of social wasps polistes major major found in dominican republic. we have also reported the synthesis of their analogues in order to investigate structure-activity relationship with respect to the antimicrobial and hemolytic activities ( ). here we report the activities of a few further analogues of one of the peptides called pmm (h-ile-asn-trp-lys-lys-ile-ala-ser-ile-gly-lys-glu-val-leu-lys-ala-leu-nh ). the parent sequence or its truncated analogues were modifi ed on the n-terminus with -aminocaproic acid, glycolic acid, palmitoic acid, and -acridinyl and -( , , , -tetrahydro)acridinyl groups. the new analogues were tested for their antimicrobial activity (determination of minimal inhibitory concentration values -micusing broth dilution method) and hemolytic activity (determination of the ic value using suspension of rat erythrocytes). the palmitoylation unfortunately did not enhance antimicrobial activity against the tested microorganisms (bacillus subtilis, staphylococcus aureus, escherichia coli and pseudomonas aeruginosa). substitution of amino acids ser or glu subsequently for alanine, serine or lysine did not infl uence the activity of the peptides signifi cantly. gramicidin s, gs, c-(val-orn-leu-d-phe-pro) , was isolated from bacillus brevis. it forms a two-stranded antiparallel -sheet fl anked by two ii' -turns. it was found that the distribution of hydrophobic and hydrophilic residues on the opposite sides of the sheet is a structural feature required for gs antimicrobial (am) activity. despite its wide gram+ and gram-antimicrobial activity gs is useless in therapy because of its high hemotoxicity in humans. it was found, however, that the analogues of gs- (gs with lys-leu inserted into each strand) got more am selective, when their amphipatic moments were perturbed by swapping adjacent lys leu/val or confi guration reversal at lys ( ) . here, we report on effects of similar perturbations put on gs original c-decapeptide, using the following examples: c-(val-lys-leu-d-his-pro) , , . as the mother compound, and its three analogs, viz. c-(val-d-lys-leu-d-his-pro-val-lys-leu-d-his-pro) -lys converted to d, c-(lys-val-leu-d-his-pro-val-lys-leu-d-his-pro) -val -lys swapped, and c-(val-leu-lys-d-his-pro-val-lys-leu-d-his-pro) -lys -leu swapped, - ; all having reduced ring-sequence symmetry. the peptides were synthesized by solid-phase methods using -fl uorenylmetoxycarbonyl (fmoc) methodology. having solved their structures by d-nmr and having tested their activities/selectivities, we confi rmed that only had relatively favorable bio-profi le, as already published ( ) cereulide, a foodborne peptide highly toxic towards the insulin producing beta-cells of the pancreas cereulide is a heat stable cyclic, lipophilic (log kow . ) peptide ( g mol/ ) produced by certain strains of bacillus cereus, a bacterium connected to emetic food poisonings. it is insoluble in water, soluble in ethanol, methanol, dmso, food oils. cereulide exposure caused collapse of mitochondrial membrane potential in all tested human cells at low (ng/ml) exposure concentration (nk cells, t lymphocytes, caco , calu , neural paju, hela). toxicity is caused by its action as ion carrier with a selectivity of k + : na + = > : . in various foods connected to human illness, concentrations of . to g/g of cereulide were measured. in a case where the remains of a meal that had caused acute serious illness of two adult persons, were obtained for analysis, . g of cereulide was found /g of food. we undertook to explore the effects of purifi ed cereulide, and cereulide containing bacterial extracts, on porcine pancreatic islet cells in culture. foetal porcine islet cells were exposed to heat killed extracts from food-borne b. cereus strains producing or not producing cereulide and to purifi ed cereulide. effects were assayed using viability staining with fl uorochromes and cellular contents of dna and insulin. exposure to ng/ml of purifi ed cereulide caused necrotic cell death of the islet cells impairing their insulin content within days. cell extracts of cereulide positive b. cereus strains connected to food poisoning or isolated from food items were toxic, corresponding to their measured cereulide content. extracts of b. cereus strains producing or not producing the b. cereus diarrhoeal toxin, but not cereulide, were tolerated by the porcine islet cultures up to concentrations fold higher compared to extracts from strains containing cereulide, produced substance toxic towards porcine fetal langerhans islets and beta cells. application of non-sequential pharmacophore concept for design of antimicrobial peptide dendrimers unique structure of dendrimeric compounds consisting of a central core and several generations of branches provides opportunity of multiple practical solutions in the area of medicine. location of a high number of functional groups at the surface, allows to present multiple pharmacophoric units to the receptors, with immediate application in the design of a new generation drugs or vaccines, tools for studying autoimmune diseases or understanding gene delivery mechanism. here we present another possible application of dendrimeric compounds -preparation of drug molecules, which mimic active conformations of various macromolecular ligands. we focused on low molecular weight basic dendrimeric peptides, which mimic active conformations of recently discovered natural antimicrobial peptides. structurally, natural compounds are linear cationic peptides consisting of - amino acids that kill a broad spectrum of microbes destabilizing ordered structure of their cell membrane. it is generally accepted that positive charge and an induced amphipathic conformation are necessary for their antimicrobial activity. the project is related to multi-drug resistance of numerous bacteria against conventional antibiotics and involves de novo design of - generation dendrimeric peptides, which mimic sequencerelated active conformations of natural compounds (non-sequential pharmacophore concept). apparently, several groups of small peptide dendrimers were synthesized and structurally characterized (nmr, cd). they are potent antimicrobials, active against broad spectrum of species including mrsa and esbl strains. interactions between model membranes and peptide dendrimers of various structure will be discussed. seawater desalination is most commonly done today by reverse osmosis (ro) using thin-fi lm composite membranes. a major problem in ro desalination is biofouling, caused by adhesion and growth of bacteria to form biofi lm on the membrane surface. recently we proposed a new approach to reduce biofi lm formation on ro membranes that is based on immobilization of antimicrobial peptides (amps) onto the membrane surface. in this study we screen amps capable of reducing biofi lm growth under conditions simulating seawater desalination, and search for mode of binding to the membrane without affecting the peptides bioactivity. a specifi c bioassay was developed for screening peptides activity in high salinity conditions in order to evaluate the inhibition of biofi lm growth, based on growing biofi lmforming bacteria in a -wells microtiter plate. we prepared various amps known from the literature by solid phase peptide synthesis (spps) using fmoc-chemistry. the bactericidal activity of amps was examined in fresh water and compared to high salinity water. most amps lost their activity in high salinity conditions; yet, few peptides possessed their bactericidal activity and were used in subsequent experiments. searching for mode of linkage was performed by evaluating the bactericide activity of amps modifi ed with numerous types of linker molecules. based on literature studies that showed no decrease in bactericidal activity of amps upon n-terminal modifi cation, we prepared the corresponding peptides with modifi ed spacers on their amino-terminal and evaluated their antimicrobial activity in solution. indeed, we obtained gly -spacers that retained activity, which were used subsequently as linkers to ro membranes. the mode of binding, as well as bactericide activity of the peptides and of the membranes will be presented and discussed. this study will lay the bases for a novel approach to decrease biofi lm formation on the surface of ro membranes during ro desalination. bioactive tripeptides ile-pro-pro and val-pro-pro protect endothelial function in vitro in normotensive and hypertensive rats milk drink containing casein-derived bioactive tripeptides isoleucylprolyl-proline (ipp) and valyl-prolyl-proline (vpp) has been shown to decrease blood pressure both in animal models and clinical studies. this effect can be attributed to the tripeptides. it has been suggested that one possible blood pressure lowering mechanism of the tripeptides could be angiotensin-converting enzyme (ace) inhibition. however, not all studies support this fi nding. the effect of tripeptides ipp and vpp on vascular function was investigated in vitro using rat mesenteric arteries. superior mesenteric arteries isolated from male wistar-kyoto and spontaneously hypertensive (sh) rats were incubated in krebs solution containing mm of the peptide (either ipp or vpp) in + ºc for , , or h. after incubation mesenteric artery rings were mounted in an organ bath chamber and extensive vascular reactivity measurements were performed. acetylcholine-induced endothelium-dependent relaxation was better preserved (p < . ) in mesenteric arteries of both strains incubated with ipp or vpp compared to the control. clear differences were not observed in sodium nitroprusside-induced endotheliumindependent relaxation. the ace-inhibitory activity of ipp and vpp was studied by measuring the response to a single administration of angiotensin i and ii in organ chambers. proportioned to kcl-induced contraction, no clear reduction in angiotensin i -contraction was seen. thus, ace-inhibition may not be the main mechanism for the long-term effects of ipp and vpp in the protection of endothelial function. we suggest that the tripeptides do not affect smooth muscle but they protect endothelium during incubation indicated as preserved acetylcholine-induced endothelium-dependent relaxation. and directly and modulate other parts of host innate immunity. today, more than cationic peptides have been identifi ed. they all have certain conserved physical features including a net positive charge, contain approximately % hydrophobic amino acids and have sizes ranging from to amino acids. however, virtually any -sheet, loop including -helix, type of secondary structure can arise including -turn and extended. the multitude of cationic peptide sources, structures and a spectra of activity is matched by a number of complex and controversial models attempting to describe and explain their modes of action. little is known about the sequence requirements of short host defense peptides like bactenecin ( mer). with help of our novel technique using a artifi cially created luminescence producing gram negative bacteria and peptide synthesis on cellulose we can investigate the sequence requirements of such peptides. hundreds of peptides are tested for their ability to kill pseudomonas aeruginosa. complete substitutional analyses of different bactenecin variants as well as a semirandom peptide library with about members were measured. the complete substitutional analysis will give us information about the importance of each single position whereas the peptide library will give us broader information which composition of amino acids results in an active antimicrobial peptide. the data will be analyzed using the quantitative structureactivity relationship approach (qsar) to identify sequence patterns that discriminate between superior activity cf. equivalently active and inactive. this will give us mechanistic cues for a better understanding of the mode of action of the short antimicrobial peptides. the results of these measurements and analyses will be discussed in detail. here we propose a simple and rapid fl ow cytometric method to assess internalization of the peptides in bacteria. the method is based on the use of fl uorescently-labeled peptides and of the extracellular quencher trypan blue to discriminate between a cell surface and cytoplasmic localization of the tested molecules. to his aim, we used bodipylabeled peptides showing different modes of action. these included some fragments of bac , a proline-rich peptide known to penetrate bacterial and eukaryotic cells without membrane damage [ , ] , and polymyxin b, a peptide antibiotic that binds to lps and to the cell membranes. by using this approach coupled to fl ow cytometric analysis, we showed that the fl uorescence intensity of e. coli and s. typhimurium cells treated with sub-inhibitory bodipy-bac concentrations did not decrease despite extensive washing and addition of the quencher trypan blue. in contrast, the fl uorescence of cells treated with bodipy-polymyxin b, as well as that of bacteria treated with a fl uorescein-labeled anti lps antibody, were promptly and almost totally quenched by addition of trypan blue, indicating their accessibility on the bacterial surface. these results confi rm the suitability of this method to rapidly infer the localization of labelled molecules in an accessible or inaccessible compartment of the treated bacterial cells. hp ( - ) is an antimicrobial peptide derived from the n-terminus of helicobacter pylori ribosomal protein l (rpl ). in our previous study, several analogues of hp ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , with amino acid substitutions that increased or decreased net hydrophobicity, were designed and showed that an analogue, a designed by substituting gln and asp with trp at positions and , respectively, caused increased antibacterial activity in minimal inhibition concentration (mic) and minimal bactericidal concentration (mbc) without having hemolytic activity. the peptide a acted also synergistically with known antibiotics including chloramphenicol against bacterial cells. fluorescence activated fl ow cytometry showed that a -treated cells had higher fl uorescence intensity than untreated cells, similar to that of melittin-treated cells. the peptide a showed a strong antimicrobial activity against antibiotic-resistant pseudomonas aeruginosa from otitis media, clinically isolated mrsa and vrsa including biofi lm-forming bacteria in vitro and in vivo. the ototoxicity of a was studied in vivo by topical application to the middle ear in guinea pig model. twenty guinea pigs ( groups, each group n= ) were each injected by transtympanic approach with ul of ug/ml, ug/ml, ug/ml, ug/ml of a , and . % gentamicin sulfate was instillated as a control. auditory brainstem responses (abr) to click were measured between st and th days after injection. histologic investigation of cochlea was performed by scanning electron microscope and light microscope. the results showed that topical application of a to the middle ear is well tolerated without cochlear damage. the present study, therefore, demonstrates that the usefulness of antimicrobial peptides for multi-drug resistant bacteria including as a new ototopical agent for crpa otitis media. structure-activity relationship study of kiss- : identifi cation of an antagonist of gpr in the absence of ccda, ccdb inhibit the cell division and can kill bacteria by a mechanism that involves the dna gyrase. bacterial dna gyrase is unique among the type ii topoisomerase with ability to negatively supercoil dna. the enzyme consists of two subunits, a (gyra) and b (gyrb) and operates as an a b heterotetramer. the mechanism of the inhibition of the gyrase activity by ccdb is still an object of many debates, but is clear that r residue of gyra and the c-terminus of ccdb (w -i ) play a crucial role in the gyrase-ccdb interactions. as an approach for a better understanding of this mechanism as well as for development of new gyrase inhibitors, we have synthesized peptide analogues of the ccdb protein and studied its activity by supercoiling assays and bacterial growth. five fragments (ccdb et , ccdb et , ccdb et , ccdb et and ccdb ss ) of the natural ccdb were designed and synthesized by spps. for the design, we considered the residue c-terminal -helix (residues e to w ), the loop that connects two strands of the wing sheet (residues r to l ) and an n-terminal region that includes the fi rst of the fi vestranded antiparallel -sheet. all peptides, except ccdb et , showed inhibition of the supercoiling activity of the dna gyrase, especially ccdb et with a mic = m. free peptides not showed antimicrobial activity, but when encapsulated in liposome (suv) were able to inhibit the bacterial growth in liquid culture medium. the growth inhibition in vitro for ccdb et was about % for gram negative bacteria. our fi ndings revealed a novel synthetic inhibitor of dna gyrase and ccdb et analogue is a good starting point for the development of a new and specifi c class of antibacterial agents based in the dna gyrase inhibition. acknowledgements: support: fapesp and cnpq synthesis and neuroprotective properties of short peptides consisting solely from glycine and proline residues now appears more and more data on biological activity of short peptides consisting solely from glycine and proline residues. it is suppose, that these peptides, named as "glyprolines" (gps), can be formed from collagen, ÅÑÌ and related proteins. however an effect of these peptides on cns is not yet understood. the aims of this work were to improve a methodology of gps synthesis and to study cytoprotective properties of some new gps in culture of neuronal cells. gps with a common structure (gp)n, (pg)n, and (pgp)n (n= - ) were synthesised by consecutive growing of peptide chain and fragment's condensation. synthesised peptides were characterised by hplc, mass-spectrometry, element analysis etc. cytoprotective activity of gps was assessed on an increase of survival of cultivated ÐÑ cells after Í Î -induced oxidative stress. it was shown, that from all tested peptides only (gp)n and pgp reveal cytoprotective activity. at the concentration m these peptides reduced an amount of damaged cells . - . times in comparison to the control. thus preliminary data received show that some gps demonstrate a strong cytoprotective activity and therefore it is advisably to put them on further study on in vivo models. in preliminary investigations we found that all the peptides inhibited to a high degree the replication of hsv- in vero cells. moreover, these compounds did not show any cytotoxic activity against the vero cells. sexual dimorphism of hldf- peptide neuroprotective action in alzheimer's models in vivo and vitro we established that hldf- peptide, biologically active fragment of human leukemia differentiation factor (hldf), restores ability for learning, and also prevents loss of long-term memory and decrease in the exploratory behavior of wistar rat-males with experimental alzheimer's disease induced by intrahippocampal injection of betaamyloid peptide a ( - ) and ibotenic acid. the hldf- peptide was shown to render protective infl uence directly on primary culture of hippocampal and cerebellar neurones, isolated from newborn male brain, under conditions of the beta-amyloid toxicity. protective action of hldf- peptide on newborn rat-male neurons is connected with poster abstracts its ability to reduce -alpha reductase mrna expression in more than times, blocking testosterone metabolism into dihydrotestosterone (dht) and thus interfering hyper activation of nmda receptors in ca areas of hippocampus. the protective action of hldf- peptide was investigated also and on female-rats with experimental alzheimer's disease. it was shown, that in contrast to males at which both forms of memory are broken: and long-term and working ones, in the case of females, injections of a ( - ) + ibotenic acid result in infringement of only working memory, at safety of long-term memory. introduction of hldf- peptide restored the broken working memory at females as effectively, as at males. however the mechanism of protective action of peptide on primary culture of hippocampal and cerebellar neurones, isolated from newborns female-rat brain, is connected not with the decrease in hyper activation of nmda receptors, but with abad ( beta-hydroxysteroiddehydrohenase of the th type) mrna expression enhancement which is a target of beta-amyloid peptide action and blocking of progesterone conversion into alpha-dihydroprogesteron due to -alpha-reductase mrna expression inhibition. in vitro and in vivo studies of p- , an antimicrobial peptide active against multidrug resistant gram positive cocci we have designed octapeptide based on the sequence of sepecin b, an antibacterial protein of sacrophaga peregrina, effective against gram + bacteria. we systematically incorporated internal hydrophobic residues for a cationic, helical amphipathic structure effective for its high antimicrobial activity. it is also modifi ed c-terminally by a dehydro leucine residue effective for its structural stability. the synthesis of dehydro amino acid was done by solution phase and other amino acids were coupled by solid phase peptide synthesis method. anti-bacterial activity of the peptide was done by standard micro broth dilution technique against clinical isolates of gpc including methicillin resistant s. aureus (mrsa), methicillin sensitive s. aureus (mssa), hlar, group a and group b streptococci cultured from pus, wound and throat swab. all these isolates were known to be responsible for nosocomially acquired infections. s. aureus atcc was used as quality control strain. invivo effi cacy of this peptide was also tested in a mouse model with epidermal lesions caused by mrsa. the mic obtained for the peptide was g/ml (average) for the tested strains. the peptide showed no hemolytic activity against human red blood cell. in the invivo studies the healing was induced early (after hrs total healing was seen) in the experimental animals. this novel peptide has a potential to evolve as a therapeutic option in infections caused by resistant gpc. , an adhesive protein, is recognized by the activated platelet integrin á ÉÉb through the arg-gly-asp (rgd) sequence resulting to platelet aggregation and thrombus formation. inhibition of this process can be achieved by rgd peptide analogues that bind to á ÉÉb receptor. however, this class of inhibitors upon binding to receptor cause an outside-in signaling which induces a further activation of the platelet. in previous studies we presented cyclic (s,s)-cdc-containing compounds (ic ~ ìm) and á ÉÉb derived sequences (y mesradr , á ÉÉb - ) (ic ~ ìm ) that exhibit a non-rgd-like inhibitory activity [ , ] . this interesting aspect could be the basis for the design and development of a new class of anti-platelet agents that could overcame the drawback of platelet activation through the outside-in signaling. to this aim we designed, synthesized and tested for their inhibitory potency various á ÉÉb - hybrid analogues incorporating the (s,s)-cdc-motif. cyclization reduces the allowed conformations, of both the backbone and the side chains, and possibly induces a favourable for the biological activity orientation of the charged side chains. the inhibition assays on the adp induced platelet aggregation revealed that incorporation of the (s,s)-cdc-motif considerably increases the inhibitory activity of the á ÉÉb - analogue. the unique toxic effect of acrebol, a novel peptide from acremonium exuviarum a novel peptaibol, named acrebol, was isolated and purifi ed from the fungal strain bmb found in water-damaged wood-based indoor building material. the strain bmb was identifi ed as acremonium exuviarum based on morphology, its sequence, and cycloheximide resistance. ms/ms analysis showed that acrebol is a mixture of two almost identical peptaibols composed of - amino acid residues with masses and da, acephe-iva/val-gln-aib-ile-thr-leu-aib-pro-aib-gln-pro-aib and acephe-iva/val-gln-aib-ile-thr-leu-val-pro-aib-gln-pro-aib, respectively. the c-termini of the peptaibols was seroh however the sequence (mass of da) between b and seroh could not be interpreted. both isoforms of acrebol had a strong toxic effect on boar spermatozoa, feline fetus lung cells, murine neuroblastoma, and mouse insulinoma min cells. we found that, unlike other peptaibols, acrebol in toxic concentrations did not increase the ionic and solute permeability of membranes of isolated rat liver mitochondria. acrebol did not disturb the ionic homeostasis and osmotic balance of mitochondria and induced no release of apoptogenic proteins (cytochrome c) from the intermembrane space of mitochondria. acrebol strongly inhibited complex iii of the respiratory chain (ic ~ ng/ml), presumably, the outer quinone-binding center and, similarly to myxothiazol but in contrast to antimycin a, decreased the production of superoxide anion in the outer compartments of mitochondria. in the boar spermatozoa, acrebol, blocking the respiratory chain, caused the atp depletion due to the oligomycin-sensitive reversion of the reaction of atp synthesis, which resulted in the inhibition of progressive movement. acrebol induced necrosis-like death of mouse insulinoma min cells whose energetic metabolism is strongly dependent on oxidative phosphorylation in mitochondria. thus, acrebol is a unique peptaibol with a specifi c pattern of the toxic effect. delta sleep inducing peptide (dsip), its analogues and deltaran®: biological activity and mode of action for the last decade we have been engaged in studies on the endogenous neuromodulator dsip (waggdasge) and a large group of its derivatives in respect of both their physiological activity and mechanism of action. a wide range of evidences confi rmed benefi cial effects of dsip and some active analogues under experimental stress models. dsip has emerged as promising and potentially effective therapeutic agent due to strong and unique adaptive and stress protective activity revealed during the study. dsip related drug deltaran® registered in russia has also showed the signifi cant effi ciency in animal test models and clinic. the peptides of dsip family often do not demonstrate any effects under normal and comfortable conditions or even cause slight prooxidative and stress promoting effects. these properties and established wide profi le of biological activities of dsip complicate the work on dsip mode of action. cellular and subcellular effects of this peptide still remain poorly studied. previously we investigated some biochemical events underlying the stress protective effi ciency of dsip and its derivatives. in continuation of these studies we have attempted to evaluate the putative dsip infl uence on classical cellular processes utilizing stressprotective heat shock proteins (hsps) and apoptosis. effect of dsip on the level of hsp expression in human erythroleukemia cell line k was detected.. we have found that dsip down regulates the increase of intracellular hsp level during incubation of cells in high density cell culture. according to our preliminary data dsip increased hsp level in murine t-cell line ctll- similar to -and ß-adrenergic receptor agonists. in murine thymocytes dsip increased both apoptosis and hsp . we propose that registered effects of dsip are mediated through adrenergic receptors. cellular mechanisms of dsip and related peptides action are under way. identifi cation, chemical synthesis, and antimicrobial activity of tbd- -the fi rst -defensin isolated from reptiles antimicrobial peptides (amps) and proteins have been discovered in single and multicellular organisms indicating the importance of this peptide class for the innate immune system. amps are active against bacteria, fungi and viruses by affecting either the microbial cytoplasmic membrane, thus increasing its permeability or interacting with specifi c targets. defensins form one of the major subfamilies of amps, among which -and -defensins play a signifi cant role in bridging innate and adaptive immunity in mammals. the cationic -defensins are cysteine-rich and vary in length from to residues. the -stranded structure of -defensins is stabilized by a characteristic arrangement of three conserved disulfi de bonds between cysteines - , - , and - . although -defensins lyse the bacterial membrane at higher concentrations it has been shown that they modulate also the immune system, e.g. being chemotactic for t-cells. we have isolated a novel mer -defensin called tbd- from leukocytes of the european pond turtle and deduced its complete sequence de-novo by combining different tandem mass spectrometry techniques (maldi, esi; cid and etd) and edman degradation. it was also possible to identify the disulfi de pattern in a tryptic digest by maldi-mass spectrometry. the deduced peptide sequence was afterwards confi rmed by solid phase peptide synthesis. thus two fragments were synthesized by standard fmoc/ t bu chemistry using three orthogonal cysteine protecting groups (trt, acm, tbu) to selectively form the right disulfi de bridges. after native chemical ligation of the two peptide fragments the fi rst two cysteines were oxidized on air followed by iodide oxidation for the second and dmso oxidation for the third disulfi de bridge. in antimicrobial activity assays, tbd- synthesized in -conformation was active against gram-positve, gram-negative bacteria, and fungi similar to the native peptide. trichogin ga iv (trga), an antimicrobial peptide of the lipopeptaibol family, continues to reveal peculiar properties since the time of its former identifi cation by rebuffat and coworkers. x-ray diffraction studies showed that trga is folded in a mixed /a-helix conformation, while nmr, cd and ir absorption experiments proved that this structure is predominantly populated in solution, although more disordered structures contribute to the conformational landscape of trga. we have recently shown by time-resolved experiments, that an equilibrium between helical conformers and more compact, folded conformers takes place in solution, with interesting transition dynamics in the microsecond time scale. in our conformational studies on trga we realized that the d geometry of the peptide chain reproduces the structural environment of the ion coordination site of calcium-binding proteins. this fi nding urged us to investigate the binding properties of trga with respect to ca(ii), gd(iii) and tb(iii), because lanthanide ions have been widely employed in biochemical studies as best substitutes of ca(ii). the binding of ca(ii), gd(iii) and tb(iii) to trga gives rise to a conformational transition, monitored by cd spectra at different ionpeptide molar concentration ratios. at high r values, the cd curves of all the ion-peptide complexes show a positive maximum at ~ nm, typical of type ii turns, suggesting the population of bent structures. the quasi-isodichroic point found for all systems between and nm, indicates that an equilibrium between extended helical and bent conformations actually takes place. fluorescence experiments on the tb(iii)/trga adduct have shown that, upon ion binding, the fl uorescence quantum yield of tb(iii) markedly increases, due to the release of water molecules from the ion coordination inner shell. molecular dynamics calculations on the peptide/ca(ii) adduct were also performed to obtain structural and dynamical information. antibiotic resistant bacterial strains represent a global health problem with a strong social and economic impact. thus, there is an urgent need for the development of antibiotics with novel mechanisms of action. castros group isolated and determined the sequence of the peptide hy-a (ifgailplalgalknlik) of skin secretion from the frog hypsiboas albopunctatus which showed antimicrobial activity. the aim of the present work was evaluated analogues to supply information poster abstracts about the relationship structure-biological activity. the peptides were synthesized by spps using the fmoc chemical approach. the biological activities were assayed by measuring growth inhibition of two types of gram-positive bacteria and others two types of gram-negative. the synthesis and purifi cation of peptides by hplc was effi cient and a high purity level ( %) was obtained. the peptide containing trp in position (for fl uorescent studies) replacing leu presented mic values comparable to wild type sequence: um, um, um and um for e. coli, p. aeruginosa, s. aureus and b. subtilis, respectively. two peptides with this modifi cation but containing at the n-terminal region one group acetyl or a residue of asp showed mic values of um for e. coli and p. aeruginosa, although um for gram-positive bacteria. different results were observed when the residue added was lys. in this case, the activity against whole bacteria was sustained or increased. conformational properties were investigated by cd techniques in water, tfe and in zwitterionic micelles (lpc). the cd experiments demonstrated that in water, the peptides have a random structure, but in tfe and lpc solutions they acquired an ordered structure, composed mainly by -helix. however, these data there is no relationship between the structure and activity against bacteria gram-positive. these results showed that the n-terminal region of the peptide hy-a develop key roles in its antibacterial action different types of bacteria. pexiganan is an antimicrobial peptide remarkably effective against bacteria causing skin infections, and has been commercially developed as a topical cream to treat infected diabetic foot ulcers. [ ] [ ] [ ] as peptide drug-based therapy often suffers with low drug stability in vivo, suitable delivery systems must be developed. with this purpose in mind, we have designed a pexiganan-chitosan conjugate to combine the exceptional bioadhesion and tissue regenerating abilities of chitosan [ ] [ ] [ ] with the excellent antibiotic properties of pexiganan. we herein wish to report our fi rst results on the successful synthesis, ft-ir and amino acid analysis of a pexiganan-chitosan conjugate prepared by covalent attachment of a cys-containing pexiganan analogue to the chitosan's amino groups, by means of the heterobifunctional cross-linker sulfo-emcs. . overexpression of e. coli oligopeptidase b confers resistance to the proline-rich antibacterial peptides scocchi, marco; de gobba, cristian; mattiuzzo, maura; gennaro, renato university of trieste, italy the proline-rich antimicrobial peptides (pramps) are a large group of cationic peptides isolated from mammals and insects, which show a spectrum of antibacterial activity limited to gram-negative bacteria and a remarkably low cytotoxicity towards eukaryotic cells. pramps are thought to act in a permeabilization-independent manner, via energydependent internalization into bacteria followed by recognition and inactivation of internal molecular targets. with the aim of investigating their mode of action, we have isolated a number of e. coli clones showing increased resistance to the bovine proline-rich peptide bac after transformation with a dna library from bac -resistant mutants. among the recombinant plasmids responsible for resistance, some of them harboured the gene coding for the oligopeptidase b (opdb), a serine peptidase belonging to the prolyl oligopeptidase family (pop) broadly distributed among unicellular eukaryotes and gram-negative bacteria, which has emerged as an important virulence factor. opdb was cloned and expressed under the control of an inducible promoter and the transformants tested for susceptibility to a panel of antimicrobial peptides, including pramps. olipopeptidase activity of purifi ed opdb was then tested in vitro against the same peptides. the results indicate that the clones overexpressing opdb are more resistant to the pramps and that the degree of resistance correlates with the expression level of opdb. in addition, in vitro incubation of pramps with the purifi ed peptidase, followed by mass spectrometry analysis, showed that it promptly hydrolyzes all the peptides assayed to short, inactive fragments. these results suggest that opdb may contribute to cleavage and inactivation of antimicrobial peptides that are internalized into the target cells and support the notion that opdb is a novel virulence factor. guan, shuwen; huang, lei; li, pengfei; wang, liping; li, wei college of life science, jinlin university, china great progresses have been made in the research on identifying molecules of therapeutical potential for delaying aging. using caenorhabditis elegans, we had tested the effects on stress resistance and life span of treatment with dhhp- (deuterohaemin-alahisthrvalglulys), synthetic mimetics of the antioxidant peroxidases, which neutralizes peroxide. to further test the mechanisms of dhhp- on life span extension, exogenous protein sod and catalase levels were measured. we show that dhhp- is able to elevate in vivo sod and catalase activity levels after two days administration. treatment with exogenous dhhp- affected endogenous protein sod and catalase levels and elevated the expression of sod- ::gfp in the head and vulva. on the other hand, dhhp- can extend life span of c. elegans lacking sod- or ctl- gene expression by rnai. this suggested that the antioxidant enzyme in c. elegans may not the only target affected by dhhp- . du, haidong; yan, yumei; wang, liping; li, wei college of life science, jinlin university, china high concentration of hydrogen peroxide (h o ) induces nuclear dna fragmentation, lipid peroxidation and has been implicated in many diseases including heart failure, parkinson's disease, and cancer. in a systematic attempt to develop an effective scavenger of h o , we have successfully synthesized an artifi cial microperoxidase, deuterohaemin -alahisthrvalglulys (dhhp- ) as core catalytic center to which amino acid peptide was covalently attached. dhhp- exhibited potent peroxidase activity, favorable membrane permeability and thermal stability.two experimental models of oxidative injury were established in order to investigate the anti-oxidative effect of dhhp- : one was induced by hypoxia-reoxygenation in cultured heart-derived h c cells bioactive peptides and another was caused by h o in cultured neonatal rat ventricular myocytes. dhhp- could protect cells against oxidative injury by determining mtt cell proliferation assay, ldh leakage and ca +-atpase activity. furthermore, dhhp- repressed the apoptosis gene expression, such as p , hsp and heme oxygenase¢ñ, that proved dhhp- had protective effect in cultured neonatal rat ventricular myocytes . taken together, this small microperoxidase exhibited excellent ¡°druggable¡± properties, and could be a promising agent for ros-associated diseases with unmet needs. trichogin ga iv is the most extensively investigated member of the class of lipopeptaibols that are linear peptide antibiotics of fungal origin, characterized by the presence of a variable, but remarkable, number of aib residues, a fatty acyl group at the n-terminus, and a , -amino alcohol at the c-terminus. several analogues of trichogin ga iv with amino acid substitutions or deletions were designed which allowed determination of the minimal inhibition concentration against gram-positive and gram-negative bacteria and various pathogenic fungal cells. the natural peptide exhibits a specifi c activity against s. aureus and only a marginal hemolytic effect. interestingly, trichogin ga iv is active also against several methicillinresistant s. aureus strains. studies on synthetic analogues demonstrated that substitution of the c-terminal leucinol by leu-ome, or substitution of one aib residues by the epr label toac do not perturb signifi cantly the biological activity of the peptide. on the other hand, removal of or n-terminal residues eliminated any antibacterial activity. finally, studies of proteolytic degradation on trichogin ga iv and analogues where the aib residues are replaced by leu demonstrated that the presence of several non-coded aib residues endows the natural peptaibol with remarkable resistance to proteolysis. the present results indicate that trichogin ga iv is a promising lead compound for the development of new, selective and protease-resistant, antibacterial drugs. the siderophore microcin family: from the genetic systems to the antimicrobial peptides vassiliadis, gaëlle; peduzzi, jean; rebuffat, sylvie muséum national d'histoire naturelle-cnrs, france microcins are low molecular weight antimicrobial peptides secreted by enterobacteria and involved in microbial competitions within the intestinal tract. they are synthesized by the ribosomal pathway. we have isolated the fi rst siderophore peptide ( ), a post-translationally modifi ed form of the chromosomally encoded microcin e (mcce ) from klebsiella pneumoniae, having a potent bactericidal activity mainly directed against escherichia coli. the post-translational modifi cation consists of a glycosylated catechol-type siderophore linked to the c-terminus. we have recently identifi ed the genes responsible for the acquisition of this modifi cation and proposed a model for its biosynthesis ( ) . in order to identify novel siderophore peptides, we analyzed the genetic systems of several microcinogenic strains. based on the genetic organization of the microcin gene clusters, three microcins which had preparation of a close mimic of the n-terminal part of the c a receptor (c ar) by selective introduction of sulfated tyrosine residues ( ) and chips in complex with our sulfated c ar-model by multi-dimensional nmr techniques. based on these interaction data and the solution structure of the complex, chips-based c ar inhibitors for use in anti-infl ammatory therapy might be designed. insulin-like peptide (insl ) was fi rst identifi ed through a search of the expressed sequence tags (est) databases. primary sequence analysis showed it to be a prepropeptide that is predicted to be processed in vivo to yield a two-chain sequence (a and b) containing the insulin-like disulfi de crosslinks. the high affi nity interaction between insl and the receptor rxfp (gpcr ) coupled with their apparent co-evolution and partially overlapping tissue expression patterns strongly suggest that insl is an endogenous ligand for rxfp . given that the primary function of insl /rxfp pair remains unknown, an effective means of producing suffi cient quantities of this peptide and its analogues is needed in order to systematically investigate its structural and biological properties. a combination of solid phase peptide synthesis methods together with regioselective disulfi de bond formation were used to obtain insl . both chains were identifi ed as being ¡ §diffi cult sequences¡¨ and were unusually resistant to standard synthesis protocols including those mediated by microwaves. the a-chain was also prone to signifi cant aspartimide formation. the b-chain, in particular, required the use of the strong tertiary amidine, dbu, for more effective nƒÑ-deprotection during its assembly. following chain combination and sequential disulfi de bond formation, the resulting synthetic insl was obtained in good overall yield and shown to possess a similar secondary structure to human relaxin- (h relaxin). the peptide was able to inhibit camp activity in sk-n-mc cells expressing the human rxfp receptor with a similar activity to h relaxin. in contrast, it had no activity on the human rxfr receptor. novel cyclic bacteriocin-like peptides from strains of anabaena (cyanobacteria) leikoski ( ) and microcystis aeruginosa ( ) . the genome sequence of fi lamentous and diazotrophic cyanobacterium anabaena revealed a putative gene cluster (acy) encoding a bacteriocin-like peptide. one of the acy genes encoded a prepeptide, which showed n-terminal homology to the known cyanobacterial prepeptides but the mature peptide product in anabaena could not be predicted. we sequenced prepeptide genes from several anabaena strains to fi nd a prepeptide containing either cysteine or methionine, since sulphur containing amino acids enable the detection of the mature peptide product through s-labelling and lc-ms. the nucleotide sequence of the prepeptide genes revealed enormous variety in closely related anabaena strains. one strain, anabaena b contained a methionine in the prepeptide, and a cyclic heptapeptide product corresponding to the precursor was discovered in this strain. since the cleavage sites were found to be conserved in all anabaena strains the products could be predicted from the amino acid sequence and identifi ed by lc-ms. in addition, the biosynthesis of the decapeptide anacyclin in anabaena was verifi ed by heterologous expression of the acy genes in e. coli and the structure of the peptide was confi rmed with a synthetic reference peptide. altogether, new cyclic peptides were found in strains of anabaena with very little sequence conservation. cyanobacteria seem to be versatile producers of peptides using both ribosomal and non-ribosomal biosynthetic pathways. it is well established that n-terminal fl ank of corticotropin-releasing factor molecule is crucial for its biological activity. little, however, is known about in vivo effects of crf-derived peptides. this study was aimed to investigate possible actions of a tripeptide crf fragment - pro-pro-ile (ppi). tripeptide ppi was found to exert effects similar to fullsize crf molecule after central administration. the tripeptide induces behavioral activation in home cage, but inhibits behavioral activity under stressful conditions. ppi increases blood pressure and heart rate, blood glucose level and body temperature, decreases pain sensitivity, increases eeg amplitude and large doses of ppi induce seizures. ppi inhibits sexual motivation and performance in mating tests in males. crf antagonist -helical crf - abolishes ppi infl uence on circulatory system, glucose metabolism, thermoregulation and pain sensitivity. adrenalectomy does not interfere with hyperglycemic and hyperthermic actions of ppi, while pancreatectomy prevents hyperglycemic effect. nonselective beta-adrenergic blocker obsidan prevents hyperglycemic and decreases hyperthermic effects of the tripeptide and ganglionic blocker hexamethonium abolishes both effects. taken together effects of ppi correspond to stress-reactions, involving corticotropin-releasing factor and evidence that ppi is either ) a part of a crf molecule active site, or ) physiologically active crf derivative, realizing its effects through activation of crf receptors, or ) an independent regulator, affecting crf-ergic neurons. acknowledgements: the work was supported by rfbr grant - - - -à huggins, kelley; andersen, niels university, united states amyloid fi bril formation is associated with at least human diseases; among these, the human-amylin(ham)-derived deposits in type ii diabetes were the discovery system and ham aggregation is one of the more thoroughly studied systems. to date, inhibitors of beta-aggregate and fi bril formation have been polyphenols, mutants of ham, or short peptide related to the ham( - ) sequence, nfgailss. we now report that stable beta-hairpin scaffolds displaying trp and tyr residues are effective inhibitors, delaying the onset of both the cd changes associated with beta structure formation and the nucleation time and net enhancement of the fl uorescence observed with added thiofl avin-t (tht). under our test conditions ( microm ham, % hfip in mm phosphate buffer, ph ), ham begins to display an increase in beta structure by cd at min with a constant maximal value from - min. tht fl uorescence also indicates a circa min onset time with a rapid (< min) rise to the full response. inhibition (delayed onset and reduction in the maximal fl uorescence enhancement) has been observed with a number of hairpins; those with two trp residues on a single face of the hairpin are more potent. of these, our best inhibitor to date, kkltvwipgkwitvsa (p = d-pro), increases the onset time more than -fold at equimolar concentrations. at molar equiv., the onset time is greater than min and tht fl uorescence levels out at < % of the control value. in analogy to the report by prof. ghosh (jacs , p. ) that a beta-sheet protein with added tyr and trp residues inhibits fi bril formation by the alzheimer-related abeta peptide; we expect that designed beta-hairpin scaffolds will be more generally applicable and will afford new insights into the recognition phenomena of amyloidogenesis. baabur, hemda; ashkenasy, gonen ben gurion university, israel the main advantage in utilizing proteins for the development of sensors and receptors, over the more often used approach that exploits small molecules, is the ability to manipulate large recognition surfaces, which expose toward the bulk solution number of different functional groups. we search for stable artifi cial proteins that can be utilized as 'universal' recognition entities. to that end, modular chemical syntheses are exploited for the preparation of repeats-proteins. leucine rich repeat proteins (lrrs) are - residue sequence motifs present in several proteins with diverse functions. internalin b (inlb), a surface lrr protein of the human pathogen listeria monocytogenes, promotes invasion into various host cell types by inducing phagocytosis of the entire bacterium. consensus design uses statistical analysis of sequence alignments of families of homologous proteins for protein engineering. we show here that using consensus design, series of protein mutants that differ in the recognition surfaces are synthesized and will be probed for their ability to bind different natural and non-natural ligands. comparative structural studies of potent neuroprotective peptides of the humanin family although the rescue activity of hn peptides has been linked to a number of signaling pathways and receptors, their mechanism of action remains unknown. in this work cd and nmr data on the above peptides are presented and compared in an effort to defi ne structural characteristics related to their function. evaluation of our fi ndings in combination with existing structure-function relationship data for this class of peptides, brings forth fl exibility as an important structural feature that may facilitate interactions with functional counterparts of the neuroprotection pathway. the ability to adopt a partial helical conformation in the presence of low concentrations of tfe is another common structural feature that may be defi ning the interactions of these peptides in the environment of cell membranes. desleu-aga(c r)hng and cl display a more complex behavior shifting from -helical to -sheet conformations depending on ph, peptide concentration, and % of tfe present in solution. this fact may be related to their high potency since in hn literature evidence for self-association ability has been linked with neuroprotection. our cd and nmr experimental data combined with theoretical modeling will hopefully provide important clues for the elucidation of the mechanism of action of the hn family of peptide the process of molecular recycling describes a dynamic mechanism by which individual amyloid fi brils are continuously dissolving and reforming ( ) . the present work aims at the study of the dynamic properties of the -amyloid, a ( - ), amyloid fi brils. since the formation of a aggregates has been suggested as a key process in the pathology of alzheimer's disease, the study of the dynamic nature of a ( the coiled coil structure is widely distributed in natural proteins, such as transcription factors, receptor proteins and enzymes. this motif has been recently used by chemists for the design of functional synthetic proteins. specifi c coiled coil sequences can be utilized as templates for self replication processes ( , ) . the stability and the activity of these replicators depend on the characteristics of the amino acid residues in the recognition interface. it has been suggested that it is possible to control protein structure and the replication process by external triggers such as light, and that the light can also be used to monitor the conformational changes and/or to follow the protein functionality( ). we show here our ability to control the folding stability of coiled coil peptides and their reversible or irreversible self replication effi ciency by light, and we demonstrate the possibility to exploit fret couples to facilitate in-situ monitoring of the folding and reactivity. we use 'caged' mutants with a photo-switchable molecule in the recognition interface of the peptidewhich disrupts its coiled coil structure -as inactive species. deprotection of the caged proteins is used as a mechanism to restore the self replication process. the ligation is followed by monitoring the changes in fl uorescence of either the donor or acceptor of the fret couple. we will describe synthesis and structural characterization of caged and cage-free peptides and measurements that show, as expected, that the cage free peptides are more stable as coiled coils and better catalyst for their own formation than the caged analogs. moreover, we discuss the reversible replication process and how we can shift and control its equilibrium staes. , a member of the rfamide peptide family, has been reported to have pronociceptive and analgesic activities as well as pro-and anti-opioid effects. these contradictory effects seem to result from npff capacity to bind two different gprotein coupled receptors (npff and npff ). although the exact role of npff and npff receptor is still unclear, this complex system appears to play an important role in pain regulation. structure-activity relationship (sar) studies using analogs of npff and derived peptides have shown that the c-terminal part of the molecule (i.e. pqrf-nh ) is crucial for affi nity and activity. however, some of the essential features for ligand recognition by npff receptors are still missing to design highly selective molecules. in order to provide further insight into ligand recognition, we have investigated the solution conformation of npff in different media using circular dichroism and nmr spectroscopy. our results showed that ( ) in the presence of methanol or trifl uoroethanol, turn-like elements are present in npff structure, and ( ) are not yet established, although genetic and animal models have shown a causal role of amyloid -peptide (a ) in ad. however, recent debate has focused on whether amyloid fi brils or soluble oligomers of a are the main neurotoxic species contributing to neurodegeneration and dementia. one approach for preventing aggregation would be the conversion of the peptide conformation. prior investigations indicate that polymeric nanoparticles offer strong advantages in modulating the secondary structure of the peptide ( , ) . these results have encouraged us to extend our work on polymeric nanostructures for conformational transformations contributing to the development of new therapies for these diseases. complexes of polyampholytes and dodecanoic or perfl uorododecanoic acid were prepared ( ) resulting in nanoparticles with hydrodynamic diameters ranging from to nm. the fl uorinated nanoparticles induced -helix rich structures in a peptide, whereas their hydrogenated analogues were less effi cient leading in most cases to aggregation orsheet formation, as determined by circular dichroism spectroscopy. the degree of fl uorination, the hydrophilic balance and the charge density of the fl uoropolymers, as well as the size of the nanoparticles in aqueous solution, are decisive for the interactions. the impact of these structures on the a -induced toxicity in cultured neurons was studied. we report that the fl uorinated nanoparticles increased the a -mediated mtt ( -[ , -dimethylthiazol- -yl]- , diphenyltetrazoliumbromide) reduction, which indicates a higher cell viability. the anti-apoptotic effect of the complexes was evaluated by determining the activation of caspase- . this assay also confi rms the decrease of a -mediated cytotoxicity in the presence of fl uorinated biocompatible complexes. alternative strategy to investigate enzymatic activity using peptides containing toac spin probe ( ), the present work extended the investigation of the specifi city of angiotensin i-converting enzyme (ace, ec . . . ), a dipeptidyl carboxypeptidase which cleaves the c-terminal dipeptide from angiotensin i (angi, drvyihpfhl) to produce the potent vasoconstrictor angiotensin ii peptide (angii). the use of paramagnetically labeled ai analogues attaching the toac ( , , , tetramethylpiperidine- -oxyl- -amino- -carboxylic acid) probe ( ) advantageous allows the monitoring of their conformation and its enzymatic hydrolysis specifi city through the epr and fl uorescent methods, the latter due to the quenching effect induced by the stable free radical toac probe upon the tyr residue of angi. the study of toacattaching ai analogues at positions , , , , , and indicated that the fi rst four analogues are substrates for ace in the decreasing order ~ > > , thus confi rming that greater the proximity of the unnatural probe to the cleavage site ( - ) of the sequence, the smaller are the substrate specifi city of analogues. otherwise the quenching effect of tyr fl uorescence by toac decreased with increasing distance between both residues, thus suggesting overall fl exible structures for most of analogues. these fi ndings were also corroborated in a combined cd and epr studies although some differences were detected among the derivatives either in the variation of ph or amount of the structuring tfe studies. finally, differences between epr spectral lineshapes of some labeled analogues and their corresponding cleavage products seems to allow a real time monitoring of the enzymatic reaction. the effect of the so called " -sheet breakers" (bsbs) on a - aggregates inhibition of aggregation of amyloid -peptide seems to be a critical step in the therapeutic approach to prevent amyloidosis in alzheimer's disease. the therm " -sheet breaker" (bsb) had been introduced by soto c. et. al. as a -residue peptide in , that inhibits amyloidprotein fi brillogenesis. we synthetized several derivatives of the "soto peptide" as well as a big number of peptidomimetics. their mechanism of action has been studied by nmr spectroscopy, cd and transmission electron microscopy (tem). all of these methods showed that the soto peptide lpffd and the similar peptides can not prevent a aggregation. these compounds bind to the surface of a aggregates and decrease bioactive peptides the specifi c surface area of a which accessible for the cell-membranebound receptors, can lead to a decreased toxicity. the -sheet breaking effect does not work up to an a peptide-small peptide ratio of : . as a consequence, these compounds are not -sheet breakers, only they modify the surface of a fi brils and rather speed up the formation of -sheet structure. designing trehalose-conjugated peptides for the inhibition of alzheimer's a oligomerization and neurotoxicity. interactions between cationic or anionic porphyrins and polypeptide templates with charges that are opposite to those of porphyrins have been extensively investigated for their possible applications in biomedicine and photodynamic therapy (pdt). the infl uence of porphyrin on the conformation of the peptide part of the complexes is studied in this work. non-covalent interactions of cationic tripeptide l-lysyl-l-alanyl-lalanine (kaa) with anionic meso-tetrakis( -sulfonatophenyl)porph yrin (tpps) and its copper(ii) (without axial ligand), iron(iii) (one axial ligand), and manganese(iii) (two axial ligands) derivatives were investigated in aqueous solutions by vibrational (vcd) and electronic circular dichroism (ecd) spectroscopies. although both the cd spectroscopies are sensitive to conformation, particularly vcd is extremely sensitive to subtle conformational changes. the vcd spectra of pure kaa in the amide i' (c=o stretch vibration) region showed the spectral patterns typical for left-handed polyproline ii helical conformation (ppii). interaction of kaa with non-metallated tpps was accompanied by change of the amide i' vcd patterns -loss of vcd intensity and arising of a new negative band at ~ cm - -that was interpreted as a partial change of ppii into less compact conformation as extended helix or -sheet segment. in case of cu(ii)-and fe(iii)-tpps, the loss of the amide i' vcd intensity of kaa was observed only. for mn(iii)-tpps having two axial ligands, the vcd pattern was unchanged compared to the pure tripeptide indicating that this derivative is not able to change the conformation of kaa in peptide-porphyrin complex. suitable models of biologically important small-sized proteins -histons -located in the chromosomes of eucariotic cells. they are able to interact with negatively charged functional groups of many biologically important molecules as dna, polyuronic acids, and porphyrins and thus infl uence a broad range of biological functions. in this work, the solution conformation of synthetic oligotripeptides (llysyl-l-alanyl-l-alanine) n [n = , , ] was investigated at the different temperatures and ph using combination of vibrational (vcd) and electronic circular dichroism (ecd) spectroscopies. vcd spectra of all the oligopeptides measured at room temperature show a negative couplet (positive to lower frequency) in amide i' region. this spectral pattern is indicative of left-handed polyproline ii helical conformation (ppii), which is stable at wide range of ph. the temperature dependence of the vcd spectra indicates that ppii conformation of all the oligopeptides remains stable even at °c, independently on length of oligopeptide chain. these results were confi rmed by temperature dependent ecd experiments, where the characteristic negative and positive bands at about and nm, respectively, were observed. taken together, vcd and ecd results suggest that ppii conformation of (lys-ala-ala) n sequence is the dominant conformation within the range of temperature from to °c. all the results including spectral data analysis are discussed in detail. acknowledgment: the work was supported by the research grant / /p from the grant agency of the czech republic. we thank miroslava Žertová, msc (iocb prague) for the oligopeptide synthesis. -peptides are probably the most thoroughly investigated peptidomimetic oligomers. to extend the fi eld of -peptides towards the construction of possible new secondary structures, the replacement of the c and c atoms of the -amino acid with heteroatoms could be an attractive modifi cation, for example c -atom of -peptides by an nr moiety, leading to hydrazine peptides. in the literature, there are only a few studies about hydrazine peptides [ ] [ ] [ ] , and hydrazine peptides with cyclic side-chain have not been studied yet. in order to determine the secondary structure preference of h-[(cis or trans)-acpc- s-aza-acpc] -nh peptides (figure ), their potential energy hypersurface were probed at the ab initio b lyp/ - g** level. the cis ( r, s) formed / -helices, while the opposite acpc enantiomer resulted strand. the trans ( s, s) formed -strand, while the opposite acpc enantiomer resulted -helix. the hybrid-peptides in question were synthetized on solid support, and their high-resolution d assignments were made by using nmr, which was supported by ecd, vcd, qls and tem methods. the hydrazino modifi cation resulted in better water solubility than that of the acpc homooligomers. this result is very important concerning the further biological applicability. mazur, adam; katarzy ska, joanna; zabrocki, janusz; jankowski, stefan technical university of lodz, poland cyclolinopeptide a, (cla, ), a cyclic nonapeptide cyclo(-pro -pro -phe -phe -leu -ile -leu -val -), possesses strong immunossupresive and antimalarial activity as well as the ability to inhibit cholate uptake into hepatocytes [ , ] . the mechanism of cyclolinopeptide a activity is similar to that of cyclosporine a. the object of our investigations are six cla analogues with pro and pro residues replaced by -isoproline or -homoproline. the immunosuppressive activity of these new cla analogues was evaluated on the basis of the mouse splenocyte proliferation assay .. nmr spectra analysis (chemical shifts assignment and structural constraints) was based on d and d nmr experiments at mhz. long ( ns) molecular dynamics calculations were carried out using gromacs program (gromos g a force fi eld) for isomers of at least % content. in woolley, andrew university of toronto, canada thiol-reactive azobenzene-based cross-linkers provide a straightforward means for introducing a photo-isomerizable unit into a peptide or protein structure. we have shown that the conformational response of the peptide in such systems is critically dependent on the manner of attachment of the cross-linker as well as the cross-linker structure. cross-linkers can be introduced in which trans-to-cis photoisomerization leads of formation of helical structure, or conversely to loss of helical structure. these effects can be understood in a semi-quantitative manner by calculating the degree of mismatch between the steric requirements of the linker and the conformational ensemble of the peptide. kinetic studies reveal that in general photoisomerization of the linker is fast (several ps) whereas subsequent peptide folding or unfolding occurs over hundreds of microseconds. such cross-linkers are thus potentially useful phototriggers for studying protein folding processes, as well as for controlling the equilibrium stability of different conformational states. applications to photo-control of bioactive peptides and proteins will be discussed. short corticotropin-like peptides with stress-protective activity it was synthesized linear è cyclic corticotropin-like peptides, which contained from to amino acid residues. the ability of each of the synthesized peptides to inhibit the specifi c binding of tritium-labelled corticotropin ( - ) to the adrenal cortex membranes of rat in vitro was investigated. on the base of the obtained results peptides with the highest inhibitory activity were selected for in vivo tests. the infl uence of the selected peptides on the level of -oxicorticosretoids and catecholamines in the adrenals and blood of rats in the experiments on acute hemorrhage and hypobaric hypoxia, cold and heat shock and low doses of g-radiation were studied. it was established that intravenous injection of short peptides at the dose of g/kg could correct disturbance of -oxycorticosretoids-catecholamines system in the adrenals and plasma of rats that were subjected to hemorrhagic shock and hypoxia. the rest of investigated peptides possessed lower stress-protective activity. it was also shown that under cold or heat shock, thrice-repeated intranasal injection of these peptides into rats at the dose of - g/animal abolished temperature induced changes in the level of -oxycorticosretoids and catecholamines in the adrenals as well as the content of free histamine and the activity of diaminoxydase in the myocardium. it has been shown that the stress-protective activity of corticotropin-like peptides is mediated by the corticotropin receptor in cortex of the adrenals. a study of immunobiological activity of the wrnwdyyk octapeptide the evaluation of the effect of japanese herbal in many cases, japanese herbal (kampo) medicines have been used in the empirical treatment of chronic hypofunction. however, the kampo medicines consist of several herbs, whose pharmacological mechanism is not clear. in western medical science, medical doctors usually treat patients according to disease diagnosis. in eastern medical science, treatment is based on diagnostics called gsho h, which is a unique concept in kampo medicines and quite different from diagnosis. the concept of gsho h is diffi cult for non-professionals to understand, furthermore, there are responders and non-responders when nonprofessionals prescribe kampo medicines. the concept of gsho h focuses on individuals, not diseases, therefore, it is diffi cult to gain a given effect for everyone by general clinical trials. in this study, we investigated the effects of prokinetic kampo medicines on plasma levels of gut-regulatory peptides (somatostatin, motilin and gastrin) and compared with that of dopamine receptor antagonists, the western prokinetics. the fi ve kampo medicines, including pinelliae tuber and zingiberis rhizoma, three dopamine receptor antagonists or placebo was orally administered. venous blood samples were taken before and till min after administration. plasma peptide levels were measured using a sensitive enzyme immunoassay. the dopamine receptor antagonists and kampo medicines caused signifi cant increase of plasma gut-regulatory peptide levels compared with placebo group. in recent years, chronic hypofunction without mechanical problem, such as non-erosive refl ux disease and functional dyspepsia, is diffi cult to cure using western medicines. the preliminary study indicated the plasma somatostatin levels of patients with any symptoms are high and plasma motilin levels of them are low compared with those of healthy subjects. to evaluate kampo medicines using bioactive peptides as biomarkers, it may be possible to cure diseases that is diffi cult to treat by western medicines. bapst, jean-philippe; calame, martine; eberle, alex n.; tanner, heidi university hospital basel, switzerland radiolabeled -msh analogs are potential candidates for melanocortin- receptor (mc -r)-mediated melanoma targeting. several short -msh peptides carrying a dota ( , , , -tetraazacyclododecane- , , , tetraacetic acid) metal chelator were designed and evaluated as potential diagnostic (e.g. with in, , ga) or therapeutic (e.g. y, cu) radiopharmaceuticals. the analogs tested to date showed high affi nity for the mc -r in vitro, excellent internalization into the tumor cells, as well as a good incorporation in tumor xenografts and a low uptake in normal tissues in vivo, except the kidneys where considerable uptake is observed. our current studies attempt to infl uence the pharmacokinetic parameters in order to address specifi c uptake (i.e. by melanoma) versus non-specifi c uptake (i.e. by the kidneys). as glycosylation had been shown to improve tumor-to-kidney ratios in the case of somatostatin and to reduce peptide re-uptake by the tubular system of the kidneys in general, we investigated glycosylated analogs of [nle , asp , d-phe ]--msh - (napamide). carbohydrate moieties such as glucose, galactose and maltotriose were introduced at various positions on the msh peptide carrying the metal chelator dota for labeling with in. the peptides were evaluated in vitro in both murine and human cell lines for mc -r binding and cellular localization, and in vivo in b f tumor-bearing mice for tissue distribution. the tumor-to-kidney ratio for gal-napamide ( - h aucs) was superior to any of the previously published msh peptides. other glycopeptides showed very good binding affi nities but lower selectivity in vivo. in additional, a series of non-glycosylated dimeric derivatives, bearing one or two moieties of the chelator complex, were developed which displayed excellent receptor affi nity but tended to result in higher kidney accumulation. by contrast, at least one negatively charged dota-napamide showed excellent tumor-to-kidney ratios. there is a critical lack of validated early biomarkers for most conditions and diseases. early diagnosis does enable treatment of less severe disease states, the use of less invasive techniques and could potentially reduce the costs of healthcare systems. however, it is most likely that peptides in tissue or blood -or their modifi cations -can be specifi cally associated with different disease states. the discovery and verifi cation/ validation of such disease markers are two distinct workfl ows. during the discovery phase, a relatively small number of samples with a high number of potential biomarker candidates are screened. the highthroughput provided by the itraq™ reagent strategy allows for simultaneous analysis of such samples. once biomarker candidates have been identifi ed with initial statistical signifi cance, these have to be validated. this validation workfl ow involves analyzing a large number of samples with a relatively small number of candidates to establish the biological signifi cance of the biomarker candidates. rather than switching to immunological techniques for this validation step, we suggest a mass spectrometry based approach. this orthogonal strategy is a novel targeted, high throughput quantitative multiplexed multiple reaction monitoring (mrm) approach. the approach relies on assay development using a combination of mrms to target specifi c peptides identifi ed in discovery, followed by ms/ms to confi rm that the quantitative mrm signal results from the target peptide. in addition to being a quantitative method, this validation approach is extremely specifi c and sensitive. several published examples of the application of this mrm-based approach utilizing the actual or hypothetical physical properties of peptides in complex mixtures will be presented. infl uence of the charge on the in vivo behavior of radiolabeled bombesin analogues prostate and breast cancers are the second leading cause of cancer death in men and women, respectively. the side effects related to the treatments that are available today have a great infl uence on the patients' quality of life. therefore, the development of new diagnostic and therapeutic strategies may have an important impact in the outcome of these cancers. gastrin-releasing peptide (grp) receptors are present in high quantities in a variety of cancers, prostate and breast tumors among them. targeting of over-expressed grp receptors with radiolabeled bombesin (bbs) analogues would offer an interesting tool for tumor imaging and therapy, depending on the radionuclide used. some analogues of bbs, based on the fragment - , were functionalized with the (n his)chelator for labeling with the m tc-and re-tricarbonyl-core. despite an increased metabolic stability, these analogues showed very low tumor uptake. additional insertion of a ala-ala linker led to increased tumor uptake but still unfavorable in vivo properties. in order to further improve the biodistribution, novel polar linkers with different charge were introduced in the molecule. a positive charge resulted in increased kidney uptake, whereas one single negative charge led to a signifi cant increase in the tumor uptake and also signifi cantly higher tumor-totissue ratios. co-injection with natural bbs importantly inhibited the uptake in the tumors and receptor-expressing tissues, which confi rmed the specifi city of the in vivo uptake. moreover, imaging of the tumor xenografts by spect/ct was also much clearer with the analogues bearing one negative charge. additional negative charges, however, resulted in a loss of binding affi nity and internalization, and unfavorable biodistribution. in conclusion, bbs analogues with one single charge in the linker hold a greater potential for imaging and therapy of grp receptor-overexpressing tumors peptide-macrolide conjugates as novel instruments for protein biosynthesis study bacterial ribosomes are targets of numerous therapeutic agents. macrolide (ml) antibiotics prevent nascent polypeptide growth by blocking the ribosomal exit tunnel (rt). availability of high-resolution structures of complexes of ribosomal s subunits with ml enabled developing novel ideas concerning their inhibitory activity of translation. formerly we obtained peptide derivatives of ml in which the peptide part modelled a growing chain, while an antibiotic moiety served as an "anchor" for positioning the peptide in the rt. the goal of this study was to synthesise tryptophan-containing peptide derivatives of -omycaminosyltylonolide (omt) to monitor location of the specifi c trp binding site that modulate the activity of the peptidyl transferase centre of the ribosome. the peculiarity of compounds designed in this study lies in that of a trp containing peptide fragment, connected to the primary hydroxyl group in position c of omt, is oriented in the rt towards the hypothetical trp binding site. the following peptides were used: boc-l-trp-gly-oh, boc-l-trp-ala-oh, boc-l-trp-abu-oh, boc-l-trp-ape-oh. variable distance between trp residue and macrolide ring was achieved by introduction of glycine, -alanine, -aminobutyric acid and -aminovaleric acid as c-terminal amino acids of the dipeptides. the peptides were obtained from boc-l-trp-oh and ethyl esters of the corresponding amino acids by condensation with bop followed by saponifi cation of the peptide esters. the reactions between peptides and omt were produced using dcc and dmap as condensation agents. as a result peptide-macrolide derivatives were obtained. all compounds were purifi ed by column chromatography on silica gel and characterized by hplc, mass-spectrometry and nmr. it was shown by means of chemical probing that trp containing omt derivatives specifi cally bound to rt of the ribosome. moreover these compounds displayed an antibiotic activity in testes with several staphylococcus strains. zwanziger, denise ; neundorf, ines ; schatzschneider, ulrich ; beck-sickinger, annette g. university of leipzig, germany; university of bochum, germany npy is a amino acid peptide amide that belongs to the pancreatic polypeptide-hormone family ( ) . it is the most abundant neuropeptide in the brain and triggers a number of central activities, such as regulation of food intake as well as stress induced reactions [ ] [ ] [ ] . npy forms a selective interaction with at least three receptors, which are called y , y and y . in the past it was shown that y -receptors are overexpressed in > % of all breast tumors as well as in % of the derived metastases. normal breast tissue expresses y -receptors, while the neoplastic tissue expresses y -receptors .. as a result, the npy-y -receptor system can be used for tumor targeting and therapy. in the fi rst step we synthesized the truncated and modifi ed npy analogue [pro ,nle ,bpa ,leu ]npy( - ) (nle-norleucine; bpa-benzoyl-phenylalanine) by solid bioactive peptides phase peptide synthesis using fmoc/tbu strategy. the npy analogue was characterized with respect to in vitro binding affi nity and selectivity at y -receptor expressing human breast cancer mcf- cells and the metabolic stability in human blood plasma, respectively. then we coupled different potential chelators to [pro ,nle ,bpa ,leu ]np y( - ), which was n-terminally modifi ed by two ß-alanines as spacer between the peptide sequence and the chelator. next, we determined their ability of metal conjugation of diverse metals, as cu + and re +. metal conjugated peptides were characterized by maldi-tof-ms (matrix assisted laser desorption/ionization mass spectrometry), rp-hplc (reversed phase high performance liquid chromatography) and ir-spectroscopy (infrared). after optimization of the metal conjugation fi rst binding affi nity studies were performed. chain is mainly expressed in skeletal muscles and peripheral nerves, and interacts with cell surface receptors such as integrin, dystroglycan, and heparan sulfate proteoglycans. biological functions of the laminin chain n-terminus including integrin binding and heparin/heparan sulfate binding were found previously. here, we focused on the n-terminal region of the mouse laminin chain (position - ) and screened the biologically active sequences using peptides. the synthetic peptides were generally amino acids in length and overlapped with neighboring peptides by amino acids. cell attachment activity of the peptides was evaluated using a peptide-coated plastic plate assay and a peptide-conjugated sepharose bead assay using ht- human fi brosarcoma cells. eleven peptides showed cell attachment activity on plate assay and fi ve peptides showed cell attachment activity on beads assay. previously we screened active sites on the laminin chain and identifi ed several active sequences. when we compared with active sequences of the and chains, a - (yydetvasrnlsln) and a - (ggklkyaiyfea) are unique active peptides only within laminin chain. these results suggest that the biological activity of a - and a - are chain specifi c and are useful for investigating the chain specifi c functions of laminin chain. novel peptide biopharmaceuticals by using phage display technology Štrukelj, borut; lunder, mojca; bratkoviè, tomaž university of ljubljana, faculty of pharmacy, slovenia libraries of random peptides displayed on the surface of bacteria, mammal cells or bacteriophages are an essential tool that enables systematic study of target molecule interactions. the identifi cation of ligands from large biological libraries by phage display has now been used for almost years. in a last few years several improvements have led to numerous high affi nity peptide ligands that express various biological activities. phage-displayed peptide libraries have been used successfully to isolate peptide ligands directed to a functional site for which the natural ligand is or is not a protein or peptide. by using a modifi ed, in-house developed selection proctocol, we successfully selected several phage clones with high affi nity to pancreatic lipase, ghrelin and cysteine protease cathepsin k as target proteins. based on their deduced animoacid sequences, twenty heptapeptides with the highest affi nity to target proteins were synthesized and characterized for their capacity to inhibit enzyme or acceptor function. the most succesful peptide candidates inhibited pancreatic liapase with the apparent inhibition constant of um, and cathepsin k with the apparent inhibition constant of , um. a set of peptidomymetic compounds were sinthesyzed based on the aminoacid sequence of selected peptides and their inhibitory activity was determined. the most potent candidates were selected for further development of peptide biopharmaceuticals aganist obesity (inhibitors of pancreatic lipase and ghrelin) and in the prevention of osteoporosis (cathepsin k inhibitors). novel approach to non-specifi c elution in phage display using ultrasound phage display is used to select and optimize peptides or protein domains binding to virtually any protein and sometimes even non-protein targets. several rounds of screening are performed, until the increase in phage output, or binding assays performed with phage pools, indicate that the population of binding phages has been adequately enriched. in cases where ligands of particular target are not known or available, targetbound virions are released by non-specifi c elution for example with acidic buffer, or competitively with free target molecule, or by addition of bacterial host directly to the target-bound phages. we used streptavidin, immobilized by adsorption, as a model target protein for affi nity selection of peptides from phage display library. the effi ciencies of a number of well-known typically used non-specifi c elution strategies in selecting and retrieving a phage clone displaying the tripeptide biotin mimetic (hpq) from a streptavidin coated surface were compared. all the commonly used elution strategies have failed to elute and select high affi nity hpq-bearing phage clones bound to streptavidin. the failure was shown to be due to the inability of eluants to break the interaction of high affi nity clones with the target, which is thus likely to be the cause for failed selection with other targets also. to surmount this, we have introduced a new elution strategy, combining low ph elution buffer with sonication which, in addition to loosening the peptidetarget interaction, also serves to detach the target molecule from the immobilization surface. this ultrasound-based method enabled single step selection of a high affi nity peptide from a library of diversity greater than , and thus represents a dramatic improvement in searching for novel, specifi c peptide ligands. apoptosis to various tumor cells but not in normal cells. using fmoc solid phase synthesis, cellulose membrane-bound octameric peptide library of trail scan was prepared and cell viability assay was directly performed on peptide disk with jurkat cells. six peptide sequences that could induce cell death were found, and particularly rnscwskd (trail - ) peptide was shown the strongest effects. then, peptides of stronger effects were found through amino acid substitution, and the cnscwskd peptide induced > % cell death in treated cells. features of apoptosis, such as dna fragmentation, activation of caspase, phosphatidylserine externalization, chromatin condensation, and competition with trail for binding to the death receptor (dr) or dr were observed, suggesting that this peptide is a trail mimic. caspase- activation was observed in various tumor cells treated with this peptide as well as with trail, while no activation was observed in human normal fi broblasts. the cnscwskd peptide is a potential candidate for use in cancer therapy. the being an incretin mimetic, exendin- exerts the same action as glp- including the glucoregulatory and the insulinotropic effects through glp- receptor. exendin- may be preferable to glp- on the aspect of stability. however it was comprised with amino acids and was not suitable for developing as an oral drug. recently, there was an upsurge in the development of exendin- mimetic as potential therapy for type diabetes. in this study, a receptor-binding region on the surface of rinm f cell was used as the target to screen peptide ligands for the receptor in a phage -mer peptide library. dna sequencing revealed a group of closely related peptides from the fourth round of selection. through the activity of decreasing blood glucose assay in vivo, the cell proliferation assay and capability against dppiv in vitro, one highest bioactivity peptide (qpsvgmkpsprh, ex- pa) was acquired. the bioactivity of ex- pa was almost identical with exendin- on the promoting cell proliferation in a dose-dependent manner. the decreasing blood glucose effect of ex- pa was up to min after administration. the stability against dppiv of ex- pa maintained good performance that % parent peptide remained after h. in summary, ex- pa as a shorter glp- receptor agonist mimicked the action of exendin- and built a good foundation for designing the oral diabetes drug. key words: exendin- , mimetic, phage display peptide library, screen over the last decades we have witnessed the emergence of bacterial resistance to virtually all clinically important antibiotic types. therefore, continuous development of antibacterial agents with completely novel modes of action accompanied by rationalization of chemotherapeutic prescription is the key strategy to adopt in a long-run struggle against the growing problem of pathogen resistance. numerous indispensable antibiotics interfere with peptidoglycan cell wall biosynthesis making this unique metabolic pathway a well validated target for antimicrobials. while nearly all of these antibiotics inhibit late stages of murein synthesis occurring on the extracellular side of plasma membrane, initial cytoplasmic steps have not been extensively exploited as drug targets. we have performed affi nity selections from random linear and conformationally constrained (disulphide cyclized) peptide libraries displayed on bacteriophage particles against two essential bacterial enzymes murd and mure, involved in the cytoplasmic synthesis of peptidoglycan monomer precursor. selected peptides were found to inhibit respective targets in an in vitro assay with ic values of m to . mm. reported inhibitory peptides should be regarded as templates for design of low-molecular-weight peptidomimetics. resulting murd and mure inhibitors with improved potency and/or physicochemical characteristics (especially membrane permeability) have the potential to act as broad spectrum chemotherapeutics. fidan, zerrin; ljeskovica, nizama; portwich, michael; volkmer, rudolf charité-universitätsmedizin berlin, germany coiled coil (cc) sequence motifs are common structural motifs and versatile protein interaction modules. the cc is composed of at least two right-handed amphipathic a-helices that wrap around each other into a left-handed supercoil such that their hydrophobic surfaces are in contact. cc can associate up to heptamers, form homomeric and heteromeric complexes at different stoichiometries, and be aligned parallel and antiparallel. a characteristic of all coiled coils is the presence of heptad repeat sequences [abcdefg]i, where i denotes the heptad number. although cc motifs can be predicted with a high degree of confi dence, predicting the association states and topologies is still a great challenge. we will report on synthetic peptide arrays useful to study cc associations at the amino acid level. in contrast to rational design, which mostly depends on short model peptides, our approach relies on the full-length homodimeric gcn -and the heteromeric cjun/cfos coiled-coil domain formation. the infl uence of amino acid substitutions on association is tested without restrictions and presumptions and the stoichiometry is examined by biophysical methods. furthermore, we generate arrays comprising hundreds of (putative) cc sequences and subsequently probe them for association to a set of several native cc sequences. an interactome can be drawn for each of the investigated cc sequences, enabling one to interpret the biological functions. we will present: (i) association analyses of all single substitution variants of the gcn -, cfos and cjun leucine zipper probed with the associated native leucine zipper; (ii) the exposure of a heteromeric cc-network deduced from gcn variants and the determination of the networkstoichiometry; (iii) the cc-interactom of several native coiled coils like the cc sequences of akap , pqbp , orai and others. almost two decades ago we used the spatial screening technology to optimize activity and receptor subtype selectivity among rgd recognizing integrins. this culminated in the avb selective superactive pentapeptide c(-rgdfv-) which gave rise to a number of peptidomimetic modifi cations. among them we studied all retro-, inverso-and retroinverso peptides of this structure. only one retro-inverso peptide exhibits full activity: the peptide c(-dgrvf-) strongly inhibits avb /vitronectin binding ( nm) but not aiibb /fi brinogen binding (> nm). all investigated retro-sequences show very low affi nity for avb . recently it was discovered that the ngr sequence in the fi bronectin domain fi after rearrangement into iso-asp-g-r exhibits high binding affi nity for integrin avb . this surprising result stimulated us to investigate cyclic peptides containing the iso-asp-gly-arg sequence .. after optimisation we obtained peptides which bind with high activity to integrin a b and lower, but signifi cant activity for avb . in the ongoing work we investigate if receptor modelling can help to understand these results. selectivity and activities for these two integrins have been obtained recently in our group using two different peptidomimetic scaffolds bioactive peptides (tyrosine based and diacylhydracine based) using a homology model of the integrin a b for which no x-ray structure is known. binding of integrin ligands involves binding of a carboxyl group to the metal ion (ca or mn) in the so-called midas region of the integrin. so far all integrin ligands contain a carboxyl group and any attempt to substitute this group by an isoster failed. we recently found that hydroxamic acids (-conhoh) allow for the fi rst time a substitution of this carboxyl group. the activities and selectivities for integrin subtypes avb , a b and aiibb have been explored and give interesting results. development of gnrh-iii-antracycline conjugates as multifunctional drug delivery systems for targeted chemotherapy targeted chemotherapy based on the cell specifi c or overexpressed receptors on tumors might be an effi cient therapeutic approach for the treatment of cancer. expression of gonadotropin-releasing hormone (gnrh) receptor was identifi ed on different types of tumors. it has been shown that gnrh-iii (glp-his-trp-ser-his-asp-trp-lys-pro-gly-nh ) isolated from see lamprey has antiproliferative activity on numerous tumor cells, and signifi cantly less potency on releasing gonadotropin hormones (lh, fsh); therefore, it is a more selective antitumor agent than the human gnrh derivatives. in our work, gnrh-iii was used as targeting moiety for the preparation of multifunctional drug delivery systems for targeted cancer chemotherapy. daunomycin (dau) and doxorubicin (dox) as antineoplastic agents were attached to the side chain of lys of gnrh-iii through amide, oxime, hydrazone or ester bonds, either directly or by insertion of an enzyme cleavable tetrapeptide spacer. stability studies of the conjugates were performed in human serum, as well as in the presence of chymotrypsin. the effect of chemical structure of the conjugates on in vitro antitumor activity was studied using different cancer cell lines (mcf- human breast, ht- human colon and c murine colon cancer cells). oxime bond linked dau-gnrh-iii conjugate was selected for in vivo experiments using c colon tumor bearing mice. the conjugate showed similar antitumor activity as the free drug, but less toxic side effect and longer survival of the animals was determined in the case of the application of the conjugate. guillon, g. ( ) . it was also shown to be highly selective for the human v b receptor ( ) . we now report the synthesis and some pharmacological properties of three fl uorescent hv br ligands (a,b,c), based on modifi cations of the lys residue in d[leu ,lys ]vp, with alexa (a), alexa (b) and antraniloyl (atn) (c). the fl uorescent peptides a, b and c exhibit the following affi nities for the hv br: (a) ki = . nm, (b) ki = nm and (c) ki = . nm. peptides a and c exhibit moderate affi nities for the hotr and very weak affi nities for the hv ar and for the hv r. on v b-transfected att cells, they activate plc coupling as evaluated by stimulation of ip levels. they also induced receptor internalization visualized by accumulation of fl uorescence in endosomial vesicles. one or more of these new fl uorescent hv br ligands promise to be useful tools for studying human v b receptor localization or traffi cking. distribution of prolyl oligopeptidase in the mouse wholebody sections and peripheral tissues prolyl oligopeptidase (pop) is a serine endopeptidase that hydrolyses proline-containing peptides shorter than -mer, including many bioactive peptides. the distribution of pop in the brain has been studied but little is known about the distribution of peripheral pop. we used immunohistochemistry to localize pop in mouse whole-body sections and at the cellular level in peripheral tissues. furthermore, we used a pop activity assay to reveal the associations between pop protein and its enzymatic activity. the highest pop protein densities were found in brain, kidney, testis and thymus, but in the liver the amounts of pop protein were small. there were remarkable differences between the distribution of pop protein and activity. the highest pop activities were found in the liver and testis while kidney had the lowest activity. in peripheral tissues, pop was present in various cell types both in the cytoplasm and nucleus of the cells, in contrast to the brain where no nuclear localization was detected. these fi ndings support the proposed role of pop in cell proliferation in peripheral tissues. the dissociation of the distribution of pop protein and its enzymatic activity points to nonhydrolytic functions of pop and to strict endogenous regulation of pop activity. the rapid cytoplasmic entry of cationic cell-penetrating peptides in the past few years, our understanding of the cellular import of cellpenetrating peptides has evolved rapidly. the initial concept of cell entry by direct permeation of the plasma membrane, was followed by endocytosis as a major route of import at least for most cellpenetrating peptide-cargo conjugates. more recently, we and others observed a rapid cytoplasmic delivery of fl uorescein-labeled analogs of the cationic cpps nonaarginine and tat-peptide at subtoxic lower poster abstracts micromolar concentrations ( ). this import originates from spatially confi ned zones of the plasma membrane and depends on the presence of heparan sulfate proteoglycans. these molecules have been proposed to interact polyvalently with the guanidinium groups of the arginine residues. moreover, the threshold for the induction of this import could be lowered by inhibition of endocytic routes of entry. recently, it was reported that the absence of serum in the medium lowers the threshold for this uptake process ( ) . in summary, these observations suggest that the concentration of peptide associated with the plasma membrane is a decisive trigger for this import process. currently, it is not known, to which degree this cytoplasmic entry of labeled conjugates at higher concentrations is based on similar molecular mechanisms as the direct permeation of unlabeled conjugates that has also been observed at lower concentrations ( ). here we summarize the current knowledge on direct transport across the plasma membrane of cationic cpps and present new data on the molecular events involved in this uptake process. plasma membranes have numerous essential functions for maintaining cellular homeostasis. however, the membranes are also barriers to intracellular delivery of various therapeutic molecules. for improvement of their translocations, we developed a novel method using gala peptide/cationic lipid complexes. gala, a -residue amphipathic peptide with a repeat sequence of glutamic acid-alanine-leucine-alanine, was designed to mimic the function of viral fusion protein sequences that mediate escape of virus gene from acidic endosomes to cytosol ( ) . when attached with bioactive cargoes, the gala peptide may thus serve as intracellular vector bearing effi cient endosomal escape function. however, because of negative charges from glutamic acids ( -residues) in the gala sequences, access of the peptide on negatively charged cell surface would be not so effi cient. to overcome this problem, cationic lipid was employed as an gadhesive h for pasting the gala peptide onto cell surface to accomplish effi cient cellular uptake. we examined the ability of gala peptide as a delivery vector using fitc as a model of membrane-impermeable low-molecular weight drugs. when fitc-gala ( ƒÊm) was administrated to hela cells, co-addition of cationic lipid, lipofectamine (lf ), drastically increased the effi ciency in uptake of fitc-gala. in a time-dependent manner, the fitc-gala escaped from endosomes, and diffuse fl uorescent signals were observed in both cytosol and nucleus. also the gala/cationic lipid system was applied for the intracellular delivery of fitc-avidin protein ( kda). when fitc-avidin was mixed with biotinylated-gala/ lf complexes, fitc-avidin ( nm) was internalized into cells effectively. in the absence of these complexes, internalization of fitcavidin was poor. these results suggest the usefulness of our approach for intracellular delivery using gala peptide and cationic lipid. the membrane repair response masks membrane disturbances caused by cell penetrating peptide uptake even though cell-penetrating peptides are able to deliver cargos of different sizes into cells, their uptake mechanism is still not fully understood and needs to be elucidated in order to improve their delivery effi ciency. recent studies have suggested that there might be a direct penetration of peptides in parallel with different forms of endocytosis. however, the direct penetration of hydrophilic peptides through the hydrophobic plasma membrane is highly controversial. three proteins involved in target cell apoptosis -perforin, granulysin and granzymes share many features common in uptake of cell-penetrating peptides e.g. they bind proteoglycans on the plasma membrane. the uptake of perforin activates the membrane repair response, a resealing mechanism triggered in cells with injured plasma membrane, due to extracellular calcium infl ux. upon activation of the membrane repair response, internal vesicles are mobilized to the site of the disrupted plasma membrane resealing it within seconds. in this study we present evidence that the membrane repair response is able to mask damages caused by cell-penetrating peptides when they internalize cells, thus preventing leakage of endogenous molecules out of the cell. the hypothalamic decapeptide gonadotropin-releasing hormone (gnrh-i; gnrh symmetric dimer derivatives ([ in vitro (cellular uptake and antiproliferative effect of the dimer derivatives) and in vivo (comparison of per os and intraperitoneal administration) antitumor effect of these symmetric dimers were investigated. the cellular uptake of dimer derivatives were studied by fl ow cytometry (bd lsr ii) on mcf- (human breast cancer) and ht- (human colon carcinoma) cell lines using carboxifl uorescein labeled symmetric dimer derivative. the cells were treated with different concentration of synthetic dimers and after the treatment were analysed by fl ow cytometry in order to investigate their cellular uptake. the antiproliferative effect of symmetric gnrh-iii dimers were studied on mcf- , ht- and t -d (human breast cancer) cell lines. ht- xenograft was applied for studying in vivo antitumor effect of gnrh-iii dimers in different administration routes. the cellular uptake and the antiproliferative effect are cell type dependent as it can be found in the literature. we found that symmetric dimer derivatives of gnrh-iii signifi cantly decreased the tumor volume in vivo ( %). iib receives intracellular signals (inside-out signaling) that allow cytoplasmic proteins to interact with the cytoplasmic domains of iib subunits, resulting in platelet aggregation. the aim of this work is to inhibit platelet thrombus formation by specifi cally disrupting the insideout signalling pathway using synthetic peptides based on the cytoplasmic region of subunit, residues - . peptide analogues derived from - region ( - , - (ptyr ), - , - and - ) were synthesized in their free, palmitoylated and/or tagged with the tat( - ) signaling sequence and carboxyfl uoresceinlabeled in order to investigate their membrane permeability, as well as their inhibition potency on the platelet aggregation. from the biological assays in prp and washed platelets we concluded that the modifi ed peptides that carry either palmitoyl-group or the tat( - ) signaling sequence penetrate platelet membrane and inhibit human platelet aggregation, in contrast to the corresponding free peptide analogues. acknowledgements: the gsrt (epan yb/ ) and e.u. for the fi nancial support. ; aib = -aminoisobutyric acid) is a potent mitochondriotoxic analogue of mp that demonstrates a signifi cant intracellular co-localization with mitochondria. through co-operation with a protein of the mitochondrial permeability transition pore vdac, mitoparan specifi cally promotes apoptosis. in contrast, cytc - , a cryptic fragment of human cytochrome c, is a moderately potent apoptogenic cpp that demonstrates a strong propensity for colocalization within the endoplasmic reticulum. using a sychnologic modular design, we have synthesized and evaluated a broad range of chimeric constructs combining apoptogenic cpp (message) and peptidyl address motifs. the overriding objective of these studies was to enhance cytotoxicity and/or develop target-selective drug delivery vectors. address motifs that target both plasma membrane and nuclear envelope protein structures included i) the integrin-specifi c rgd sequence, ii) a fas ligand mimetic wewt, iii) heptagastrin (aygwmdf) that targets a novel membrane binding site on high grade astrocytoma and, iv) a mimetic of fg nucleoporins (nups). incorporation of peptidyl address motifs, by simple n-terminal acylation or via a fl exible aminohexanoic acid (ahx) linker, produced chimeric constructs with enhanced cytotoxic potencies. most signifi cantly, ld values readily achievable in vivo were demonstrated by the modular apoptogenic cpp z-gly-rgdf-mitoparan, (ld = . m) and ac-nfkfglss(ahx)cytc - (ld = . m). in conclusion, targetspecifi c modular design of apoptogenic cpp is a promising strategy for the study and therapeutic induction of apoptosis. phage display screening of geminin-binding peptide and validation of its therapeutic use in tumors yoshida, kenichi meiji university, japan dna replication is controlled by the stepwise assembly of a pre-replicative complex (pre-rc). geminin is a component of the pre-rc and plays a role in preventing incorporation of the minichromosome maintenance protein complex into the pre-rc via binding and inhibiting cdt function. it may be possible to design low-molecular-weight chemicals that would bind to geminin to suppress the aberrant cellular proliferation in tumor cells. a random -mer peptide phage library was used for the selections. phage was selected by panning on immunotubes coated with recombinant geminin. positive clones were identifi ed by a screening phage produced from single colonies for specifi c binding to gst-geminin in elisa. fluorescent-labeled peptide linked to the sv nuclear localizing signal was synthesized by solid-phase synthesis, using fmoc chemistry. by detecting fl uorescein fl uorescence to mark specifi c transfected cells, we examined the effects of the peptide transfection on the synthesis of new dna in hct cells. by screening a random peptide phage library, we identifi ed a certain peptide sequence bound to geminin. using a series of mutant geminin, elisa test revealed the amino acid residues - are responsible for the peptide binding. we found fewer brdu positive cells following transfection of the geminin-binding peptide than following that of the control peptide. this study suggests that the chemical peptidomimetics of this peptide might form the basis for the development of drugs that could be used to prevent tumor progression. acknowledgements: this study was supported by kakenhi. it has been previously demonstrated that n -( -metoxyfumaroyl)-l- , diaminopropanoic acid (fmdp) is a strong inhibitor of glucosamine- phosphate synthase, a potential target for antimicrobial chemotherapy. fmdp is transported into the cells when incorporated in a peptide chain and hydrolyzed by intracellular peptidase releasing free inhibitor as the "warhead" component, which can react with the target enzyme. the concept of utilizing of peptide transport system for delivery of toxic amino acids into the microbial cells is characterized by authors as "illicit transport". unfortunately, peptides are not stable in physiological fl uids, due to activity of peptidases. analogs with modifi ed amide backbone are more resistant towards enzymatic degradation. it has been shown that enzymatic hydrolysis of endothiopeptides is often signifi cantly slower than natural peptides. in our studies we synthesized nva[csnh]-fmdp, an analog of nva-fmdp, which is a strong antimicrobial peptide. because of the fact that the "warhead" component in this peptide contains an amide backbone, fi rstly we decided to synthesize free fmdp with thioamide backbone. the results of enzymatic kinetics studies showed that in this way modifi ed compound is much weaker inhibitor of glucosamine- -phosphate synthase than fmdp. it was the reason why we planned and carried out a multi-step synthesis in order to obtain a potential more resistant towards enzymatic degradation, antimicrobial peptide containing a thioamide backbone only between nva and fmdp. activity of nva[csnh]-fmdp nva-fmdp against selected microorganism in medium containing blood serum was determined. neuroprotective effects of cortexin and cortagen in rats with focal brain ischemia and brain trauma there is a growing body of evidences that natural peptides and their smaller synthetic analogues have good outlooks for medical use. three compounds were studied in this research -cortexin, cortagen and nîleylcortagen. cortexin is a balanced combination of oligo-peptides isolated from calf/porcine cortex. the number of non-clinical and clinical studies confi rmed cortexin effi ciency as nootropic and neuroprotective drug. in spite of benefi cial results the natural source of the drug implies some diffi culties for it general use. to overcome this issue a number of putative cortexin synthetic analogues were developed. among them is tetrapeptide cortagen. in order to improve the peptide transport to the brain and lipophility the hydrophobic oleyl radical was added to the peptide chain. the aim of research was to study the effects of these peptides on recovery of conditional refl ex in active avoidance paradigm after heavy brain trauma and neurologic defi cit dynamics induced by focal brain ischemia in rats. males albino rats were involved in experiment. closed craniocerebral trauma was modeled by weight-drop method. focal cerebral ischemia was induced by cortical phototrombosis. experimental therapy started in . hours after mechanical action and continued for days. synthetic peptides were administrated i.p. once-a-day in dose of - mcg/kg. cortexin was used at the same schedule in dose of mcg/kg. cortagen and cortexin administration in dose of and mcg/kg respectively resulted in pronounced neuroprotective effect in rats with focal cerebral ischemia. the observed effects were proved by limb-placing test. cortexin experimental therapy resulted in earlier restoration of conditional refl ex starting from the rd day after brain trauma. nîleylcortagen had weaker benefi cial effect with signifi cant improvement in conditioned response recovery by the th day. probiotics such as lactobacillus rhamnosus gg provide health benefi ts beyond their mere nutritive value, and are useful in the treatment and prevention of several diseases. isolation and use of the anti-apoptotic factor(s) would ultimately culminate in the development of novel therapeutic agents in enteropathy resulting from chemo-and radio-therapy in the treatment of cancer patients, which could in turn be administered in a consistent and pharmacologically sound manner. in present study, intestinal epithelial cells were isolated from wistrar rats (n= ), and were grown in dmem medium supplemented with % foetal bovine serum at °c in a humidifi ed % co atmosphere. after h, these cells were treated with bioactive peptides isolated from lgg ( - ìmol/ lit) for h, and were incubated with camptothecin ( ìmol/ lit) for and h, and cell viability was measured by mtt assay. caspase activity assay was carried out to determine caspase and caspase activity. moreover, cell lysates were also pretreated with caspase inhibitor ( ìm). dna fragmentation assay was carried out to determine the increase in dna fragmentation. bioactive peptides signifi cantly prevented camptothecin induced apoptosis in rat intestinal epithelial cells. the treatment of intestinal epithelial cells with camptothecin for h resulted in fi ve-fold increase in dna fragmentation, and resulted in three-fold increase in caspase activity, compared with controls. pre-treatment with bioactive peptides ( ìmol/ lit) signifi cantly attenuated the camptothecin-induced dna fragmentation by % (p< . ), and signifi cantly inhibited caspase and caspase activity by % and %, respectively, after h of camptothecin exposure (p< . ). hence, it was concluded that novel bioactive peptide from lgg were found to be potential anti-apoptotic agents and hence could be administered as therapeutic molecule for treatment of enteropathy resulting from chemo-and radiaton-therapy. cisplatin belongs to the most powerful and useful anticancer agents. it binds strongly to dna in regions containing several guanine units, forming pt-dna links within strands. through disrupting base-pairing guanine to cytosine cross-links lead to unwinding of the dna. as a result cisplatin works against both types of cells, destroying cancer and normal type ones. therefore more selective delivery system of platinum to cancer cells is still needed. based on the evidence of the presence of -and -opioid receptor types in carcinoma cells we proposed to use opioid peptides as selective carriers for delivering platinum ions to the cancer cells. we designed hybride molecules which combine two fragments. one part of the molecule contains the opioid pharmacophore and the other fragment is designed to form a complex with platinum ion. such molecule can serve not only as carrier for platinum, but also give a strong analgesic effect. as a result such hybride molecule should express analgesic properties and provide anticancer activity. we will present synthesis of a few opioid peptide-platinum(ii) complexes, the binding affi nity at the opioid receptors and effect on the proliferation of the human glioblastoma cells. peptides derived from the c-terminal heptad repeat region of the hiv fusogenic protein gp are potent inhibitors of viral infection, and one of them, t (enfuvirtide), is used for the treatment of therapy-experienced aids patients. the mechanism of action of these peptides is to interfere bioactive peptides with the fusion of the viral and target cell membranes, and it is known that hiv entry takes place in membrane microdomains ('lipid rafts') enriched in cholesterol and sphingolipids. therefore we explored the advantage of targeting a peptide fusion inhibitor to membranes by addition of a lipid moiety, specifi cally a cholesterol group. since derivatization with lipids is also known to improve the half-life of peptide therapeutics, we chose the antiviral peptide c , which is more potent than t in cell-based assays, but unlike t has a very short half-life in vivo. we show here that attachment of cholesterol to c (c -chol) dramatically increases its antiviral potency against a panel of hiv strains, including primary isolates: for strain hxb , ic = pm for c -chol, versus pm for c , and pm for enfuvirtide). consistent with the anticipated mechanism of action, c -chol accumulates at the site of action, since washing of the target cells after incubation with the peptide, but prior to triggering fusion, increases ic only -fold, relative to -fold for c . moreover, cholesterol must be strictly positioned at the c-terminus of c , in line with the need of an antiparallel orientation of the c-peptide relative to n-peptide trimeric coiled-coil, present in the fusion-active conformation of gp . in addition to boosting antiviral potency, derivatization with cholesterol has the expected benefi cial effect on the peptide pharmacokinetics. we believe that these fi ndings may be of general utility for viruses, which share with hiv the dependence on a type i fusogenic machinery. multiple sclerosis (ms) is an autoimmune demyelinating disease mediated primarily by cd + t cells of the th subset. the design of peptide mutants of disease-associated myelin epitopes to alter immune responses offers a promising avenue for the treatment of ms. we designed and synthesized a number of peptide analogues by mutating the principal tcr contact residue based on mbp - epitope. the synthesis of the linear peptide agonist mbp - , as well as of the cyclic analogue was carried out by the fmoc/tbu methodology, utilizing the -chlorotrityl chloride resin. cyclization was achieved using obenzotriazol- -yl-n,n,n',n'-tetramethyluronium tetrafl uoroborate (tbtu) and -hydroxy- -azabenzotriazole, , , collidine allowing fast reaction and high yield cyclization product. the purifi cation was achieved using hplc reversed-phase chromatography and the peptide purity was assessed by analytical hplc and by mass spectrometry (esi-ms). agonist and antagonist (linear and cyclic) peptides were conjugated to reduced mannan. immune responses were diverted from th to th in sjl/j mice and generated antibodies which did not cross react with native mbp protein. ( ) . due to its high effi cacy and specifi city, hn represents a potential lead to new therapeutic approaches of ad. however, the molecular mechanism(s) of hn function(s) are not yet fully elucidated. wild-type hn and hn-derivatives were synthesized by spps and amino acid sequences and homogeneities of the rp-hplc purifi ed peptides ascertained by esi-and maldi-mass spectrometry (ms). the complex formation between hn and a ( - ) was studied by affi nity-chromatography and high resolution ms (fticr-ms), as well as by immunoanalytical (elisa) techniques. the binding sites between the two peptides were identifi ed by applying proteolytic epitope extraction/excision procedures [ , ] integramide a, an effi cient inhibitor of the coupled reaction of hiv- integrase, is a -mer linear peptide characterized by c -methylated -amino acids ( iva, isovaline, and aib, -aminoisobutyric acid, residues) that was isolated from fungal extracts of dendrodochium sp. the amino acid sequence was fully elucidated by the merck group a few years ago (s. b. singh et al., org. lett. , , - ) . on the other hand, the chiral sequence was only partially determined. in particular, the precise stereochemistry of the iva -iva dipeptide (known to contain one d-and one l-residue) near the c-terminus was not reported. to solve this unsettled issue and to assess integramide a primary structure-bioactivity relationship we performed by solution methods the total chemical independent syntheses of both l-d and d-l -mer diastereomers and compared their properties with those of the natural inhibitor. for an unambiguous, complete stereochemical assignment of integramide a we relied heavily on hplc and nmr techniques. our results clearly indicate that the chirality sequence of the iva -iva dipeptide of the natural product is l-d. the two integramide a diastereomers were also evaluated as inhibitors of hiv- integrase in the coupled reaction of proviral dna into the host cell dna. . a prototype peptide, r - , spanning rantes residues to , contains two hydrophobic clusters of fundamental biological importance connected by a nonessential hydrophilic linker. r - has been rationally modifi ed to improve its ccr binding and its antiviral potency. nmr studies on the modifi ed peptides revealed important similarities and differences with the three-dimensional organization of rantes. although these peptides are consistently more active as dimers, an increase in anti-hiv- activity of the monomeric forms was observed in parallel with their molecular evolution. in addition, no tertiary interactions could be detected by nmr, indicating an autonomous folding of the monomers also in the context of dimeric peptides. the most potent peptide designed so far, rmax, shows anti-hiv- activity in the low nanomolar range (vangelista et al, in preparation). strikingly, a similar potency was observed in the same assay with t , an hiv- gp -derived peptide currently licensed for use in aids therapy. these results provide a rationale for the use of rantes-derived peptides as new candidates for treatment and prevention of hiv- infection. % of the population older than years and % exceeding the age years are affected by alzheimer's disease (ad).( ) a factor in the pathogenesis of ad is the cerebral deposition of amyloid fi brils as senile plaque. bace initiates the pathogenic processing of app by cleaving at the n-terminus and the resulting c membrane bound c-terminal peptide can then be hydrolyzed by -secretase to form apeptide (a or a ). bace is not only a promising target cause it is a key player in formation of a but also because bace knock-out mice are viable and free of the gross phenotypic changes. our group was recently able to shown that the phosphino dipeptide (pdp) isostere is a suitable replacement of the hydroxyl ethylene isostere in om - ( ) resulting in pseudo peptidic inhibitors of about same potency. [ , ] in our search for conformationally restrained pdp isostere bace inhibitors we speculated that the p and p cyclization would lock the active conformation of the cyclic linear pseudo peptidic inhibitor in the n-terminal region. first the detail analysis of the crystal structure of om - bound to bace ( ), revealed that the ideal macrocyclus should consist of a -membered heterocycle. on this basis we developed a macrocyclic inhibitor with p and p cyclized side chains containing a pdp isostere with improved serum stability. specifi c tumor-targeting, via tumor-associated antigens (taa), selectively expressed or over expressed on tumor cells, is the goal of modern cancer therapy aimed at overcoming non-specifi c toxicity of most anticancer drugs. the expression of taa varies among different tumors and patients, resulting in highly variable response to tumor targeted therapies. therefore, diagnosis should provide information on the expression of the targeted antigen in each patient, thus allowing to predict possible effi ciency of a therapy mediated by targeting agents directed to the same tumor antigen. in this approach, the molecule used for tumor cell tracing should be as close as possible to that used for therapy. the use of peptides as tumor targeting agents was envisaged years ago with the fi nding that receptors for different endogenous regulatory peptides are over-expressed in several primary and metastatic human tumors, and can be used as tumor antigens (reubi jc. j nucl med ; : - ). in previous works, we demonstrated that peptides synthesized in a branched form, result in molecules that are resistant to proteolytic activity and can retain (or even increase, through multivalent binding) peptide biological activity. a branched peptide that targets neurotensin (nt) receptors, known to be over-expressed in a number of tumors, including colon, pancreas and prostate carcinoma (reubi jc. endocr rev ; : - ) and was conjugated to effector units and proved to be stable and active in vivo (falciani c, et al. mol cancer ther. ; : - ). here, we created new nt-based molecular tools conjugated to different fl uorophores and chemotherapeutics and demonstrated that branched peptides can be modulated either as tracers for measuring the presence of the specifi c target in primary tumor or metastasis on human surgical resections, or as specifi c drug-carriers, which use the same target to enter and eventually kill tumor cells in vitro and in vivo. aquaculture has become an increasingly important activity, as an immediate source of animal protein required for several countries growing population. vaccination of fi sh against bacterial and viral diseases is one of the best strategy for controlling certain infectious bioactive peptides diseases in aquaculture worldwide, thus decreasing the need for antibiotics, and increasing cost-effectiveness and net profi ts. infectious pancreatic necrosis virus (ipnv) is a pathogen of farm fi sh with a worldwide distribution. peptides derived from ipnv protein (vp ), a major structural protein, could be used for development of effective vaccine against ipnv, according to a search of vp antigenicity we selected two peptide sequences, k pyvrledetpqg (vp - - ) and n fslaeqpanetk (vp - - ), that are expected to be highly immunogenic. these peptide sequences were synthesized in their n-terminus iodoacetylated form and conjugated to ac-(lys-aib-cys) -nh , a cell penetrating sequential carrier (cpsc). conjugation of four copies to cys side chains of the carrier was realised by the chemoselective ligation method. the resulted constructs were purifi ed by hplc and characterized by esi-ms. immunizations are in progress in order to test their immunoprotective potency. acknowledgements to the gsrt (greece-egypt project -å) for the fi nancial support. for more then two decades, the rgd sequence is known to bind integrins, e.g., , v , and iib among many others. it is present in the natural ligands for these integrin receptors, as in fibronectin, vibronectin, or fibrinogen. recently, it was shown that fibronectin in which the rgd sequence has been mutated into an rge sequence (which is known not to be recognized by integrins) still has the ability to interact with its receptor. however, it forms fn fi brils only with a slightly different phenotype than wild-type fibronectin ( ). a hypothetical model for these unexpected results was proposed by curnis et al. ( ) . the ngr (asn-gly-arg) sequence, present at four positions in the fi bronectin molecule, is able to undergo a rearrangement to isodgr (isoasp-gly-arg), which shows activity on v and -with less potency -on . the mechanism of asn-deamination is already known for a long time and has widely been considered to be a process of degradation, acting as a biochemical clock that limits protein lifetimes in vivo ( ). curnis et al. were the fi rst to show that the deamination process increases protein function instead ( ). these fi ndings stimulated us to create a library of cyclic peptides containing the isodgr sequence. a screening of this library showed various new peptides with high activity and selectivity towards the integrin receptor . the correlation between the intensities of muramyl peptides adjuvant effect and nod activation. it is well known that essential activity of muramyl peptides -minimal structures of bacterial cell wall -are adjuvanticity. the comparison of the adjuvant effect of these compounds with the ability to activate nf-kb pathway through nod was examined. the adjuvant activity of di, tetrasaccharide peptides and stearoyl containing derivatives has at least two peaks in dose-response curves and greater of them correlates with respective dose-response data for nf-kb stimulation through nod . introduction of stearoyl moiety, with the aim of improving muramyl peptide interaction with the cell membrane and subsequent intracellular delivery, infl uenced the corresponding activities in vitro, but did not correlate with improved effects in vivo experiments. the comparison of the adjuvanticity in vivo and the nod activation in vitro revealed clear correlation between two responses. these fi ndings confi rm the view that nod pathway activation should account, at least in part, for the adjuvant effect of these compouds. the correlations of the nf-kb pathway induced by muramyl peptides with the aim of enhancing their adjuvant activity were investigated. the thymus gland plays an important role in overall immunomodulation. the studies in early s by several authors have established that thymus is necessary for the normal development of immune response. it is thought to be responsible for the development and regulation of tcell immunity, acting through endocrine mechanism ( ). it is known that thymus peptides play an important role in the development, maturation, differentiation, and activation of t-lymphocytes. investigation of the function and properties of this gland shows that the thymus contains pharmacologically active components with immunological properties. when the immune system is challenged, thymic peptides seem to regulate the expression of various cytokine and monokine receptors on t-cells and induce secretion of il- , interferon alpha, and interferon gamma. up to date several thymic extracts have been isolated using different biochemical methods of extraction, homogenization and purifi cation. these are, usually, semipurifi ed aqueous and lipid calf thymus extracts. the goal of this study was to determine the biological activity of peptide components, isolated from the calf thymus. extract of calf thymus was prepared and fractioned into lipid and nonlipid (peptides) fractions. the nonlipid fraction was isolated and characterised by biuret, ir , nmr and hplc methods. analyses of ir and nmr spectra indicated the presence of charasteristic bands and peaks for peptides. results estimated from hplc analyse showed that molecular masses of isolated peptides were below daltons. biological activity was accesed by using proliferation of thymocites and splenocites in vitro in the presence of mitogen, and obtained results showed signifi cant imunomodulatory effect. keywords: thymus, peptides, immunomodulators, thymocites, splenocytes, proliferation according to contemporary knowledge about proteins, every protein can play the role of a precursor of biologically active peptides. bioactive peptides isolated from food proteins display various activities, for example: antihypertensive, antithrombotic, immunomodulating, opioid and antibacterial. the database of protein and bioactive peptide sequences designed in chair of food biochemistry (www.uwm.edu.pl/biochemia) contains information on proteins which are precursors of bioactive peptides. the database, biopep, enables an evaluation of food proteins according to the following criteria: the frequency of the occurrence of fragments with the given activity in a protein chain, a potential activity of protein fragments, and profi les of a potential biological protein activity i. e. the type and location of a bioactive fragment in the protein chain. the biopep database was used to determine the profi les of potential biological activity of food proteins and classify them into families and subfamilies. results that we obtained indicate that the greatest number of bioactive peptides can be released from milk proteins. we also analysed structural properties of bioactive fragments encrypted in protein chains which are predicted to be accessible for endopeptidases. the application of biopep and msblast software enabled us selection of a fragments with high degrees of identity to the celiac-toxic peptides in food proteins sequences. based on the biopep, we are able to design the processes of the release of bioactive peptides from protein sequence and with the use of mass spectrometry -to identify released peptides. the detailed results of authors in silico food proteins analysis will be presented. the computational methods and the above-mentioned tools can be applied for designing food with the special designed and desired properties (functional food) as well as production of nutraceuticals i. e. food with therapeutic properties. neurotensin analogs with high affi nity and selectivity at human neurotensin receptor . neurotensin (nt), pglu -leu -tyr -glu -asn -lys -pro -arg -arg -pro -tyr -ile -leu -oh, exerts numerous physiological actions in the central nervous system and in the periphery. studies in animal models have suggested its involvement in the modulation of dopamine transmission, hypothermia, analgesia, locomotor activity, cardiovascular function, and others. at present, three receptors are known to bind nt; two of them are g-protein coupled-receptors (ntsr and ntsr ). the physiological effects of ntsr have been extensively studied but the functions associated with the nt binding to ntsr are less well defi ned. the c-terminal fragment of nt, arg -arg -pro -tyr -ile -leu -oh, has been long recognized as an essential and suffi cient segment for the effective interactions of nt with the receptors; a short peptide, arg -arg -pro -tyr -ile -leu -oh, designated nt( - ), displays binding affi nity similar to that of the full-length nt at both ntsr and ntsr . in this study, the role of arg in the interactions of nt( - ) with the human ntsr and ntsr was examined through ligand structure-function studies. the side chain of arg was determined to not be critical for binding to hntsr but essential for binding to hntsr . several analogs of nt( - ) are reported which are high affi nity hntsr ligands (ic = . to nm) and more than -fold selective versus hntsr . human umbilical vein endothelial cells (huvecs) form tubular networks when cultured on top of the matrigel basement membrane preparation, refl ecting the ability of the cells to form blood vessels. using this model, we have previously shown that psa (also known as klk ) inhibits endothelial cell tube formation( ), indicating reduced angiogenic potential. furthermore, we have shown that the antiangiogenic activity of psa is related to its enzymatic activity [ , ] . we have developed peptides that stimulate the enzymatic activity of psa towards a small chromogenic substrate. these peptides also enhance the anti-angiogenic activity of psa both in vitro and in vivo. this supports our hypothesis that enhanced psa-activity by our peptides could be used to reduce tumor angiogenesis and, thus, to reduce tumor growth. the most extensively studied tads are those that contain acidic tads and one extremely important protein is the human tumour suppressor protein p . given the presence of repetitive stretches of acidic amino acids tads are generally disordered in the free state and this has also been demonstrated for p tad. using a combination of nmr spectroscopy, isothermal titration calorimetry and site-directed mutagenesis studies we have recently characterized the interaction of the p tad with the pleckstrin homology (ph) domain of the tfb / p (yeast/human) subunit of tfiih. the nmr structure of the tfb / p tad complex demonstrates that p forms a short alpha-helix in complex with tfb ( ). this structure is an important step towards developing a sequence code for acidic tad binding to the ph domain of tfb /p . such detailed structural information is essential to design molecules that modulate transcription activators such as p . we will present the structure of the p /tfb complex, structural and biophysical characterization studies with peptides designed to mimic acidic tads and compete with p for binding to tfiih. integrins are a family of transmembrane cell surface receptors, which mediate cell-cell and cell-matrix adhesion. the binding of integrins to their natural ligands is the molecular basis of physiological processes such as cell adhesion, migration and signal transduction of cells, as well as of patho-physiological processes. thus, small molecules capable of interfering with this integrin-natural ligand binding process have pharmacological potential in the therapy of cancer and infl ammatory diseases. the amino acid sequence rgd, present on many of the natural ligands, is a prominent recognition motif of integrin ligands. synthetic rgd-containing peptides are an excellent starting point for the identifi cation, synthesis and development of selective integrin ligands. the affi nity and selectivity of the peptide ligands towards different integrins depend strongly on the secondary structure of the sequence and the overall three-dimensional shape. cyclization is frequently used as a method to reduce the accessible conformational space. additionally, the incorporation of non-natural conformationally constrained amino acids can greatly affect the secondary structure of the peptide, in such a way that the synthetic ligands prefer to adopt a particular conformation. the aim of this investigation are small cyclic peptides containing the rgd motif and constrained amino acids (such as -methylated amino acids, -amino acids or dehydroamino acids) that exhibit well-defi ned conformational properties. the present communication describes the synthesis of different cyclic rgd peptides with the general sequence c-(-arg-gly-asp-xaa-yaa-) and the evaluation of their activity as ligands for the v and integrins, present on human cells. acknowledgments: this work is supported by a marie curie intra-european fellowship from the th framework programme. in recent years there has been increasing interest in the design of chemical agents capable of inducing dna interstrand crosslinking (isc), which, shutting down the dna replication process, represents by far the most cytotoxic of all the alkylation events. quinone methides (qms) are interesting compounds that have been proposed as intermediates in a large number of chemical and biological processes. the asymmetry introduced by the presence of two electronically different substituents, carbonyl and methylidene, on the cyclohexadiene ring imparts a strong dipolar character to quinone methides not found in benzoquinones and quinodimethanes. qms have been successfully used to accomplish nucleoside alkylation and dna isc by photochemical and fl uorideinduced activation. apart from their involvement in biological processes, o-qms react with a variety of nucleophiles (michael addition), resulting in substituted hydroquinones. they also function as effi cient heterodienes in diels-alder reactions and undergo a variety of self dimerization reactions. here we present the synthesis of a new series of synthetic o-qms (bis-napthalene type) conjugated with rgd analogue peptides. the biological activity of these compounds is under investigation. aknowledgements: we thank the european social fund (esf), operational program for educational and vocational training ii (epeaek ii) and particularly the program pythagoras ii for funding the above work. platelets aggregation causes clotting in the blood vessels during the blood circulation due to several reasons. factor viii (fviii), a blood coagulation glycoprotein, is a key component of the blood coagulation system. fviii in its activated form (fviiia) acts as a cofactor to the serine protease fixa in the conversion of the zymogen fx to the active enzyme (fxa). the role of fviiia is to increase the catalytic effi ciency of fixa in the activation of factor x (fx). the target of this research is the synthesis of biologically active peptides, which are expected to inhibit selectively the maximisation of thrombin production depended on factor ix (fix) and accordingly the additional activation of platelets. these peptides are based on the regions in which the fviii interacts with fix. glycoprotein fviii is composed of three distinct domain types in the arrangement, a -a -b-a -c -c . the sequence - of a subunit is the following: ser -val-asp-gln-arg-gly-asn-gln this work covers the synthesis and biological evaluation of linear and cyclic head to tail peptides, analogs of the loop sequence - of the a subunit, aiming at the inhibition of interaction of fviiia with fixa. substitutions have been taken place at asn , which are related to the pharmaceutical groups at the side-chain, like asp(r) . all the synthesized analogs are purifi ed (rp-hplc) and identifi ed (esi-ms). the synthesized peptides analogs were investigated for their inhibitory activity and tested for clotting defi ciency by measuring their activated partial thromboplastin time (aptt) in vitro. these results will be discussed in relation with their biological activity against the fviiia factor of blood coagulation. nerve growth factor (ngf) is an homodimeric protein that binds two different cellular receptors: ) the transmembrane tyrosine kinase receptor trka, a member of tyrosine kinase family; and ) p , a member of the tumor-necrosis-factor receptor superfamily. both receptors are involved in the activation of different intracellular signal transduction cascades. small peptides retaining the most essential elements of ngf may be useful either as agonist or competitive antagonist in the treatment of several neurodegenerative desease and nerve injuries. the n-terminal fragment of ngf was previously demonstrated to be an important determinant for affi nity and specifi city in the binding to trka. crystal structure of ngf-trka complex, site-directed mutagenesis studies and substitution of individual amino acids, contributed to identify within the ngf molecule the most relevant domains for its biological activity in the loop- ( - ), loop- ( - ) and in the nterminal region ( - ). we synthesized twenty peptides mimicking the loop and linked together with aminoacid-based spacers (n glycines) or with two unit of -ammino- , -dioxaottanoic acid with or without the n-terminal region. l l and l l homodimeric loops, moreover l and l were also synthesized, as single loop, and their biological properties were compared to l l and n-l l activity . the n-l l (hpifhrgefsvadsvsvwvgdctdikgkctgacdgkqc) and l l (ctdikgkctgacdgkqc) peptides showed a good ngf agonist activity at concentration as low as um. both were able to induce differentiation of chick dorsal root ganglia (drg) and to stimulate the tyrosine phosphorylation of trka but not of trkb receptor. in addition the l l peptide was able to induce the pc cells differentiation into sympathetic-like neurons. moreover the l l peptide was shown to reduce neuropathic behaviour and restore neuronal function in a rat model of peripherical neuropathic pain, thereby suggesting a potential therapeutic role for this peptide. short proline-rich peptides interact with sh domains, exhibiting little or no secondary structure before their binding to the cognate proteintargets. under these conditions the binding process of a proline-rich peptide with the sh domain shows unfavorable binding entropy, likely resulting from a loss of rotational freedom on the formation of the ppii helix. with the aim of stabilizing the ppii helix conformation in sh binding motifs, in the previous years, we replaced the proline residues of the hpk proline-rich decapeptide, ppplppkpkf (p ), either with -r-(fp) or with -s-(fp) fl uoro-l-proline at different i, i+ positions. the interactions of the fl uoroproline-peptides with the sh domain of cortactin protein were analyzed quantitatively by non-immobilized ligand interactions assay by circular dichroism (nilia-cd), whereas cd thermal transitions were measured to correlate their propensity to adopt ppii helix with their affi nity for sh . results show that although the introduction of the fp residue stabilizes the ppii helix conformation of peptides in a position-dependent manner, the induction of a stable peptide conformation does not increase the ligand affi nity towards the sh domain of cortactin. to explore the effect of electron-withdrawing substituent in the pro residue, the fp residues of p were replaced by the natural amino acid -r-hydroxy-proline (hyp). unexpectedly, nilia-cd and cd thermal transitions results showed that the hyp-containing peptides exhibit a stable conformation in aqueous buffer and k d values lower than the corresponding fp peptide-analogues. in particular, hyp peptide containing hyp residues at i, i+ and i+ positions adopts the greater percentage of ppii helix conformation over the entire studied temperature range and shows a k d value comparable to that of the parent p peptide. we are now searching for inhibitory peptides able to reduce survivin function in breast cancer cells. we take advantage of the observation that survivin forms homodimers in vivo and have derived short peptides representing the dimerization domain. to enable intracellular expression and visualization of these short peptides, they will be fused to a fl uorescent carrier protein. alternatively, the peptides comprising the dimerization domain will be inserted into a scaffold protein. this allows the presentation of the peptides in a constrained and stabilized conformation. we will analyze the effects of lentiviral expression of these fusion peptides in cancer cells expressing high levels of survivin. the peptides will be analyzed for their ability to inhibit proliferation, cell-cycle progression and/or to induce apoptosis in cancer cells. cd c/cd ( x ) is a member of the leukocyte integrin (cd or integrin) subfamily of adhesion molecules and it is expressed on monocytes, macrophages and granulocytes. its ligands include fi brinogen, ic b, lps, collagen type i and denatured proteins as well as intercellular adhesion molecules icam- and icam- . icam- is a red cell specifi c membrane glycoprotein that was fi rst described as anerythrocyte blood group antigen lw (landsteiner-wiener) and later it was found to belong to the family of intercellular adhesion molecules. the fi rst reported receptors of icam- were leukocyte integrins cd a/cd and cd b/cd . later it has been reported to bind to several other integrins as well. latest reported interaction is to cd c/cd . the binding of icam- to integrins expressed in macrophages might clarify some of the controversy concerning the recognition and uptake of senescent red blood cells in spleen. another function of icam- / macrophage integrin interactions could be in retaining the maturing red cells in bone marrow until they are ready to be released in the circulation. adhesion between icam- and integrins have also been found to be important in different pathological situations and inhibition of these adhesion events could be of important therapeutic value. we have studied the interaction between icam- and cd c/cd using peptides derived from both binding partners using pepspot method and synthetised peptides. the inhibitory or activating role of these peptides could beof clinical importance in pathological conditions where abnormal red cell adhesion is observed. the regulatory effects of relaxin in mammals realize via receptors of the serpentine type. in this work we studied the relaxin activating effect on the adenylyl cyclase (ac) activity in the rat tissues and muscle tissues of invertebrates using a peptide strategy. the strategy involved the synthesis of the peptides - and - , as well as the palmitatemodifi ed peptide - , all corresponding to the c-terminal region of the third intracellular loop (c-icl ) of the human type relaxin receptor lgr . the peptides - and - -lys(palm) had a dose-dependent activating effect on the ac activity and gtp-binding in myocardium, brain and, to a smaller extent, skeletal muscles of rat. the - -lys(palm) had a stronger ac effect than the longer peptide - , because of the possibility to be anchored in the membrane by the hydrophobic radical and, hence, to interact with the g protein more effectively. in mollusks and earthworm muscles, the stimulatory effects of both peptides were weak. competitive inhibition of the stimulating effects of relaxin by the lgr -derived peptides indicates that the relaxin signal in myocardium and brain is transmitted to ac via lgr receptor. in skeletal muscles of rat and muscle tissues of invertebrates, the inhibition of ac and gtp-binding stimulating effects of relaxin by peptides was almost indiscernible. it can be concluded that relaxin controls the ac of these tissues via the receptor different from the relaxin receptor lgr . the stimulating effects of lgr -derived peptides also decreased in brain and myocardium in the presence of Ñterminal peptide - of mammalian g s -subunit and after cholera toxin treatment. thus, relaxin stimulates ac via lgr receptor and g s protein in myocardium and brain, and the coupling between receptor and g s protein is mediated by the interaction of receptor Ñ-icl and Ñ-terminal segment of g s . acknowledgements: supported by rfbi (grant - - ) and «russian science support foundation». normal tissue function depends on adequate supply of oxygen through blood vessels. angiogenesis is a fundamental process by which new blood vessels are formed and which is highly regulated in healthy individuals. however, many diseases are driven by unregulated angiogenesis. excessive angiogenesis is associated with cancer, rheumatoid arthritis, psoriasis, while insuffi cient angiogenesis results in ischemia or artherosclerosis. many new angiogenic modulators have been developed in the last years, mostly to inhibit angiogenesis but only few peptide-based angiogenic stimulators have been reported. there are data in the literature suggesting roles of small peptides or basichexa peptides as angiogenic modulators like ringseis et al. reporting effects on endothelial cell function (such as ec proliferation) and fazekas et al. describing effects for the latter. endostatin, an endogenous inhibitor of angiogenesis, among its proteolitic fragments contains both, an inhibitor and a stimulator of angiogenesis. we considered it possible that fragments of basic heptapeptide d-phe-cys(-)-tyr-d-trp-lys-cys(-)-thr-nh (tt- ), a strong antitumor agent, could also act as angiogenic modulators. the heptapeptide was developed in our laboratory and has been recently in a clinical trial (phase ii). we synthesised, characterized and tested partially protected di-and tripeptide fragments of it such as boc-d-phe-cys(acm)-tyr-ome, d-phe-cys(acm)-tyr-ome, boc-tyr-d-trp-cyclohexilamide and h-tyr-d-trp- -adamanthylamide. the biological activity of the compounds was tested in vitro using an immortalized kaposi sarcoma cell line to determine their pro-anti-angiogenic character. to test the angiogenic potential of the best compound, the aorta ring assay was used, which by using intact vascular explants reproduces more accurately the environment in which angiogenesis occurs. in this study we report the synthesis and angiogenesis modulating effects of the peptides. somatostatin is a neuropeptide that regulates several functions of the endocrine and exocrine systems. it also affects cell proliferation and neurogenic infl ammation through a family of g protein-coupled receptors. neurogenic infl ammation plays signifi cant role in the pathogenesis of numerous infl ammatory diseases (such as asthma, arthritis, allergy and migraine). inhibitory effect of somatostatin on infl ammation is well known but the pharmaceutical use of the native peptide is limited due to its broad spectrum of anti-secretory effects and short plasma half-life time. tt- , a heptapeptide analogue of somatostatin (developed by our research group and it is in clinical phase ii trials) has selective antitumour and anti-infl ammatory effect without regulating other endocrine or exocrine processes. receptor-ligand binding experiments with various analogues of the somatostatin as well as tt- [d-phe-c(cys-tyr-d-trp-lys-cys)-thr-nh ] verifi ed that the side chains of tyr, d-trp and lys are important pharmacophoric groups for somatostatin-like biological activity. linear, cyclic and branching derivatives were designed and synthesised using above amino acids to selectively inhibit infl ammatory actions. unnatural moieties also were applied to enhance their enzyme resistance. linear and cyclic peptides consist tyr and d-trp, while ring closed ones contain lys too. branching peptidomimetics have the same, fl exible core [tris( aminoethyl)amine] and three protected or unprotected amino acids situated in equal positions. the biological activity of the compounds was evaluated by in vitro assay of substance p release and in vivo assay of plasma protein extravasation. the most potent agents strongly inhibited substance p release by - % and one of them showed strong anti-infl ammatory activity when administered orally. the structure of the novel compounds and the relationship between their structure and biological activity will be discussed. urotensin ii (u-ii), a potent vasoconstrictor, is found in diverse species, including human. several biological studies indicate that u-ii is the most potent mammalian peptide vasoconstrictor reported to date, and it appears to be involved in the regulation of cardiovascular homeostasis and pathology. in order to elucidate the importance of trp residue for receptor interaction and biological activity recently we have designed, synthesized new analogues where trp was replaced with constrained analogues ltpi or dtpi ( , , , -tetrahydronorharman- -carboxylic acid). the tpi residue was replaced in both agonist p u and antagonist urantide sequences. on these new ligands we performed biological and nmr conformational studies. the new ligands will be used in further biological investigations of the ut receptor. gnrh analogs as carriers for targeted suicide gene delivery suicide gene therapy represents one of the promising approaches to the cancer treatment. an application of herpes simplex virus thymidine kinase (hsvtk)/ganciclovir system possesses additional advantage due to bystander effect on neighboring cancer cells. overexpression of gnrh receptors in the case of most adenocarcinomas creates the basis of gnrh analogs use as carriers for targeted suicide gene delivery. we investigated different manners of gnrh molecule modifi cation; their infl uence on peptide/dna complex formation and its penetration into cancer cells. analogs, containing nls from large antigen of sv were synthesized using combination of boc-and fmoc-chemistry. depending on peptide structure (agonist or antagonist) nls was attached via position or of the natural molecule. the competition experiments demonstrated that internalization of peptide/ dna complex into hepg cells is mediated by specifi c receptor binding. moreover, nls/dna complexes, lacking gnrh moiety were unable penetrate cellular membrane. subsequent studies permit to identify the infl uence of analog structure on in vitro effi ciency of suicide gene therapy, followed by acyclovir treatment. it was shown that application of reference peptide gene delivery system damaged about % of tumor cells. use of cationic peptide conjugated with rgdf sequence provides high effi ciency of treatment, however can not ensure selective action on cancer cells. gnrh analogs completely suppress tumor growth in vitro due to specifi c interaction of peptide/dna complex with correspondent receptor. the effi ciency of suicide gene therapy depends on peptide structure and is in favor of agonists as compared to antagonists. preliminary data of experiments in vivo on laboratory animals demonstrated practical utility of tested peptides in the course of intravenous administration. thus, it was shown that gnrh analogs containing nls moiety represent promising candidates for the delivery of hsvtk gene into the cancer cells. the structural-functional study of putative grape (vitis vinifera) uncharacterized protein sequences was performed. we developed a special method of computer analysis ( ) for this. this method has allowed to reveal new potentially active regulatory oligopeptide sequences yet not investigated experimentally. information on grape amino acid sequences of public databases [ , ] , computer database erop-moscow (endogenous regulatory oligopeptides) . containing the information on structure and functions of known natural oligopeptides and specially created computer programs were used for this. protein amino acid sequences were compared with all known oligopeptide sequences in this method. as a result several tens of grape oligopeptide sequences were elucidated. the similarity of their sequences with the known oligopeptide structures of other biological species was the basis for the prediction their potential functional properties. it has been shown that grape contain putative regulatory oligopeptides possessing functions of antibacterial and antifungal agents, enzyme inhibitors, calmodulin binding structures, rapid alkalinization factors, etc. the primary structure similarity of grape sequences was found not only with plant species but with bacteria, fungi, and animals also. cyclotides are plant derived mini-proteins with compact folded structures and exceptional stability. their stability derives from a headto-tail cyclised backbone coupled with a cystine knot arrangement of the three-disulfi de bonds. taking advantage of this stable framework we developed novel vegf-a antagonists by grafting a peptide epitope involved in vegf-a antagonism onto the stable cyclotide framework. antagonists of this kind have potential therapeutic applications in diseases where angiogenesis is an important component of disease progression, including cancer and rheumatoid arthritis. a grafted analogue showed biological activity in an in vitro vegf-a antagonism assay at low micromolar concentration and importantly the in vitro stability of the linear epitope was markedly increased using this approach. in general, the stabilization of bioactive peptide epitopes is a signifi cant problem in medicinal chemistry and in the current study we have shown the cyclotide framework is ideally suited for such stabilization. cystine rich scaffolds are emerging as valuable templates in drug design and in the current study we have shown that the cyclotide scaffold, with the advantageous features of a knotted disulfi de core and a cyclic backbone, has signifi cant potential in stabilizing a wide range of bioactive peptide epitopes. gonadotropin releasing hormone (pglu-his-trp-ser-tyr-gly-leu-arg-pro-gly-nh , gnrh) plays a signifi cant role in the controlling of gonadotropins and steroids hormones. a large number of linear gnrh analogues has been synthesized and tested for several medical uses. leuprolide acetate (pglu-his-trp-ser-tyr-(d)leu-leu-arg-pro-nhet, lpa) is a potent gnrh agonist and is used to treat a wide range of sex hormone related disorders, including prostatic cancer, endometriosis and precocious puberty. despite its widespread use, only limited information based on spectroscopic evidence regarding the solution conformation of leuprolide are known. moreover, non crystallographic data is available for the receptor of gnrh (g protein-coupled receptor). the aim of this study was to characterize the conformation of leuprolide and its modifi ed linear analogue (pglu-his-trp-ser-tyr(ome)-(d)leu-leu-arg-aze-nhet) in dmso solution (which simulates better the receptor environment) using nuclear magnetic resonance (nmr) and molecular modeling techniques. by using both nmr and molecular modeling we have characterized the secondary structural preferences of these gnrh analogues. structural determinants of binding to the mu-opioid receptor -an important target in analgesia -attracts great scientifi c attention. many natural and synthetic peptides and peptidomimetics were shown previously to bind to the mu-opioid receptor selectively but there is no consensus about what structure is responsible for such biological activity. no high resolution structure of this receptor is available and the binding site of ligands is not exactly known despite numerous site-directed mutagenesis studies. this suggests that the determination of structural aspects of mu-opioid activity should focus on the ligands. mu-opioid ligands with similar affi nity and selectivity should possess at least one common structural feature in which they differ from other ligands of different affi nity and selectivity. comparative structural analysis of such ligands, considering adequate representation of binding conditions may reveal key features of bioactivity. in this study ten mu-opioid receptor ligands, damgo, tyr-w-mif- , morphiceptin, endomorphin- and and their analogues, possessing different affi nity and selectivity were examined using molecular dynamics. conformational preference of these molecules was determined in aqueous and dmso media which were meant to model different possible binding environments. no structural trend, correlating with previously measured bioactivities was observed in aqueous media. in dmso it was found, that a preference for trans orientation of the tyr side chain and gauche (-) orientation of the third aromatic side chain, the free rotation of the phe side chain and a high propensity of bent backbone structure is favorable for high affi nity binding to the mu-opioid receptor, while deviations from these criteria results in variable loss of bioactivity. constellation of these four key conformational parameters may be a guiding principle in the future for the design novel mu-opioid receptor ligands. in cui-catalyzed azide-alkyne , -dipolar huisgen's cycloaddition -prototypic "click reaction"-is a recently developed synthetic procedure to obtain cyclopeptides carrying, as a rigid linking unit, the lactam bioisoster, , -disubstituted [ , , ] triazolyl ring ( ). we have recently reported the synthesis and conformational analysis of the , -disubstituted- [ , , ] triazolyl containing cyclopeptide derived from the sequence of the potent i-to-i+ side chain-to-side chain lactam-containing antagonist of parathyroid hormone-related peptide (pthrp) .. the conformational properties of triazolyl-containg peptide were compared to those of the corresponding lactam analog. cd and nmr studies revealed that, despite a slight difference of the backbone arrangement, triazolyl containing cyclopeptide and lactam-containing cyclopeptide share a common orientation of the side chains ( ). here we present the structural study of a new library of disubstituted- [ , , ] triazolyl containing cyclopeptides designed to obtain different cycle dimensions and different triazolyl-ring positioning. cd and nmr analysis in different solvent systems shows that both, the dimension of the cycle and the specifi c positioning of [ , , ] triazolyl ring are critical to fully resemble the conformational properties of the potent lactamcontaining antagonist of parathyroid hormone-related peptide (pthrp). the somatostatin (srif) is a cyclic tetradecapeptide, which exerts inhibitory effects on the secretory processes in the endocrine and exocrine systems, and the cell proliferation through somatostatin receptors (sstr - ). sstrs are distributed throughout human body, not only in normal cells but also in tumor cells. most of the somatostatin analogues developed for clinical use, such as octreotide which act longer than somatostatin are being used in the diagnosis and treatment of endocrine tumors. the use of these analogues as antitumor agents has been limited because of their antisecretory effects and poor oral bioavailability. tt- [d-phe-c(cys-tyr-d-trp-lys-cys)-thr-nh ], was reported by keri et al. to have potent antiproliferative activity without antisecretory action .. based on the above, we aimed to design and synthesize somatostatin analogues with more potent antiproliferative activity and high oral bioavailability. we synthesized pyrazinone ring containing cyclic peptides ( ) and linear peptides which are substituted at the c-terminus lys with hydrophobic and rigid groups. our focus was on the active sequence: tyr-d-trp-lys. we also examined their antiproliferative activity and found that boc/h-tyr-d-trp- -adamantylamide exhibited the most potent antiproliferative activity higher than that of tt- . furthermore, on the best analogues we studied dna fragmentation by facs analysis and cellular morphology. the results demonstrated that these somatostatin analogues induced cell death by apoptosis. kobe gakuin universit, japan background and aims: endomorphin- (em- : h-tyr-pro-phe-phe-nh ) has high affi nity and selectivity for the mu opiod receptor ( ). we focus on the pro residue, which imposes strong restraints on the conformation of the peptide chain or induces cis-trans isomerization of x-pro bonds. we substituted the pro with -azetidinecarboxylic acid (aze) or piperidinecarboxylic acids (pip) to increase or decrease the conformational fl exibility. in this paper, we deal with the synthesis of em- analogues containing aze and pip and the evaluation of the biological functions of the analogues on the opioid receptors. methods: the synthesis of peptides was achieved according to the procedure of okada y. et al. ( ) the fi nal products were identifi ed by maldi-tof mass spectrometry and elemental analyses. the receptor binding activity of peptides was assessed by radio-ligand receptor binding assay using mu and delta opioid receptors from cos- cell membranes expressing each opioid receptors. for the evaluation of the biological function of peptides, the gpi and the mvd tests were performed. the new analogue of csf (glc) ., [pro ,asn (glc),thr ]csf is characterized by a type i -turn around the minimal epitope asn(glc), and it was demonstrated to show the highest antibody affi nity in competitive elisa in multiple sclerosis (ms) patients' sera and thus it appears as a promising tool for the detection in patients' sera of specifi c autoantibodies. in previous studies, we synthesized and tested different fragments of this new glucosylated peptide, [pro ,asn (glc), thr ]csf , identifying the shortest sequence [pro ,asn (glc),thr ] csf ( - ) able to detect antibodies by elisa in ms patients' sera. this heptapeptide is characterized by thr in position generating a characteristic n-glycosylation consensus sequence. according to the results obtained with the linear heptapeptide in ms, we applied the backbone cyclicization method to develop two backbone cyclic libraries of glycopeptides based on the sequence of the linear hepta active peptide. backbone cyclization is a method that allows obtaining cyclic peptides without changing the natural sequence or the chemical character of the amino acid residues, in order to enhance activity, stability to metabolic degradation, selectivity and bioavailability ( ) . the fi rst library contains a gly building unit at the c-terminus and is connected to the n-terminus by linker of various lengths (n= , , , ) . in the second library, his of the heptapeptide is replaced by gly building units with various alkyl chains (n= , , , ) [fmoc-n (n alloc(n-alkyl))gly-oh] that is connected to the n-terminus by linker of various lengths. twenty cycloglycopeptides were synthesized, and screened by competitive elisa s on ms patients' sera to select the most bioactive cyclic glycopeptide. peptide nucleic acids have become, arguably, one of the most interesting of dna mimics. owing to their high chemical stability and resistance towards nucleases and proteases, they are very attractive as antigene/ antisense agents, molecular biological tools and for genetic diagnosis. the lack of charge and polar groups in the backbone decrease their solubility in aqueous environment and their ability to cross cell membranes, reducing their performance in in vivo applications. in order to overcome these problems -to improve solubility, increase affi nity and specifi city of binding, a number of analogues were synthesized. this study describes the synthesis of pna-monomers on the base of non-protein amino acids analogues of basic amino acids lys and arg. nucleobases are the second residue we have selected. studies will include replacement for example of the uracil, with the fl uorinated analogue. growing evidences indicate that n-glycosylation is a co-translational modifi cation that, either native or aberrant, may play a fundamental role in a large number of biological events. in particular among postand co-traslational modifi cations glycosylation plays a crucial role in the immune system. in fact, almost most of all the key molecules involved in the immune response are glycoproteins. there are growing evidences of defects in glycosylation with diseases that assets to pathway oligosaccharides as code words. in previous studies, we demonstrated that the presence of a -dglucopyranosyl moiety on an asn residue at position of csf (glc) is fundamental for auto-ab recognition resulting the fi rst multiple antigenic synthetic probe (msap) able to detect autoantibodies in ms patients' sera. up to now we have investigated the carbohydrates infl uence and specifi city in ms antibody recognition introducing several glycosilated building blocks in the msap sequence (i.e. glc, man, glc glc, gal, glcnac on the side chain of ser, thr, asp, glu, hypro) .. due to microheterogeneity and the extremely high specifi city of carbohydrateprotein interaction we included in our library screening, the ribose. we report the synthesis of new asn-derivatives bearing on the side chain ribose rib and rib linked by an n-glycosidic bond and protected for spps.these building blocks introduced in the csf -turn scaffold lead to the new ribosylated peptides contributing to the library of glycopeptides to fi shing out families of autoantibodies specifi c for different autoimmune diseases. fraczyk, justyna; kujawska, nina; kaminski, zbigniew, j. we designed and prepared supramolecular structures formed from nlipidated oligopeptides immobilized in the regular pattern on the cellulose surface which are able to specifi c binding of ligand molecule. due to the conformational fl exibility of the fragments forming the supramolecular structure, the shape and prosperities the binding cavities are adjusted the most effectively to requirements of the guest molecules. the previous studies documented that process of binding guest molecules is highly selective, reversible and competitive. therefore, we supposed that under favorable circumstances the structures could operate as catalysts if suitable molecular fragment are included inside the binding pocket. in order to verify this hypothesis we prepared library of supramolecular hosts with catalytic triade: his asp(glu) ser, incorporated into the binding pocket .. for the fi rst generation library the rate of hydrolysis of p-nitophenyl esters of n-protected amino acids was measured by spectrophotometric determination of liberated p-nitrophenole in buffered, aqueous methanol and compared with appropriate data obtained in the absence of catalytic structures. the most active catalyst were selected from the library and their stability, selectivity and ability for re-use was studied. for the second generation of library the stereoselectivity of artifi cial esterase we present here a new technique for identifying very small quantity of peptide mixtures that are selected out from a peptide library in solution, by using multi-component fl uorescence labeling. the technique is basically a modifi cation of positional screening method associated with a : correspondence between the amino acid at the i-th position and the type of the fl uorescence lebel at the n-terminal. -dimensional fl uorescence spectroscopy was employed for identifying the fl uorescence labels in the mixture of peptides that bound to target cells or target proteins. the results of the new screening method will be presented for peptides that specifi cally bind to human cancer cells. show increased biological activity in a dimeric form . in recent years there have been signifi cant efforts to obtain minimized versions of naturally occurring proteins such as dimeric dna binding proteins which retain their function. the gcn basic region peptides were connected trough a disulfi de bond to give a dimer which specifi cally bound the ap -dna sequence. dimeric peptides and proteins were obtained also by non covalent interactions. in this work we propose a strategy for obtaining by expressed protein ligation (epl), one pot protein homodimers covalently connected at the c-terminus. the synthetic strategy was extended also to the synthesis of heterodimers. epl is a protein engineering tool for the chemo and region-selective modifi cation of proteins based on the use of intein containing constructs. in this work dimers were obtained by reacting a new bi-functional linker with carboxyl-activated polypeptides. we synthesized a linker containing two cysteines in a n-terminal-like position, separated by an ethylendiamine spacer, and obtained thioester proteins by intein mediated splicing reactions. this strategy affords chemically stable dimeric proteins. the linker can be easily modifi ed at need, changing the lenght and rigidity of the spacer between the cysteines. this strategy has potential in biochemical and bioorganic applications, for obtaining minimized and/ or modifi ed natural proteins and for joining two different proteins at the c-terminus position. this technique will be extended to the synthesis of dimeric proteins mimicking the transcription factor ap- . previous studies showed that csf (glc), , a designed glycopeptide characterized by a -d-glucopyranosyl moiety, can detect and isolate specifi c autoabs in sera of a signifi cant number of ms patients. this synthetic ag could be considered a mimetic of aberrantly glycosylated myelin proteins triggering autoimmunity in ms. myelin oligodendrocyte glycoprotein (mog) is considered a putative autoag in ms. our aim is to obtain mog properly glycosylated to characterize the molecular mechanisms of ab-mediated ms and to design new antigenic probes to detect autoabs as biomarkers. production of specifi c glycoproteins may benefi t from a chemical approach, such as expressed protein ligation (epl) a protein engineering strategy useful to introduce noncanonical amino acids and biological probes into proteins. epl allows synthetic and recombinant polypeptides to be chemoselectively and regioselectively joined together. the recombinant rmog ed ( - ), obtained as c-terminal thioesther by protein splicing, will be ligated to the peptide fragment [gly ,a sn (glc)]mog ed ( - ) bearing a cys residue at the n-terminus. an alternative strategy exploits the selective reaction between a glucosyl iodoacetamide derivative and the cys free thiol of a protein. a site directed mutation has been performed on rmog to introduce a cys residue at its native site of glycosylation. the semi-synthetic proteins will be tested by elisa using ms patients' sera. automation is an identifi ed goal in the peptides r&d at lonza. this approach would facilitate and accelerate peptide production. this is highly desired within peptides r&d, where the need exists for rapid synthesis of peptides to fulfi l iso and gmp projects requirements. automated peptide synthesisers are available on the market but many have limitations which make them inappropriate for lonza (e.g., low scale, coupling systems limitation, pre-activation procedure at low temperature). furthermore, there is no obvious standard instrument for scalable spps equipped with pat. ge healthcare provides scalable and complete solutions for automated solid-phase synthesis of oligonucleotides from small research amounts to full commercial production ( mol- mol) based on the Äkta, oligopilot and oligoprocess™ platforms. the unicorn™ software provides control and monitoring of the processes. ge healthcare synthesisers are designed around fl ow-through column reactors, giving faster kinetics and lower solvent and reagent peptide biotechnology and diagnostics consumption compared to batch synthesisers. based on experience with scale-up of oligonucleotides, ge healthcare and lonza are confi dent that the fl ow through-column technology can be scaled up for peptides to a cost effi cient process. this is a strong argument that the potential of the Äkta platform as a peptide synthesiser should be explored. ge healthcare was approached by lonza to develop a peptide synthesiser based on the Äkta platform instrument to compete with the state of the art in the peptide fi eld. the aim is to develop the fl uidics, programming and chemistry methods of the Äkta system to lonza's needs for routine production of . g to g of crude peptide, and later scale up to kg. this synthesiser, controlled by the unicorntm software, has the capability to perform synthesis using on-line mixing or pre-activation at different times and temperatures of amino acids, coupling reagents, solvents and additives. results from peptides will be presented. neuropeptides are produced from precursor proteins by selective cleavage at specifi c sites. classical biosynthetic cleavage occurs at basic residues due to the activity of a small number of well-known proteases. however, with the discovery and characterization of new neuropeptides, a new non-classical pathway has been described with cleavage occurring at tryptophane, leucine and other residues. neuropeptide-processing peptidases involved in this new non-classical pathway are completely unknown but essential for correct processing of certain neuropeptides. therefore, we are interested in identifying proteases involved in the non-classical pathway using activity-based proteomics. for this purpose, we fi rst designed and synthesized some peptides based on the sequence the mature neuropeptides described in the literature but with an active functional group able to bind the active site of the proteases of interest. bound peptides were labelled with biotin or rhodamine by click chemistry, and the brain and pituitary proteome was then characterized by in-gel analysis and multidimensional nlc-ms/ms. several proteases that may be involved in neuropeptide processing have been identifi ed in this experiments. further studies concerning cloning, protein expression and activity assays will confi rm their role in biological tissues. the incidence of autoimmune diseases continues to increase. although an enormous research effort has been directed in understanding this increment, etiology of human autoimmunity remains enigmatic. primary biliary cirrhosis (pbc) is a chronic cholestatic liver disease characterized by destruction of bile ducts and the presence in the serum of antimitochondrial antibodies (ama positive in - % of patients), directed against the e , which is one of the three component enzymes of the -oxo acid dehydrogenase multienzyme complex family chiefl y pyruvate dehydrogenase complex (pdc-e ). environmentally induced co-or post-translational modifi cations of autoantigens are hypothesized to break immune tolerance leading to self reactivity in pbc .. in this context it is possible to take advantage of a unique technology that allows the monitoring of peptide epitope modifi cations that will lead to the identifi cation of altered autoantigens that the human host will recognize as foreign, similarly to what recently reported in multiple sclerosis ( ) . our approach will lead to new diagnostic tools that can be used in earlier stage patients and possibly to monitor disease activity. herein we report the synthesis of lipoamide and glyco-peptides characterized by a -hairpin structure with the modifi cation on the tip of the -turn as peptidomimetic of native antigens involved in pbc. in order to identify the best synthetic antigens and to clarify the role of the -turn in exposing the modifi cation we compared the antibody recognition in pbc sera by elisa using the modifi ed peptides in comparison with the proposed autoepitope of pdc-e [pdh ( - ) ]. the synthetic antigens were able to detect by elisa autoantibodies in % of ama negative sera of pbc patients confi rming that ptm-peptides are useful diagnostic/prognostic tools. alzheimer fs disease is characterized by the abnormal accumulation of amyloid peptide (a ) into extracellular fi brillar deposits known as amyloid plaques. a can self-assemble into soluble oligomers, protofi brils, and amyloid fi brils, and all of these aggregated forms contain signifi cant -sheet structure. the core of a ( - residues) containing hydrophobic region is a key to promoting a aggregation. in the previous study, we constructed green fl uorescent protein (gfp) variants which have the core part of a sequence on the surface -sheets. it has been demonstrated that the gfp variants inhibit a aggregation .. in this study, we have utilized a small protein, insulin-like growth factor ‡u receptor domain (igf r-d ) as a scaffold, and a part of a sequence was incorporated into the -sheet surface of igf r-d . igf r-kk and igf r-ka were designed by substituting two a derived sequences for some amino acids in igf r-d as parallel and anti-parallel -sheet models, respectively, of the a aggregates. these insoluble proteins expressed by e coli. were solubilized by denaturing buffer, and the denatured proteins were refolded in conditions permitting formation of native proteins. after refolding, the proteins were purifi ed as monomers by size exclusion chromatography. we have investigated the interaction between a and the designed protein variants by surface plasmon resonance studies. as a result, igf r-kk bound tightly to a more than igf r-ka. inhibitory activities of a fi brillization in the presence of igf r-kk, igf r-ka, or wild type igf r-d were evaluated by thiofl avin t (tht) binding assay. it was demonstrated that igf r-kk inhibited a fi brillization effectively. because of their high structural fl exibility. on the other hand, a stranded -structure is presumed to be more stable than a single strand as a fi bril forming intermediate. therefore, the aggregation mechanism of prion protein remains to be further studied on their precursor structures. recently, we have established a novel strategy to determine the amino acid sequence for amyloid formation, based on their essential interactions. a number of fi bril forming peptides at positions from to in the amino acid sequence were obtained by our calculation method. in order to confi rm whether aggregates of the candidate peptides consist of the -stranded -structure, a series of peptides consisting of g residues-turn- residues h were prepared. several candidate peptides, of which regions are - , - , - , and - , showed the typical enhanced fl uorescence intensities in the thiofl avin t-binding assay, suggesting the amyloid formation. ir spectra of these peptides showed the typical bands corresponding to the -structure. in addition, the peptide ( - ) exhibited a shoulder band at cm - in ir spectrum, refl ecting a turn structure. these results revealed that the amyloids of the peptide ( - ) are constructed with the -stranded -structure, which may be formed by intra-molecular hydrogen bonds. in conclusion, the results obtained here indicate that the sequence from to could be a key region for the transition from a normal to an abnormal structural state. single-molecule force spectroscopy provides a powerful tool to investigate biomolecular interactions. a different approach measures forces required for breaking a bond in a differential format by comparison with a known reference bond of dsdna ( ). here we apply this molecular force balance to the integrin v , which is over expressed in ovmz- cells. this protein interacts with ligands containing an -arg-gly-asp-(rgd) motive. ligands containing the rgd sequence were made by peptide synthesis and linked to a gcn -peptide using a peg-spacer. for detection carboxyfl uoresceine was introduced to a lysine side chain between both peptides. the gcn -peptide can be recognized by specifi c anti-body fragments ( ) and can act as a reference bond. these force balances was linked to a pdms-surface, whereas the reference bond is attached to the surface and the rgd-peptide is accessible to the integrin. when the pdms-stamp gets in contact with the ovmz-cells, the interaction of ligand and receptor can occur. by applying a force at the stamp the force balance is stretched and the weaker bond brakes. investigation of the fl uorescence-level on stamp and cells enables the localization of the balance construct and thus an estimation of the bond force. first stamp-experiments on living cells showed that the gcn anti body interactions are too strong to act as a reference system. since the binding forces of dna are well investigated by afm and small dna-molecules were already used as force sensor, dna can act as a more sensitive reference. thus in a further approach the rgd-peptide was linked to a biotinylated peg. this enables the application of short double-stranded dna as a reference bond via a fl uorescence-labelled streptavidin. using this system the force balance could be transferred to the v integrines located in the cell membrane of ovmz-cells. nanogaps, which allow making electrical contact to structures on the nanoscale, are increasingly used for the preparation of biosensors. the positioning of the synthetic or biological species inside the nanogap must be controlled to reach optimal electrical or detection properties. ( ) ( ) ( ) in this context, the chemical properties of the layer between the electrodes is of prime importance, since they will impose the imbibition of the nanogap and permit the formation of chemical bonds between the molecules of interest and the substrate. thin fi lms can be characterized by a variety of physical or chemical methods. however, the characterization of the chemical properties of nanogaps is complicated because of the small size of the substrate delimited by the electrodes. in this context, novel experimental tools are needed for probing rapidly the chemical reactivity of nanogaps. we show here for the fi rst time that a specifi c functional group in a - nm nanogap can be detected by combining peptidecapped gold nanoparticles and electrical detection.( ) a semicarbazide layer and semicarbazone chemoselective ligation was used in this proofof-concept study, which thus required the preparation of stable peptidecapped gold nanoparticles modifi ed by aldehyde groups and control gnps derivatized by amide groups. the chemoselective insertion of gold colloids into the nanogaps led to current increases from to orders of magnitude, in accord with the number of gold nanoparticles in the nanogaps detected by scanning electron microscopy. we present the electrical detection of immunoglobulin g (iggs) from human serum using a nanogap-based biosensor. the detection method is based on the capture of iggs by a probe immobilized between gold nanoelectrodes of to nm spacing. the captured iggs are further reacted with secondary antibodies labelled with gold nanoparticles (gnps). insertion of gnps into the nanogap resulted in increasing the conductance through the nanogap. the use of a chip with ninety nanogaps enabled the calculation of a quality factor for the detection which, coupled with a non-linear regression analysis of the data, easily discriminated specifi c and differential capture of human antibodies by arrayed probes. we obtained a -fold higher quality factor with protein a compared to goat anti-murine antibodies. this method can be applied, through these proof-of-concept experiments, to the detection of protein-protein interactions in biological samples. in the past several decades, hundreds of peptides have been identifi ed which have specifi c biological activity and are highly potent in in vitro assays but lack the prerequisite pharmacokinetics to become effi cacious human therapeutics. we have developed a novel antibody-based platform technology that provides an improved pharmacokinetic profi le for biologically active peptides, resulting in a long duration of action. one such mimetibody™, cnto , is a novel erythropoietin (epo) receptor agonist. although cnto bears no sequence homology to erythropoietin, it is a potent erythropoietin receptor agonist, rescuing epo dependent cells from apoptosis in vitro and stimulating erythropoiesis in vivo. studies were done in normal rats to explore the pharmacodynamics and pharmacokinetics of cnto in normal rats and to demonstrate its effi cacy in rat models of anemia. in vitro, cnto was approximately fold less potent than rhepo in stimulating the growth of ut- epo cells. despite this lower in vitro potency, when compared to rhepo and darbepoietin in normal rats, a single subcutaneous dose of cnto resulted in a longer-lived reticulocytosis and longer-lived increase in hemoglobin. also, cnto caused only minor changes in red cell distribution width (rdw) or mean cell volume (mcv) and led to the release of mature reticulocytes. we have also shown that cnto was effi cacious in rat models of anemia and in a rat model of pure red cell aplasia. taken together, our data show that cnto is a novel stimulant of erythropoiesis in rats. this platform has been applied to other biologically active peptides as well and has proven to be a robust platform for enhancing the pharmacokinetics of peptides that would otherwise be rapidly cleared. cell penetrating peptides (cpps) have been recognized as promising tools for the delivery of different therapeutic molecules. previous studies in our laboratory have shown that the s ( )-pv peptide accumulates inside cells very effi ciently through a rapid, dose-dependent and nontoxic process. formulations based on the s ( )-pv cell penetrating peptide presented great potential for the delivery of plasmid dna, which may prove useful for gene-based therapies. in the present work, we aim to ) investigate the relevance of the dermaseptin-derived sequence and of the nuclear localization signal to the effi ciency of the overall process of plasmid dna delivery by the s ( )-pv and related peptides; ) compare the potential of the s ( )-pv peptide to mediate plasmid dna delivery with that of the extensively studied tat cpp. a comparative analysis of the transfection effi ciency mediated by the systems based on the s ( )-pv, reverse nls and scrambled peptides was performed in tsa and hela cells. in general, for both cell lines, the reverse nls peptide mediated transfection at effi ciencies comparable to those observed for the s ( )-pv peptide. however, transfection mediated by the scrambled peptide was signifi cantly less effi cient than that obtained for the s ( )-pv and reverse nls peptides. to compare the biological activity of the s ( )-pv with that of the tat peptide, we transfected tsa cells with various s ( )-pv-and tatbased formulations. our results have shown that both peptides enhanced the activity of cationic liposome-based systems. above a threshold peptide/cationic liposome/pdna charge ratio ( / / ), the enhancing effect was independent of the peptide used, although for the lowest charge ratios, this effect seemed to be more relevant in the case of the s ( )-pv peptide. higashi, nobuyuki; kawamura, yoko; koga, tomoyuki doshisha university, japan the aim of tissue engineering is to replace failed organs with new functional tissue and organs. to realize this, materials are needed, which can direct the growth of cells to generate new tissue. to control and direct cell behavior, a defi ned biomimetic environments is needed, which surrounds the cells and promotes specifi c cell interactions. the rgds sequence has been recognized as the cell attachment site of the natural extracellular matrix. the purpose of this study is to fabricate rgdscarrying nanoscaffords towards cell adhesion using a self-assembling technique. here we synthsize two types of rgds-based materials, one of which is an amphiphilic triblock peptide composed of leu and lys (rgds-l k l ) that has been revealed to self-assemble into ƒÀ-sheet nanofi ber under specifi c conditions ., and another one is a rgdsended polystyrene (pst-rgds) that is a typical artifi cial polymer. these peptide and peptidomimetic were prepared by solid phase synthesis using fmoc-chemistry and by coupling fmoc-chemistry and atom-transfer radical polymerization (atrp) method, respectively. rgds-l k l was found to self-assemble into ƒÀ-sheet-based nanofi ber at ph . , and by lowering ph the conformational change from ƒÀ-sheet to random coil structure was induced, giving no aggregation of the peptide. when mouse nih/ t cells were seeded on the rgds-l k l nanofi bercoated plate, successful cell adhesion and spreading were observed, but not for the rgds-l k l random coil-coated plate, indicating the importance of the rgds sequences densely and regularly located at the nanofi ber surface. the utility of pst-rgds nanofi lms will be discussed, in comparison with that of nanofi bers. the role of deiminated protein antigens in the diagnosis of rheumatoid arthritis autoantibodies directed against citrulline-containing peptide (acpa) have high specifi city of rheumatoid arthritis (ra). citrullinated proteins are formed by posttranslational modifi cation, namely by deimination of arginine residues in protein sequences by peptidylarginine deiminase enzymes (padi). autoantibodies to deiminated (citrullinated) proteins are the most specifi c serological markers of rheumatoid arthritis. rheumatoid arthritis symptoms develop gradually, and it is diffi cult to precisely date the beginning of the disease. these antibodies are detectable already years before the fi rst clinical symptoms of the disease. the aim of our study was to identify the epitopes of vimentin and fi laggrin derived-peptides targeted by ra specifi c antibodies to provide further information about the nature of the initial autoantigenic substance. we used conventional solid-phase peptide synthesis (fmoc strategy) carried out on "multipin ncp" (chiron mimotopes peptide system) non-cleavable kit. identifi cation of epitope structure of antigenic proteins represents one of the major applications of these technologies. the peptides were prepared in duplicates. citrullinated peptides and the nonmodifi ed counterparts containing arginine instead of citrulline residues were synthesized in order to compare their respective reactivities. in the "indirect" elisa experiments the presence of acpa was determined using serum samples of ra patients and healthy blood donors. this series of experiments effi ciently identifi es citrullinated epitopes of fi laggrin and vimentin as a potential antigenic target for ra specifi c antibodies. the determination of epitopes of these proteins could be important for the development of appropriate diagnostics in this most frequent human systemic autoimmune disease. at the present time the human lysosomal enzymes cathepsins b and l, cysteine proteinases of c family, attract great attention. they not only take part in protein degradation but also appear to play role in other important physiological processes, for example, antigen presentation, caspase-independent cell death and involved in different diseases such as osteosarcoma, acute pancreatitis, tumor invasion and metastasis. but selective detection of these enzymes is diffi cult because determination of their enzymatic activity is carried out using substrates also correspond to specifi city of trypsin-like enzymes. the chemo-enzymatic synthesis of selective substrates of cysteine proteinases of c family was developed. these compounds are chromogenic glp-phe-ala-pna (i), glp-val-ala-pna (ii) and fl uorogenic substrates abz-phe-ala-pna (iii), glp-phe-ala-amc (iv). peptides were obtained in preparative quantities and were characterized by the data of amino acids analysis, hplc, mass-spectrometry, spectrophotometry (i and ii) and fl uorimetry (iii and iv). the specifi c activities of cathepsins b, l and similar enzymes of plants papain, bromelain and fi cain were determined using synthesized substrates and commercial available substrates z-phe-arg-pna, z-arg-arg-pna and bzl-arg-pna. the specifi c activities of enzymes of other classes -serine (chymotrypsin, trypsin and subtilisin), aspartic (pepsin) and metalloproteinases (thermolysin) -were also defi ned with these substrates. it was shown, that glp-phe-ala-pna, glp-val-ala-pna and glp-phe-ala-amc are not detectable cleaved by enzymes of other classes. acknowledgements: this work has been supported by rfbr grant ¹ - - à. to implement a new electrochemical biosensor for autoantibody detection in ms we used csf (glc) analogues, properly modifi ed at n-terminus with ferrocenyl and ferrocenyl-thiophosphine derivatives, as "electrochemical probes" in cyclic voltammetry ( ) . the electrochemical properties of ferrocene, coupled to thiophosphine ability to build simple monolayers on gold surfaces, allow peptides to be anchored on the working electrode used for detection. in particular, -fcphp(s)abu organometallic amino acid was specifi cally designed to be used directly in solid phase peptide synthesis. the organometallic moiety introduced in the new glycopeptides did not affect autoantibody recognition as demonstrated both in sp-elisa and in inhibition experiments. an electrochemical monitoring was able to detect interactions of the modifi ed glycopeptides with isolated antibodies from ms patients' sera. we demonstrated a detection sensitivity comparable to elisa method. therefore, the new electrochemical probes can be proposed to characterize autoantibodies as biomarkers of multiple sclerosis by a simple, rapid, and reproducible cyclic voltammetry-based diagnostic methodology. troponin is a structural protein complex, located on the thin fi lament of the contractile apparatus. it is composed of three protein subunits: troponin i ( kda), troponin c ( kda), and troponin t ( kda) and exists as isoforms specifi c to the cardiac and skeletal muscle cells, respectively. cardiac troponins are released in the peripheral blood during irreversible cardiac muscle damage in a time-specifi c manner. rapid troponin elisa assays based on the production of specifi c antibodies against the whole complex or individual subunits have been shown to possess suffi cient sensitivity and specifi city for use in the emergency departments. however, their usefulness sometimes is limited by various factors, such as the selection of epitopes for antibodies production, derived from cardiac troponins representing high homology against the skeletal isoforms, interfering blood factors etc. aiming to contribute in the fi eld of developing highly sensitive and specifi c reagents for the detection and isolation of cardiac troponins in the sera of patients with cardiovascular diseases, we selected epitopes derived from the cardiac isoforms for production of antibodies, mainly based on their predicted immunogenicity, the minimum homology against the skeletal isoforms and the lack of interferences of the produced antibodies with various blood factors. the selected sequences were conjugated to the tetrameric sequential oligopeptide carrier (soc ), either by the classic solid phase step-by-step methodology or by chemoselective ligation reactions and the resulted conjugates were used as immunogens for releasing anti-troponin specifi c antibodies. the performed elisa experiments revealed the high affi nity and specifi city of the produced anti-troponin antibodies against the native protein. a chemical reverse approach for autoimmune diseases diagnosis alcaro an increasing number of individuals throughout the world is affected by autoimmune diseases, a large and diverse group of disorders that are categorized by tissue injury or pathology. although the incidence and prevalence of individual autoimmune diseases are not high, the population burdens of the disease are large and underestimated. thus, reliable diagnostic/prognostic tools are necessary for an early diagnosis and for monitoring disease activity. in this scenario, we proposed a 'chemical reverse approach' based on the use of patients' sera to screen synthetic modifi ed peptides. we showed that this approach could lead to the effective identifi cation of specifi c probes able to characterize highly specifi c autoantibodies as disease biomarkers of highly relevant autoimmune diseases such as multiple sclerosis and rheumatoid arthritis [ , ] . toscana biomarkers is a r&d company involved in the application of the "chemical reverse approach" to different autoimmune conditions for the development of innovative diagnostic/prognostic tests. as a number of autoimmune diseases have been associated with posttranslational modifi cations, which alter the function and immunogenicity of protein/peptide antigens, we are synthesizing and screening focused peptide libraries based on post-translational modifi ed amino acid, conformational and minimal epitope diversity. lead compounds can be thus used as antigenic probes for specifi c recognition of autoantibodies as biomarkers of diseases in the set up of diagnostic/prognostic assays ( ). autoimmune diseases are considered now as a plague. in fact some autoimmune diseases previously considered rare are actually increasing their frequency because of an earlier diagnosis (e.g. celiac disease). possibly also an environmental factor (bacterial and/or viral infection) should contribute to autoimmune diseases. the idea we have been investigating for years and more recently proposed by others, is that aberrant post-translational modifi cations, i.e. glycosylation, deimination etc., create neoantigens triggering autoantibodies. this could explain why proteins (both recombinant and isolated), components of target organs or tissues, are failing. anyway, it is evident that one single biomarker will never enable to reach successful diagnostic & prognostic tools. on the contrary, synthetic peptides specifi cally modifi ed (with sugars, citrulline, lipoyl moieties, etc.) are interesting tools to fi shing out of patients' sera these autoantibodies. we have recently reported that this can be effi ciently done following a "chemical reverse approach" .. we successfully applied this strategy in the development of the fi rst multiple sclerosis antigenic probe [msap] : an n-glucosylated peptide characterised by a -hairpin structure exposing at the best the minimal epitope asn( -glc) involved in antibody recognition ( ) . a wider application of our sap is obtained by its citrullinatation and/or galactosylation ( ) useful for rheumatoid arthritis or lipoylation for investigating primary biliary cirrhosis. in addition to being able to show the viral infection from a serum sample within a few minutes, at the point of care, the poc assays are inexpensive, easy-to-perform and do not require special equipment or laboratory. as antigen in the poc assay we used a -amino acid peptide in four branches of a lysine core, with the kyvtgin sequence in the middle. the peptide antigen was conjugated to gold particles and absorbed to a fabric ribbon and dried. in the test, a serum sample is added together with buffer, and the solution dissolves the conjugate. a positive result is obtained when the antibodies together with the gold conjugate bind to anti-human-igg on the nitrocellulose, and form a specifi c coloured line which can be detected in the test window. serum samples were collected from patients with acute b infection, and control samples were drawn many years after infection; additional control sera came from subjects devoid of b antibodies. the assay was shown to be stable in accelerated stability study. the conditions for industrial scale manufacturing were evaluated and the sensitivity and specifi city were addressed, whereby the assay proved to be highly specifi c for acute b infection. alzheimer's disease (ad) is a chronic neurodegenerative disease characterized by a progressive loss of memory and cognitive decline, for which the aggregation and plaque-formation by the -amyoild (a ) polypeptide has been identifi ed as a key event. recently, unpaired variable domains of llama single chain antibody (vhh) fragments against a have been found to exert considerable therapeutic potential for ad. vhh represents the smallest antigen-binding unit with a molecular size of ~ kda, compared to ig-heavy and light chain variable domains, fab fragments and complete igg antibodies. human cystatin c (hcc) is a cystein protease inhibitor present in all human body fl uids which has a propensity to co-associate with a -plaques/fi brils, and has plaque-inhibitory properties. using proteolytic extraction and excision of the llama-vhh-a ( - ) immune complex (e.g., trypsin, glu-c protease) in combination with electrospray ionization (esi)-and maldi-mass spectrometry, the a -binding epitope was identifi ed at the middle-carboxyterminal domain of a , a ( - ). an analogous mass spectrometric approach was employed for the identifi cation of the binding epitopes of the hcc-a -complex, using immobilized hcc and a -affi nity matrices. an almost identical minimal epitope to that of the vhh-anti-a -antibody ( a ( - ) ) was found, which binds to a specifi c c-terminal domain of hcc, hcc( - ) .. the identifi ed hcc epitope peptide was found to specifi cally inhibit a -oligomerization in vitro, in agreement with the a -epitope domain interefering with the a -aggregation. the identifi ed a and hcc epitopes represent new lead structures for designing neuroprotective inhibitors of the a -aggregation process, and for molecular ad diagnostics. using peptide arrays to reveal mechanisms of apoptosis aspp is a pro-apoptotic protein that stimulates the p -mediated apoptotic response. the c-terminus of aspp contains ankyrin repeats and an sh domain (aspp ank-sh ), which mediate its interactions with apoptosis-related proteins such as p and bcl- . we have used a combination of membrane-bound peptide arrays and biophysical methods to study the protein-protein interactions of aspp at the molecular level in order to reveal their possible role in apoptosis. using peptide arrays, we have mapped the binding interfaces of aspp with its partner proteins such as bcl- , nf-b and other proteins that are involved in apoptosis. we then applied the peptide array results to study profoundly the interactions of aspp with proteins from the anti-apoptotic bcl- family. we found that aspp ank-sh binds to bcl- , bcl-xl and bcl-w at two homologous sites in all three bcl proteins tested: (i) the conserved bh motif (ii) a binding site for pro-apoptotic regulators. quantitative biophysical analysis of the interaction with the free peptides revealed that the binding was selective, and the bh domain of bcl- binds tightest to aspp . we propose a mechanism in which aspp induces apoptosis by inhibiting functional sites of the anti apoptotic bcl- proteins. the array screening results also served as a basis for docking studies that resulted in binding model for the complex between the full length protein bcl- and aspp ank-sh . we conclude that the use of combinatorial methods such as peptide arrays, combined with quantitative biophysical techniques, can signifi cantly contribute to better understanding of biological pathways. micron-sized monodispersed polystyrene for synthesis, tagging and biological screening of small molecules peptide arrays are useful tools to characterize antibodies, to determine sequence specifi cities of enzymes (e.g. kinases) or to fi nd interaction partners to given peptide sequences. one popular format for such arrays is a cellulose sheet with hundreds of synthetic peptides bound to it. these spot-arrays have been used successfully in a broad range of applications since their invention years ago .. a drawback is the use of large reagent volumes and the limited throughput with only one copy of the library. celluspots™ represent a new method [ , ] that allows the production of hundreds identical peptide arrays from a single synthesis run on individual membrane disks. the peptides are synthesized on a modifi ed cellulose support which is dissolved in a cleavage-mixture after the synthesis. resulting solutions of peptide-cellulose-conjugates are then spotted onto coated microscope slides by conventional spotting techniques. the identical arrays are useful tools for large, parallel sera screening projects. there is a great importance in biological macromolecules integration within electronic devices. this is since these molecules are expected to be suitable both as the active components of electronic devices, or as guides for bottom-up assembly of hybrid structures. the goal of this research is to study the assembly and electronic properties of novel devices that exploit synthetic protein molecules. here we show the fabrication of de-novo protein based diode-like devices. we have chosen coiled coil protein structures, which can adopt two conformational states that differ in their internal molecular dipole. the proteins have been equipped with surface binding groups, typically cysteine thiols, on both ends. dithiol bridges at one side facilitated self assembly on gold surfaces. protected cys residues were placed on the other end that after deprotection will allow successive binding processes in order to complete the device assembly process. characterization of the correct folding in solution has been achieved by hplc and cd. the dependence of the dipole in the dimeric protein has been shown using large scale kelvin probe and high resolution kelvin force microscopy (kfm) measurements. we believe that this work will contribute to the understanding of electronic processes in proteins and may serve as foundation for their exploitation in device confi gurations by making use of the ability to trigger conformational changes in order to control device activity. synthesis of new tetracyclic rgd peptides for specifi c tumor cell targeting. the integrin family of adhesion molecules participate in important cell-cell and cell-extracellular matrix interactions in a diverse range of biological processes. the avb integrin is overexpressed in several types of cancer cells and play an important role in angiogenesis as well as in tumour cell migration by interacting with vitronectin on the extracellular matrix mainly through the recognition of the tripeptide sequence rgd. the search for highly selective ligands to target the endothelial cell integrin avb is currently the focus of many research groups as they may represent new therapeutics or valuable diagnosis in a number of areas such as metastasis, angiogenesis, arthritis and retinopathy. to date, the cyclic (-rgdfx) peptides and related n-methylated analogues developed by kessler's group are among the most active and selective compounds for the avb integrin receptors. for instance, the use of c(-rgdf(nme)v-) is actually evaluated in several clinical trials. therefore, we designed a new class of cyclic tetrapeptides. rgdk peptides were fi rst synthesized by solid phase synthesis then cyclized in solution through a urea bond using n,n'-carbonyldiimidazole. using skmel and a cells, expressing and non-expressing avb respectively, we demonstrate that one of our peptide showed a better internalization than the reference peptide crgdfe. our strategy allowed the coupling of the best cyclic recognition peptide through its extracyclic acidic group to any molecular moieties containing an amino group that makes him a great cadidate for easy multimerization. along this line, different applications are currently developped in our group. design in this study, we described a simple method for aminocylation of unprotected saccharides with mildly activated amino acids esters as potential ace inhibitory activity. as a model reaction, we investigated esterifi cation of sucrose -" royal carbohydrate" with cyanomethyl ester of benzyloxycarbonyl-phenylalanine.the difference of reactivity between the eight hydroxyl groups is a key factor in chemical exploration. in polar solvent as dmf or dmso the primary positions might react faster than secondary ones if the reaction is essentially sensitive to steric interactions. on the other hand, the reaction might be more sensitive to the activity of the alcohol functions. in this respects, g-oh has been shown to be the most reactive among eight hydroxyl groups of sucrose. after removal of benzyloxycarbonyl group, the products possess groups which can accommolated in the hydrophobic s and s subsites of angiotensin i converting enzyme. the free amino group in the amino acid-carbohydrate esters can also serve as good ligands for zn + in the ace active site. carbohydrates possess both hydrophobic and hydrophilic groups in their structure and could also bind with enzyme subsites. the measurements of ic value of newly amino acyl esters of carbohydrates are in progress. synthetic polyelectrolytes (pe) have been widely used to modify proteins via complex formation and covalent attachment, increasing (or reducing) the immunoreactivity and/or immunogenecity of originally antigenic proteins and improving their in vivo stability with prolonged clearance times. such conjugates seem to be great importance for medicine and immunobiotechnology in particular with respect to drug delivery and vaccine innovation. synthetic peptides are promising candidate vaccines for the control of viral diseases. previous studies with foot-and-mouth disease virus (fmdv) have identifi ed fragments of isolated vp protein and synthetic peptides from vpi which stimulate antibody production, albeit of poor neutralizing activity, or are recognized by antivirus antibodies. fmdv is an attractive model with which to study the potential of peptide-based synthetic vaccines. in this study, we sought to evaluate the immunogenecity of a candidate containing - synthetic peptide epitops of vp capsid protein of fmdv. the immunogenic properties of the conjugates were also investigated and the relationship between immunogeneticity and structure formation in the solutions is analyzed. a new high immunogenic protein-polymer complex and conjugates with antibody production, processing relatively prolonged times was obtained. it was obtained that a single immunization of mice with pepeptide bioconjugates without classical adjuvant increased the primary and secondary peptide-specifi c immune response to fmdv. polyacrylic acid (paa) is a well known bioactive polymer (bioadhesive nano-and microparticles, ph-sensitive paa grafted poly(vinylidene fl uoride) membrane bags, ultrafi ne cellulose fi ber surfaces grafted with paa for enzyme immobilization, paa-polyvinylpyrrolidone biopolymeric systems for the treatment of the dry eye, etc. paa, their alkyl-esters and non-toxic copolymers with vinyl pyrrolidones are strong adjuvants for primary and secondary responses and that they are promising alternatives to the mineral oil-based adjuvants presently used in various veterinary vaccines. in this study, the interaction between peptide epitops of vp protein of foot-and mouth disease (fmdv) and polyacrylic acid (paa) will be discussed on the basis of the experimental results obtained by the size-exclusion chromatography (sec) with online quadruple detection system: uv absorption (uv), refractive index (ri), right angle light scattering (ls) and viscosity (vis) detectors and the binding coeffi cient, i.e., the binding ratio of peptide to polyacrylic acid. human igg-specifi c binding peptides to distinguish normal and abnormal conformers: applications for igg purifi cation and detection in recent years, human immunoglobulin g (igg) attracts attention as protein of the main format of the antibody medicine. in the purifi cation of igg, protein a originated from bacteria is frequently used as a affi nity ligand, but several problems such as the contamination of endotoxin and the deterioration of protein a by the repeated use were pointed-out in the use of protein a. on this account, the development of low molecular ligands and mimic peptides which can be used instead of protein a has been performed. we have searched for the peptides which specifi cally bind to human igg using a t random peptide phage library and, as a result, succeeded in isolation of two kinds of peptides called type i and type ii. type i peptide recognize normal structure of human igg and can be used for the purifi cation and the detection of igg. however, it not only binds to human igg but also to mouse and rabbit igg with comparatively high affi nity. on the other hand, type ii peptide is extremely specifi c only for human igg. a more important thing is that type ii peptide does not bind to igg in serum, but get possible to bind to igg antibody purifi ed by a protein a column. from this result, we elucidated the abnormal structure of igg is generated by acid condition used in the elution from a protein a column and type ii peptide recognizes this specifi c structure. in this presentation, we report the results that the type i peptides functioning as an affi nity ligand instead of protein a can be applied for the purifi cation system of the antibody by improvement by amino acid substitutions and chemical conversion. furthermore, we also report the generation condition of an abnormal conformer of igg structure by ph and the temperature and the effective removal of the generated specifi c conformer by the type ii peptideimmobilized column. acknowledgements: this research was partially supported by a grant of practical application research from japan science and technology agency. the isolation and detection of low abundant enzymes and proteins is one of the most challenging tasks in bioanalytical and pharmacological fi elds, especially if information about their state of activity is required. for this purpose tailored solutions for addressing the members of a protein family are required. peptide chemistry provides established methods to assemble building blocks to construct such molecular probes. we chose matrix metalloproteinases (mmp) for validation of such an approach. this protein family processes and degrades various extracellular matrix proteins and possesses a highly conserved catalytic site with a zinc ion in its centre. most of the known and potent inhibitors are peptidomimetics containing zinc chelating hydroxamate groups (e. g. marimastat). although it binds reversibly, it is potent and active against a wide range of metalloproteinases. it was chosen as synthetically available binding group to target the protein in its active state. it was modifi ed by peptide chemistry in order to introduce multiple functions. depending on the purpose, different reporter groups, photoreactive or cleavage sites were chosen. the probes were tested positively to inhibit various human recombinant mmps using activity assays. mmp were isolated using streptavidin coated magnetic beads and a biotinylated probe. furthermore a photoreactive group was introduced to enhance the binding to the target protein. a covalent interaction was achieved by irradiation of a probe protein mixture, isolation with magnetic beads and cleavage from the beads. pyclock superior coupling reagent for biosensors construction based on peptides coupling onto solid phase polymer atias, danit; abu-rabeah, khalil; marks, robert s. electrochemically biosensors are valuable analytical tools for the monitoring of various biological/ chemical molecule levels, and tremendous research effort has been put into the development of such accurate analytical devices. one crucial aspect in the fabrication of a biosensor is the deposition of biological macromolecules in high amounts with retention of their specifi c activity. among the various deposition methods of biomolecules, such as direct adsorption onto the surface, cross-linking, covalent attachment by carbodiimide chemistry is one of the few methods that allows a stable deposition into a biocompatible environment. hence the urgent need for proteins coupling with high yield to solid surface in the aim of biosensors construction. however the use of carbodiimide as peptide coupling agents was found to be limited in various aspects. thus, during the activation of hindered carboxylic components, such as those involved in bulky protein coupling reactions to solid phase fi bers pyclock was found to be very effi cient for slow coupling reactions and it can be used in excess to assure a complete activation of the carboxylic function. in this study -chloro-benzotriazole- -yl-oxy-tris-pyrrolidino-phosphonium hexafl uorophosphate coupling reagent (pyclock) was shown to have better performance than -ethyl-[ -(dimethyl¬amino)propyl]- ethylcarbodiimide (edac) and hydroxysulfosuccinimide (nhss) in peptides large sequence coupling to solid phase fi bers. cantel, sonia; valmalle, charlene; subra, gilles; enjalbal, christine; martinez, jean general approaches used in protein identifi cation and characterization involve consecutive purifi cation steps. then the desired protein extract is submitted to mass spectrometry analysis (lc-ms/ms) after enzymatic digestion. technical diffi culties are involved in determining the pmf of a protein particularly in relative low abundance. a typical protein will give rise to at least twenty to thirty peptides after trypsin digestion. not all of these peptides will appear in maldi analysis. one factor that is believed to cause incomplete detection is competition for protonation during the ionisation process inducing ion discrimination. we have recently developed a new technology allowing specifi c labeling of lysine residues in proteins and easy maldi-ms detection and identifi cation of labeled peptides following protein hydrolysis ( ) . nhydroxysuccinimide ester of -cyano- -hydroxycinnamic acid (chca) was used as a labeling reagent to increase maldi signal of lysinecontaining peptides in cytochrome c proteolytic mixture. this original approach enables to discriminate labeled peptides of interest among other abundant peptides. herein, we report the optimization process to investigate the limits of this tool. a defi ned receptor-binding domain (rbd) on the viral spike protein (s) mediates the attachment of sars-coronavirus to its cellular receptor, angiotensin-converting enzyme (ace ). we have synthesized peptide libraries for identifi cation of rbd binding epitopes by surface plasmon resonance (spr) and saturation transfer difference (std) nmr spectroscopy. three dodecapeptides were identifi ed to have signifi cant affi nity to ace ; the best of them had a kd = m . further refi nement yielded a hexapeptide from y to l (ykyryl) of s that bound ace at kd= m. std nmr spectroscopy reveals close contacts of the aromatic tyrosine residues to the receptor. the peptide was also analyzed for antiviral activity using an in vitro assay that measures sars-cov infection of vero cells. at a concentration of mm virus replication was reduced about fold. no replication occurred at peptide concentrations above mm. the peptide blocks the binding site on ace that is necessary for the virus to infect the cells. it can be used to design peptidomimetic compounds as entry inhibitors for sars-cov. to exclude other effects that were due to unspecifi c inhibition of viral replication, the peptide was tested during an alpha virus infection of vero cells. no signifi cant inhibition was observed. however, for the human corona virus nl that causes severe colds in humans and that uses the same receptor a clear inhibition could be observed comparable to the results obtained for sars-cov. additionally, we have synthesized a hexapeptide library with amino acid substitutions of the chemical lead ykyryl and measured their binding affi nity to ace via spr to analyze the importance of the individual amino acids. spr studies indicate an important role of r . prostate cancer is one of the most common malignancies in men and is responsible for more deaths than any other cancer, except for lung cancer. in the last decade we have developed bradykinin antagonist peptides (b , b ), peptide dimer (b , suim-(d-arg-arg-pro-hyp-gly-igl-ser-d-igl-oic-arg) , suim: suberimidyl; hyp: trans- -hydroxyproline; igl: -( -indanyl)glycine; oic: s, as, asoctahydro- h-indole- -carboxylic acid) and four generations of our small molecule, bkm- which showed high inhibition against lung cancer in vitro and in vivo. some of the compounds also showed inhibition against prostate cancer. it is well known that prostate cancer metastasizes into bones. while prostate cancer itself has many treatment options, no drugs currently exist for the treatment of bone metastatic prostate cancer. we modifi ed our potent anti-cancer small molecules by incorporating an aminobisphosphonate group to target these compounds to bone. bkm- , f c-oc y-atmp (f c: , , , , pentafl uorocinnamoyl; oc y: (o- , -dichlorobenzyl)-tyrosine; atmp: -amino- , , , -tetramethylpiperidine) is the fi rst generation of our small molecules. this compound consists of three parts: a, an acyl group; b, a tyrosine amino acid residue and c, an amide group. we introduced aminobisphosphonate or aminobisphosphonate derivatives at position c, and the new n-(bis-phosphonatoalkyl)amide small molecules had anti-cancer activity against prostate cancer metastases in mice. one of these compounds has been shown to be effective against the growth of pre-established human prostate tumors in mouse skeleton through the induction of apoptosis and blockade of survivin expression. the synthesis of these aminobisphosphonate small molecules, the structure relationship studies and the biological activity of these compounds in cultured human prostate cancer cells and animal models will be presented. hayouka novel, potent and selective angiotensin iv short analogues the hexapeptide angiotensin iv: h-val-tyr-ile-his-pro-phe-oh (ang iv) mediates a wide range of physiological actions, including control of blood fl ow and cognitive enhancement. it exerts its effects by binding to at receptors, which are widely distributed across tissues. the at receptor has been identifi ed as the insulin-regulated aminopeptidase or irap. it has been proposed that ang iv exerts its action by inhibiting the catalytic activity of this enzyme. we have reported that the -homo amino acid containing analog h- hval-tyr-ile-his-pro- phe-oh (al- ) is a potent, selective and stable ang iv antagonist, in which the hval is responsible for stability and the hphe for selectivity. in this study we report the new ang iv short analogues. the incorporation of erythro-mephe or tic resulted in analogues which have great ability to inhibit the hydrolysis of leu-p-nitroanilide by irap or by ap-n. our data showed that the full ang iv sequence is not necessary for high potency. peptides with pro deletion containing tic or erythro-mephe were even more potent than full ang iv sequence. moreover a peptide design and synthesis of a non -peptide par thrombin receptor antagonist, using cyclohexane as template receptors that mediate thrombin action are attractive drug discovery targets because of their involvement in cardiovascular pathophysiology (dysregulation of platelet aggregation and endothelial cell function). the cellular actions of thrombin are, in large part, caused by the activation of proteinase-activated receptors (pars) , and (pharm. rev. : ). the serine proteinase thrombin cleaves and activates cellular par in many pathophysiological settings associated with hemostasis, tissue injury and the proliferation of vascular smooth muscle and tumor cells. in the present study, we synthesized a novel non-peptide par mimetic, based on a conformational analysis of the s fllr tethered ligand sequence of par in order to inhibit the cellular actions of thrombin. the rational design, based on nmr constraints and molecular dymamics, led to compound (fig. ) containing the spatially closed key pharmacophoric guanidyl and phenyl groups, attached to cyclohexane as a template. compound , inhibited both tfllr-amide ( m) and thrombin ( . and u/ml)-mediated calcium signaling in a cultured human hek cell assay (j pharm. exp ther. : ). aboye, teshome leta ; clark, richard j ; craik, david j. ; göransson, ulf biomedical centre, sweden; institute for molecular bioscience, australia the cyclic cystine knot motif, as defi ned by the cyclotide peptide family, is an attractive scaffold for protein engineering ( ). however, to date the utilization of this scaffold has been limited by the inability to synthesize members of the most diverse and biologically active subfamily, the bracelet cyclotides. here we describe the synthesis and fi rst direct oxidative folding of a bracelet cyclotide, cycloviolacin o , and thus provide an effi cient method of exploring the most potent cyclic cystine knot peptides ( ) . the linear chain of cycloviolacin o was assembled using fmoc solid phase peptide synthesis and cyclized by thioester-mediated native chemical ligation, and the inherent diffi culties of folding bracelet cyclotides were successfully overcome in a single step reaction. the folding pathway was characterized and included predominating fully oxidized intermediates that slowly converted to the native peptide structure. angiogenesis, the process of new blood vessel formation, is important in both physiologic and pathologic situations, and its inhibition can be useful in the fi ght against several diseases, such as tumor development, diabetic retinopathy, etc. one of the key factors in promoting angiogenesis is the vascular endothelial growth factor (vegf), which exerts its biological activity through interaction with specifi c receptors, vegfr- (flt- ), vegfr- (kdr, flk- ) and vegfr- (flt- ) . vegf is over-expressed in all examples of pathologic angiogenesis, and its effects on tumor growth are mainly mediated by kdr. our approach to novel anti-angiogenic drugs is centered on the search for inhibitors of vegf-kdr interaction. directed mutagenesis studies allowed the identifi cation of a surface of vegf recognized by kdr, with residues arg , ile , lys , his and glu , located in a -hairpin of loop , identifi ed as essential for the interaction. most residues in this region are also located in the interface of interaction between vegf and some none-humanized phage-displayed antibodies with potent antiangiogenic activity, suggesting a binding to vegf similar to that of vegf receptors. starting from vegf - fragment (mrikphqgqhi), we have designed different hydrocarbon-bridged analogues to preserve the -hairpin native structure. this communication will describe the solid-phase synthesis of olefi n-bridged (c=c) peptides and their saturated (c-c) analogues. considering that the -turn of native vegf - is slightly distorted, due to the presence of an extra amino acid residue, in addition to the bridged undecapeptides, two series of decapeptide analogues have also been prepared, just by removing the residues glu or gly . the conformational behavior of linear and bridged-peptides has been analyzed by nmr (noe, h and c chemical shifts). the ability of these peptides to adopt the native vegf -hairpin structure will be compared with their anti-angiogenic activities. integrins constitute a family of heterodimeric, transmembrane cell adhesion receptors which connect cells to the scaffolding proteins of the extracellular matrix. the pioneering observation that integrins -especially v and -are hallmarks of metastatic cancer and seriously involved in the process of tumor angiogenesis turned them into attractive targets for cancer therapy. out of that, the inhibition of integrin function is a major challenge in medicinal chemistry. potent ligands are currently in different stages of clinical trials for the antiangiogenic therapy of cancer and age-related macula degeneration (amd). especially the subtype has recently been drawn into the focus of research due to its genuine role in angiogenesis.( ) in here, we describe the rational design and the synthesis of high affi nity binders and the optimization of their activity and selectivity against v by means of extensive sar-studies and docking experiments.( ) starting from a tyrosine scaffold( ) we succeeded in getting compounds with affi nities in the low and even sub-nanomolar range and selectivities of fold against v . the insights about the structure-activity-relationship gained from the tyrosine based ligands could then be successfully transferred to ligands bearing an aza-glycine( ) scaffold to yield ligands with affi nities of even sub-nanomolar range and selektivites exceeding . fold. modeling studies and biological activities of a nonpeptide at receptor angiotensin ii antagonist the octapeptide angiotensin ii (h-asp-arg-val-tyr-ile-his-pro-phe-oh) is the major factor of the renin-angiotensin system (ras) and plays a signifi cant role in the regulation of arterial blood pressure. in the present study, we have modeled a losartan analogue, the non-peptide angiotensin ii at antagonist, -butyl- -hydroxymethyl- -{[ '-( htetrazol- -yl)biphenyl- -yl]methyl} imidazole (v ). structure activity relationship (sar) and molecular modeling studies indicate close proximity of hydroxymethyl and tetrazole pharmacophoric groups of antagonist v and losartan. conformational analysis was performed using a grid scan search in order to derive all the possible conformations from which six energy local minima (syn and anti) were extracted after a cluster analysis. furthermore, these different conformations were superimposed with losartan, where a spatial correlation among the pharmacophoric groups is observed. antagonist v showed similar potency with losartan in our in vivo model with anesthetized rabbits and in vitro binding studies to at receptor. at specifi c concentrations compound v showed higher affi nity compared to losartan indicating that reorientation of butyl and hydroxymethyl groups on imidazole template allows a better binding to at receptor. h-benzotriazolium -[bis(dimethylamino)methylene]- -chloro-,hexafl uorophosphate ( -), -oxide (hctu) is a non-toxic, non-irritating and non-corrosive coupling reagent [ ] [ ] . seven biologically active peptides (ghrp- , - acp, oxytocin, g-lhrh, c-peptide, hamylin - , and -amyloid - ) were synthesized with reaction times reduced to deprotection times of minutes or less and coupling times of minutes or less using hctu as the coupling reagent. no expensive coupling reagents or special techniques were used. total peptide synthesis times were dramatically reduced as much as . hr ( . days) without reducing the crude peptide purities. it was shown that hctu can be used as an affordable, effi cient coupling reagent for fast fmoc solidphase peptide synthesis. human sdf- contains sixty-eight amino acids and is a member of the chemokine family of peptides. this long peptide was synthesized step-wise using our quality control conditions in hours. the reaction times were then reduced to deprotection times of x min and coupling times of x . min, resulting in a total synthesis time of hours. the effect of different resins, resin substitutions and deprotection reagents on the crude peptide purities were compared. a small portion of crude peptide was purifi ed using an rp-hplc column and the mass of the fi nal product was confi rmed with maldi-tof mass spectrometry. references: . steven l. kunkel and nuria godessart, chemokines in autoimmunity: from pathology to therapeutics, autoimmunity reviews, , - ( ). pedersen, søren l.; sørensen, kasper k.; jensen, knud j. despite the development of new coupling reagents and solid supports, spps is still often faced with diffi culties in the assembly of long or "diffi cult" sequences, e.g. due to aggregation and steric hindrance giving rise to incomplete reactions. the use of convenient and precise heating with microwaves for spps has gained in popularity as it for many syntheses has provided signifi cant improvement in terms of speed, purity, and yields, maybe especially in the synthesis of long and "diffi cult" peptides. thus, precise microwave heating has emerged as one new parameter for spps, in addition to coupling reagents, resins, solvents etc. we have previously reported on microwave heating to promote a range of solid-phase reactions in spps. here we present a new, fl exible semi-automated instrument for the application of precise microwave heating in solid-phase synthesis. it combines a slightly modifi ed biotage initiator microwave instrument, which is available in many laboratories, with a modifi ed semi-automated peptide synthesizer from multisyntech. a custom-made reaction vessel is placed permanently in the microwave oven, thus the reactor does not have to be moved between steps. mixing is achieved by nitrogen bubling. washing steps are automated, however the activated amino acid derivatives have to be added manually. first, we developed optimized protocols for short cycle times in semi-automated spps with general fmoc chemistry. we utilized a microwave-compatible temperature probe for exact temperature measurements during microwave heating. then we developed protocols for on-resin reductive amination for anchoring of the fi rst amino acids to a bal handle. finally, we used the new instrument and the optimized protocols to assist in the synthesis of a range of diffi cult and long sequences. we believe that these successful syntheses demonstrate that this semi-automated instrument and the methods developed for it, can be an effi cient starting point for spps with microwave heating. investigation of polyelectrolyte-antigenic peptide conjugates by fl uorescence spectroscopy in proteins or peptides, it is possible to localize the interaction between protein and pe at certain protein domains. we investigate covalent binding mechanism of synthetic peptides which include tryptophan residue in the peptide sequence with copolymers of acrylic acid and n-vinylpyrolidone (vp\aa) depending upon the weight concentration ratio of components by fl uorescence method. antigenic peptides are synthesized by microwave assisted solid phase peptide synthesis method. characterization of these peptides are performed with lc-ms. subsequently these crude peptides are purifi ed by preparative hplc system. peptide-polymer covalent conjugation are performed in organic and pbs media. covalent conjugation are carried out by carbodiimide method. carbodiimide is used for the activation of carbonile groups of synthetic polymer which is the binding area of peptides that includes free amino groups. from the analysis of peptide-pe conjugates, it is possible to discuss the mechanism of the conjugate formation and structure of forming particles. according to the fl uorescence analysis results, free peptide which containing trp residue, gives fl uorescence spectrum. however, after the conjugation reaction it is observed that products max decreased and it is characterized as blue shift of max. this indicates that when conjugates formed, trp is completely isolated from aqueous solution. antiangiogenic thrombospondin type repeat analogs: synthesis, structure, and biological activity the thrombospondin type repeat (tsr) has been shown to inhibit angiogenesis and tumor growth in a number of in vivo models of cancer, but the details of this activity, including structure-function relationships, are not well understood. we explore these tsr elements in further detail via structural and biological evaluation of a series of tsr analogs. the tsr domain has a characteristic fold consisting of a two-stranded -sheet and a third 'rippled' strand. three tryptophans on the 'rippled' strand form cation-stacking interactions with two arginines on the adjacent sheet, forming an extended cation-network. in addition its structural importance, it has been postulated that the surface formed by this interaction is a key site for protein-protein recognition. a synthetic approach to the generation of novel tsr analogs, utilizing spps and native chemical ligation, has allowed us to rationally introduce unnatural amino acids in an effort to probe the structural and functional landscape of this clinically relevant protein domain. we have synthesized analogs of the second tsr domain of human thrombospondin- (tsr ) designed to probe the importance of the cation-stack. the arginine residues have been replaced by ornithine and citrulline, and thermal denaturation experiments by circular dichroism have been used to estimate stability differences between the mutant tsr and the native. taken as a set, these thermodynamic measurements provide insight into the stability afforded by a cationinteraction in the context of a native protein fold. synthetic access to the tsr domain also provides the opportunity for the introduction of various modifi cations, including the introduction of aminooxy groups as chemical handles, pegylation for in vivo stability, and biotinylation to create an affi nity caputure reagent for identifi cation of protein-protein interaction partners. the tetradecapeptide bombesin (bbs) has a high affi nity for the gastrin-releasing peptide (grp) receptor. these receptors are overexpressed in human tumors such as breast and prostate cancers. therefore they can serve as targets for in vivo imaging and therapy of these tumors with m tc-and re-radiolabeled bbs analogues, respectively. for the radiolabeling, a chelator is attached to the n-terminus of the bbs analogues. our group developed the (n his)ac chelator, which is easy to synthesize and has a very high affi nity for the m tc-and retricarbonyl complexes. however, an important part of the injected radiolabeled bbs analogues accumulated in healthy organs, such as liver and kidneys. to improve the pharmacokinetic properties, a bbs analogue was glycated via the lys side chain of the spacer using the maillard reaction. unfortunately, during glycation, overalkylation on the secundary amine of the chelator was observed. therefore, two alternatives for this glycation were examined. the fi rst one was based on the chemoselective reaction between a hydroxylamine (incorporated into the spacer) and an aldehyde (glucose). the second alternative was the cu(i) catalyzed cycloaddition between an alkyne (incorporated into the spacer) and an azide (azido-glucose). these approaches for carbohydration circumvented the problem encountered during glycation via the maillard reaction. glycation by the cu(i) catalyzed cycloaddition was, by far, the easiest way to incorporate the glucose moiety. moreover, after m tc labeling, this analogue showed the best biodistribution and the best diagnostic properties of all glycated analogues. an alternate approach to improve the pharmacokinetics consisted of including different polar spacers such as ³hglu, ³hasp, ³hlys, ³hser and -nh(ch ch o) co-between the (n his)ac chelator and the bombesin sequence. in particular the analogues with a negative charge ( ³hglu, ³hasp) in the spacer showed a favorable effect on the pharmacokinetics. determination of binding ratio of hydrofobic peptidepolymer conjugates by using fl uorescamine assay budama battal, yasemin; derman, serap; mansuroðlu, banu; mustafaeva, zeynep yildiz technical university, bioengineering department, turkey non-fl uorescent -phenylspiro-[furan- ( h), -phthalan]- , '-dione (fl uorescamine) reacts readily with primary amines in amino acids, peptides and proteins to form stable, highly fl uorescent compounds (fl uorophors). fluorescamine has been used to detect free amino groups on peptides or completion of coupling reactions in solid phase peptide synthesis and not only in aqueous solution but also in organic solvents and on solids. in this study, peptide epitops of vp capsid protein of foot-and-mouth-diseases virus - amino acid residues (val-lys-ile-asn-asn-thr-ser-pro-the-his-val-ile-asp-leu-met-gln-thr-his-gln-his-gly) were synthesized by sigma. we synthesized the covalent conjugate of - amino acid residues with copolymers of acrylic acids and nvinylpyrolidone (vp\aa) at different ratio of components (npeptide/ npolymer) and investigated the mechanism of condensation reaction by using different physicochemical analyses as hplc, fluorescence spectroscopy and fluorescamine assay. for determination of binding ratio of hidrofobic peptide-polymer congutages, fl uorescent measurements were performed in the presence of fl uorescamine at the wavelengts of excitation nm and emission nm. when conjugation reaction completed, the amount of free amino groups had decreased and observed that fluorescence intensity had decreased and the estimated degree of primary amino group after conjugation, calculated from fl uorescence spectrums. ( -phenylspiro-[furan- ( h), -phthalan]- , '-dione) which heterocyclic dione is a reagent for the detection of primary amines on peptides or completion of coupling reactions in the picomole range. its reaction with amines is almost instantaneous at room temperature in aqueous media, organic solvents ect . fluorescamine reacts with primary amines (in peptide, protein ect.) to form highly fl uorescent product (fl uorophors) whereas the reagent and its degradation products are nonfl uorescent ( ). this is the basis of a fl uorescent protein assay [ , ] . fluorescamine is used in many sensitive detection methods, e,g., characterization of poly-l-lysine (pll)/dna complexes post-modifi ed with a multivalent hydrophilic polymer ( ), or synyhetic peptide-polymer conjugates. in this study, foot-and-mouth-diseases virus vp capsid protein's synthetic peptide epitope which containing tryptophane (trp), - (p ) amino acid residues (try-ser-lys-tyr-ser-thr-thr-gly-glu-arg-thr-arg-thr-arg-gly-asp-leu-gly-ala-leu-ala-ala-arg-val-ala-thr-gln-leu-pro-ala) were synthesized by using the solid-phase methods. we synthesized the covalent conjugate of copolymers of acrylic acids and n-vinylpyrolidone (vp\aa) with - amino acid residues at different npeptide/npolymer ratio and investigated the mechanism of condensation reaction by using fluorescence spectroscopy and fluorescamine assay. in the fl uorescamine assay the conjugate solution was measured on a pti qm- steady state fluorescence spectrometer with an excitation wavelength of nm and emission at nm. after the conjugation reaction, the amount of free amino groups had decreased because of binding carboxyl group and observed that fluorescence intensity had decreased. determination of free amino group degree after conjugation, calculated from fl uorescence spectrums. newly developed high strength and chemically stable silica gel based preparative reversed phase packing materials kuriyama, naohiro; shoji, noriko; morishita, kiyoshi; omote, masakatsu ymc co., ltd., japan a new high strength silica gel and a bonding technology based on preparative bulk packing materials for hplc have been developed to provide improved recovery, selectivity, and longer life time for the preparative peptide separations. the novel preparative silica particle was successfully prepared by the new generation process, which allows the higher gel density than typical silica gel and the particle size distribution would be practically mono-dispersed character. for the effective reversed phase peptide separations, pore size and pore volume of these new particle were optimized depending on the molecular weight of peptides. to enhance chemical stability and selectivity under the typical peptide purifi cation conditions, the combination of chemical bonding method and functional group density was optimized for maximum performance. by repeated packing and unpacking of this synthesized gel with large dynamic axial compression column, it was demonstrated that no fi ne has appeared and no back pressure increasing has occurred comparing to commercially available packing materials. also cost effective peptide purifi cation with high loadability, productivity, and recovery was achieved with signifi cant small and large peptides. calcitonin gene-related peptide (cgrp) is a amino acid neuropeptide produced by tissue-specifi c alternative mrna splicing of the calcitonin gene. the cgrp peptide signals through a seven transmembrane g protein-coupled receptor (gpcr) belonging to the secretin receptor family. co-expression of the calcitonin-like receptor (clr) with receptor activity modifying proteins- (ramp ) forms a mature cgrp receptor on the cell membrane surface. cgrp is widely distributed in the peripheral and central nervous systems. in the later, cgrp is expressed in trigeminal ganglia nerves and when it is released has potent dilator effects on cerebal and dural vessels. through this mechanism, cgrp is involved in the regulation of blood fl ow to the brain and painsensitive meninges. the pathology of migraine has been associated with the vasodilation effects of cgrp on cerebal circulation. it has been reported that the cgrp peptide consist of an alpha-helical n-terminal region, a fl exible center region, and two putative c-terminal beta-turns. truncation of the fi rst seven residues in cgrp results in antagonists of the cgrp receptor (ic nm), however, cgrp( - ) is rapidly degraded in plasma. here, we report the iterative design and synthesis of new high affi nity cgrp antagonists with signifi cantly increased plasma stability, and higher affi nity for the human cgrp receptor. cordopatis, paul; pappa, eleni; zompra, aikaterini; magafa, vassiliki; diamantopoulou, zoi; lamari, fotini; katsoris, panagiotis university, greece mammalian gonadotropin releasing hormone (gnrh-i) and the sea lamprey gonadotropin releasing hormone type iii (gnrh-iii), belong to the class of conserved gonadotropin releasing hormone peptides. in addition to the classic hypophysiotropic action of gnrh-i, it has been shown that many malignant cells, such as prostate cancer cells, secrete gnrh-i and express the gnrh-i receptor/s. gnrh-iii has no endocrine activity in mammals even at high doses, but has been shown to suppress directly the growth of breast and prostatic cancer cells in vitro. in a continuation of our previous work and in order to study the effect of modifi cations in positions and of leuprolide, an agonist of gnrh-i, on prostate cancer cell proliferation, we synthesized ten new conformationally restricted analogues. d-leu of leuprolide was substituted by d-lys, d-or l-glu and ser by n-me-ser. we also synthesized fi ve new analogues of gnrh-iii and studied their effect on prostate cancer cell proliferation. asp in position of gnrh-iii was substituted by asn and glu, pro was substituted by á-aminoisobutyric acid (aib) and trp and/or trp by d-trp. peptides were synthesized by the solid phase methodology and fmoc/but chemistry in high yields. results show that the inhibitory effect of gnrh-i analogues on the proliferation of human prostate cancer cells depends on the nature of the substituted amino acid in position . incorporation of d-trp in positions and of gnrh-iii preserved its antiproliferative activity, whereas all other modifi cations reduced its activity. mastoparan (mp) is an antimicrobial cationic tetradecapeptide with following primary structure inlkalaalakkil. this amphiphilic -helical peptide was originally isolated from the venom of wasp paravespula lewisii. however some mp analogues (tetradecapeptides with different amino acids sequence) have been also isolated from the venom of hornets, yellow jackets and paper or solitary wasps. they are rich in hydrophobic amino acids such as leu, ile, ala and commonly posses lys residues. mp shows a variety of biological activities such as inhibition of the growth of gram-positive bacteria, activation of mast cell degradation and histamine release, activation of phospholipase a and c or erythrocyte lysis. mp is also turned out to enhance the permeability of artifi cial and biological membranes and activate gtp-binding regulatory proteins by a mechanism analogous to that of g protein-coupled receptors. nowadays many mastoparan analogues have been synthesized. some of them contains in primary structure the mastoparan sequence but they loss some of the properties of mp, such as transportan (a cell penetrating peptide, consisting n-terminal fragment - of galanin and mastoparan at the c-terminus linked with lys residue) or its truncated analogue, transportan- . in present study we have designed and synthesized several new chimeric mastoparan analogues composed of mp and other biologically active peptides (e.g. galanin, rnaiii inhibiting peptide) and containing natural or unnatural structures. next we examined each of these hybrid constructs as well as natural peptides for they antimicrobial activities. acknowledgement: this work was supported by the university of gdansk grand bw - - - cinnamic acid derivatives as esters, amides and glycosides are known to have antibacterial, antiviral, antiinfl ammatory, antiproliferative, immunostimulatory etc. properties. some of them (amide and ester analogues of caffeic acid with natural amino acid esters) exibit stronger antioxidative activity. in particular thiazole, oxazole and imidazole amino acids that may play a key role in biological activities of unusual peptides are also important intermediates for natural product synthesis and peptidomimetics. here we report the synthesis of novel cinnamic acid amides with oxazole containing glycyl-methyl ester as potential antioxidants. the amides were synthesized from sinapic, coumaric, ferulic acids and the corresponding glycyl-methyl ester hydrochloride form using n-ethyl-n-( -dimethylaminopropyl) carbodiimide hydrochloride (edc) and -n,n-(dimethylamino)-pyridine (dmap). their antioxidant activity was evaluated by , -diphenyl- -picrylhydrazyl dpph ( , diphenyl- -pycrylhydrazyl) test. the venoms of marine cone snails contain a complex mixture of peptide neurotoxins known as conotoxins ( ) . they are a diverse class of biomolecules that exhibit exquisite selectivity and potency for a wide range of pharmacological targets in the central nervous system, making them valuable tools for studying the mechanisms of neurotransmission and pain. despite their diversity, conotoxins have evolved from a relatively small number of rigid disulfi de bonded frameworks that give rise to a series of intervening loops of amino acids that project outwards from the framework and interact with the receptor binding site. receptor targets are generally determined by the shape of the framework, with the amino acids within the framework infl uencing receptor subtype specifi city. this work aims to use positional scan libraries ( ) based on / -conotoxin frameworks to study the interactions of amino acids with the binding site of neuronal nicotinic acetylcholine receptors, and to discover new ligands that possess greater potency and selectivity for different subtypes these receptors. the disulfi de bond frameworks were formed by oxidizing each mixture in dilute aqueous buffer and their correct formation monitored by cd spectroscopy. an á nachr functional assay was used to screen each mixture to determine the activity of each mixture and the results of this assay were used to design a series of individual candidates for use in further structure activity relationship studies. melittin effect on the sensory neurons prelevated from double transgenic vs. normal mice melittin is a aa peptide. it is the active compound of bee venom and a powerful stimulator of phospholipase a . its antimicrobial effect was extensively studied in the literature, but little is known about its neuroactive properties. melittin increases the cell membrane permeability in excitable and non-excitable tissues, as a result of its interaction with the negatively charged phospholipids. our interest was focused on the algesic properties of melittin and on its potency against the electrophysiological parameters of sensory neurons from dorsal root ganglia. in our study, we have performed patch-clamp studies, by the whole-cell confi guration. primary cell cultures were obtained from double transgenic mice tcr-ha+/ins-ha+ with type i diabetes. this animal model is well-suited for the study of neuropathic pain and it enables us to test the analgesic effects of melittin ( . - m). our recordings indicate that the active-peptide modifi es the algesic profi le of the sensory neurons, in particular by acting against the capsaicinactivated ionic currents. in addition, we have monitored the action of melittin on the i h currents, na + voltage -dependent currents, delayed rectifi er k + currents. a particular interest was focused on the recordings of ca + voltage-dependent currents. the maximal effect was obtained at a dose of m melittin, and at higher doses the active-peptide strongly disturbs the lipid membrane order. in conclusion, the algesic profi le of the sensory neurons from double transgenic mice is signifi cantly modifi ed by the neuroactive melittin in comparison with the profi le of neurons from balb/c mice. these electrophysiological data are important, and are very well corroborated with the increased thermal and mechanical hypersensitivity induced by melittin itself and the bee venom extract, that have been proved their effi ciency in behaviour tests . the toxic effects of amps tested on mammalian cell cultures the amps (antimicrobial peptides) is a class of molecules that belongs to the innate immunity. the ability of some of these peptides to penetrate the microorganism's external membrane and to induce their death suggests that this class of molecules may represent a new generation of antibiotic pharmaceuticals. in our work, we focused on the amps side effects on mammalian cells. the target was to characterize the toxicity of several amps evaluated on in vitro cellular cultures. the following amps were used in our experiments: melittin, magainin i and ii, cecropin a and mastoparan. the viability of cells in cultures was assessed by mts test on chinese hamster lung fi broblasts v and on human lymphocytes primary cultures. concentrations in the range of . - m were used. the ic value for each type of amp was evaluated from the viability versus concentration curves. haemolysis assays were also performed for these active-peptides. based on ic values, melittin has a stronger citotoxicity than magainin i and magainin ii. on the other hand, magainins appears to posses higher haemolytic properties than melittin. the less cytotoxic peptide seems to be mastoparan. these types of results are very useful to characterize the ability of amps to induce toxic effects in human body during the treatment. acknowledgements: financial support by the cnmp research grant pnii number - / . the melanocyte-inhibiting factor (mif- ), which is the tripeptide pro-leu-gly-nh , was isolated in the conventional way from bovine hypothalamus tissue. mif- represents a class of naturally occurring opiate antagonists with varying activities in independent situations. the purpose of the present study was to investigate the analgesic activity of mif- and its analogues modifi ed at position with unnatural amino acids cav, slys, sleu, sile and snle. the experiments were carried out on male wistar rats ( - g). the changes in the mechanical nociceptive threshold were measured by the paw-pressure test using an analgesiameter (ugo basile). mif- and analogues (all in dose mg/ kg) were administered intraperitoneally (i.p.). mif- exhibits a weak analgesic effect and selective affi nity for the -opioid receptor. mifanalogues -mif-cav, mif-sleu, mif-ile and mif-nle were found to have a naloxone-reversible analgesic effects. pacap (pituitary adenylate cyclase-activating polypeptide) exists in two biological isoforms, i.e a -amino acid peptide and a shorter form of residues corresponding to the n-terminal part of pacap . previous structure-activity relationships studies revealed that the n-terminal segment is essential for the activation of the specifi c and selective type i pacap receptor (pac ). in order to clarify the molecular requirements for pac activation, we initiated structure-activity investigations of the n-terminal part of both pacap isoforms. in particular, an important library of analogs focusing on the fi rst seven residues (his -ser -asp -gly -ile -phe -thr ) was rationally developed and pharmacologically evaluated for their capacity to bind the pac receptor and to induce ca + mobilization in chinese hamster ovary (cho) cells stably expressing the human pac receptor. ala scan demonstrated that the carboxylic acid function of residue asp and the benzyl group of phe are essential feature for proper pac receptor activation. the inversion of chirality of residues ile , phe and thr caused a loss of affi nity whereas the incorporation of d-amino acids in positions , or did not affect the binding properties of pacap. moreover, using a n-methyl scan, we showed that the n-terminal domain of pacap is not tolerant to back-bone modifi cations. however, replacement of ser by pro did not decrease signifi cantly the potency of the peptide while substitution of this same residue by d-pro totally inhibited the biological activity of pacap. furthermore, other structural constraints reduced the potency of pacap, suggesting that the n-terminal domain needs fl exibility to bind and activate the pac receptor. finally, residues - of c-terminally extended peptides played a favorable role for the binding affi nity towards the pac receptor as pacap analogs demonstrated highest binding affi nity compared to their pacap counterparts. neutrophil elastase-dependent synthetic host defence pro-peptides for the treatment of bacterial infections in cystic fi brosis patients chronic infection and infl ammation play a major role in the pathophysiology of lung disease in cystic fi brosis (cf). besides pseudomonas aeruginosa, there is increasing evidence that other multidrugresistant organisms, such as methicillin-resistant staphylococcus aureus (mrsa) are implicated in the activation of the infl ammatory response in cf patients. cationic host defence peptides, multifunctional mediators of the innate immune system, have been recognised as promising candidates for the development of novel antimicrobial agents ( ). most of these macromolecules are produced as inactive prepropeptides and are proteolytically activated to release a c-terminal cationic peptide with antimicrobial activity. we thought to mimic this natural mechanism to target cf pathogens with synthetic host defence propeptides. saltresistant, all-d cationic antimicrobial peptides ( ) were conjugated at their n-termini to a polyglutamic acid sequence to compensate the net positive charge of the parent peptide, through a linker selectively degraded by a disease-associated enzyme (neutrophil elastase). in vitro effects of the all-d p peptide candidate on planktonic and biofi lm forms of pseudomonas aeruginosa and staphylococcus aureus clinical isolates were assessed. reactivation studies of the propeptide were performed in presence of purifi ed neutrophil elastase and in physiologically relevant conditions, using bronchoalveolar lavage fl uids from cf patients. the nmr structures of the propeptide and active p sequences were also compared. these studies confi rmed that the propeptide remains inactive in the absence of neutrophil elastase and that the latter enzyme can potentiate its antimicrobial activity. synthesis and biological activity of a series of aza pseudopeptides related to rfa, the endogenous ligand of gpr rfa, a novel neuropeptide of the rfamide family, is the natural ligand of the previously orphan receptor gpr . both rfa and gpr mrnas are highly expressed in hypothalamic nuclei of rodents, and icv injection of rfa induces a potent orexigenic effect in mice. recently, it has been shown that gpr -defi cient mice suffer from osteopenia. analysis of the rfa precursor reveals that it may generate several additionnal rfa-peptides including an n-terminally extended form ( rfa) and a truncated form ( rfa ( - ) ). rfa and rfa increase dose-dependently [ca + ]i in gpr -transfected cells while rfa( - ) is about times less potent than rfa. molecular modeling under nmr constraints of rfa shows that the n-terminal region encompasses an -helix and the c-terminal region adopts a -turn in dpc micelles. the c-terminal peptide rfa( - ) exhibits major distortions of this turn that may be responsible for its weak potency. the aim of this work was to introduce an aza residue in rfa( - ) in order to stabilize the -turn. sequential aza -counterpart substitution of amino acids at positions and enhanced by and folds the potency of rfa( - ), respectively. the aza -phe analog was times less potent than rfa( - ). replacement of the native ser by the aza surrogate of (ho)homothr (aza -hth) to favor hydrogen bonding, generated an analog that was folds more potent than rfa( - ). in contrast, substitution of residues to led to analogs totally devoid of effect on calcium mobilization. as the -turn is centered on the ser of rfa, we can assume that the aza -hth moiety partially mimics the -turn in the rfa( - ) sequence. these data constitute the fi rst step towards the development of new gpr analogs that could prove useful for the treatment of feeding disorders and/or osteoporosis. acknowledgements: supported by inserm (u and pnr-re) and the région haute-normandie. olm is recipient of a fellowship from mesr. it is well known that microtubule targeting agents (mtas) are used in clinical treatments as anticancer agents. recently, it has become clear that some mtas also function as gvascular disrupting agents (vdas), which induce tumor-selective vascular collapse. as one of such candidates, a natural cyclicdipeptide, phenylahistin (plh) exhibiting colchicine-like anti-microtubule activity, has been our focus. we have succeeded in synthesizing plh, and performed the structure activity relationship study of its derivatives. from the biological evaluations, according to these results, a highly potent derivative npi- (ic = nm, ht- ) was selected, which is now in phase i clinical trial as an anticancer drug in the us. furthermore, we have established the synthetic route of npi- and its derivatives. about analogs were prepared so far and screened by ht- citotoxicity assay. as a result, several highly potent derivatives ware developed. although npi- and its derivatives are believed to be recognized around the colchicine binding site on tubulin, the three-dimensional structure of npi- could not be superimposed over that of colchicine. in order to understand the precise binding mode of npi- , we developed a highly potent cytotoxic derivative, kpu- (ic = . nm, ht- cells) with a benzophenone structure. because benzophenone is recognized as a photo-reactive group, we synthesized biotin-tagged kpu- derivative as a photoaffi nity probe. since this probe has biological activity enough to function to tubulin, photoaffi nity labeling was performed to analyze the binding site. as a result, irradiation-time-dependent labeling towards tubulin was observed. the labeling was also dose-dependently inhibited by colchicines addition, suggesting that the probe specifi cally recognizes around the colchicine binding site on tubulin. chemical biology study for determining the precise binding site is now in progress. conformational analysis of the new temporin analogues: gln ta and pro tl. temporins are antimicrobial peptides (amp €™s) isolated from the skin of red european frog rana temporaria. they are active particularly against gram-positive bacteria, candida species, fungi. they have the ability to bind and permeate both artifi cial and biological membranes. we have recently investigated by spectroscopic means two members of this amp family temporin l (fvqwfskflgril-nh ) and temporin a (flpligrvlsgil-nh ) ( ). based on the nmr results, we developed two new analogues of these peptides named pro tl (fvpwfskflgril-nh ) and gln ta (flqligrvlsgil-nh ). biological data indicate that pro tl has a higher antimicrobial activity and a lower hemolytic activity than the native peptide tl. in contrast, gln ta more hemolytic than the parent peptide ta. the conformational behavior of the new analogues was investigated in membrane mimetic environment (sds and dpc micelles) by spectroscopic and computational methods. diagnostic nmr parameters observed for glu ta and pro tl indicate a conformational propensity toward helical structure. for glu ta, bturn structures are also observed along the n-terminal fragment of the peptide. we are currently studying the conformational behavior of the four peptides also by molecular dynamics simulation in explicit solvated dodecylphosphocholine and sodium dodecylsulfate systems. these simulations would provide a realistic picture of the interactions between the peptide and models of bacterial and mammalian membranes. where xaa was selected form a collection of cyclic and acyclic nonaromatic amino acids. the peptides were prepared by standard spps methods using either the fmoc or boc strategy and tested in vitro for their agonistic potency and effi cacy at the v a and the related receptors. unlike avp, which has aromatic amino acids in positions and , these analogues contain only non aromatic residues, but are still potent v a agonists. compounds with cyclic aliphatic residues (e.g. cha) in position were found to be generally more potent v a agonists than the ones containing acyclic residues. the ala analogue ([ala ,ile ,dab ]vp) was totally inactive (no signifi cant v ar agonism up to nm) as pharmacology and medical peptide chemistry reported previously for [ala ]avp ( ). the cha containing peptides are slightly less potent as v ar agonists than their phe counterparts, but the overall in vitro pharmacological profi le of the two classes of compounds is similar. selected new analogues were also tested in vivo in a rat pressor model. the compounds were found to be effective in raising arterial blood pressure and appeared to be longer acting in vivo when compared to avp and related compounds, as demonstrated by slower disappearance of their pressor effect at equieffective doses. detailed experimental procedures, the results of biological testing, and sar discussion will be presented. the impact of lithium cations on the peptide bond lithium ions play an important role in some biological processes, e.g. in the treatment of neuronal dysfunctions and some metabolic pathways. however, the mechanisms for these actions are not known up to now. recently it has been shown that lithium cations infl uence the isomerisation state of peptide bonds which is essentially pronounced in the case of the amino acid proline. such an isomerisation goes hand in hand with conformational changes possibly resulting in altered folding and structuring. thus, lithium cations might essentially infl uence the biological function of peptides and proteins. to shed light on the underlying mechanism we carried out detailed studies on several model peptides. thus, the isomerisation as well as the kinetic properties of the peptides have been determined employing various techniques of nmr spectroscopy. besides, quantum chemical studies on li + -peptide complexes were performed to get insight into the structural aspects of the cation-peptide interaction. enhanced screening and understanding of hypolipidemic peptides assisted by informatics hypolipidemic peptides targeting the bile acid to inhibit cholesterol absorption in intestine has been indicated by several naturally obtained short peptides. these bile acid-binding peptides are known to disrupt the micellar formation of bile acid for capturing intestinal cholesterol, and lead the aggregates pass the intestine without absorbance. such effect also disrupts the hepatic circulation of bile acids, which accelerates the conversion of stored cholesterol into lacking bile acids. however, traditional extraction procedure of peptides form natural products is time-consuming. therefore, to enhance the screening of such functional short peptides, we have combined the array-based screening strategy with bioinformatics strategy, to design effective experiments by prediction of physicochemical peptide structures. as a result, we resulted in signifi cantly higher screening effi ciency and high bile acid affi nity compared to random screening. we hare report the introduction of one of supervised learning algorithms, fuzzy neural network, and unsupervised algorithm, hierarchical clustering scheme for such functional peptide screening. analogues of the kinin b receptor antagonist r- bearing n-terminal lipid moieties. adrenomedullin (am) is a -amino acid peptide that shares structural similarities with regulatory peptides belonging to the calcitonin family (calcitonin, cgrp and amylin). am is known to produce a potent vasodilation via the coupling to the calcitonin receptor-like receptor (crlr) associated with a chaperone protein, the receptor-activity modifying-protein (ramp). binding studies performed in mammalian tissues revealed that the largest distribution of am binding sites was found in the heart and lungs. in fact, the pulmonary blood vessels display a vast abundance of am binding sites that act as clearance receptors. pulmonary arterial hypertension (pah) is a condition associated with obliteration of pulmonary arterioles which carries a very poor prognosis, in part because of delayed diagnosis due to the lack of specifi c noninvasive diagnostic tools. likewise, there currently exists no molecular imaging agent to diagnose pulmonary embolism, a pathological condition that occurs when a blood clot blocks a lung artery. therefore, due to the high density and incidence of am receptors in the cardiorespiratory system, am could represent a key-target for diagnosis with radiolabeled am-related drugs. in this study, we looked at structureactivity relationships and synthesized various am fragments exhibiting high binding specifi city for am receptors but reduced biological activity. moreover, we introduced different chelating moieties into these am peptides to evaluate the possibility of labelling those molecules for imaging purposes. using cell binding assays, we observed that a cyclic moiety combined with the am( - ) sequence is able to maintain a good affi nity for am receptors. furthermore, we showed that the incorporation of a -amino acid sequence into the peptide chain was the best chelating moiety that allows high effi ciency labelling with mtc. hence, our study strongly suggests that am derivatives are promising leads for lung specifi c imaging. m tc-labeled analogs with pansomatostatin properties may lead to clinically useful radiotracers and further search for analogs displaying improved biological profi le is warranted. and for the rat (r) sst by competition binding assays in ar - j cell membranes with [ i-tyr ]octreotide as radioligand. after m tc-labeling, radiopeptide internalization was studied in ar - j cells. biodistribution of [ m tc]demotates was studied in healthy swiss albino mice and for [ m tc]demotate , , and in ar - j tumor bearing mice. results: demotate showed the highest affi nity binding for the hsst and the rsst , while introduction of one asp linker(s) at the n-terminus diminished the affi nity for the hsst and the rsst and led to lower internalization rates. substitution of thr by asp in demotate resulted in good affi nity for the rsst and lower affi nity for the hsst . single (d)phe -substitution/ or thr substitution by asp combined with asp introduction at the nterminus/ led to inferior binding affi nity, poor internalization capacity and poor uptake of the respective radiopeptides in target-organs and, in the case of [ m tc]demotate , , and , in ar - j tumors in mice. conclusion: introduction of asp residues in the original [ m tc]demotate chain exerted a negative effect on receptor affi nity, internalization capacity and targeting of somatostatin binding sites in mice precluding their use as hydrophilic pharmacokinetic modifi ers. the family of secreted aspartic proteinases (saps), which are encoded by ten distinct genes, is an important virulence factor of the human pathogen candida albicans.( ) due to an increase in the number of candida strains that are resistant to the drugs currently used in therapy, these proteinases are highly promising new drug target candidates. based on the knowledge of sap -substrate specifi cities( )and x-ray structural studies of sap in complex with inhibitors,( ) we designed and synthesized a series of sap inhibitors by modifying the structure of the pentapeptide pepstatin a, which is a potent yet non-selective aspartic proteinase inhibitor. these inhibitors were synthesized manually via a spps methodology using a -cl-tritylchloride resin and fmoc-protected amino acids. in addition, the key residue of the designed peptide inhibitors, the -amino acid statine (sta), which was prepared according to a slightly modifi ed literature procedure, . was further protected at its hydroxyl group as a silyl ether for the purpose of avoiding competitive side reactions during amino acid couplings. we prepared new inhibitors by varying the pepstatin a structure at the p , p , or p ' position. all variants showed effi cient inhibition when screened against the isoenzymes sap , sap , and sap , and some of them exhibit ic values similar to or even lower than that observed for pepstatin a. peptide library is a general technique for screening specifi c peptides that bind to a target protein. but, in a standard screening method, peptide libraries need to be fi xed on large carriers like phage, beads and so on. the large carriers would affect the binding between peptides and the target protein. in this work, we develop a new method for screening peptide libraries without carriers. we attached a variety of fl uorescent amino acids to peptide libraries and the peptides that bind to target proteins were identifi ed through -dimensional fl uorescence spectroscopy in aqueous solution. in this presentation, -different fl uorescent amino acids were synthesized or purchased. of those amino acids, we newly synthesized fl uorescent amino acids. -d fl uorescence spectra of those amino acids were measured. they were different in fl uorescence excitation/emission wavelengths. excluding those of weak fl uorescence intensities, fl uorescent amino acids are selected. those fl uorescent amino acids are mixed in methanol/water (ph . ) (= / (v/v)) and the -d fl uorescence spectrum was measured. then, concentrations of the component amino acids were evaluated by a linear least-squares method. the results suggested that all fl uorescent amino acids can be quantifi ed. the set of fl uorescent amino acids combined with the -d fl uorescence spectroscopy technique will be a powerful tool for screening peptides and other drug compounds without using any carriers. n-methyl phenylalanine-rich peptides as potential blood brain barrier shuttles malakoutikhah, morteza; teixidó, meritxell; giralt, ernest institute for research in biomedicine, barcelona, spain several peptide families containing n-methylated amino acids were designed and synthesized using solid-phase peptide synthesis (spps). the permeability of these compounds was studied by parallel artifi cial membrane permeability assay (pampa) and immobilized artifi cial membrane chromatography (iamc) so as to select the best peptides in terms of length, terminal groups and amino acid replacement to be used as carriers that pass through a model of blood-brain barrier (bbb) by passive diffusion for non-permeating agents. furthermore, their enzymatic stability in human serum and their cell viability were tested by mtt assay. these peptide families showed great stability and nontoxicity. the three best peptides were coupled to levodopa and assessed. these peptides transferred levodopa through an artifi cial membrane and transformed it from a non-passive permeating drug into a compound able to cross the membrane by passive diffusion. the structure-activity study of insulin-like peptide (insl ) requires the design and synthesis of various analogues. in order to test these for their receptor binding affi nity, a high-throughput receptor binding assay is needed. we have therefore developed an effi cient solid phase synthesis protocol to prepare specifi cally mono-labeled human insulin-like peptide (insl ) for the study of its interaction with its g-protein-coupled receptor, rxfp . a commercially available chelator, diethylene triamine pentaacetic acid (dtpa), was coupled to the n-terminus of solid-phase bound insl a-chain and then a coordination complex between eu + and dtpa chelator was formed. after combination of the purifi ed aand b-chains together with sequential formation of the three insulinlike disulfi de bonds, the labeled peptide was purifi ed at high yield using high-performance liquid chromatography with high ph buffer to prevent the liberation of europium from chelator. saturation binding assays were undertaken to determine the binding affi nity (pk d ) of labeled insl for rxfp in hek stable cell line expressing rxfp . the binding affi nity of dtpa-labeled insl ( . ± . ) was comparable to that of i-labelled insl ( . ± . ). the effi cient solid-phase synthesis has provided a novel lanthanide-coordinated, dtpa-labeled insl with excellent sensitivity, stability and high specifi c activity and which is superior to the traditional i-insl . this labeled peptide can be used in a high-throughput screening of insl analogues in structure-activity studies. multimodal tumor imaging using mri and pet/spect provides comprehensive diagnostic information as it combines anatomical information (by mri) and functional information (by pet or spect). development of dota-derived imaging probes is advantageous for multimodal imaging as it accommodates many metal ions employed in different imaging modalities including gd(iii) for mri. however, high relaxivity and enhanced specifi c up-take at the targeted site is important for the gd-based mri contrast agents. the dota-based prochelators required for the multivalent vectorization of targeting ligands were synthesized by functionalizing cyclen or do a-tertbutyl ester with modifi ed glutamic acid. multivalent conjugation of bombesin peptides, which specifi cally target tumors expressing gastrinreleasing peptide (grp) receptors, yielded the corresponding mono-, di-, and tetravalent bombesin analogues. the conjugates showed excellent chelating properties with gd(iii) (for mri applications) and also with in, lu and ga (for radiopharmaceutical applications). the in and lu labeled divalent conjugates showed rapid internalization and slower externalization rate compared to their corresponding monovalent analogues as studied with prostate tumor cell lines. the gadolinium complexes of monovalent and divalent conjugates showed signifi cant relaxivities at mhz ranging from . to . mm- s- at °c. the values represent the to fold enhancement of relaxivity compare to clinically employed dotarem® (gd-dota). further, the relaxivities increased signifi cantly from monovalent to divalent conjugates. this is highly promising as we would expect much higher relaxivities in case of tetravalent conjugates, which are currently under investigation. in conclusion, the work demonstrates the development of high relaxivity multivalent bombesin analogues, which could be employed for the multimodal imaging such as pet/mri or spect/mri with improved tumor targeting capabilities. a new highly potent dota-conjugated bombesin antagonist for grpr-positive tumor targeted imaging peptide receptors are very promising targets for tumor imaging. the somatostatin receptors were successfully targeted with peptides labeled to -emitters ( in, ga and m tc) and positron emitters ( f, ga, y) for diagnostic imaging. for tumor targeting, radiolabeled agonists were developed as they usually trigger the internalization of the peptidereceptor complex. we have recently shown that somatostatin-based radiolabeled antagonists may not only have a higher tumor uptake than equipotent agonists but also a longer lasting tumor uptake. among the most promising receptors to be targeted, the bombesin receptors are of great interest as they are overexpressed in major human tumors such as prostate and breast. the aim of this study was to develop a radiolabeled bombesin-based antagonist conjugated to the macrocyclic chelator dota which allows effi cient and stable labeling with a variety of two-and three-plus charged radiometals for imaging (pet, spect). we compared its in vitro pharmacologic properties and biodistribution in the pc- mouse model side-by-side with the potent agonist [ nat, in]-do a-ch co-g- -aminobenzoyl-q-w-a-v-g-h-l-m-nh (amba). in(iii)-amba was shown to be a potent agonist by ca + fl ux and immunofl uorescence studies and in(iii)-rm was effi ciently antagonizing the activity of in(iii)-amba. the pharmacokinetics showed a distinct superiority of the radioantagonist with regard to the high tumor uptake as well as to all tumor to normal tissue ratios. [ ga]-rm showed similar pharmacokinetic than [ in]-rm with a lower initial kidney uptake and a faster wash out from the kidney. the pet/ ct scintigraphic studies of [ ga]-rm in the animal model confi rm the signifi cant high tumor uptake and tumor to background ratio. as we found for somatostatin receptor-targeting radiopeptides,bombesin-based radioantagonists also appear to be superior to radioagonists for in vivo imaging and potentially also for targeted radiotherapy of grpr-positive tumors cell penetrating peptides (cpp) are a special class of peptides that possess the property to traverse the formidable barrier of the plasma membrane and deliver cargos into cells. using cpp as vectors and dna, mrna or proteins/enzymes as potential intracellular targets, a new generation of intracellular contrast agents (cas) can be developed. these agents have prospective use for molecular imaging (both optical and magnetic resonance imaging) by targeted labeling of cells. aiming to image the presence of specifi c mrnas or enzymes, two mrna targeting (contains a pna sequence antisense or non-sense to the target mrna of dsred) and one enzyme targeted (contains a unit cleavable by -galactosidase) cas were tested for their activity in the presence and absence of respective targets. the antisense targeting ca, their nonsense derivative and the enzyme targeted ca were taken up effi ciently into cells by an exclusively endosomal mechanism as observed by fl uorescence microscopy. cell free binding assays proved a specifi c interaction with a synthetic target for the antisense but not for non-sense ca. magnetic resonance studies showed a higher uptake in transgenic dsred expressing cells than the parent cells. however, no difference was observable for antisense versus non-sense ca in dsred cells, due to the vesicular entrapment which is preventing the specifi c interaction between ca and cytosolic target. since a comparable cellular distribution was visible for the enzyme targeted agent, a specifi c accumulation in -galactosidase containing cells is also unlikely. the results show that even though the designed cas were effi ciently taken up into cells, they can interact specifi cally with the target only if colocalization is achieved. however, a lack of specifi city is caused by the endosomal entrapment. further modifi cations are required to achieve the release from endosomes or a direct uptake into the cytosol. the infl uence of pegylation on the tumor accumulation of frop- , a tumor specifi c peptide identifi ed by phage display the pool of natural peptides suitable for the tumor targeting is limited and therefore novel lead structures identifi ed by screening of phagedisplayed libraries are highly warranted. however, transfer of the novel peptide sequences to clinical application is often diffi cult. we had shown that the coupling of dota to frop- , a peptide identifi ed in phage display libraries resulted in a fundamentally improved in vitro binding capacity. however, a biodistribution study revealed that the slow binding kinetics frop-dota (h-glu-asn-tyr-glu-leu-met-asp-leu-leu-ala-tyr-leu-lys(dota)-cys-nh ) allowed the excretion to forestall a signifi cant tumor accumulation. the aim of this study was to investigate whether the conjugation of peg to frop-dota results in a derivative with a prolonged residence time in the blood. frop- bearing a c-terminal dota residue to allow labeling with in- and a cysteine to attach a maleimido-modifi ed da peg oligomer was obtained by solid phase synthesis. the conjugate was purifi ed by size exclusion chromatography. several attempts to couple different peg derivatives on the solid support did not proceed satisfactorily and therefore the peg residue was attached in solution. the breast cancer cell line mcf- showed a relative low accumulation of the pegylated peptide in the cells. in contrast, biodistribution studies of the labeled conjugate in mice bearing human fro - showed a time dependent increasing uptake of the pegylated peptide with a high retention (at h p.i. % of the maximal activity concentration persisted in the tumor). the highest uptake values were determined at min. p.i. reaching . %id/g tumor as compared to . % %id/g observed for the non-pegylated derivative at min p.i. apparently pegylation provides a substantially improved stabilization in the circulation which allows a stable tumor accumulation. in vivo spect/ct imaging of intracranial human glioblastoma xenografts with indium-labeled homing peptide. huhtala, tuulia* ; enbäck, julia* ; rautsi, outi ; weisell, janne ; laakkonen, pirjo* ; närvänen, ale* *equal contribution. university of kuopio, finland; university of helsinki, finland phage display libraries provide a powerful tool to identify new biologically active peptides that bind specifi cally to their target molecules. different tumors express a distinct range of molecular markers on their vasculature providing a possibility to use peptides for targeting. tumor-homing peptides offer an opportunity to target, image and destruct the target tissue. glioblastomas represent the most aggressive form of brain tumors. we have identifi ed a novel peptide, coop, using an ex vivo / in vivo phage display screen. this peptide homes specifi cally to the early stage astrocytoma model. in addition, the peptide homes to the u human glioblastoma xenografts (enbäck and laakkonen, unpublished data). we have studied the biodistribution and tumor homing properties of indiumlabelled peptide in mice bearing intracranial human u glioblastoma tumors using spect/ct imaging. coop peptide was conjugated with the dtpa (diethylenetriamine pentaacetic acid) via amino terminus and labeled with indium. imaging was performed in four different time points ( minutes, hour, hours & hours), and the organ and tissue specifi c radioactivity was measured after the scarifi cation of the animals. intravenously injected in-labeled coop peptide accumulated in the u braintumors. the amount of the peptide increased with time and a clear radioactive accumulation was seen in / animals at hour and in / animals at hours after injection. due to the small size of the dtpa-peptide conjugate the majority of the molecules were secreted via the kidneys during the fi rst minutes. at h timepoint the tumorto-brain tissue ratio was , . we report here that the coop peptide, which specifi cally homes to the brain tumor vasculature and tumor cells, strongly accumulated in vivo to the xenografted human glioblastoma tumors in mice. this is the fi rst time when a synthetic peptide has been used in spect imaging of brain tumors in animal mode. failure of the development of a novel peptide tracer for molecular imaging of cancer therapy response - ( ) have shown that fl uorescently labeled derivatives of the hvggssv motif accumulated in tumors undergoing therapy. this peptide motif had been identifi ed by the in vivo screening of phage-displayed peptide libraries. the ability of the peptide to differentiate between responding and nonresponding cancers after treatment could be utilized for the development of promising tracers to monitor cancer therapy. the goal of this study was to investigate whether radiolabeled derivatives of the hvggssv motif can be used for the noninvasive determination of therapy response in vivo. therefore three different conjugates were synthesized by solid phase synthesis using fmoc chemistry. the fi rst peptide was n-terminally modifi ed with the chelator dota (dota-ggghvggssv-conh ) to allow radiolabeling with metallic radioisotopes such as in or ga. a glycine linker was placed at the n-terminus to separate the peptide motif from the chelator. in order to obtain derivatives accessible to radioiodination a tyrosine was introduced at the n-terminus (h n-yggghvggssv-conh ) of the second peptide. the third peptide was biotinylated at the n-terminal amino group and bound to radioiodinated streptavidin (sa-biotin-ggghvggssv-conh ). the biodistribution was monitored by scintigraphy in mice bearing a -tumors treated with a vegf receptor-specifi c inhibitor (su ). this study revealed that the in labeled hydrophilic dota conjugate shows a rapid renal clearance, the iodinated peptide accumulates mainly in the liver and kidneys. all of the peptide derivatives do not specifi cally accumulate in treated tumors after i.v. injection. in contrast to the promising results shown by han z. et al. our study shows that the hvggssv motif can not be easily adapted to radiolabeling approaches with clinical relevance. in conclusion, the peptide motif is not attractive for further clinical evaluation as new diagnostics for the imaging of cancer response. a novel approach to improve cellular delivery of -aminolaevulinic acid: new ala-containing peptide prodrugs for photodynamic therapy. photodynamic therapy (pdt) is a binary therapeutic modality which is currently under investigation for the treatment of several kinds of malignancies. it relies on the interaction of two individually harmless components: a photosensitiser and an external radiation. the interaction of the photosensitiser with light of the appropriate wavelength and molecular oxygen results in the generation of cytotoxic species, namely singlet oxygen and/or radicals, and localized destruction of tumours or infected tissue. in -aminolaevulinic acid photodynamic therapy (ala-pdt), exogenous administration of ala is employed to generate elevated intracellular levels of the natural photosensitiser protoporphyrin ix (ppix), via metabolism through the haem biosynthetic pathway. however this approach suffers from several drawbacks associated with ala's lack of stability at physiological ph and its highly hydrophilic nature, which prevent it from crossing biological membranes and hence limit tissue penetration. ala delivery with synthetic peptide prodrugs is a promising way to address these problems. in this work we report the synthesis and characterisation of a series of peptide prodrugs of general structure ac-xaa-ala-or, where xaa is an alpha amino acid, chosen to provide a prodrug with appropriate lipophilicity and water solubility. the uptake of the compounds and metabolism to ppix in pam keratinocytes, relative to ala is evaluated by fl uorescence spectroscopy, and further quantifi ed by recovery and chemical derivatisation of intact/ partially metabolised prodrugs. in a parallel study, we have also explored the possibility of coupling of ala and ala prodrugs to cell penetrating peptides (cpp). the conjugation of one or more molecules of ala to a cpp sequence represents an interesting approach to enhanced topical delivery of ala. preliminary results of these studies are reported herein. acknowledgements: thanks are due to biotechnology and biological sciences research council (grant bbd ) bioshuttle as a carrier for temozolomide transport into prostate cancer cells if metastatic prostate cancer gets resistant to antiandrogen therapy, there are few treatment options, because prostate cancer is not very sensitive to cytostatic agents. temozolomide (tmz) as an oral applicable chemotherapeutic substance has been proven to be effective and well tolerated with toxicity especially for brain tumors. unfortunately tmz was ineffi cient in the treatment of symptomatic progressive hormone-refractory prostate cancer. this may have different reasons like the short plasma half-life of tmz, a non adapted application schema and as a result, an insuffi cient bioavailability. to improve the specifi city, we built our so called tmz-bioshuttle-construct with cathepsin b (ctsb) mrna specifi city. this complex combines, a transmembrane transporter molecule connected via a disulfi de bridge to an antisensepeptide nucleic acid (pna) against a docking site in exon of the ctsb mrna. furthermore this part is connected via a ctsb cleavable peptide substrate to a nuclear localization sequence coupled with tmz. inside the target-cell, the pna recognizes the cytoplasmic ctsb mrna and after annealing (the pna/rna hybride is not a substrate for rnase h) results in a cell-specifi c retention of the tmz in the cytosol especially of ctsb-expressing cells. then, after the cathepsin b-mediated cleavage in the cytoplasm, the nls-sequence is separated and activated for an ran/importin-mediated transport of the tmz-cargo into the nucleus of the target cells. this bioshuttle-mediated tmz transfer could be a step forward to a successful therapy with higher specifi city, avoiding adverse reactions and circumventing the previous therapy limiting situation. the intracellular delivery of protein segments and other bioactive molecules using membrane -permeable peptides has been investigated in multiple aspects. most of the currently recognized cell penetrating peptides (cpp) are of cationic nature and derived from viral, insect or mammalian proteins endowed with membrane translocation properties. these peptides enter the cell by a receptor independent mechanism, which is poorly understood and carry only one epitope. our target was to achieve a cell-penetrating carrier able to transport more than one bioactive molecule. to this aim we designed a cell penetrating sequential carrier (cpsc) formed by the repetitive -lys-aib-cysmoiety, which incorporates the cysteine residue for anchoring the bioactive molecules through a thioether bond. the lysine free side chain possesses the cationic nature of the construct while the á-amino isobutyric moiety could induce a helicoid peptide backbone with amphipathic characteristics. to test the ability of the cpsc to penetrate the cell membrane we synthesized the carboxyfl uorescein -labelled cpsc, cf-[lys-aib-cys(-ch conh )] -nh , while the cf-tat-cys(-ch conh )-nh analogue, which is a small basic peptide that has been shown to deliver a large variety of cargoes into the cells, was used as a positive control. the results indicated that cpsc had a homogeneous distribution into the cytoplasm suggesting that cpsc may provide a new powerful biological tool for drug transportation. acknowledgements: the gsrt and eu (pened ÅÄ ) for the fi nancial support. amphipathic pro-rich peptides as intracellular drug delivery systems pujals, silvia; fernandez-carneado, jimena; giralt, ernest institute for research in biomedicine, spain shared among many of the known cpps. in our laboratory described a novel group of cpps: amphipathic proline-rich peptides. the principal advantages of these compounds are non-cytotoxicity, nonviral origin and high solubility in aqueous media. the best candidate for cpp among the amphipathic pro-rich peptides was sap (sweet arrow peptide), (vrlppp) .( ) derivatives with hydrophobic moieties, such as fatty acids or silaproline, have shown highly improved internalisation effi ciency; an all d-amino acid version of the cpp sap was shown to be completely protease resistant and was evaluated in a preliminary in vivo study. cd and tem studies regarding the self-assembly properties of this family of peptides highlight the possible role of aggregated species in the internalisation process. finally, these cpps have shown to be internalised via caveolae or lipid-rafts mediated endocytosis, which circumvents the lysosomal route of degradation. in order to test a challenging cargo for sap, gold nanoparticles were conjugated to sap. thereafter, the transport of au np by sap in hela cells has been studied by tem. while np alone are not internalised, np-c-(vrlppp) are clearly uptaken. studies with qds (quantum dots) and egfp as sap cargo are currently being done in our laboratory. . pujals, sílvia; giralt, ernest. proline-rich, amphipathic cell-penetratingpeptides. addr ( ), , - . novel cysteine-rich cell penetrating peptide: effi cient uptake and cytosolic localization introduction: crossing the plasma membrane is a prerequisite for intracellular targeted drug delivery. cell penetrating peptides are actively used as the delivery tool for intracellular delivery of various cargos. however, confi nement of biomolecules into endosomes limits their use for intracellular targeting. therefore, there is a need for vectors capable of transferring cargo molecules directly into the cytoplasm. herein, we focus on the development of a novel cpp (derived from polypeptide crotamine .) which shows an effi cient uptake at low concentrations ( . m) and cytosolic distribution along with vesicular uptake. methods: series of peptides were synthesized by fmoc strategy, introducing mutations in cro ( - ) (proposed cpp sequence in crotamine). all were n-terminally labeled with fl uorescein isothiocyanate for optical imaging. structure activity relationship (sar) studies were done by substitution and/or deletion of amino acid residues in the sequence observing the uptake behaviour by fl uorescence spectroscopy and microscopy. results: amongst synthesized peptides, one of shorter length showed the best intracellular delivery and cytosolic distribution at lower concentration ( . m) when compared to other cpp. replacing or deleting cysteines had negative impact on internalization. results also displayed the involvement of tryptophans in cellular uptake indicating, along with cationic amino acids, the importance of each residue in this optimized sequence. conclusions: sar studies identifi ed a novel cell penetrating peptide showing, besides of endosomal uptake, also an effi cient delivery into the cytoplasm at low concentrations. thus, this peptide might prove useful for effi cient transmembrane delivery of agents directed to cytosolic targets. peptide nucleic acid (pna) is a dna mimic consisting of the four common bases of dna on a pseudopeptide backbone that makes it extremely stable in biological fl uids. antisense pna is targeted against mrna in cytoplasm in a sequence specifi c manner. however, the main hindrance to the effective use of pnas has been their relatively poor uptake by cells. endosomal release or direct uptake into cytosol of agents is mandatory for attaining mrna based targeting. there are reports on the cell penetrating peptide (cpp) based delivery system. it has also been reported that conjugates of cholesterol and sirnas facilitate cellular import ( ) . the aim of this study was to synthesize different sequences of cholesterol coupled antisense pna and to compare its uptake characteristics with a cpp-pna conjugate. the synthesis of pna (anti-dsred pna (agcgcctgtacc), specifi cally targeted to mrna of dsred, a red fl uorescent protein) conjugated to cpp (d-tat) or cholesterol was performed in fully automated synthesizer (prelude, protein technologies, inc.) using continuous solid phase chemistry. to increase the solubility in water, linkers (aeea) and additional charged amino acids were coupled or the sequence of peptide, pna and cholesterol was changed. all compounds were labelled with fitc to confi rm the cellular uptake by fl uorescence microscopy and spectroscopy. cell uptake studies showed that the cpp bound pna was located predominantly in vesicles indicating an endosomal uptake mechanism and subsequent entrapment in vesicles. cholesterol bound pna was also effi ciently internalized. however, it was also located inside vesicles without detectable cytosolic distribution. pna-cholesterol has fewer synthetic steps than pna-cpp. however, it was also located inside vesicles restricting its applicability for mrna targeting. the effi cient uptake might make it a promising cellular delivery agent after further improvements. opioids are gold standards in pain treatments. unfortunately, fast development of tolerance and dependence creates limitations in application of these drugs in chronic pain treatment. over twenty years ago we have proposed development of multitarget medicines as a new avenue of drug discovery. identifi cation of numerous endogenous components that participate in the formation, transmission, modulation and perception of pain signals offers various strategies for the development of new analgesics. neurotensin is an endogenous neuropeptide that play important regulatory role of pain transmission. therefore, it was interesting to develop chimeric analogues that hybridize opioid and neurotensin active components. in such chimeric compounds the possible structural interference may signifi cantly infl uence on interaction of both components with target receptors and/or biological barriers transport systems. in this communication we present synthesis, pharmacological profi le and structural analysis of new potent chimeric compounds. acknowledgements: presented studies have been supported in part with eu grant normolife (lshc-ct- - ) artifi cial ribonucleases based on short peptides. the synthesis of molecules that are capable of nonrandom rna cleavage has found a variety of important applications in molecular biology. for instance, such molecules are used as structural probes for nucleic acids in solution, or in rational design of novel anti-infectives, since rna is the genetic material of many pathogenic viruses. here we represent design and synthesis of peptide-like molecules mimicking the catalytic site of natural ribonucleases (a and t ): series f aa -aa -aa -phe -oalkyl; aa -glu/lys, aa -thr/ser/lys, aa -glu/ lys/thr/arg; series r glu -x -arg -gly -oalkyl; series k glu -x -lys -gly -oalkyl; -gly, -ala, -aminobutyric acid, aminohexanoic acid, p-aminobenzoic acid; series l aa -aa -aa -l (l )-aa -aa -aa , l - , -dioxa- , -dodecanediamine, l - , -diaminododecane; aa -aa -glu, lys, ser, arg. ability of artifi cial rnases to rna cleavage was shown in experiments with mer rna hiv- . the effi cacy of rna cleavage depends on artrnases structure (for example, on location of negative and positive charged amino acids (glu, arg lys) in peptide). the effi cacy of the most active compounds was - % at h incubation. anti-infl uenza activity in vitro of such artrnases was investigated by inhibition of reproduction virus a/hong kong/ / (h n ) in tissue cultures of chorioallantoic membranes (ccm) of - days age chick embryos. acknowledgements: this work was supported by rfbr ( - - a, - - -bel_a), pharmamed ruxo- -n - , the grant of novosibirsk region government for the best young scientists. (ziagen)-( s,cis)- -[ -amino- -(cyclopropylamino)- hpurin- -yl]- -cyclopentene - -methanol is a synthetic carbocylcilic nucleoside analogue with inhibitory activity against hiv. serious and sometimes fatal hypersensitivity reactions have been associated with abacavir. a possible way to increase the side effects is by modifying the known antiviral drugs with various amino acids. the aim of this study was to design and to synthesize of new amino acids and peptide (gly, gly -gly) and thiazole containing (gly, gly -gly) esters prodrugs of abacavir and to explore their activity on the hiv. chemical stability of some purine analogues in the search of new prodrugs effective against herpes simplex virus, series of acyclovir- -[( -hydroxyethoxy)methyl]guanine (acv) esters with peptidomimetics, as well as abacavir-( s,cis)- -[ -amino- -(cyclopropylamino)- h-purin- -yl]- -cyclopentene- -methanol derivatives have been synthesized and tested for antiviral activity. the chemical stability of some of them is studied at ph and . and temperature of °c. a high-performance liquid-chromatografi c (hplc) method was developed for quantifi cation of the unchanged ester concentration. the promelittin represents a readymade cancer pro-toxin; the prodomain inhibits cytolytic activity and the sequence of the prodomain could be manipulated into a substrate for activating proteases. here we present the development of a novel promelittin pro-toxin that can be activated by the serine protease fi broblast activation protein (fap). fap is over expressed on the surface of reactive stromal fi broblasts present in the stroma of human epithelial tumors. fap is not expressed by tumor epithelial cells or by fi broblasts in other tissues, thus making fap a pantumor target for pro-toxin development. peptides containing truncated pro-domain sequences were tested on erythrocytes to determine the optimal prodomain length for inhibiting cytolytic activity. once the length was optimized, modifi ed promelittin peptides were generated that contained previously identifi ed fap substrate sequences in the prodomain. these peptides were digested with fap and tested in vitro against fap+ and fap-cell lines in order to determine their fap cleavage potential and toxicity. our lead promelittin peptide was found to be effi ciently activated by fap and selectively toxic to fap+ cell lines with an ic value in the low micromolar range that is similar to that of melittin. pharmacology and medical peptide chemistry region of highly hydrophobic and the antigenic part of hbsag by using microwave assisted solid phase peptide synthesis (spps). tryptophan at the n terminus of the sequence was added by our research group for fluorescence dedection analysis. this peptide was conjugated with synthetic polyanions. different conjugates comparised each other. the synthesized bioconjugates analysized by choromatographic and fl uorometric methods. the fi nal product peptide was characterized by lc-ms and purifi ed by rp-hplc. the bioconjugates were synthesized using different activation mechanisms and the different initial molar ratios (npeptide/npolymer = , , , , ) of the polyanions and the peptide. also physicochemical properties of the bioconjugates were investigated. the structure and characterization of the synthesized conjugates was analyzed by various choromatographic and fl uorometric methods such as hplc, gpc and fluorescence spectrometer. the reaction of high temperature solid-state catalytic isotope exchange (hscie)( ) between insulin and spillover tritium was studied. hscie reaction with the solid mixture containing mg recombinant human insulin was performed for min at o c using % pd/baso catalyst. the product of the reaction was purifi ed by hplc on waters delta-pak c a . x mm column. tritium labeled insulin with specifi c radioactivity ci/mmol and radiochemical purity of more than % was obtained. performic acid oxidation of cysteine residues and subsequent acid hydrolysis were used to analyze the distribution of tritium. it was demonstrated that specifi c radioactivity of À and b chains amounted to . and . ci/mmol respectively. all the amino acids contained tritium and its content in his residues of the b chain was equal to %. experiments for the evaluation of the biological activity of tritium labeled insulin were performed on cd- - -week-old awake male mice. activity of labeled insulin was compared with the activity of standard insulin. insulin was injected subcutaneously at a dose of . u/kg. twofold statistically signifi cant lowering of glucose level was observed min after injection ( , + , and , + , mmol/l for standard and tritium labeled insulin, respectively). lowering of the glucose level in animals obtaining labeled insulin was the same, as in animals obtaining standard insulin. thus, hscie reaction of insulin with tritium doesn't change its hypoglycemic activity. cortexin is polypeptide (up to kda) brain extract accepted for clinical use in several countries including russia due to its positive effects on memory, attention, and cortical processes. a synthetic peptide analog of cortexin, cortagen (ala-glu-asp-pro), was recently developed. the heptapeptide semax (met-glu-his-phe-pro-gly-pro) is an analogue of the acth( - ) fragment, which is completely devoid of any hormonal activity associated with the full-length acth molecule. both cortexin and semax are currently effectively used for treatment of brain hypoxia and ischemia with comparable clinical benefi ts. however, the cellular and molecular mechanisms underlying the action in the brain are mainly unknown. in present work we studied effects of cortagen, cortexin, semax, its analogue pro-glu-pro (pgp) on glutamate-induced neuronal death and intracellular ca + homeostasis deterioration in cultured cerebellar granule cells. peptide drugs gain increased interest as a new supra-group of therapeutics between the classic-organic, small-molecule drugs and the large biotechnology-derived bio-drugs. they are used in different therapeutic areas like allergy, anti-infection, oncology, obesity, etc… due to their particular structure and biochemical origin, pharmaceutical development of a peptide drug poses special challenges which will be discussed here, exemplifi ed by own research as well as literature data. currently, there are about a dozen classical peptides described in pharmacopoeia, excluding the derived peptidomimetics like ace-inhibitors. these pharmacopoeial peptide-drugs, together with the approved marketing authorisations of new peptidic entities, are a good starting point to look at the desired characteristics. last, the regulatory developmental guidelines are in a general way seldom taken peptide drugs into account, leaving an interpretational gap between the two current main supragroups of therapeutics. after the initial active pharmaceutical ingredient (api) synthesis, analytical characterisation is aimed at integrity and purity evaluation of the api, which is also required for biomedical experiments. in this analytical characterisation, sample treatment issues like solubility and adsorption are considered as well. the chemical and plasma-metabolic stability, a critical parameter for peptides, is to be assessed to obtain kinetic and mechanistic information. functionality is tested in vitro using cell-and organ-based protocols, including ligand binding studies, as well as in vivo encompassing adme and target-organ confi rmation like brain. the pharmaceutical drugability information thus obtained allows further development decisions including required api modifi cations and proof-of-principle drug delivery formulations. lysine dendrimers and starburst copolymers as new carriers of anticancer drugs based on the complexes of platinum and gold. though the complexes of metals such as platinum and gold are very promising compounds for the treatment of different types of cancer, the low solubility complicates their application as medicines. in this work a series of lysine dendrimers of different structure and starburst copolymers of lysine and glutamic acid was studied as solubilizing carriers of anticancer drugs based on the complexes of platinum (cisplatin) and gold. cd analysis showed the small changes in the structure of the polymers upon metal binding. the esem study revealed that the presence of the amino groups on the surface of the carriers is crucial for the metal ligation. the highest content of metal bound was found in the samples with high amount of lysine residues. while the content of gold was rather low in all the samples (about %), the copolymer of lysine and glutamic acid with the ratio : as well as lysine dendrimer of fourth generation had to % of platinum linked to the carrier. the absence of the bromine in the samples in the case of gold complex allowed to assume the covalent bond between metal and polymer. in the same time all the conjugates were highly soluble in water. the peculiarities of the synthesis and the anticancer activity in different cell lines will be discussed. acknowledgements: the work was supported by the "russian science support foundation". prospects for the development of synthetic peptide vaccines against hepatitis c no vaccinoprophylaxis of this disease or cardinal treatment means has been developed till now because of the problems produced by high genetic variability and heterogeneity of hepatitis c virus (hcv) isolates, lack of both hcv infection models and effi cient expression systems for the virus and its components. the problem of anti-hcv vaccine development can be solved via a principally new approach, the creation of artifi cial synthetic immunogenic constructs with the help of bioinformatics and high-throughput screening technologies. two approaches exist towards the development of anti-hcv peptide-based vaccines: one is to develop vaccines that stimulate cytotoxic t-cell response (peptides loading dendritic cells; the so-called "cell vaccines") and the second is to construct immunogenic peptides raising protective antibodies. however, the fi rst approach may lead to the massive hepatocyte death and the development of the heavy hepatic injury during hcv infection. in order to develop peptide immunogenic constructs able to raise antibodies against whole native hcv envelope proteins analysis of amino acid sequences of these proteins belonging to different hcv genetic variants has been performed. this analysis has allowed to choose the most conserved and presumably functionally important protein fragments. immunogenicity of these fragments in the whole protein molecules has been determined and interaction sites for one of the putative hcv receptor, heparansulfate, have been revealed with the help of peptide scanning. despite of the low immunogenicity of the most fragments inside the whole protein, antipeptide antibodies against them have been obtained via immunizing by conjugates of the peptides with certain carriers. two synthetic peptide immunogenic constructs have been developed that have raised antibodies against whole hcv envelope proteins. synthetic immunoactive fragments of endogenous proteins: selection and application for diagnostics and immunotherapy we have selected and synthesized the immunoactive fragments of four endogenous proteins: nucleophosmin, survivin, prion and alpha- subunit of acetylcholine receptor (achr). nucleophosmin and survivin are overexpressed in tumor cells and exhibit an antiapoptotic activity. their detection in tumor cells could be used for tumor differential diagnostics and selection of optimal chemotherapy scheme. accumulation of pathogenic isoform of prion protein in brain causes the neurodegenerative prion diseases and antiprion antibodies as known could be used for immunotherapy of prion disorders. alzheimer's disease (ad) development is accompanied by an accumulation ofamyloid peptides ( a) in brain and destruction of neurons . one of the ad development hypotheses assumed that a when binding to the alpha- achr forms a complex that penetrates into the cells, resulting in the neurons death. we have proposed that alpha- achr could be a new target in the ad therapy and antibodies against alpha- subunit could prevent its binding to a. selected synthetic fragments of these four proteins were able in a free nonconjugated to a protein carriers state induce antibody response in experimental animals. obtained rabbit antibodies against fragments of nucleophosmin and survivin were affi nity purifi ed. antibodies against nucleophosmin detected monoand oligomeric forms of this protein in immunoblotting of hela cells lysates. antibodies against survivin were able to detect this protein in immunohistochemical test of the breast cancer tumor samples. rabbit antiprion antibodies interfered with patigenic prion isoform in scn a neuroblastoma cell culture. it was shown that synthetic alpha- achr fragments induced the antibodies formation in mice with symptoms of ad which penetrated the blood brain barrier, regenerated the spatial memory and normalized the beta-amyloid level in animal's brain. acknowledgements: supported by ras program mcb and rfbr grants - - , - - . investigation peptide-based cyclin a inhibitors: new tools to understand and to regulate the cell cyle-apoptosis signalling pathway the knowledge of the aetiology of cancer has increased considerably in the last two decades. the objectives have moved from unspecifi c cytotoxic chemotherapeutics to the identifi cation of small molecules (and antibodies) with a well defi ned molecular target. proliferation of eukaryotic cells is under control of a series of concerted molecular mechanisms defi ned as the cell division cycle whose progression is tightly governed by members of the cyclin-dependent kinase family. the protein-protein complexes formed between different cyclins and cdks (cdks) are central to cell cycle regulation. these complexes have been object of extensive research in cancer programs. considerable effort has been focused on the development of atp-competitive small molecule inhibitors of cdks. however, in general, these compounds have several alternative protein targets that compromise their demanded selectivity. cdk- was recently suggested to be dispensable for cell proliferation, although this does not appear to be the case for cyclin a, which therefore is defi ned as a more appropriate target for drug design. we have identifi ed an hexapeptide (nbi ) that inhibits the kinase activity of the cdk -cyclin a complex through selective binding to cyclin a ( ). the characterization of the inhibitory mechanism revealed that the hexapeptide does bind neither to the atp site nor to the cyclin recruitment site. a cell permeable derivative of the nbi peptide induces apoptosis and inhibits proliferation of tumor cell lines. we believe that the current structural and mechanisms of action studies on nbi will be useful for characterizing and controlling the important relation between cell cycle and apoptosis signalling pathways. the average size and size distributions of protein, polymer and peptideprotein or polymer-protein mixtures (complexes) and conjugates was investigated using photon correlation spectroscopy with a zetasizer nano zs instrument. the zeta potential measurements of this complex and conjugates were carried out in the folded capillary cell of the zetasizer nano zs instrument the same as used for dynamic light scattering measurements. in this study we investigate that size and zeta potential of synthetic peptide-carrier protein covalent conjugates. synthetic peptides are small antigenic molecules and not strong immunogens because of their small size for this reason, synthetic peptide must be coupling to a suitable carrier proteins or polymers (bsa, klh, ova) for increasing their immunogenity. most of the vaccine development studies are focused on the preparation of a good effective adjuvant. in this study we prepare carrier-protein (bovine serum albumin protein)-synthetic peptide conjugates whit carbodiimide method by using -ethyl - -( dimethylaminopropyl) carbodiimide hydrochloride (edc). for one protein molecule we studied with different ratios of peptides (npeptid/ nbsa) and synthesized the conjugates at these ratios. conjugates are purifi ed by using different column systems. purifi ed conjugates are characterized and the mechanism of the binding process was investigated comparatively with synthetic peptide, bsa and bsa-synthetic peptide physical mixture by using zeta-sizer nano zs depent on the time. an analysis of deletion mutants of the pld d domain defi nes short regions within the pld interacting with ped/pea : implications for the development of peptides-specifi c antagonist. ( ) . several studies have revealed that it regulates cellular functions by binding components of intracellular transduction pathways. recent reports also evidenced that it binds to phospholipase d (pld ) and enhances its stability, resulting in increased intracellular levels of diacylglycerol( ), deregulating protein kinase c signalling and generating resistance to insulin action on glucose transport ( ) . thus, disrupting the interaction between these proteins by a cell-penetrating compound represents a novel strategy for improving insulin sensitivity in target cells. the expression of d domain, (the shortest ped-interacting region with pld ) in l skeletal muscle cells stably overexpressing ped, reduces the interaction of this protein with pld ( ), suggesting that this region could bind ped preventing its interaction with the whole pld and restoring insulin action. aim of this work is the identifi cation of d crucial residues for ped interaction and the development of specifi c antagonists. we expressed three soluble truncated d domains, determining whether binds to ped like the d wild type, by carrying out dose-response elisa. only one of these regions exhibited an effi cacy similar to d -wt and functional cellular data support this evidence. to further investigate the d residues involved in ped binding, we prepared a set of overlapping peptides covering the d -region identifi ed. our results suggest that the n-terminus region of d encompassing residues - is involved in ped/pea recognition and, currently, cellular experiments on peptides are underway. ew-peptide family. one family, two drugs. two dipeptide analogs -l-glu-l-trp ( ) and -d-glu-d-trp ( ) -had been registered in russia as immunomodulator thymogen ( ) and immunosuppressor thymodepressin ( ). these two drugs possessed reciprocal activities in vivo [ , ] . the individual peptides were initially separated by preparative hplc from the crude thymus homogenate and sequenced. dipeptide l-glu-l-trp was the most active in the majority of in vitro and in vivo tests. in the process of sar studies, the "signal" role of l-glu-l-trp in the immune response was discovered, as well as the critical role of manifestation of in vitro effects as well as the infl uence of precise chemical and optical structures on biological activity of different ewpeptide analogs will be discussed. the experiments were conducted on blood neutrophiles, monocytes, thymic epithelial cells, lymphocytes, thymocytes, endothelial cells of human blood vessels and newborn blood cord. we evaluated the peptide effects on the phagocyte activity of cells, on the expression of functionally important membrane molecules and cell activation markers, and on the capacity of cells for mutual adhesion. the dose dependence data of and on such activities as cytokine production, the processes of stimulation and suppression of apoptosis and proliferation of immunocompetent cells will be presented. ion exchange chromatography (iec) is widely used for analysis and purifi cation of biomolecules. we have newly developed polymer-based iec column, named ymc-biopro, specially designed for separation of proteins, peptides and nucleic acids. ymc-biopro iec columns are based on micron @porous and non-porous hydrophilic polymer beads with low nonspecifi c adsorption, and they show higher binding capacity and higher recovery of biomolecules compared to conventional iec columns. the completely spherical and monodispersed beads, with optimal packing technology, provide high theoretical plate number and symmetrical peak shape. excellent resolution is achieved from the high column effi ciency coupled with the excellent selectivity of qa and sp ion exchangers. in this poster, we will show benefi ts of ymc-biopro iec columns and some example cases of superior separation of important biomolecules, such as monoclonal antibody and dna. the analysis of proteins and peptides by rp-hplc is important in the development of well-characterized biotechnology pharmaceuticals. since polypeptides interact only slightly with the stationary phase after desorption, short columns are suited for fast separations. also, by decreasing the particle size of hplc packings, column effi ciency is increased. we demonstrate the use of short hplc columns packed with . um, large pore c silica for ultra-fast biomolecule analysis. due to the short column format, high fl ow rates and optimal separations are possible, with moderate backpressures (< psi), using a conventional high-pressure gradient, binary pump system. narrow-bore lc and lc-ms analyses were performed on a binary or a quaternary hplc system, with detection by uv or ms (ab/mds sciex q trap). the solvent system comprised of acn/water or n-propanol/water plus one or two ion pairing agents (formic acid, tfa, hfba) at . to . %. a fl ow rate of . ml/min ( cm/h), x higher than typical for a . mm id column, allowed for ultra-fast one minute separations of proteins with broad physicochemical characteristics. closely related insulin variants (bovine, sheep, human) may be separated in under . minutes. while the typical hplc run time for the separation of hemoglobin chains on conventional columns ranges from to minutes, runs under two minutes may be realized with the large pore, sub-two micron column. accordingly, fi ve different hemoglobin samples (from different animal species) may be compared after a total of only minutes (includes run and equilibration time). highly reproducible runs of a synthetic peptide mixture can be completed in < minutes, including equilibration with column volumes between runs. intact igg in sheep serum may be separated from albumin in < min. using a n-propanol/water solvent system containing . % tfa. new peptides and triterpene derivatives as dimerization inhibitors of hiv- protease protein-oligopeptide fragmentomics zamyatnin the substantiation and defi nition of the term "fragmentomics" is given. within the framework of this scientifi c direction the theoretical structural-functional analysis of all possible fragments of protein molecule can be performed with the purpose of determination of its sites which could be potential sources of the regulatory oligopeptides. the data on the primary structure of proteins from public protein databases, information from the erop-moscow database ( ) that contains data on the structures and functions of the natural oligopeptides, and special computer program complex were used for it. as a result the natural regulatory oligopeptides both representing exact structures of protein fragments and containing protein fragments were found. the method has allowed also to reveal new potentially active sites of protein amino acid sequence yet not investigated experimentally. it was shown that different fragments of the food protein molecules are involved in amino acid sequences of many natural antimicrobial oligopeptides, toxins, neuropeptides, and hormones. these results confi rmed deep relationship between the basic regulatory systems. in connection with the obtained data, the process of oligopeptide biogenesis, the possibility of natural formation of regulatory oligopeptides from different protein molecules, formation of exogenous oligopeptides pool, and correspondence of the obtained results with the conception of oligopeptide continuum are discussed. a possible practical importance of active fragments of proteins in regulatory processes of a living organism is noted. the binding of peptides containing tyrosine residue to -cyclodextrin cyclodextrins form inclusion complexes with various organic molecules. aromatic amino acid residues bind to -cyclodextrin with deep penetration of the cyclodextrin cavity. in case of oligopeptides binding depends on the peptide conformation. the formation ofcyclodextrin inclusion complexes with the tyrosine residues within three peptides was investigated using steady-state fl uorescence spectroscopy and molecular dynamic simulations. the free energy along the reaction pathway delineating the inclusion of tyrosine's aromatic ring into -cyclodextrin was computed using umbrella sampling molecular dynamic simulation. the association constant and the corresponding association free energy were derived by integrating the potential of mean force over a representative ordering parameter. the three peptides studied consist of eighteen amino acids and the tyrosine residues are located at the position , or . selected sequences are fragments of notch receptors. (notch =ykieavqsetveppppaq, notch =tlsyplvsvvsesltper, notch =pyplrdvrgepleppeps). out of three peptides only notch binds strongly to -cyclodextrin with binding constant similar to that of actyrnhme. the binding of cyclodextrins with phenolic compounds involves nonspecifi c van der waals and hydrophobic interactions and depends on accessibility of tyrosine sidechain. role of carboxyl groups in the secondary structure and function of sturgeon gonadotropin free negatively charged carboxyl groups were selectively modifi ed (neutralized) in sturgeon (acipenser güldenstädti br.) gonadotropic hormone (gth) and subunits. carboxyl groups, in and in subunit, were neutralized by the reaction with glycine ethyl ester. investigation of re-associated -dimers (recombinants) comprising one or both modifi ed subunits showed that specifi c hormonal activity was completely lost while immunoreactivity was lowered in comparison with that of the standard -dimer. cd-spectroscopy of the modifi ed subunits did not indicate considerable changes in their spatial structure. conclusion was made that free cooh-groups of gth are important as bearers of the negative charge necessary for the hormone activity on the level of the hormone-specifi c membrane receptors. wagner, nathaniel; dadon, zehavit; yishay, eliya; ashkenasy, gonen ben gurion university of the negev, israel living cells can process rapidly and simultaneously multiple extracellular input signals through the complex networks of evolutionary selected biomolecular interactions and chemical transformations. recent approaches to molecular computation have sought to mimic or exploit various aspects of biology. a number of studies have adapted nucleic acids and proteins to the design of molecular logic gates and computational systems, while other works have affected computation in living cells via biochemical pathway engineering. we described recently the graph structure and experimental analysis of a self-organized synthetic peptide network ( - ). the system was designed rationally to operate in neutral aqueous solutions based on sequence selective auto-and cross-catalytic template-directed coiled-coil peptide fragment condensation reactions. here we show that such de novo designed synthetic networks can also mimic some of the basic logic functions of the more complex biological networks. consequently, we describe experimental and simulation studies that highlight the possibility of segments of the networks to express all sixteen two-input boolean logic functions ( , ) . many studies deal with the folding of double helices in nucleic acids. in contrast, much lesser attention is given to double helices in peptides. nevertheless, the investigation of peptides with alternating l-and d--amino acids, like gramicidin a, shows various double helix patterns with antiparallel and parallel arrangements of the strands. in this study, we want to give a systematic overview on the possibilities of double helix formation in -peptides on the basis of ab initio mo theory. according to general principles, double helices with characteristic intermolecular hydrogen bonding patterns were generated and verifi ed as minimum conformations at the hartree-fock level employing the - g* basis set. the calculations show several types of antiparallel and parallel double helices with different stability, which is strongly infl uenced by backbone substituents. the dengue virus causes a spectrum of clinical symptoms, ranging from mild dengue fever to the severe forms of dengue hemorrhagic fever and dengue shock syndrome. there are four dengue virus serotypes (den - ). the dengue virus ns protease is an attractive target for development therapeutics against dengue. the aim of the study was to investigate the prime side specifi city of dengue virus ns protease substrates using proteochemometrics, a new technology for drug target interaction analysis. a set of internally quenched peptides were designed using statistical molecular design (smd), synthesized and assayed with proteases of four subtypes of dengue virus for michaelis (k m ) and cleavage rate (k cat ) constants. the obtained data were subjected to proteochemometrics analysis, concomitantly modeling all peptides on all the four dengue proteases, which yielded highly predictive models for both activities. the interpretation of the models suggested that considerably differing physico-chemical properties of amino acids (aa) contribute to k m and k cat activities. it was found that for k cat activity only p ' and p ' prime side residues played important role, while for k m all four prime side residues, p '-p ', were important. high cleavage rate (characterized by k cat ) was obtained for peptides having small aa (ser, gly, ala) at the p ' position and small or fl exible aa at the p ' position. for high affi nity (low k m ), at the p ' position aa should be small and moderately hydrophilic, while acidic residue is highly unfavorable. for the p ' position, the most favorable was trp; however, this position allowed a broader diversity of aa. for the p ' position, cys was more benefi cial than the aa present in native cleavage sites of the dengue polyproteins. for the p ' position, the best affi nity would be obtained with ala, gly or cys. the models may be used to modify each p' substrate position to optimize separately substrate affi nity and cleavage rate for den - proteases. identifi cation of novel bioactive peptide sequences from human proteins for the development of potential therapeutics many protein-protein interactions are facilitated by the binding of peptides recognition modules to short linear motifs within the target proteins's primary sequence. therefore, synthetic peptides based on parent sequences of human proteins containing functional motifs are useful tools to uncover protein signaling and interactions. to date, peptide studies typically derive sequences from a single identifi ed protein or (at the other extreme) screen random combinatorial peptides, often without knowledge of the signaling pathways targeted. the objective of the novel bioinformatic approach presented here was to determine whether rational design of oligopeptides specifi cally targeted to potentially signaling-rich juxtamembrane regions could identify modulators of human platelet function ( ) . the aim of this project was to identify peptides that span residues strongly conserved in the corresponding proteins in other species (orthologues), but that differ from those in related human proteins (paralogues). synthetic selection rules to avoid unfavorable amino acid combinations and excessively hydrophobic peptides were devised to reduce the risk of by-products during synthesis, postsynthetic degradation and solubility problems. high-throughput in vitro platelet function assays of fatty acid-modifi ed cell-permeable peptides corresponding to these regions identifi ed many agonists and antagonists of platelet function. the combined bioinformatic and experimental screens of human protein subsequences can therefore be used to validate functions of candidate proteins and provide templates for the development of potential therapeutics. denessiouk, konstantin; denesyuk, alexander; johnson, mark s. Åbo akademi university, finland many small molecule ligands (or parts thereof) with important roles in the biochemistry of the cell are recognized by different proteins with different cellular functions. these proteins do not necessarily share a common fold since their three-dimensional structures can differ completely such that they are considered to be unrelated proteins. surprisingly, we see on numerous occasions that similar ligands are often recognized in a very similar way by means of a common structural motif formed from main-chain elements of several consecutive amino acids within the ligand-binding site of the proteins. these short polypeptide segments can be found in many protein folds, and variations in their structure can often explain different elements of protein function. in particular, we have characterized the presence of a nucleotide binding motif present in more than protein folds that function in part to recognize the adenine ring system in many essential biological ligands (e.g. atp, nad(p), fad, fmn, coa, etc.). furthermore, a study of unrelated folds among pyridoxal phosphate dependent enzymes led to the characterization of a c nn structural motif for protein recognition of phosphate ions common to fold-representative protein structures that belong to different folds. interestingly, amino acid side chains play little or no role in ligand recognition, explaining the evolutionary independence of such binding mechanisms (i.e. the motifs are found in unrelated folds), while possibly refl ecting the types of interactions that were characteristic of the earliest protein-ligand interactions during early stages of molecular evolution. bioinformatics meets medicine: structure-based peptide design as a basis for drug development here, we present different structure-based approaches on the design of peptides mimicking the whole surface, a specifi c region of a protein or even tumor markers. the potential of such methods stretches from the development of peptide-derived drug candidates to immunological screenings. mimicry of binding sites: we have developed superficial, a program that proposes peptide libraries representing the entire surface or just regions of proteins. these peptides can be synthesised, e.g. using the spot-synthesis technique, and tested for their ability to mimic linear as well as non-linear binding sites. as a proof of principle, a library of peptides representing the surface of lysozyme was generated starting from a crystal structure of a lysozyme-hyhel- complex. adjacent sequence segments were linked by spacers to conserve local conformations. disulfi de bonds were generated to tether the peptides. binding assays against the hyhel- antibody identifi ed several peptides representing the non-linear recognition site of the lysozyme. translation of a tumor marker into peptides: the tumor-associated carbohydrate antigen gd is an established target for immunotherapy in neuroblastoma. we proposed peptidic mimics of this ganglioside for active immunisation against the tumor marker gd using molecular modelling. the ability of these peptides to mimic the carbohydrate moiety was verifi ed with in silico docking experiments. in a next step they were successfully tested as mimotopes in a mouse model. design of switchable peptides: photo-switchable compounds are becoming increasingly popular for a series of biological applications. goal is the reversible photo-control of structure and function of biomolecules. a required death domain for the binding of proapoptotic proteins (e.g. bak) to the hydrophobic groove of anti-apoptotic proteins is the bh helix. inserting the photo-reactive compound hemithioindigo into this short peptide, stabilization towards proteolytic degradation is achieved. the identifi cation of peptides that bind to antibodies is an important step in characterizing antibody specifi city in order to study molecular recognition. numerous approaches have been developed to select peptides that bind with desired specifi city and affi nity to antibodies of interest. in our study peptide arrays produced by spot technology were used to develop and optimize binders to antibody derived by mutations from the anti-p (hiv- ) single chain fv antibody, cb - . three mutations were introduced in the complementarity determining region of the light chain being in close proximity with epitope-homologous peptide. this mutated antibody had completely lost its affi nity for the epitope-homologous peptide. a substitutional analysis of epitopehomologous peptide was performed on cellulose, in which all positions of the peptide sequence were substituted by each of the naturally occurring amino acids. the peptides representing the binding activity in spot membranes were synthesized using solid phase synthesis and their binding activity was confi rmed by fl uorescent polarization method. a substitutional analysis indicated that binding to mutated antibody could be restored and improved by combining favourable substitutions at the n-and c-terminal residues of the epitope-homologous peptide. this approach has allowed us to generate binders from non-binders to mutated antibody in high micromolar range. it is well known that physiological regulatory peptides that act as hormones and neurotransmitters are produced by the specifi c cleavage of their precursor proteins that per se have no biological functions. during these processes, many fragmented peptides are also produced from the same precursor proteins. it is expected that they may have various unexpected biological activities, but their biological functions have not been investigated in depth. the neutrophil-activating peptides we recently purifi ed turned out to be the peptides that are cleaved from mitochondrial proteins by proteolysis, suggesting that fragmented peptides produced by maturation and degradation of functional proteins may also have various biological functions [ , ] . therefore, we named such functional cryptic peptides hidden in protein sequences cryptides, and those cryptides that are derived from mitochondrial proteins "mitocryptides" in particular ( ). we then identifi ed many mitocryptides by the combined investigation with "dry" and "wet" experiments, i.e., the sequences of mitocryptides that activate g proteins were predicted based on the distribution of charged and hydrophobic residues and their activities were examined on granulocytic cells. receptors for these peptides were also characterized by the direct cross-linking experiments between peptides and their targeted proteins. moreover, it is demonstrated the presence of a novel signaling mechanism involving mitocryptides. the comprehensive identifi cation of cryptides is expected to lead to the elucidation of various cryptic signaling mechanisms. the -benzoylphenylalanine (bpa) residue is widely used as a photoaffi nity label for the study of intermolecular (peptide) ligand-protein (receptor) interactions, where it is thought to function by hydrogenabstraction from met residues followed by covalent c-c bond formation of the resulting radical pair. we are carrying out detailed studies of this reaction in a series of fi ve, structurally rigid, hexapeptides of general sequences boc-u x bu y mu z -ome and boc-u x mu y bmu z -ome, where b = l-bpa, u = aib, m = l-met, and u x +u y +u z = , with a view to determining the effects of spacer length (u y = - ) on the rate of the intramolecular excited state reaction and the chemical and d-structures of the resulting products. the triplet state lifetimes of the bpa residues in the compounds, determined in a deoxygenated, dilute, acetonitrile solution by laser fl ash photolysis, vary in the following order: ubu mu, = ns; umu bu, = ns; ubumu , = ns; ubmu , = ns; and ubu m, = ns. in addition to the information these data provide on the structural requirements for intramolecular excited state quenching in the molecules, they also serve to defi ne the conditions necessary for optimal intramolecular reaction in preparative photolysis experiments. accordingly, the products resulting from ubu mu and ubumu have been prepared in high yields, isolated, and structurally characterized by hplc, mass spectrometry, nmr, and cd techniques. for each of the two photoinduced macrocyclizations, two diastereomers, arising exclusively from the bpa diradical regioselective attack on the met s-methyl group, were found. gattin, zrinka; van gunsteren, wilfred eth, switzerland during the past decades, the dependence of the secondary structure of peptides on their amino-acid sequence has been the focus of numerous investigations. in addition to naturally occurring peptides based onamino acids, peptides based on -amino acids, have raised particular interest because of their potential for pharmaceutical use. these peptides often have high folding propensities and it has been found that peptides with as few as four residue may fold into a stable secondary structure. -peptides offer the possibility to investigate the folding-unfolding equilibrium in the context of very small systems, therefore they have also been the subject of a number of investigations based on atomistic molecular dynamics (md) simulations which revealed that the foldingunfolding equilibrium typically occurs on time scale of nanosecond to tens of nanoseconds. how the backbone bound heteroatoms (oh, nh ,f) infl uence the structure of a peptide is little known by now, both experimentally as well as theoretically. we performed several md studies on the conformational behavior of centrally placed hala( -met) fl uoro and hydroxy analogues, hala( -f) and hala( -oh), as function of chirality and level of substitution to investigate the infl uence of these factors on secondary structure formation and stability. all -peptides were simulated in methanol solution at temperature of k and a pressure of atm using the gromos biomolecular simulation software and the gromos a force fi eld. the ensembles of trajectory structures were analyzed in terms of conformational space sampled by the peptide, folding behavior, structural properties such as hydrogen-bonding and in terms of the level of agreement with the available experimental nmr data, noe's and j-coupling constants. the nitroxide spin-labelled , -cyclic amino acid poac was synthesized, resolved and its absolute confi guration assigned (k . wright et al., tetrahedron, , , - ) in order to be used as a spin-probe to evaluate the -helicalpeptide secondary structure. a series of -hexapeptides, b o c -a c p c -p o a c -p o a c -a c p c -a c p c -a c p c -o m e , b o c -a c p c -p o a c -a c p c -p o a c -a c p c -a c p c -o m e , boc-acpc-poac-acpc-acpc-poac-acpc-ome, and b o c -a c p c -p o a c -a c p c -a c p c -a c p c -p o a c -o m e , based on the ( r, r)-poac enantiomer, combined with ( s, s)-acpc for confi gurational homogeneity of the amino acid components, was designed. in these hexapeptides two poac residues are incorporated at positions i, i+n (n = - ) to observe conformation-related spin-spin interactions. the peptides were synthesized by n-to -c chain elongation of n -boc protected peptide segments in solution. their conformational analyses, performed by cd, ft-ir absorption and esr spectroscopic techniques, will be also described. computational design has been successful in creating new proteins. we focus on the design of a -sandwich protein which is challenged by general problems as h-bonds between neighboring strands being dependend on a tightly packed hydrophobic core and aggregation. an improved version of our program propac was tested to repack the backbone of several natural protein structures with high reproducibility. starting with backbone coordinates we found amino acid sequences by packing amino acid side chains with defi ned conformations (rotamers) into the core of -sandwich proteins. in a fi rst approach we have assembled four synthetic -hairpins on a cyclic peptide template (tasp) to form a -sandwich with two identical four-stranded antiparallelsheets. improvement of surface residues reduced the aggregation of the proteins. spectroscopic analysis shows a fold with a free energy near - kj/mol and confi rms the -structure by cd and ftir. we improved the design to a partial asymmetric -sandwich assembled from three different -hairpins. the synthesized molecule was the fi rst -sandwich molecule showing a defi ned fold in d-nmr. this data and results from the symmetric betabellins ( ) suggest that symmetrical sheets do not fold into defi ned structures. to synthesize completely asymmetric sheets we simplifi ed the synthesis of -sandwiches by directed coupling of two four-stranded antiparallel -sheets. the computed proteins were selected for tight packing of the hydrophobic core and analyzed for a stable fold during dynamic simulations. one of our goals is to fi nd a correlation between the experimentally determined protein stability and the parameters used for the selection of the residues in the hydrophobic core and at the surface. biocatalyst-catalyzed peptide synthesis using inverse substrates as acyl donor sekizaki previously we reported that the p-amidinophenyl esters behave as specifi c substrates for trypsin and trypsin-like enzymes. in these esters the site-specifi c group (charged amidinium) for the enzyme is included in the leaving group portion instead of being in the acyl moiety. such a substrate is termed an inverse substrate ( ) . inverse substrates allow the specifi c introduction of an acyl group carrying a non-specifi c residue into the trypsin active site without recourse to a cationic acyl moiety characteristic of conventional substrates. we also showed in an earlier study that inverse substrates were applicable to enzymatic peptide synthesis. therefore, a general method for the preparation of a variety of inverse substrates would be valuable. we designed two series of new type inverse substrates, p-(amidinomethyl)phenyl and m-(amidinomethyl)phenyl esters derived from n-(tert-butyloxycarbonyl)aminoisobutylic acid, were prepared. we also analyzed the kinetic behavior of trypsin towards these synthetic esters ( ) . they were found to be readily coupled with an acyl acceptor such as l-alanine p-nitroanilide to produce dipeptide. the optimum condition for the coupling reaction was studied by changing the organic solvent, ph, and acyl acceptor concentration. the optimum standard condition was selected as follow: acyl donor (inverse substrate), mm; acyl acceptor (l-alanine p-nitroanilide), mm; enzyme, m; % dmso-mops ( mm, ph . , containing mm cacl ); Ž. an -aminobutyric acid containing dipeptide was obtained in high yield. streptomyces griseus trypsin was a more effi cient catalyst than the bovine trypsin. it was found that the enzymatic hydrolysis of the resulting product was negligible. peptides derived from nk- selective for phosphatidylserine as novel cancer agents chemotherapeutic agents, commonly used as anti-cancer drugs, have severe side effects, also affecting healthy human cells. natural antimicrobial peptides, and their derivatives, have gained interest as potential anti-cancer agents. under normal conditions, due to the asymmetric distribution of plasma membrane lipids across the bilayer, mammalian cells comprise phosphatidylserine (ps) only in the inner leafl et. in the case of malignant transformation inner leafl et ps can move to the outer leafl et and act as a surface marker. the surface exposure of negatively charged ps on various tumour cells makes these cells susceptible to killing by cationic membranolytic peptides such as nk- ( ).the aim of this study is to develop short peptide sequences still acting selectively towards ps exposed on cancer cells without damaging healthy cells. in order to use this new strategy with ps-specifi c peptides it is necessary to analyze the lipid composition of mammalian cancer and non cancer cell membranes. further as a basis for peptide activity studies the biophysical characteristics of cancer cell membranes and healthy counterparts with respect to lipid composition were determined by investigation of liposomal mimics composed of phosphatidylcholine and/or phosphatidylserine by dsc and x-ray. these model systems were also used to screen the activity of a series of peptides derived from nk- . fluorescence spectroscopy was applied to test the release of fl uorescence marker molecules from liposomes composed of solely ps, pc or pc/ps mixtures in the presence of various concentrations of peptides. data revealed that some nk- derived peptides have a high affi nity towards ps causing signifi cant leakage of liposomal content, whereas healthy mammalian cell mimicking pc liposomes were not affected. optimized peptides resulting from these experiments will be used for in-vitro studies on prostate cancer cell lines. in most moths, the sex pheromone production is regulated by pheromone biosynthesis-activating neuropeptide (pban), a - amino acid neuropeptide that is released from the subesophageal ganglion into the hemolymph in response to physiological and environmental cues. pban exerts its pheromonotropic effects by binding to pbanr, a member of the rhodopsin-like family of g protein-coupled receptors (gpcr) that is predominantly expressed in the pheromone-producing cells of the female pheromone gland. the structure-activity relationship studies for bombyx mori pban have revealed that the shortest peptide with pheromonotropic activity is the c-terminal pentapeptide-amide, pban( - )-nh (fsprl-nh ), and that the c-terminal amide group is required for the pheromonotropic activity of pban. in this study, we have analyzed the solution structures of the c-terminal decapeptides derived from bombyx mori pban with an amidated and a free c-termini by two-dimensional nmr. the pheromonotropically active decapeptide-amide, pban( - )-nh (srtryfsprl-nh ), and its inactive counterpart, pban( - )-oh (srtryfsprl-oh), were dissolved in three kinds of solvents: ( ) mm sodium phosphate buffer (ph . )/ mm nacl/ . % nan in %(v/v) h o/ %(v/ v) d o (buffer a), ( ) mm dodecyl phosphocholine (dpc)-d in buffer a, and ( ) %(v/v) , , -trifl uoroethanol (tfe)-d in buffer a. the nmr data indicated that these decapeptides did not adopt specifi c conformations in buffer a, but they adopted specifi c conformations in the presence of dpc micelles or tfe. these peptides exhibited different chemical shifts for some protons in all the solvents used, and they took similar but different conformations in the presence of dpc micelles. the conformational difference between these peptides may refl ect their difference in pheromonotropic activity. structuring and membrane interactions of the human antimicrobial cathelicidin ll cathelicidins are a family of vertebrate host defence peptides with both a direct capacity to inactivate microbes and to modulate components of the innate and adaptive immune systems. they are characterized by a conserved pro-region carrying individual, highly variable antimicrobial sequences, that become active only after proteolytic release, and with quite different structural and aggregational features that lead to differential biological effects on prokaryotic or eukaryotic cells. ll- is the only human cathelicidin, and displays a broad-spectrum, medium sensitive antimicrobial activity in vitro that is accompanied by some cytotoxicty towards eukariotic cells at antimicrobial concentrations, and a strong capacity to modulate host immune and healing processes at lower concentrations. these activities likely all involve interaction with biological membranes at some point, and are related to its amphipathic, helical structure and strong tendency to aggregate in specifi c conditions. the latter is an evolved feature absent in some primate orthologues, whose activities appear more limited to a direct antimicrobial action. the structural and aggregational behaviours of ll and selected primate orthologues were systematically investigated by biophysical and biochemical methods. these included cd spectroscopy in different buffers, sds micelles or anionic or zwitterionic luvs, transmission ftir spectroscopy, dye release from liposomes, and atr-ftir spectroscopy on supported lipid monolayers, followed by atomic force microscopy to probe morphological effects on lipid order. data was also collected from antimicrobial, cell lysis and cytofl uorimetric assays, giving a more complete picture of the possible mode of action of these helical peptides, and highlighting the importance of biochemical parameters such as structuring and dimerization/oligomerization processes in the selective interaction with biological membranes, leading to cytotoxic or immunostimulatory effects. oligomeric structure of fowlicidin- , an antimicrobial/ anti-endotoxic peptide from the family of cathelicidin, in lipopolysaccharide bilayer a recurring theme in the structure-function correlation studies of the cationic antimicrobial peptides (amps) is that the formation of oligomeric structures in lipid environments by amps. self-assembly of amps may result disruption of the membrane (outer/inner) structures of the microorganisms by forming pores or ion channels. however, high-resolution oligomeric structures of amps in lipid environments are diffi cult to obtain. to-date, oligomeric structure at atomic resolution of any antimicrobial peptide has not been reported. secondary structures of amps are usually obtained in perdeuterated sds or dpc micelles, as a mimic to the inner cytoplasmic membrane, by nmr spectroscopy. cathelicidins comprise a major family of host-defense antimicrobial peptides in vertebrates. these peptides are synthesized as a part of large precursor proteins containing a well conserved cathelin domain and an extremely variable, in terms of length and amino acid compositions, cterminal region. the c-terminal part of the cathelicidins is bestowed with antimicrobial and lps neutralizing activities. here, we report a tetrameric structure of a -residue active fragment of fowlicidin- or vk , one of the fi ve cathelicidins found in chicken, in lipopolysaccharide by nmr spectroscopy. the tetrameric structure of the vk determined from trnoe is highly helical. a large number of trnoe cross-peaks were observed connecting residues far apart in the sequence. calculated structures reveal an anti-parallel arrangement of four helices. the interface of the tetramer appears to be rich in polar or charged residues delineating a plausible pore or channel. most of the non-polar residues are found to be exposed indicating plausible interactions with non-polar fatty acyl chains of lps or with membrane in general. the oligomeric structure of vk peptide may be useful to understand structure/activity relationship, in particular pore formation, by the helical antimicrobial peptides. the interaction of the antimicrobial peptide nk- with different lipid systems antimicrobial peptides are important components of the natural defense system of most living organisms against invading pathogens. these are relatively small (below kda), cationic and amphipathic peptides of variable length, sequence and structure. in this study, the antimicrobial peptide nk- , which is a amino-acid residue derivative of the cationic core region of nk-lysin, has been used. the used membrane mimetic model systems have different dimensionality: two-dimensional (monolayers at the air-liquid interface) as well as three-dimensional (vesicles, micelles) systems of different phospholipids. the aim of this study was to investigate the infl uence of peptide-lipid interactions on the lipid structure and vice versa on the secondary structure of the peptide. the peptide secondary structure was measured by cd (circular dichroism spectroscopy) in bulk and irras (infrared refl ection-absorption spectroscopy) at the air-liquid interface. the structure of lipid langmuir monolayers has been examined by numerous techniques such as pressure-area isotherm measurements, fl uorescence and brewster angle microscopy and x-ray techniques. charged as well as zwitterionic phospholipids have been used to study the adsorption of nk- . the peptide adsorbs at the air-buffer interface due to its amphiphilic character. it also inserts into uncompressed phospholipid monolayers. the peptide reorients from random coil in bulk to -helix lying fl at at the interface. the incorporation of nk- into a dppg monolayer leads to a different orientation (either -helix with an oblique orientation or random coil) and to the fl uidization of the aliphatic chains. nk- infl uences also the structure of ordered dppg domains. to assess the location of the peptide nk- in the lipid matrix, specular x-ray refl ectivity studies were carried out. wadhwani, parvesh; reichert, johannes; buerck, jochen; ulrich, anne s. karlsruhe institute of technology, germany antimicrobial peptides (amps) constitute an essential part of innate immunity and act against an external microbial invasion where as cell-penetrating peptides (cpps) can carry a biologically functional molecule through the cell membrane and are relevant in drug delivery developments. fusogenic peptides (fps) are active on membraneinterfaces and are instrumental in fusion of membranes which is vital for various fertilization and viral processes. membrane fusion properties of short amps and cpps have been seldom tested and have therefore eluded the attention of most investigators, instead only their cytotoxicity is investigated. there is general lack of understanding if these peptides are fusogenically active. we have investigated the ability of amps and cpps to trigger membrane fusion. as an example, hiv- -fusion peptide fp is used as benchmark to compare fusion activity of various amps and cpps. most fps are believed to be unstructured or conformationally fl exible ( -helix or -sheet); therefore the secondary structure of the peptides before and after the fusion reaction is investigated and correlated to their respective ability to execute membrane fusion. our results show that various amps and cpps which are capable to switch their secondary structure are also able to promote fusion to an extent that is even higher than the known hiv- fusion peptide fp . these results will be presented in the poster. interaction of the minimal active peptide sequence of human growth hormone releasing factor with negatively charged liposomes zschörnig, olaf; thomas, lars; weigelt, heiko; köhler, guido university of leipzig, germany the most bioactive peptides such as hormones and neurotransmitters do not exist in an ordered structure in aqueous solution. the conformation of the peptide changes from this fl exible unordered structure into an inherent one, only when it reaches a biomembrane. that's why it is important to investigate the such peptides like growth hormonereleasing factor (ghrf) in the presence of lipid bilayers. human ghrf is an amidated peptide consisting of amino acids residues. a lot of studies has show that the residues n-terminal part are the active core of the peptide. we studied the interaction of human ghrf ( - ) with phospholipid membranes. the interaction of ghrf ( - ) with the liposomes was investigated fl uorescence spectroscopy. to detect a fl uorescence signal the peptide were labelled at fi rst, th, th and th position with dansyl. the fl uorescence intensity of ghrf ( - ) at different lipid-peptide-ratios were analysed using a model, which includes the hydrophobic and the electrostatic interaction between the peptide and the phospholipid membrane. the hydrophobic binding constant of ghrf( - ) and its effective charge were determined using this model. further the peptides position in the membrane were investigated using spin labelled phospholipids, which are able to quench fl uorescence over large wavelength arrays. the quenching effi ciency will decrease by increasing the distance between fl ourophore and spin probe. the use of mol% tempo-pc, -doxyl-pc, -doxyl-pc and -doxyl-pc in the phospholipid composition of the liposomes allows the determination of the peptides membrane penetration depth. all results indicate, that the binding of ghrf (aa - ) to phospholipid membranes is increased strongly by increase of the surface charge density of the liposomes. the peptide is arranged parallel to the membrane surface and is localized in membrane-water-interface of the liposomes. folding propensity and biological activity of selected antimicrobial peptides bozzi, argante ; di giulio, antonio ; aschi, massimiliano ; rinaldi, andrea c. university of l'aquila, italy; university of cagliari, italy the innate immunity of multicellular organisms relies in large part on the action of antimicrobial peptides (amps) to resist microbial invasion. crafted by evolution into an extremely diversifi ed array of sequences and folds, amps do share a common amphiphilic -d arrangement. this feature is directly linked with a common mechanism of action that predominantly (although not exclusively) develops upon interaction of peptides with cell membranes of target cells. it is generally agreed that amps are essentially unstructured in the aqueous phase and fold upon contact with the membrane, adopting an amphiphilic fold. this favours absorption of peptides onto lipid bilayer and their subsequent integration into the membrane with expansion of the outer leafl et, which in turn leads to membrane thinning and permeabilization. however, recent observation suggest that things might be more complicated than previously believed. indeed, md simulations coupled to cd spectroscopy, studies with model membranes, and antimicrobial assays, have shown that, at least for some peptides, a signifi cant correlation exists between the conformation adopted by the peptide in solution, i.e. before the interaction with membranes, and its antimicrobial activity. the linear peptides we have studied following this approach include two members of the frog skin-derived temporin family, namely temporin a and temporin l, and two members of amphibian bombinins h, i.e. bombinins h and h . we observed that the presence of a partially folded structure in water solution may facilitate, both thermodynamically and kinetically, the peptide folding in the microbial membrane, and thus favour biological activity. looking for such built-in conformational characteristics could well help to rationalize the different spectrum and level of activity recorded for cationic alpha-helical amps on membraneenveloped targets, and assist the design of improved analogs and biomimetic synthetic peptides with antibiotic properties. investigation and computational modelling of antimicrobial peptides linser the uprising resistance of pathogenic bacteria against treatments with conventional antibiotics emerged an acute search for alternatives. one class of promising alternatives are naturally occurring antimicrobial peptides. we present a comparative study of computational modelling of peptide properties with structural characterization of the interaction and antibacterial or haemolytic activity of three peptides (nk-cs, nkcs-[lp] and nkcs-[aa]). we compared computational interaction models with measurements of antibacterial and haemolytic activity, small angle x-ray and surface plasmon resonance data, and structure predictions by circular dichroism (cd). all peptides were active against escherichia coli (gram negative) and staphylococcus carnosus (gram positive) bacterial cultures, but the haemolytic properties against human red blood cells were found to be poor and indicated the peptides' selectivity. cd studies of the peptide secondary structure confi rmed the prediction of peptide helicity. the antibacterial activity can be correlated with a change of the hexagonal phase transition temperature of -palmitoyl- -oleoyl-snglycero- -phosphoethanolamine (pope) as determined by small angle x-ray scattering (saxs). the calculated peptide membrane affi nity is not related in linear way with the antibacterial activity. the reason for this might be aggregation as shown by surface plasmon resonance. the inverse hexagonal phase transition temperature was increased by the peptides and this promotes a positive curvature of the membranes. we assume that this curvature fi nally leads to the disruption of the model membranes. in summary an over all helical structure, electrostatic and hydrophobic parameters as well as strong amphipaticity are good measures to describe antibacterial peptide interaction. kier, brandon; andersen, niels university, united states over the last decade, -hairpins have emerged as excellent model systems for probing the intrinsic stabilities of portions of -sheet structure and for studying the sequence dependence of the folding dynamics of -strand association and alignment. for many decades, it has been established that peptide helices can be substantially stabilized ( - kj/mol) by starting the sequence with an n-cap (acetyl, asp or sede). no comparable strategy has appeared for reducing the end-fraying of -hairpins. fully folded models of hairpins have typically been constructed by conversion to a cyclic system: inserting another turn favoring sequence (e.g. d-pro-gly, pg) at the end of the hairpin strands. we now report a set of favorable through-space interactions that can serve to cap -hairpins. the discovery followed from nmr studies of ac-w-ixgk-wtg (x = p, n). these studies indicated a favorable face-to-edge (fte) w /w interaction which also allows for a g -hn to w indole ring h-bond and the sequestration of the thr hydroxyl by h-bonding to the acetyl. these interactions produce spectroscopic diagnostics: a cd exciton couplet together with extreme upfi eld shifts for he of the c-terminal trp ( . - . ) and hn of the c-terminal gly ( . - . ppm). these feature disappear when x = l-pro and the gly-hn shift returns to its coil value (as in ac-wtg) upon removal of the n-terminal ac. in its most favorable application, ch ch co-wipglwtgps, we were able to establish that dgu at k was . kj/mol ( . % folded) by backbone hn h/ d exchange protection measures. we now report that these interactions are retained in longer hairpins. peptides of the general formula, ac-w-(zz)ningk(zz)n-wtg (n = , , or ) display enhanced fold stability. in each case, the ac is essential to prevent end-fraying and to elicit the full set of chemical shift diagnostics of the " -cap". the unique structural features of polyproline peptides to adopt an extended, relatively rigid polyproline ii (ppii) backbone conformation in aqueous solution, has led to their widespread application as a "molecular ruler" in biological and biophysical investigations. in the left handed ppii helix the peptide bonds show the all-trans conformation resulting in an interterminal distance, which increases by . to . Å per proline residue. the same backbone conformation has been identifi ed in important proline rich protein-protein recognition motifs involved in the regulation of physiological processes like transcription, cell growth, cytoskeleton rearrangement and postsynaptic signal transduction via modular sh , ww or evh domains. to investigate the structural features of polyproline and proline rich sequences by fl uorescence resonance energy transfer (fret) and other spectroscopic methods, these peptides were labelled n-and c-terminally with different fl uorescent dyes. for synthesis of homopolymeric polyproline peptides with a sequence length of more than amino acids a fragment condensation strategy has been applied to prevent the occurrence of shorter side products which can perturb distance measurement by fret. spectroscopic analysis of these peptides indicated that even for short polyproline sequences (< residues) there is a remarkable deviation from the expected ppii conformation. fret measurements revealed that polyproline peptides show an increase of the mean interterminal distance of about . Å per proline residue. single molecule fret measurements of polyproline peptides with a length of amino acids point to a heterogeneous ensemble of different conformations caused by randomly distributed cis conformations resulting in diverse species with different interterminal distances. similarly the structural consequences of the integration of non-proline amino acids in a polyproline sequence have been investigated by fret. the unusual helix stability of a vegf mimetic peptide understanding helix stability and formation is a prerogative to elucidate mechanism of protein folding and design helix peptide with specifi c activity. peptide helix is a simple model system in which various contributions to helix formation can be dissected and understood qualitatively. many strategies have been pursued to design peptide helices and notable results have been achieved even with very short sequences, but mainly these methods rely on the use of non natural amino acids or introducing constraints. in this communication, we report the stability characterization, via cd, nmr and md studies, of a designed, -helical, -mer peptide, composed only of natural amino acids, which activates the vegf-dependent angiogenic response ( ). this peptide shows an unusual thermal stability whose structural determinants have been determined. two factors, the n-terminal region and an hydrophobic interaction i, i+ , are found as playing a mayor role of this remarkable stability ( ) . these results could have implication in the fi eld of protein folding and in the design of helical structured scaffolds for the realization of peptides to be applied in chemical biology. adiponectin, a cytokine secreted by adipose tissue, which has been shown to affect lipid and glucose metabolism, attracts interest because it is a target for therapeutics in the metabolic syndrome. the molecule has a tendency to associate to form various multimers. although this multimer formation could modulate its biological function, the details of this mechanism are still unclear. thus studies on this system from the aspect of molecular assembly are interesting. the molecule consists of three domains, i.e. a variable (v), a collagen-like (c) and a globular (g) domain. the structure of the g domain determined by x-ray crystallography showed that it exists as a trimer but the remaining c and v domains have not been well characterized. we synthesized various parts of the molecule, c, g, vc, cg, and full length vcg in e. coli. the cd spectra of cg and vcg showed a positive peak around nm which is the hallmark of collagen structure. this may be attributed to the repeats of typical collagen type triplet, x-y-gly. the temperature dependency of these cd spectra showed the three states conformational changes of these peptides. the lower transition, where the positive peak disappeared corresponds to the melting of collagen like structure and the higher one corresponds to that of globular structure of the g domain. the apparent molecular weights determined by ultra-centrifugal analysis showed that these peptides exist in trimeric form at the intermediate state. however, neither c nor vc showed such transitions. these results showed that, contrary to our expectations, the contribution of the triplet of x-y-gly is not the dominant one for stabilizing the trimeric state. in order to explore the stabilizing factor, we are carrying out various experiments, such as mutation analysis, titration of ca ++ , redox reaction of disulfi de bonds and so on. one result is that ca ++ was shown to be a factor for increasing the thermal stability of the trimer. are v mutants of amyloidogenic protein -human cystatin c more or less resistant to denaturation conditions? jankowska, elzbieta; orlikowska, marta; radulska, adrianna; szymanska, aneta university of gdansk / faculty of chemistry, poland cystatins are natural inhibitors of cysteine proteases -enzymes widely distributed in animals, plants and microorganisms. human cystatin c (hcc) has been also recognized as an amyloidogenic protein directly involved in formation of pathological fi brillar aggregates which deposit in the brain arteries of elderly persons causing cerebral amyloid angiopathy ( ). our studies were performed to explore possibilities of preventing this lethal disease. the overall d architecture of monomeric human cystatin c is not known but its fold could be anticipated from the structure of the dimer which has been already determined ( ) . the dimeric cystatin c is created through the exchange of 'subdomains' between two molecules ( d domain swapping) and consists of two identical subunits in great extent reconstituting the fold of the monomeric chicken analogue of hcc. it is possible that similar mechanism is involved in cystatin c oligomerization and fi brilization process. the most signifi cant structural changes during dimerization process are observed in the l loop ( - , qivag). this loop occurred to be a hinge region in the d domain swapping event. with the aim to check implications of greater or decreased stability of this loop for dimerization and aggregation propensity of human cystatin c, we designed and construct hcc l mutants with val residue replaced by asp, asn or pro, respectively. the structural studies of these mutants and their thermal denaturation process have been performed by means of cd and ftir spectroscopies. results of these studies will be presented. azobenzene-mediated photomodulation of a collagen triple helix monitored by ir spectroscopy ( ), our most recent efforts were addressed to the design and synthesis of a photoswitchable collagen triple helix. by replacing in suitable positions of the ac-(gly-pro-hyp) -nh a pro and hyp residue with ( s)mercaptoproline, respectively, a side chain-to-side chain crosslinking of the collagen peptide was afforded with a purposely designed azobenzene derivative. as expected from modeling studies, self-association of the modifi ed collagen model peptide into a stable triple helix was observed with the trans-azobenzene clamp, while its photoisomerization to the cis isomeric state leads to unfolding processes as well assessed by nmr structural analysis ( ) . unfolding pathways can be studied by comparing ftir difference spectra induced by temperature or light. we found the photomodulation of the triple helix to be reversible and the effi ciency of the photoisomerization increased with temperature reaching a maximum value shortly below the melting point. ir spectroscopy was thus used to identify the optimal temperatures required for structure destabilization at suffi cient extents to enable unfolding by the weak driving force of the azobenzene clamp. the results confi rmed the correctness of the design and the ability of the azobenzene switch to photocontrol this complex tertiary structure, thus allowing for time-resolved monitoring of the triple-helix unfolding process. studies on various collagen model peptides (x-y-gly) where x and y are often imino acids, pro or hyp r ( (r)-hydroxyproline), have shown that hyp r at the y position plays an important role in stabilizing the collagen triple helix. however substitution of non-natural (s)-hyp (hyp s ) at both positions decreases the thermal stability of triple helix as (pro-hyp r -gly) exists in a stable triple helical state, whereas (hyp s -pro-gly) and (pro-hyp s -gly) are in a single coil state. similar effects on the stabilities of triple helices are provided by the substitution of fl uoroproline. however there is an exception that (fpro s -pro-gly) takes a triple helix at c whereas the counterpart, (hyp s -pro-gly) , is in a single coil state. although the difference could be explained by the steric hindrance of hydroxyl group in s confi guration, it is still controversial how much this hindrance could obstruct the triple helix formation. therefore, even though apparently the tripeptide with the hyp s -pro-gly sequence is not capable of triple helix formation, we could expected that (hyp s -pro-gly) n forms a triple helix as n increases. the transition temperature of (pro-pro-gly) n was shown to have the chain length dependency. here, we synthesized (pro-pro-gly) n and (hyp s -pro-gly) n (n= , ) and investigated their thermal stabilities. the cd spectra of (hyp s -pro-gly) showed the conformational transition from collagenlike triple helix to random coil with the melting temperature at c. the apparent molecular weight, determined by the sedimentation equilibrium method, showed that (hyp s -pro-gly) exists as trimer at c and as monomer at c. the melting profi le was also clearly detected by dsc. @thus, it is concluded that the existence of hyp s at the x position is not essential to interfere the triple helix formation. we are on course to investigate the stabilizing mechanism of the collagen structure. the infl uence of o-glycosylation on the folding and stability of -helical coiled coil peptides glycosylated proteins have been shown to play a key role in many biological events like cell-cell communication, immune response, cell adhesion, intracellular targeting, protease resistance, and many other processes. recently, there is a growing interest in the effect of glycosylation on the secondary structure of proteins, because of the association with the so called conformational diseases that arise from the dysfunctional aggregation of proteins in not native conformations. in this study we used -helical coiled coil based peptides as model systems to investigate the effects of serine-linked -galactose on both the secondary structure and amyloid formation tendency. thehelical coiled coil structural motif consists of two to seven -helices which are wrapped around each other with a slight superhelical twist. its simplicity and regularity have made it an attractive system to explore fundamentals of protein folding.( ) the importance of the coiled coil motif is obvious with the amount of % of all amino acid residues in the protein data bank being part of a coiled coil motif. at fi rst we examined to which extent and at which positions a glycosylation is possible without destroying the coiled coil structure. therefore, we systematically incorporated one to six serine-linkedgalactose units into several solvent exposed positions of a amino acid long coiled coil peptide. the hydrophobic dimerization face was not modifi ed. the preformed l-ser(ac --d-gal)-oh building blocks were introduced by convenient solid-phase synthesis following the fmocstrategy. folding and stability of the glycopeptides were monitored by cd spectroscopy. the amyloid formation tendency was investigated by a tht fl uorescence assay. aging, associated with decreasing protein homeostasis (proteostasis) capacity and increasing oxidative stress, is a prominent risk factor for amyloid diseases. alzheimer fs disease (ad), which is one of the most common amyloid diseases, involves intra-and extracellular amyloid formation by the amyloid bata peptide (abeta). however, how abeta can aggregate in vivo even though its physiological concentration (pc) is much lower than its critical concentration (cc) for aggregation is a mystery. we have proposed that covalent modifi cation of abeta by small molecule oxidation products can explain how abeta can form amyloid at pc. the aldehyde-bearing cholesterol oxidation product ( ), which can modify abeta by schiif-base formation, is an example of the products that could affect ad onset. however, signifi cant questions about the modifi cation of abeta by ( ) persist, including: does modifi cation by ( ) lower the cc of abeta aggregation into the pc range? and, is abeta modifi ed by ( ) able to aggregate at low concentrations on a biologically relevant time scale? in this study, these questions are answered by studying chemically synthesized analogs of abeta that are site-specifi cally modifi ed by ( ) at asp , lys , or lys . modifi cation at the different sites has a similar effect on the thermodynamic propensity for aggregation. in contrast, the effect of metabolite modifi cation on aggregation kinetics depended strongly on the modifi cation site. abeta modifi ed at lys formed amorphous aggregates fastest and at the lowest concentrations. in contrast, the appearance of thiofl avin-t positive aggregates at higher concentration was fastest for abeta modifi ed at asp( ). the infl uence of modifi cation site on the nature of the aggregates suggests that amorphous aggregation and fi brillization place different conformational demands on abeta. furthermore, these studies may partially explain how abeta can aggregate at nm pcs when the cc of unmodifi ed abeta is in the ƒÊm range. code, christian; domanov, yegor; kinnunen, paavo k.j. antimicrobial peptides are ubiquitous in nature and consist of several families of cationic amphiphilic peptides. they partition into lipid membranes and promote the segregation of anionic phospholipids ( , ) . we have shown several antimicrobial peptides, e.g. temporin b and l, indolicidin, and magainin to activate secretory phospholipase a (pla ) ( , ) and we concluded that this could represent synergistic action of these peptides/proteins in defense against microbes. the fact that the sequences of these amps are very different suggest rather non-specifi c mechanism to be involved. to pursue the latter in more detail we used förster-type resonance energy transfer (fret) between labeled pla and temporin b. interestingly, fret coincides with concentration dependant activation of pla hydrolysis of dipalmitoylphosphatidylcholine (dppc) liposomes. accordingly, temporin b and pla interact forming a supermolecular complex terminating into amyloid-like fi brils ( ). homo-oligomeric fi bers are formed on a slower time scale when pla interacts alone on dppc liposomes ( ) . a general mechanism of peptide induced pla activation forming heterooligomeric cofi brils is suggested. human islet amyloid polypeptide forms lipid-encased amyloid fi brils on supported lipid membranes domanov, yegor; kinnunen, paavo university of helsinki, finland islet amyloid polypeptide (iapp) forms fi brillar amyloid deposits in the pancreatic islets of langerhans of the patients with type diabetes mellitus and its misfolding and aggregation are thought to contribute to -cell death. increasing evidence suggests that iapp fi brillization is strongly infl uenced by lipid membranes and, vice versa, the membrane architecture and integrity is severely affected by amyloid growth. we performed direct fl uorescence microscopic observations of the morphological transformations accompanying iapp fi brillization on the surface of supported lipid membranes. within minutes of application in submicromolar concentrations, iapp caused extensive remodelling of the membrane including formation of defects, vesiculation, and tubulation. the effects of iapp concentration, ionic strength, and the presence of amyloid seeds on the bilayer perturbation and peptide aggregation were examined. growth of amyloid fi brils was visualized using fl uorescently labelled iapp or thiofl avin t staining. two-colour imaging of the peptide and membranes revealed that the fi brils were initially composed of the peptide only, and vesiculation occurred in the points where growing fi bres touched the lipid membrane. interestingly, after - hours of incubation iapp fi bres became "wrapped" by lipid membranes derived from the supported membrane. progressive increase in molecular level association between amyloid and membranes in the maturing fi bres was confi rmed by förster resonance energy transfer (fret) spectroscopy. the possible role of lipid wrapping in stabilization of iapp amyloid in vivo is discussed. fibrinogen-derived peptides that mediate cell adhesion: structure and activity studies interaction of human islet amyloid poly peptide with phospholipid membrane vesicles dannehl, claudia; zschörnig, olaf university of leipzig, germany amylin, also known as human islet amyloid polypeptide (hiapp), is a -residue peptide, suspected to play a major role in the malfunction of insulin secretion in diabetes mellitus type ii. co-secreted with insulin in the beta-cells, hiapp, in higher rates destroys the barrier function of the beta-cells, leading to a failure in insulin production. because of its amyloidogenity, aggregates of fi brils can be observed in the islands of langerhans to indicate its overexpression. we studied the physico chemical properties of hiapp by observing changes in its structure depending on time and the surrounding media using maldi-tof-ms, atr ft-ir-spectroscopy. to understand the process of penetration and toxicity to cells, we performed leakage measurements of carboxyfl uoresceine containing phospholipid large unilamellar vesicles by means of fl uorescence spectroscopy. moreover we determined membrane binding of dansyl-labeled hiapp. at physiological ph value, hiapp is positively charged and thus negative charges at the phospholipid membrane surface accelerate the process of peptide folding. being random coil as initial state, a mixture of anti-parallel betasheet and alpha-helices emerges in time. in the presence of negatively charged phospholipids, hiapp aggregates can be seen within a few minutes after titration. also in absence of any free charges, as seen in water, fi brils grow and after an incubation for hours at °c, some alpha-helices are twisted and after two weeks, no random coil is detected anymore. titration of hiapp to carboxyfl uoresceine fi lled liposomes, showed different results concerning equilibrium time and maximal extent depending on age and preparation of the peptide. in particular the composition of the vesicles seems to determine their stability in the presence of hiapp. nanoparticle induced folding and fi bril formation of a coiled coil peptide nanoparticles present large surface areas and are capable to catalyze fi bril formation of peptides. ( ) . one assumes that the nature of surface controls which of the peptides will interact with the nanoparticles and that an enhanced local peptide concentration reduces the lag-time for aggregation. in previous studies, we reported the design of a coiled coil peptide that can adopt a random coil, -helical structure, and -sheet folding in dependence of ph and peptide concentration. ( ) here, we expose this peptide to au nanoparticles that are either negatively or positively charged. the interaction of peptide and nanoparticle was investigated by cd and uv/vis spectroscopy, gel electrophoresis, and transmission electron microscopy. we found that electrostatic interactions with au nanoparticles can affect the peptide folding resulting in more than one secondary structure, namely, a competition between the -helical and -sheet structures. the latter folding is very likely a consequence of the high local peptide concentration on the surface of nanoparticle that facilitates a -sheet fi brillation of peptide. moreover, several factors such as ph, peptide concentration, and size of the nanoparticle have a strong infl uence on the nanoparticle-mediated folding. these results provide valuable information on pharmaceutical applications of nanoparticles and will help to reduce possible adverse effects. antimicrobial peptides are synthesized by all living organisms as a part of their innate immune system. those molecules are endowed with a broad spectrum of activity against pathogens. they can be divided into two classes differing in the mechanism of killing: membrane disruptive antibiotics cause a dysfunction of the membrane and subsequent cell lysis; non-membrane disruptive peptides are focused on the intracellular targets. in both situations the initial stage consists in the interactions with the cytoplasmic membrane, in most cases without the exploitation of any receptors. a non-receptor type of interactions decreases the possibility of development of microbial resistance and makes peptides a very potent alternative to conventional antibiotics. in the face of the reduced effi ciency of traditional drugs there is an urgent need for the design of new therapeutic agents with the optimized activity. monte carlo simulations of peptide-membrane interactions can be one of the strategies. that method takes in the account biophysical properties of a membrane as well as structural and physicochemical nature of peptides. in our work we are focused on the development of new analogues of nkcs, a derivative of potent antibiotic peptide nk- . in the present study the outcome of monte carlo simulations is compared to experimental results of antibacterial tests performed against escherichia coli. the insight into the molecular mechanism of peptides activity is obtained in vitro using saxs method and artifi cial systems mimicking a bacterial cytoplasmic membrane. the results indicate that monte carlo modelling is a good tool to predict the peptide -membrane interactions and can be very useful for the design of novel antibiotics. a - peptide: structural features in membrane mimicking systems amyloid (a ) peptides, the hallmark of alzheimer disease, depending upon conditions, undergo conformational transition from soluble monomers to the highly toxic -sheet oligomers which form the mature fi brils. conformational and biological analyses were carried out on several different a fragments, to understand the role of the single residues in the fi brillation process. a - , is considered the shortest fragment exhibiting large -sheet aggregates and retaining the toxicity of the full-length peptide. we recently reported the conformational analysis of the synthetic a - under several solution conditions, and analyzed the modulation of the conformational behaviour of a - peptide, by interaction with nicotine-like molecules. a - is the -mer a peptide, composed of the a - fragment, and additional n-terminal residues, known to be endowed with fi bril disaggregating activity. a - encompasses part of hydrophobic ( - ) and hydrophilic ( - ) a -regions. due to the amphipathic character of this fragment, common to the full a - and a - , a tossicological mechanism involving a -peptide-membrane interaction may be hypothesized. on these bases we decided to perform the structural investigation of the fragment a - in membrane mimicking systems including micelle and vesicles aggregates, differing for composition and structural complexity. high resolution three dimensional structure was solved by nmr spectroscopy in negatively charged sds and in dpc/ sds mixed micelles. fluorescence and epr spectroscopies were used to monitor the peptide lipid interaction. the data agree on the critical role played by the membrane composition on the stabilization of different conformers, potentially driving to different oligomeric and/or polimeric toxic species. machan, radek; jurkiewicz, piotr; benda, ales; hof, martin j.heyrovsky institute of physical chemistry, czech republic charge and hydrophobicity are important determinants of membrane activity of antimicrobial peptides. the model peptide lah whose charge can be easily controlled by ph of the medium ( ) is a very convenient tool to study the relationship between physical properties of peptide molecules and their membrane activity. it was shown that the changes in the charge of lah molecules result in changes in their orientation with respect to phospholipid bilayers ( ). in the present study, effects of lah on the properties of phospholipid bilayers at different ph values are studied by several methods. its infl uence on the lateral mobility of lipids within an spb and on the stability of suspensions of large unilamellar vesicles were characterized by fl uorescence correlation spectroscopy, showing evidence of peptide induced aggregation of lipids at basic ph. changes of hydration and local viscosity within the bilayer following changes in orientation of peptide molecules were measured by solvent relaxation technique. the effect of phospholipid charge was also taken into account in each case. several bioactive peptides, such as antimicrobial, cell-penetrating or fusogenic peptides, exert their biological function by interacting with cellular membranes. therefore, structural data on the location of these molecules inside lipid bilayers are very important for a detailed understanding of their mechanism of action. fluorescence spectroscopic methods are particularly suited to the study of peptide-membrane association, but give only low-resolution information on peptide position in the lipid bilayer. molecular dynamics simulations, on the other hand, can provide a very detailed picture of the peptide-membrane interaction, but need to be validated by quantitative comparison with experimental data. we applied several fl uorescence approaches, together with md simulations, to the investigation of two antimicrobial peptides: the lipopeptaibol trichogin ga iv, and pmap- , a member of the cathelicidin family. to perform the spectroscopic studies, a variety of peptide analogues containing a single fl uorophore were synthesized. fluorescence spectra, depth-dependent quenching experiments, and peptide-translocation assays were employed to determine the location of the two peptides inside lipid bilayers, in particular as a function of peptide/lipid ratio. molecular dynamics simulations were performed by a "minimum bias" approach, starting from a random mixture of water, lipid and peptide, and following the spontaneous self-assembling of the lipid bilayer. the fi nal membrane-bound d-structure is in quantitative agreement with the position of the fl uorescent labels determined by depth-dependent quenching experiments. for both peptides investigated, the atomic details of md simulations provide new insights on the mechanism of membrane destabilization. acknowledgements: with the support of the ministry of education, university and research, and of foreign affairs of italy. cavaco-paulo, artur university of minho, portugal surfactant proteins (sp-) are found in lungs of mammalians. most surfactant proteins have a carbohydrate binding domain (crd) and neck domain. crd have defense function in mammalian mucosa's, by eliminating microbes, due to strong binding to sugars in the cell walls. neck domains are found to order phospholipids allowing the exchange of oxygen between the alveoli and blood. x-ray structures indicate that neck domains are alpha-helixes, but circular dicroism indicates that in the presence of phospholipids, those peptides present a disordered structure. neck domains with residues from natural sequences of sp-a, sp-b, sp-c, sp-d have been tested to be delivered in lipophilic media. only disordered structures would have the ability to cross the lipid barriers. the results obtained here indicated that neck domains possibly can be used as carriers to deliver molecules across skin and hair. fraternal twins ! -peptides and oligoureas are isosteric, isostructural foldamers endowed with yet distinct biomolecular recognition properties. in the fi eld of peptidomimetics, there has been a sustained interest towards the design of non-natural oligomeric backbones with new folding patterns. over the past years, the amide linkage has become the quintessential motif to elaborate folding oligomers. aliphatic and aromatic oligoamides (peptoids, -, -peptides) have provided numerous helical-folded structures, many of which have shown interesting biological activities ( ). interestingly, substituting urea for the ch -co-nh units in the -peptide backbone represent a spectacular case of isosteric and iso-structural replacement. detailed nmr studies of the resulting oligoureas revealed a helical fold very similar to that reported for the cognate -peptides. defi nitive confi rmation of this isostructural relationship came with the recent x-ray structure determination of the canonical . -helix of oligoureas. how such isosteric and isostructural oligoamide and oligourea backbones compare in biomolecular recognition events is an interesting question that we attempted to address in the present work. notably the two systems were compared for their antimicrobial activity, membrane interaction and disruption properties. both -peptides and oligoureas designed to mimic globally amphiphilic alpha-helical host-defense peptides have been synthesized and tested. the results showed a surprising dichotomy in bactericidal activity between the two isostructural systems and enlightened the unique antibacterial of amphiphilic oligourea helices. to question whether this functional difference results from differential membrane disruption activities, we have undertaken detailed physicochemical investigations using negatively charged phospholipid membranes as model systems. understanding of the cellular uptake of an amphipathic cell penetrating peptides complexed with sirna konate, karidia; divita, gilles; heitz, frédéric crbm umr -cnrs, france the effi ciency of delivery of macromolecules into living cells is very important for therapeutic purposes. the discovery of a class of peptides, known as cell-penetrating-peptides (cpps), with the ability to mediate translocation of various cargoes both in vitro and in vivo has provided new perspectives in the fi eld of delivery. we have designed a new secondary amphipathic cpp, caddy, which can transfect sirna. many studies have tried to elucidate cpp internalization mechanisms and there is currently still much controversy between endocytosis phenomena and direct interaction with membranes. however, elucidating the interactions between peptides and lipid membranes is essential to understand how caddy delivers cargoes in cells. to highlight these interactions, we have applied biophysical method to phospholipids monolayer and bilayer as model plasmic membranes. regarding the increase of the phospholipids monolayer surface pressure in presence of caddy, it is obvious that this peptide have high affi nity with lipids. while cholesterol presence does not induce anything in peptide insertion on a phospholipids monolayer, the cargo and a widely studied partner of the internalization: heparan sulfate of the gag' s family, do not induce the same behaviour. the intrinsic probe of caddy, tryptophan residues, and the fitc probe bound to the cargo allowed to follow and identify interactions between these two entities by fl uorescence spectroscopy. adding a bilayer vesicular solution unsettle these interactions, tryptophan residues interact with phospholipids while fitc probe are less embarrassed by peptides. caddy alone in solution is not structured but cd measurements show a typical spectrum of helical conformation in presence of phospholipids vesicles. and when the peptide complex its cargo, cd spectrum show a contribution of the sirna relevant of a conformational change inside both partners interacting together. high membrane coverage as the basis of antimicrobial peptide activity the interaction of the antimicrobial peptides (amps) omiganan (h-ilrwpwwpwrrk-nh ) and bp (h-kklfkkilkyl-nh ) with model bilayers was characterized. the activity and selectivity of these peptides could be attributed to a strong preference towards anionic membrane model systems, which mimetize bacterial membranes. regarding the interactions with bacterial membrane models, there were marked differences in the interaction patterns, as well as in functional properties of the peptides at high peptide:lipid ratios. these differences occurred for both peptides, despite their being unrelated in sequence and in occurrence in nature. such events at high membrane coverage could represent the equivalent at the molecular scale of the conditions at which the antimicrobial activity of the peptides is triggered. although the lipid: peptide ratios at these transitions are lower than phospholipids per peptide molecule, the plausibility of this hypothesis was demonstrated taking into account an estimate of the amount of lipid per bacterium, and the bacterial concentration in minimum inhibitory concentration (mic) assays. according to the partition constants obtained towards bacterial membrane models, these peptides are expected to reach, at the mic, precisely those high concentrations in the membrane. in addition, surface charge neutralization was shown to occur in these conditions. activity at high membrane coverage is thus likely not only for these peptides but also for any peptide displaying high membrane affi nity and micromolar mics, which is common amongst amps. biosensors based on artifi cial membrane system that permits the functional reconstitution of transmembrane receptors have been studied for the past decade. immobilized liposome encapsulates intracellular chemicals in the internal water cavity and provides a potential advantage over supported planar lipid membrane. to prepare a stable liposome adlayer, we have proposed an immobilizing method based on small-peptide modifi cation of solid surface. in the present study, we investigated kinetics of liposome adsorption on the surface-bound synthetic peptides which have several alanine, lysine and tryptophan residues as amphiphilic segment with a cysteine at terminus. the quartz crystal microbalance studies showed that the peptide sequences can be divided into two groups according to liposome-size dependence of langmuir adsorption constant. while the initial adsorption processes could be satisfactorily described by simple langmuir adsorption kinetics, the amounts of liposome adsorbed irreversibly were increased as time advances. the results from afm reveal that most of the liposomes are bound to peptidic surfaces as single fl attened particles without fusion together for several hours. we next performed the detection of ganglioside gm -lectin interaction on the liposome surface. all the binding constants and binding amounts, as observed by qcm, were fairly consistent with each other and and showed the effectiveness of our approach. herce, henry rensselaer polytechnic institute, united states recently we have proposed theoretically an energy independent pathway for the uptake of cell penetrating peptides (cpps) that challenges fundamental concepts associated with protein membrane interactions, h. d. herce and a. e. garcia, pnas, , ( ) . this mechanism involves strong interactions between cpps peptides and the phosphate groups on both sides of the lipid bilayer, the insertion of charged side chains that nucleate the formation of a transient pore, followed by the translocation of cpps peptides by diffusing on the pore surface. this mechanism explains how key ingredients, such as the cooperativity among the peptides, the large positive charge, and specifi cally the arginine amino acids, contribute to the uptake. we will describe the details of this mechanism and present novel experimental results that directly validate the model. a comparative studies on lipid affi nity of cell penetrating peptides in presence or absence of cargo a growing number of natural and /or synthetic peptides with cell membrane penetrating capability have been identifi ed and described in the past years. these molecules have been considered as targeting structures for the delivery of bioactive compounds into various cell types. although the mechanism of uptake is still unclear, it is reasonable to assume that the relative contribute of each proposed mechanism could differ for the same peptide, depending on experimental protocol and cargo molecule composition. in this work we try to connect the capability to interact with model lipid membrane of cpp and their structural and chemical characteristics in order to obtain a biophysical classifi cation that predicts the behavior of cpp-cargo molecule in cell system. indeed, the interaction with cell membrane is one of the primary step in the interaction of cpp with cells, and consequently the studies on model membrane could become important for understanding peptide-membrane interaction on a molecular level, explaining how cpps may translocate a membrane without destroying it and how this interactions come into play in shuttling cpps via different routes with different effi ciency. we analyzed by fl uorescence spectroscopy the binding properties of six different cpps (kfgf, antp and tat derived peptides, and oligoarginine peptides containing , or residues) in absence or presence of the same cargo peptide (the [ - ]ptyr fragment of hs protein). the binding properties were correlated to the conformational and chemical characteristic of peptides, as well as to the cell penetrating properties of the cpp-cargo conjugate. results show that even if certain physico-chemical properties (conformation, positive charge) govern cpp capability to interact with the model membrane, these cannot fully explain cell-permeability properties. great deals of data show that alzheimer's pathology lead to the loss of neuronal functionality and synaptic plasticity. these effects seem to be the consequence of a toxic effect of a peptides related to their ability to modify cell membrane homeostasis. in particular, a peptides, as full length or in fragments, being in oligomeric or polymeric form, could alter membrane fl uidity and compromise its functionality. we have previously demonstrated that the - fragment of a peptide a ( - ) is able to penetrate into the outer leafl ets of the membrane. furthermore, it is able to alter membrane fl uidity changing membrane response to cholesterol, and thus affecting its ability to form low fl uid membrane regions named lipid rafts. a ( - ) is considered the shortest fragment exhibiting large -sheet aggregates and retaining the toxicity of the full-length peptide. flavonoids are a group of naturally occurring, benzo--pyrone derivatives, ubiquitous in plants. they are endowed with tumor prevent activity and they act as antioxidants through a membrane mediated molecular mechanism involving the membrane ion transport. on the basis of the common ability of fl avonoids and a peptide of altering membrane fl uidity, we carried out an epr spectroscopic analysis of several fl avonoids -specifi cally quercetin, naringenin, rutin and naringin -in , -dioleoyl-sn-glycero- -phosphocholine (dopc) vesicles. the modifi cations of the dopc physio-chemical environment were analyzed in presence of fl avonoids and beta-amyloid fragment a ( - ). our results indicate that the addition of fl avonoids induces a decrease in membrane fl uidity. this effect is retained in presence of a ( - ). thus fl avonoid compounds could be able to antagonize the effect induced by amyloid peptide on membrane fl uidity and in this respect they could be therapeutically useful as neuroprotective agents. analyse lipid peptide interactions of p and lipo-p with membrane mimicking represented by micelles and vesicles. nmr structures of p and lipo-p in mixed sds/dpc micelles are reported; the positioning of p and lipo-p peptides with respect the lipidic surface is analyzed using -doxyl stearic and -doxyl stearic acid. epr analysis is carried out in the zwitterionic dimyristoyl phosphatidylcholine and the anionic dimyristoyl phosphatidylglycerol vesicles using several different lipid spin labels. both peptides bind to lipid bilayers and trp residues and lipidic chain of lipo-p show a important role in the process of peptide adsorption onto the membrane, to exert its antiviral activity. studies on human cell membranes were necessary to further establish the role of membranes in these peptides mode of action. this interaction was assessed by evaluating the effects that these peptides have on the membrane dipole potential of human erythrocytes, using the fl uorescence probe di- -anepps. in the presence of enfuvirtide or t- , a decrease in the di- -anepps fl uorescence excitation ratio dependent of peptide concentration was observed. these results show that t- has ten times more affi nity to the erythrocyte membrane than enfuvirtide, a factor that can be associated with the adsorption of t- on cholesterol rich membranes observed on the studies with membrane model systems. moreover, as a fraction of hiv associates with erythrocytes in vivo, these cells can have a role in delivering these peptides to the viral surface. the improved clinical effi ciency of t- relative to enfuvirtide may be related to its higher partition coeffi cient and ability to adsorb to rigid lipid areas on the cell surface, where most receptors are located upon membrane fusion. moreover, adsorption to the sterol-rich viral membrane helps to increase the local concentration of the inhibitor peptide at the fusion site. the platelet receptor iib plays a critical role in the process of platelet aggregation and thrombus formation. upon platelet activation its conformation changes leading to an increased affi nity for fi brinogen, which forms bridges between adjacent platelets and assembles them into an aggregate. the iib activation is regulated by "outside-in" and "inside-out" signaling. among the protein-protein interactions, which contribute to «inside-out» signaling, that of talin with the cytoplasmic tail is the most important. it has been recently suggested that talinmediated iib activation relies on the cooperative interaction of the membrane proximal (mp) and the membrane distal (md) regions with talin f domain and that the n ply motif of , which can be phosphorylated at y , plays a critical role in this process. to evaluate the interaction of talin with the tail of integrin we designed and synthesized two peptides corresponding to the md and mp parts of in their carboxyfl uorescein-labeled form (md: cf-r akwdtannplyke and mp: cf-k llitihdrke ). emission and anisotropy fl uorescence spectroscopy was used to quantitatively assess the affi nities of these peptides for talin. furthermore, to challenge the role of the y phosphorylation in talin-iib interaction we also studied the binding of talin to the modifi ed analogue of md, cf-r akwdtannpl(ptyr) ke . our experiments revealed that the md and mp parts of bind tightly to talin and that y phosphorylation has an inhibitory effect on this binding. finally, circular dichroism studies of all peptides in aqueous solutions and mixtures with tfe were also performed in order to characterize structure-affi nity relationships. acknowledgements: gsrt and eu (epan yb/ ) for fi nancial support. the infection of human cells with hiv requires two initial steps: binding of the viral glycoprotein gp to the receptor cd followed by the interaction between the v loop of gp and a seven helix transmembrane coreceptor (ccr on macrophages or cxcr on t cells). the n-type glycosylation at asn of the v loop is assumed to play a crucial role in this process. in order to understand the interaction in detail, it is important to analyze the binding of v -glycopeptides to the membrane integrated coreceptors on an atomic scale. here, we present binding studies between multiple v -peptides and -glycopeptides and the coreceptor ccr by stdd nmr and surface plasmon resonance (spr). spr experiments were carried out by immobilizing the (glyco)peptides on an spr chip by amide coupling. several concentrations of ccr overexpressing human osteosarcoma (hos-r ) cells were passed over the sensor surface. the role of individual amino acids in the binding process to ccr can be analyzed by using different v -peptides and -glycopeptides. additionally, the observation of interactions between the glycopeptide ligands and the receptor proteins embedded in liposomes is possible by using the saturation transfer double difference (stdd) nmr technology. stdd nmr allows removal of all unwanted signals resulting from native binding processes in cells. ( ) . we used ccr liposomes derived from hos-r cells. substraction of an std spectrum of ccr liposomes from an std spectrum of the same concentration of liposomes incubated with a ligand allows recording of clean stdd nmr spectra presenting only signals of protons of the ligand interacting with the transmembrane receptor.( ) k d values of the ligand with respect to ccr have been determined in series of experiments with varying ligand concentrations. we could show that the binding epitope between hiv and ccr is formed by the carbohydrate at asn as well as the peptide. this knowledge is important for the design of new inhibitors. temperature dependant methionine proximity assays highlights conformational variations occurring through the mechanism of peptidergic gpcr activation arsenault, jason; clément, martin; leduc, richard; guillemette, gaétan; lavigne, pierre; escher, emanuel university of sherbrooke, canada g protein coupled receptors are invaluable for cell signal transduction mediated by external stimulus. pharmacological efforts towards these targets are of primordial importance albeit few efforts have resulted in structural characterisation, although rational drug design necessitates such information. recent advances in crystallisation of the -adrenergic receptor have been highly insightful and such a structure corroborates our results on the human angiotensin ii type receptor (hat ) using the methionin proximity assay (mpa) in identifying ligand receptor contact points. unfortunately, physical methods such as crystallography are still far from routine procedures and are limited to a static picture of a given receptor. in the present contribution we propose a more accessible method that permits analysis conformational variations of different receptor states through an energy landscape, spanning from low energy conformations to conformations at physiological temperatures. photolabelling, mpa mutants constructed on the wt receptor, the constitutively active receptor (cam) and a corresponding non-activable mutant across the temperature range reveal activation-status dependent labelling patterns. as an example, position becomes signifi cantly less accessible in the cam receptor compared to the wt receptor. ligand accessibility can be classifi ed from easily accessible (low temperature photolabeling) to less accessible residues (higher temperature photolabeling). this labelling pattern can be associated to the activation status of the receptor and may allow quantifying and identifying structural changes occurring during receptor activation. such structural understanding is crucial for future endeavours in rational drug design. capillary electrophoresis for diffi cult characterization of hardly soluble polypeptides recently, we have designed and synthesized polypeptides able to stimulate the natural plant defenses. these homopeptides were obtained by ring opening polymerization of n-carboxyanhydride (nca) with several initiators. ratio nca/initiator is determinant for the length of the polymers. on the bases of elicitor activity, we identifi ed a leader, called lapp , which results from l-ala-nca polymerization initiated by alaninol. the mixture of poly(alanine) with different lengths is diffi cult to analyze considering important solubility problems. malditof confi rmed polymerization despite limitation due to molecular discrimination during ionization. nmr in deuterated tfa was possible but only afforded the number-average degree of polymerization (dp). to obtain more detailed characterizations, we developed separation by capillary electrophoresis in new solvents mixtures based on hexafl uoroisopropanol (hfip) and water. this technique allowed us to separate the oligomers with baseline resolution. different parameters infl uencing electrophoretic separation were investigated and optimized conditions were established. this technique enabled a full characterization of the polymer distribution, with determination of number-average dp, weight-average dp and polydispersity index. the pertinence and the reliability of capillary electrophoresis for characterization of non-water soluble polypeptides have been confi rmed, with analysis of short and long peptide chains. weakly polar interactions support polypeptide structures although the sequence of a polypeptide appears to determine the secondary and tertiary structure, weak inter-residue interactions such as between aromatic side chains and other aromatic side chains, aliphatic side chains and the peptide backbone may contribute signifi cantly to the stability of the fi nal folded structure. recent advances in structural bioiformatics and computational chemistry made it possible to study precise strurctural features and energetics of these interactions in model peptides and miniproteins such as tc b, app and c-vhp. the interaction energies of the weakly polar interactions are of the same order as of the hydrogen bonds which occur in biopolymers ( - kj/ mol). furthermore, these interactions not only stabilize local secondary structures but also entire tertiary folds of miniproteins. acknowledgements: this work was supported by the nih-inbre grant (p rr ) and the carpenter endowed chair in biochemistry, creighton university trusova, valeriya; gorbenko, galyna v.n. karazin kharkov national university, ukraine a number of so-called conformational diseases including neurological disorders (parkinson's, alzheimer's and huntington's diseases), type ii diabetes, spongiform encephalopathies, systemic amyloidosis, etc., are associated with the deposition in tissue of highly ordered aggregates of specifi c peptides and proteins. despite the main structural elements of amyolid fi brils (particularly, cross--structure) are well-characterized, the mechanisms of fi brillogenesis remains poorly understood. accumulating evidence substantiates the idea that formation of fi brillar structures can be initiated and modulated by peptide/protein-lipid interactions. membrane-related determinants of fi brillization are thought to involve conformational changes of the peptide/protein, increase of its local concentration at lipid-water interface, specifi c orientation of aggregating species, neutralization of the peptide/protein surface charges by anionic lipid headgroups, particular arrangement of the inserted and solvent exposed segments of the peptide/protein molecule, etc. the present study was undertaken to explore the formation of lysozyme (lz) amyloidlike fi brils in the model lipid-protein systems. lipid component of the model system was represented by liposomes prepared from zwitterionic phosphatidylcholine (pc) and anionic phosphatidylglycerol (pg) lipids in the molar ratio : . fluorescence microscopy studies performed with fl uorescein-labeled and rhodamine-labeled lz showed the presence of long lz fi bers (length > ìm). the to elucidate the nature of the events preceding lz self-assembly into amyloid-like structures, the protein adsorption onto pc:pg vesicles was examined by monitoring fl uorescence changes of fl uorescein-labeled lysozyme. the observed sigmoidal shape of the adsorption isotherm is strongly suggestive of oligomerization of membrane-bound protein. it seems highly probable that such oligomers serve as nuclei in the membrane-assisted lysozyme fi brillogenesis structure and dynamics of photosystem ii lightharvesting complex revealed by high-resolution fticr mass spectrometric proteome analysis structure and dynamics of membrane-bound light-harvesting pigmentprotein complexes (lhcs), that collect and transmit light energy for photosynthesis and thereby play an essential role in the regulation of photosynthesis and photoprotection, were identifi ed and characterized using high-resolution fticr mass spectrometry. lhcs from photosystem ii (lhcii) were isolated from the thylakoid membrane of arabidopsis thaliana leaves after light stress treatment using sucrose density gradient centrifugation, and separated by gel fi ltration into lhcii subcomplexes. tsekova, daniela university of chemical technology and metallurgy, bulgaria formation of fi brous supramolecular complexes from l-val derivatives and their arrangement in spherulites was studied applying spectrophotometric approach and electron-microscopic observations. experiments show that boiling one and the same compound with different initial concentrations in water to completely dissolving and posterior cooling to room temperature lead to formation of stable supramolecular complexes with different sizes and properties. they behave like polymer molecules which specifi c numbers of monomers depend on the initial concentration of the boiling solution, moreover at higher supersaturations they crystallize in sperulites which is typical for crystallization of polymer molecules. metal -organic gels based on the self-assembly of peptidomimetics and cu (ii) ions peptidomimetics constructed from l-val and nicotinic/isonicotinic acid are good low molecular weight gelators and their supersaturated solutions in a number of solvents turn into gels. they make complexes with some metal ions. mixing of pure solutions of the peptidomimetic and some cu (ii) salts lead to immediate gel formation in some solvents. this kind of compounds, named metal-organic frameworks (mofs), are new class of nanoporous materials and are very promising ones for applications in catalysis, pharmaceutical industry, etc. the stability and molecular structure of these complexes in some solvents is in the process of investigation. peptide metalloconstructs often possess particular conformations and hence display increased activities and metabolic stabilities. we investigated new rgd peptide analogs cyclized through oxorhenium / oxotechnetium coordination. the rgd sequence is known to bind specifi cally to of the known integrins, a family of integral proteins that plays an important role in tumor neoangiogenesis, development and proliferation. several cyclic rgd pentapeptides bearing an exocyclic tc- m oxotechnetium core have been proposed for molecular imaging, however few of them have displayed attractive selectivities and metabolic stabilities. structure, biological activity and metabolic stabilities of metallated peptides cannot be predicted. therefore, we preferred a combinatorial approach to generate a panel of tracers that may be evaluated by tumor imaging in mice. tracers were constructed from a rgd model of general formula : ns -x -x -x -rs where x , , are respectively arginine, glycine and aspartic acid analogs and r is a series of linkers that feature various lengths and geometries (ns is a n-bis(ethylthio) moiety). first attempts for synthesizing these peptides by the versatile ugi multicomponent reaction did not yield the peptides with suffi cient purities. a representative library of peptides was obtained by standard parallel peptide synthesis and purifi cation of all members. peptides were metallated either with rhenium or technetium using standard procedures. their resistance towards mice serum, glutathione and tumor extracts was evaluated and showed that compounds containing an aminoethanethiol linker displayed higher stabilities. infl uence of the chemical environment on isomers ratios of the metallated peptides was also investigated. finally, oxorhenium peptide coordinates were assayed as specifi c ligands of integrins. their oxotechnetium equivalents were evaluated for tumor imaging in mice. degradation products of desmopressin in phosphate/ citrate buffer it shows, that introduction of unsaturated residue to the peptide chain could be useful tool to design bioactive compounds with desirable structure [ ] [ ] . therefore full knowledge about relation between presence of dehydroamino acid and peptide's conformation is necessary to predict biological proper and to design newdrug. for that reason we have undertook conformational investigations of numerous peptides containing two dehydroamino acid residues ( z phe, e phe, ala) in peptide chain, in different position. the investigations were based on nmr measurements (standard d techniques and d experiments, typical for detection of hydrogen bonding) and theoretical calculations. conformational preferences of investigated systems were obtained on base of roesy and noesy experiments and calculations by use of x-plor and quantum chemical calculation is concentration overload or volume overload the best strategy for synthetic peptide purifi cation? as the complexity of synthetic peptides increases so the demands on the synthesis and purifi cation increase. whilst improvements in synthesis resins and techniques enables higher purity peptides to be produced, % purity is not achieved. for many applications post-synthesis purifi cation is still required. methods for the purifi cation at the mg level can be relatively straightforward but where there is a need to develop methods which may be used for larger scale production there are a number of additionly considerations. the method developed must be scaleable and the economics of the process must be compatible with the fi nal product costs. when looking to develop a purifi cation method the loading will be critical to the throughput and fi nal production costs. the selection of the purifi cation media and the purifi cation conditions are two of the major infl uences on loading as these determine capacity and resolution. however, it is also important to consider the the physical properties of the pepitide -especially its solubility. as part of the purifi cation method development a loading study must be performed -increasing the amount of peptide loaded onto the column. the most common way of doing this is to increase the concentration of the sample and keep the injection volume constant, concentration overload. but this does require that the peptide is readily soluble in a solvent compatible with the hplc purifi cation conditions. alternatively volume overload can be used where the concentration of the peptide solution is kept constant and the volume purifi ed increases. the most commonly used method is concentration overload. the work presented in this poster compares the two overload strategies, concentration overload and volume overload, for the purifi cation of synthetic peptides. the suitability of the two strategies for large scale manufacture will be explored. expansion of human stem cells by passive transmembrane transfer of homeoproteins . these results clarify the effect of hoxb in the early stages of human lymphopoiesis, emphasizing the contribution of this homeoprotein to the intrinsic lympho-myeloid differentiation potential of defi ned lymphoid progenitor subsets. finally, this supports the potential use of hoxb for hsc and hpc expansion in a therapeutic setting, by means of direct transmembrane protein transfer. tumor selective delivery by cell-penetrating peptides systemic delivery of therapeutic agents used for cancer chemotherapy today lead to undesired side effects and toxicity. increasing the selectivity of the anti-cancer agent will not only facilitate lower dosage for equal therapeutic effect, but also decrease the frequency of drug resistance. we have studied two different targeting approaches both based on cellpenetrating peptides. the fi rst approach is a chimera between cancer homing peptides and a cpp the other is a enzyme activated prodrug design. the cyclic peptide ccpgpegagc (pega) is a homing peptide that has previously been shown to accumulate in breast tumor tissue in mice. pega peptide does not cross the plasma membrane per se; however, when attached to the cell-penetrating peptide pvec, the conjugate is taken up by different breast cancer cells in vitro. additionally, the homing capacity of the pega-pvec is conserved in vivo, where the conjugate mainly accumulates in blood vessels in breast tumor tissue and, consequently is taken up. matrix metalloproteinases (mmps) are over-expressed in a variety of tumor tissues and cell lines, and their expression is highly correlated to tumor invasion and metastasis. to exploit these characteristics, we designed a tumor cell-selective prodrug, by constructing modifi ed version of the already shown to be functional mtx-yta conjugate. it is an inactive pro form of a cell-penetrating peptide (nope) conjugated to the cytostatic agent metothrexate (mtx), selectively cleavable and thereby activated by mmps. characterization of hantavirus envelope structure hepojoki, jussi; strandin, tomas; vaheri, antti; lankinen, hilkka university of helsinki / haartman institute, finland hantaviruses are zoonotic viruses carried by different rodent species and if transmitted to man cause two severe diseases, the hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome to which there is no specifi c therapy. the hantavirus envelope consists of spike structures formed by surface glycoproteins gn and gc which anchorage to the viral lipid bilayer. the quaternary complexes formed by glycoproteins have not been solved. to begin with, it is important to understand the structure of virus particle in order to treat infection for example by prevention of membrane fusion. using pepspot technique the interaction site between gn and gc proteins was mapped to peptide level. to interpret interaction mapping results three dimensional ( d) models of gc protein were created. the structure of hantavirus was also investigated in this study by cryo-electronmicroscopy (cryo-em). according to our results, hantavirus spike consists of either four or eight glycoprotein units and the spike would consist of equimolar associations of both glycoproteins. overall it seems likely that the glycoproteins of hantavirus form a heterodimeric base unit analogous to semliki forest virus e -e glycoprotein complex, where the interaction between e -e proteins hides the fusion peptide in e protein and thus prevents premature fusion. isolation of a novel kda protein from ginger rhizomes having anti-fungal and anti-proliferative activity gill medicinal properties of ginger have been known for long. ginger has been used in traditional indian and chinese medicine and is effective on a wide range of ailments including diarrhea, nausea, respiratory disorders, infl ammatory diseases, arthritis etc. a number of constituents and active ingredients are present in ginger. recent studies have shown the role of ginger extract in the modulation of biochemical pathways involved in chronic infl ammation and thus providing evidences for the anti-infl ammatory role of ginger. mainly the medicinal properties and anti-proliferative activity of the ginger is because of the presence of certain pungent vallinoids, viz. .-gingerol and .-paradol, as well as some other constituents like shogaols, zingerone etc. we have identifi ed and purifi ed a novel anti-fungal and anti-proliferative protein with a molecular mass of kda from the crude extract of ginger rhizobium (zingiber offi cinales), belonging to the zingiberaceae family. the isolation procedure involved ion exchange chromatography using deae-cellulose and affi nity chromatography using affi -gel blue gel. crude extract was loaded on the deae-cellulose column equilibrated with mm tris-hcl buffer (ph . ). the unadsorbed protein fraction from deae-cellulose was further loaded on affi -gel blue gel column equilibrated with mm tris-hcl buffer (ph . ), from which the elution of adsorbed protein was done with the same buffer. the purifi ed protein of kda exhibited a potent anti-fungal activity against the mycelial growth in different fungal species, for example aspergillus fumigatus. in addition, the antifungal activity is also seen against important fungi, viz, fusarium and candida species. further, the purifi ed protein also showed % inhibition of cell proliferation at m concentration. the anti-proliferative activity was checked on human oral cancer (kb cells). search for native interacting partners of fl uorinated amino acids using phage display the introduction of fl uorine has proven to be a successful concept for improving the biological and pharmaceutical properties of drug candidates. the unique properties of fl uorine as well as its absence from the pool of canonical amino acids make fl uorinated amino acids a promising tool in the development of peptide based drugs.( ) however, the application of fl uorinated amino acids for rational protein design requires a comprehensive knowledge of their properties within a native protein environment. our research focuses on the effects caused by single substitutions by fl uorinated amino acids within a polypeptide environment.( ) based on a parallel, heterodimeric -helical coiled coil peptide we applied phage display technology ( ) to screen for preferred interaction partners of fl uorinated building blocks within the pool of the twenty canonical amino acids. three fl uorinated amino acids were introduced either at an a-or a d-position into one of the coiled coil monomers. the second coiled coil monomer was randomized at the four positions that represent the direct interaction partners of the fl uorinated amino acid within the dimerization domain. coiled coil pairing selectivity was used to determine the best binding partner out of the library. using the acylation method, fatty acyl chains are covalently linked to the free amino residues forming a stable amide bond. in this study, a novel method for acylation of proteins was developed using in situ prepared fatty acyl chloride dispersion in aqueous acetonitrile solution, which allows protein modifi cation under very mild conditions. chicken cystatin, a reversible kda inhibitor of papain-like cysteine proteases, was selected as a model protein due to its high potential to inhibit intracellular cathepsins. the protein was modifi ed using fatty acyl chlorides with , , , , , , and carbon atoms. based on the cell culture assays, we examined the transport properties of fatty acylated cystatin, the effectiveness of its internalization and effi ciency to inhibit intracellular enzymes, which was measured indirectly by cathepsin b inhibition. the experiments showed that acylated cystatin quickly internalized into the cells and effectively inhibited cathepsin b. in contrast, non-acylated cystatin didn't cause inhibition as it was unable to enter the cell. the permeability enhancement effect was shown to depend on the length of the attached fatty acyl chain as the strongest inhibition was caused by cystatin acylated with carbon atoms long stearoyl chloride. additionally, chemical modifi cation did not infl uence the protein's immunogenicity. the results of our study provide clear evidence that fatty acylation greatly improves membrane permeabilization properties of proteins. daisogel wing takes your process separation to a higher level. custom made solution for your process peptide purifi cation problem can be easily delivered using the daisogel wing polymer grafted silica based platform. "silica or polymer?"-it used to be an evergreen debate when it came to choose the adequate stationary phase for process scale separation or purifi cation. silica based stationary phases feature much higher mechanical strength and stability, better controlled porosity and as a result higher separation effi ciency, while polymers boast wider ph range. the attempts to bring the good aspects of these opposite methods together failed so far, or failed to provide fl exible solutions. the new daisogel wing platform for silica surface polymer grafting preceding chemical modifi cation is presented. the revolutionary new technique offers high versatility: most variations of the base silica (different pore sizes or particle sizes of choice) can be combined with your choice of familiar chemical surface modifi cations to tailor make the perfect stationary phase for your given chromatography challenge. the polymer graft on the silica surface provides extreme shielding effect, the produced stationary phase displays outstanding ph stability and durability. any of your preferred and trusted regular silica surface modifi cations can be done on the top of the grafted polymer layer. you may enjoy the usual high performance separation, excellent effi ciency, high plate numbers, mechanical strength of silica based stationary phases with a dramatically extended ph range you expected so far only from polymer resins. you have the full range of choices of particle and pore sizes with the full choice of desired bonding chemistries. finally daisogel wing is here to deliver you the most versatile polymer grafted silica hybrid platform to provide the best solution for your demanding process scale peptide separations. przybylski, józef; rogala, piotr; siemaszko-przybylska, krystyna melanin, a natural compound formed in the tyrosine metabolic process. according to occurrence, melanin is divided into that present in true skin and in internal organs. the melanin synthesis and secretion process takes place in melanocytes under the control of two neurohormons: msh -melanocyte stimulating hormone and mch -melanocyte concentrating hormone. research has shown the signifi cant role of melanin in the process of breeding and storing, in regulating metabolism of fat tissue, and in carbohydrates regulation. melanin biopolymer fi nds application in it technology as neurotransmitters for non cellular intelligence. biologically active collagen constitutes the architectonics of all tissues and organs. due to its piezoelectric and dielectric properties it also provides storage and relay functions. for the needs of medical practice, transplants, pharmacy, cosmetology and information technology the key issue is to obtain a compound of melanin with biologically active collagen. this compound we named melanocollagen type i. melanocollagen type i is formed in result of protonating collagen amino acids with melanin, which yields coloured gel ranging: grey, graphite and black. /melanin (h+) + collagen melanocollagen type i/. the therapeutical capacity of biologically active collagen type i with melanin is an ideal formulation inhibiting aging and mitigating related neurovegetative and dementia symptoms, and in vivo transplantology. freeze dried melanocollagen type i retains the features of both collagen type i and melanin. it can be a formulation on its own or a supplement for in vivo and in vitro tissue engineering, biotechnology, information technology, pharmaceutical industry and cosmetology. a patent application for melanocollagen was placed with the polish patent offi ce on . . as p- , entitled: melanocollagen type i -method of obtaining. [ , ] . , -dhp lipid structure was calculated with ab initio quantum mechanics to obtain the charges for molecular dynamics with amber . force fi eld. dhp-lipid molecules were subjected to molecular dynamics from the initial structure of a periodic lipid bilayerwater box, with a small amount of excessive water on the lipid edges to ensure the mobility of lipid molecules. after ns of md simulation the lipid molecules with the fatty acid tails started to squeeze from one bilayer layer to another one. after ns few lipid molecules turned with their charged heads to the side of the lipid bilayer and after ns a profound tubular micelle structure began to form. the tubular micellae structure becomes more perfect during the course of simulation of ns. conclusion is that one of the gene transfection agent , -dhp lipid structures is a tubular micellae, and we could expect that such the micellaes are capable to form lipoplex for the dna transfection or peptide delivery. introduction a newly developed hydrophilic polymer-based ion exchange chromatography column for biomolecules. ion exchange chromatography (iec) is a widely used for analysis and purifi cation of biomolecules. we have newly developed polymer-based iec column, named ymc-biopro series, specially designed for separation of proteins, peptides and nucleic acids. the completely spherical and monodispersed porous / nonporous beads ( m), optimally packed with advanced technology, provide high theoretical plate number and symmetric peak shape. excellent resolution is achieved from the high column effi ciency coupled with the excellent selectivity of qa and sp chemistries. in this paper, we will show features and benefi ts of ymc-biopro series. james p. tam, school of biological science, nanyang technological university, nanyang drive, singapore . viral entry requires fusion of the viral and cellular membranes. in all class- envelope viruses including hiv- , fusion is mediated by envelope glycoprotein which has a homotrimeric quaternary structure which forms a hairpin-like assembly of six-helix bundle ( hb) during the fusion event. the hb employs a -on- locking mechanism in which hrc (heptad repeating-carboxyl region) chains crosslink hrn (heptad repeating-aminal region) chains to enable membrane fusion. this locking mechanism confers avidity due to multi-chain interactions and a high genetic barrier to mutations in the viral entry strategy despite the high mutation rate of their envelope protein. this mechanism also provides inspiration to our approach in designing mutation-resistant entry inhibitors of hiv- . t /enfuvirtide, a synthetic hrc-peptide monomer designed to interrupt membrane fusion, is the fi rst and only fda-approved entry inhibitor against hiv- . however, t becomes ineffective in % of aids patients due to acquired resistance to t- resistant during the treatment course. our inhibitor design is based on a novel quaternary protein mimetic approach. key elements include a covalent-link parallel-chain construct mimicking the quaternary structure of gp in its pre-fusion state and an inhibition mechanism mimicking the multimeric interactions found in the highly conserved hb formation. our hypothesis is that covalent-linked, -chain quaternary mimetics (called mimetics) of hrc peptides can confer multi-chain binding to the hrn region in a multi-chain locking mechanism that may lead to mutation-resistant hiv fusion inhibitors. we also extend our design to -chain hrc mimetics (called mimetics) which may bind to the hrn as a -on- locking mechanism. in contrast, t and other single-chain peptide inhibitors with a -on- binding mechanism lacks the advantages offered by the quaternary mimetics and is susceptible to gp mutations. this report will describe all three types of inhibitors as a model to further our understanding of gp mutations and viral fusion mechanism in developing mutation-resistant entry inhibitors. references: . lain et al. cancer cell costentin, beauvillain, vaudry, proc. natl. acad. sci pnas , , . . references: . a.a.zamyatnin. fragmentimics of proteins and natural oligopeptides. biofi zika the fi rst genome sequence of an elite grapevine cultivar (pinot noir vitis vinifera l.): coping with a highly heterozygous genome the grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla the erop-moscow oligopeptide database press references: . alcaro galactosylated peptides, their preparation and use in autoimmune disease diagnosis new coupling reagents: development and industrial aspects assessment of new -cl-hobt based coupling reagents for peptide synthesis. part : coupling effi ciency study fast conventional synthesis of chemokine sdf- ( - ) on the symphony® references: . pardridge, w. m. drug discov.today alberto ; rondina, maria ; rosato maura ; quintieri synthesis and biological activity of new linear and cyclic shp- n-sh -ligands the sequence specifi city for most sh domains is dictated by the amino acids surrounding the py-residue (position ) and the c-terminal positions relative to py ( ). in contrast, the sh domains of shp- in addition to py+ and py+ depend on position py- ( ). it was further demonstrated that beyond this minimal consensus sequence the positions py+ and py+ also signifi cantly affect the binding affi nity and specifi city of the shp- sh domains ( ). for the investigation of shp- mediated signaling pathways, inhibitors of this phosphatase are of great interest. therefore, we focused our research on linear and cyclic peptide ligands based on the previous studies [ - ]. the new ligands were c-terminally prolongated according to the recognition determinants for py+ and py+ . except, an additional motif designed to occupy a basic gap on the surface of the ptp-domain was introduced. the latter was predicted to impair the n-sh -ptp dissociation process design of a directed molecular network restructuring artifi cial peptide networks by external triggering the road to non-enzymatic molecular networks boolean logic functions of a synthetic peptide network systems chemistry: logic gates, arithmetic units and network motifs in small networks peptides: the wave of the future references: . d'andrea l.d.; iaccarino g.; fattorusso r.; sorriento d.; carannante c.; capasso d.; trimarco b.; pedone c proc. natl. acad. sci agnieszka ; gofman nir ; willumeit, regine gkss research centre, germany; hasylab/desy, germany urtti novel cationic amphiphilic , -dihydropyridine derivatives for dna delivery dioleoyl phosphatidylethanolamine and peg-lipid conjugates modify dna delivery mediated by , -dihydropyridine amphiphiles masako ; kuriyama, naohiro poster abstracts lacking his-pro residues and stabilized by introduction of hval resulted in an analog h- hval-tyr-ile-tic-oh (al- ) that was very selective for irap versus ap-n and at( ) . the targeting properties of cell penetrating peptides cell penetrating peptides (cpps) offer the ability to penetrate across the plasma membrane of mammalian cells and to carry cargoes such as proteins, oligonucleotides and liposomes. due to this, cpps have gained high attention to improve the cellular uptake of the delivery of 'biologicals'. however, most of the research that has been performed with cpps restricted to in vivo studies. preclinical investigations are rare and the clinical validation of potential of cpps is required. a series of cpps were synthesized by solid phase peptide synthesis (spps) on an abi a synthesizer. the stability of these peptides in human serum was determined. subsequently, the uptake was studied in the following cell lines: sw , pc- , mh wt, hno in summary, the in vitro uptake studies did not reveal great differences that might have revealed a tumor type specifi city. in vivo studies revealed neither a distinct tissue uptake or tumor specifi city. design and synthesis of a tripartate paclitaxel prodrug for melanoma therapy therapy of melanoma continues to be a challenge since, regardless of the treatment used, long-term survival is quite uncommon. in an attempt to improve the effectiveness and decrease the toxicity of anticancer chemotherapy, one useful approach might be to administer a prodrug that specifi cally releases the active cytotoxic drug at the tumor site. in this work, we describe the synthesis of a peptide conjugate of paclitaxel potentially useful in the treatment of human melanoma, and characterized by the simultaneous presence of three functional domains: a "targeting domain", an "activation sequence", and the antitumor drug paclitaxel. the "targeting domain" of the prodrug is represented by an rgd-containing cyclic peptide, able to bind selectively to alpha-v beta- integrin, which is known to be highly over-expressed by both metastatic human melanoma cells, and endothelial cells of tumor vessels. the "activation sequence", responsible for the selective release of the drug, is a short peptide which is cleaved specifi cally by cathepsin b, a protease highly up-regulated in malignant tumors. the results of nmr conformational studies, as well as those of biological experiments aimed at evaluating the plasma stability of the prodrug and its ability to inhibit alpha-v beta- -mediated tumor cell adhesion to vitronectin, will be also presented. angiogenesis is a remodeling process characterized by the sprouting of new blood vessels from pre-existing ones. it occurs during embryogenesis and to a limited extent in the adult. vegf is a homodimeric protein and has been characterized as a prime regulator of angiogenesis and vasculogenesis; when cells lose the ability to control the synthesis of vegf, angiogenic disease ensues ( ) . in vitro studies show that vegf is a potent and specifi c angiogenic factor involved in the development of the vascular system and in the differentiation of endothelial cells ( ) . vegf biological function is mediated through binding to two receptor tyrosine kinases: the kinase domain receptor (kdr) and the fms-like tyrosine kinase (flt- ), which are localized on the cell surface of various endothelial cell types. this binding activates signal transduction and can regulate both physiological and pathological angiogenesis ( ) . vegf and its receptors are overexpressed in pathological angiogenesis, making this system a potential target for therapeutic and diagnostic applications ( ) . the extracellular portion of vegf receptors is comprised of immunoglobulin-like domains; deletion studies have shown that the ligand binding function resides within the fi rst three domains of flt- and in domains and of kdr. actually, no structural data are known on the extracellular portion of these receptors except for the second domain of flt- .. so, our aim is the cloning and the expression of part of extracellular domains of both vegf receptors for structural characterization and to be used in interaction studies with peptide ligands or small organic molecule. we are interested in an activation mechanism of receptor tyrosine kinases (rtks), especially, how the transmembrane (tm) and the intracellular juxtamembrane (jm) region couple ligand binding to tyrosine phopsphorylation. one of the rtks that we are working on is neu. neu (erbb ) is one of the four members of erbb receptor family. this rtk with a mutation in the transmembrane region (v e) has been recognized as a potent transforming oncogene product. structure of the tm region dimer and its structural difference between the wild type and v e mutant have been reported by smith et.al. ( ) , providing structural constraints for modeling the tm dimer. recently, we have performed a structural study on a tm-jm region of egfr (erbb ) showing that the tm helix breaks at the membrane interface and the unfolded jm region binds electrostatically to the membrane. we also have shown that ca + complex with calmodulin (ca +/cam) binds to the positively charged jm region and pulls this portion off the membrane. these results are consistent with an electrostatic engine model, postulated by mclaughlin and coworkers ( ) , for the autoinhibition and activation of the egfr. in this research, we are revisiting the structure of tm-jm region of neu to see if we can apply the electrostatic engine model to neu and to see if there is a structural difference between the wild type and the mutant tm-jm sequence. here we describe structural studies on the tm-jm sequence, neu( - ), reconstituted into bilayer vesicles. a combination of solid state nmr, infrared and fl uorescence measurements are used to draw conclusions that the unfolded jm binds electrostatically to the membrane and binding of ca + -calmodulin to the positively charged jm sequence can, under some conditions, reverse its charge and release it from the membrane. @the collagen triple-helix consists of three polypeptide chains, each of which takes the poly-l-proline ii form in a left-handed helix and undergoes a transition to a single coil state as the temperature increases. this characteristic structure is ascribed to the unique amino acid sequence x-y-gly, where x and y are commonly pro or (r)-hydroxyproline (hyp r ). @so far, various nmr studies have been done to explain the mechanism of folding and the thermal stability of the triple helical structure by using model peptides rich in imino acids. however, the complexity of nmr spectra imposed severe limitations on detailed analysis. the spectrum is complicated because of a large number of conformational isomers due to cis/trans isomerization around each imino acid causing various chemical shifts with small differences in h-nmr spectra. it has been demonstrated that the site directed n enrichment allows real time nmr monitoring of the folding of residues at specifi c locations of collagen model peptides ( ) . @to approach this problem, we have applied f-nmr study on various collagen model peptides containing (r)-fl uoroproline (fpro r ). compared to h and c, f chemical-shifts are extremely sensitive to small environmental changes around the nuclei and show a wide dispersion of chemical shifts. as a result, we not only distinguished the signals from the triple helix and unfolded states, but also differentiated the signals at intermediates states in d f-nmr spectrum of (pro-fpro r -gly) . @here, in this study, a combination of d nmr experiments including exsy, hoesy, and tocsy has been used to investigate detailed equilibrium properties of collagen model peptides. especially, in the d f-exsy spectra, we successfully observed many exchange cross peaks at high temperature. the f-nmr method shown here provides a clue to analyze the conformational transition, dynamics and stability of collagen triple-helix. by contrast to well-folded natural peptides, oligomers consisting of nonnatural building blocks, so-called foldamers, are highly stable towards proteolytic degradation which is a key feature in the development of peptide-based drugs. aside from the fact that foldamers consisting ofand -amino acid building blocks are able to display different secondary structures, the combination of different homologous building blocks to hybrid peptides has recently shown some promising results. [ , ] the ab initio mo theoretical studies have shown that / -hybrid peptides composed of alternating -and amino acid building blocks might be very well suited to mimic helical conformations. ( ) here we report the fi rst examples of -helical coiled coil formation with synthetic foldamers. based on the computational studies, we synthesized heterogeneous peptides by automated solid phase peptide synthesis (spps). purifi cation was carried out by hplc, and products were confi rmed by esi-tof-ms. furthermore, temperature-dependent cd spectroscopy was applied to investigate the stability of hetero-oligomers formed between, either / -or / / -hybrid peptides with a naturalhelical coiled coil peptide. in addition, the interaction potential with their natural -helical counterparts in aqueous solution were investigated by cd, and förster resonance energy transfer (fret). using reversed phase high performance liquid chromatography and two-dimensional gel electrophoresis, the lhcii proteins, lhcb - and fi brillins were effi ciently separated, and identifi ed by fticr-ms proteome analysis. some of the lhcii subcomplexes were shown to migrate from photosystem ii to photosystem i as a result of short term adaptation to changes in light intensity. in the mobile lhcii subcomplexes, decreased levels of fi brillins and a modifi ed composition of lhcii protein isoforms were identifi ed compared to the tightlybound lhcii subcomplexes. in addition, fticr-mass spectrometric analysis revealed several oxidative modifi cations of lhcii proteins ( ) . a number of protein spots in d-gels were found to contain a mixture of proteins, illustrating the feasibility of high resolution mass spectrometry to identify proteins that remain unseparated in d-gels even upon extended ph gradients. phosphinic analogue of the homophenylalanyl-phenylalanine dipeptide was demonstrated to be a potent, competitive inhibitor of cytosolic leucine aminopeptidase (lap, e.c. . . . ), mimicking the transition state of the reaction catalysed by the enzyme. exhibiting ki value at low nanomolar range it was ranked among the most potent inactivators of lap reported so far ( ) . recently, the compound has been also successfully employed to inhibit leucyl aminopeptidase of p. falciparum, a potential target protease for the development of new antimalarials ( ) . thus, its structure represented an attractive lead for the design and construction of next generations of modifi ed analogues. they were targeted towards both lap as well as a related metalloprotease -microsomal leucine aminopeptidase (apm, e.c. . . . ). these achievements, including recent results of studies on synthesis and activity, will be summarized here. we recently described the synthesis of some cyclic tetrapeptides bearing imidazole side chains and we analyzed, in detail, their copper(ii) binding properties in aqueous solution. the copper(ii) species obtained are of interest in relation to copper-protein active-site biomimetics. in particular, a -membered ring cyclic tetrapeptide c(lys-dhis-ala-his) (dk ) was synthesized by the solid-phase peptide synthesis method and its copper(ii) coordination properties were studied. surprisingly, all collected data strongly support the presence, at alkaline ph, of a stable peptide/copper(iii) complex that is formed in solution by atmospheric dioxygen oxidation. in order to clarify the mechanism of the copper oxidation through the average cu-n bond distance and to get information on the local geometry around copper, depending on the peptide sequence, we have collected experimental xanes and exafs spectra. the investigated cyclopeptide/copper complex shows pre-edge peak energy position and integrated intensity consistent with those of cu + model compounds. also, edge energy is consistent with the presence of trivalent copper. then, calculations performed on the exafs measures should give information on the local geometry, confi rming the square planar geometry proposed by us for this dk /cu(iii) complex. improved expressed protein ligation method for consecutive coupling of polypeptide fragments in the post-genomic era, to support the structural and functional studies of proteins, there is a growing demand for novel methods with which a wider range of selective protein modifi cations achievable. expressed protein ligation (epl) can be the method of choice for the preparation of unique protein derivatives not available by recombinant methods when the protein fragments extend the size accessible by solid phase peptide synthesis, and when extra chemical information (i.e. a special covalent modifi cation) is introduced into a well-defi ned part of the sequence. however, this method is not generally applicable, especially in the case when the target protein derivative is assembled from three or more polypeptide fragments. we demonstrate here an improved epl method that facilitates the effective ligation of large fragments in a consecutive way. the method employs a novel protection-deprotection scheme. supported by otka f and jános bolyai fellowship (cs.t.). protein ligation using protein trans-splicing , in order to facilitate protein ligation using synthetic peptides. highly effi cient fl uorescent labeling of proteins has been demonstrated in a site-specifi c manner by protein ligation using the newly designed intein. this approach could be applied for sitespecifi c modifi cation of proteins in vivo because protein trans-splicing is an autocatalytic process requiring no additional cofactor. moreover, protein trans-splicing approach could be extended to dual site-specifi c modifi cation using an additional intein. la doppel (dpl) is a glycosylphosphatidylinositol-anchored protein that exhibits % sequence homology with prion protein but lacks the octarepeat region. the role of prion is not yet completely known but prpc seems to be involved in cu (ii) traffi cking from synaptic clefts and in preventing neuronal oxidative damage. doppel is expressed in heart and testis and has been shown a regulator of male fertility. therefore, despite a high sequence homology and a similar three-dimensional fold, it's possible to hypothesize that the function of the two protein is not correlated. in the literature is reported that both prpc and of dpl are able to bind cu (ii) but the characterization of the complex species is well defi ned only for prpc. several binding sites of dpl are involved in the complexation with cu (ii). [ , ] in the present work we have focused our attention to the third alpha-helix of the dpl which is the preferential binding site for the metal ion. we have synthesized two peptide fragments relative to the - sequence of the dpl. the fi rst peptide has the native sequence whereas in the second fragment the asp was replaced by a asn residue. this substitution was performed to understand the role played by the carboxylate group of the asp residue in the complexation of cu (ii). cd ad nmr spectra were carried out on the two peptides in absence and presence of cu (ii) to investigate conformational features upon cu (ii) binding. finally, the complex species formed were completely characterized by using spectroscopic (nmr, cd, uv-vis, epr) and thermodynamic measurements (potentiometric titrations). three-dimensional ( d) domain swapping of proteins is a phenomenon associated with formation of oligomers and fi brils of several amyloidogenic proteins ( ) . the propensity of a particular protein to undergo domain swapping depends on factors like changes in environment or presence of specifi c mutations, but also on some topological determinants. it was proposed that domain swapping-prone proteins have some common "hot spots" in their structures that facilitate the process. one of these motives are so called "hinge loops" -turns showing enhanced propensity for unfolding due to conformational constraints ( ). cystatin c (hcc), main inhibitor of cysteine proteases in human body was shown to dimerize ( ) and oligomerize ( ) through domain swapping. hcc contains highly conserved throughout the cystatin family loop l , connecting two beta strands which, together with the only helix in the protein fold create a structural unit that undergoes the d process. in order to confi rm important role of distortions in l loop, which are centered around valine residue in position in the dimerization/ oligomerization process of hcc, we designed point mutations which should inhibit or promote aforementioned processes. as it was expected, substitution of val with either asparagine or aspartic acid residues abolished dimerization process almost completely. in contrast, v p mutant shows enhanced dimerization propensity. above observations are in good agreement with theoretical studies, performed on entire hcc and its fragment encompassing the hairpin structure centered on loop l . the expansion of the cag trinucleotide that produces polyglutamine (polyq) segments in several proteins is responsible for at least eight neurodegenerative diseases. a possible therapeutic approach would be to inhibit the polyq self-assembly process using disrupting agents. although, screening studies have identifi ed several polyq aggregation inhibitors, their mechanism of action remains unknown. this is due to the poor aqueous solubility and fast aggregation of uncharged polyq peptides, which complicate their study. the most common strategy to resolve these problems is to produce polyq stretches fl anked with charged residues. however, this strategy was found to modify the aggregation behaviour of polyqs, their aggregate structure and their affi nity for disrupting molecules. to circumvent these problems, polyq peptides containing morpholine moieties, whose charge is ph-dependent, were produced using fmoc-based chemistry on spps. following an acid treatment, the designed peptides were soluble in acidic aqueous solutions and their aggregation rate could be controlled by ph variations and followed by dynamic light scattering. furthermore, aggregation initiated at physiological ph provided uncharged fi brils allowing the evaluation of the effects of charged disrupting agents. the actions of known polyq aggregation inhibitors such as polyq-binding peptide (qbp ), trehalose and congo red were studied. peptide-protein and protein-protein interactions play key roles in most biological processes, and therefore represent valuable targets for drug discovery. an approach in the search of new chemical entities able to interfere with these interactions is the use of small molecules able to mimic or to induce precise aspects of specifi c peptide secondary structures. among the secondary structure elements, reverse turns have been shown relevant in many biomolecular recognition events. thus, different efforts have been directed to the design of turn mimetics or inducers. in this sense, -substituted azetidine- -carboxylates, synthesized by our group through a versatile procedure from amino acids, possess the dihedral angle restricted to approximately º or - º, depending on the absolute confi guration at the asymmetric carbon. these values are similar to those reported for the central residues of main types of -and -turns, and in fact, recent studies have shown the ability of the , -disubstituted azetidines to induce -turns when incorporated at the i+ position of model peptides. to know if this conformational behavior is distinctive of these restricted amino acids, we now investigate the infl uence of the incorporation of these amino acids at the i+ position of model peptides. with this aim, we have synthesized a series of simplifi ed tetrapeptide models, rco-l-ala-azx-nhme, in which the rco and nhme groups are simplifi cations of the i and i+ residues of the turn, respectively. for comparative purposes, dipeptide derivatives incorporating azetidine- -carboxylate, pro and -mepro have also been prepared. the turn inducing capacities of all these model tetrapeptides have been analyzed by molecular modelling, ¹h rmn, ft-ir and x-ray methodologies. the results have shown the importance of the , -disubstitution at the heterocyclic amino acid for stabilizing reverse turns, and the infl uence of the ring size on the induced turn type infl uence of position of dehydroamino acid in peptide chain on conformation of dehydropeptides containing two dehydroamino acid residues it is known that presence of c -c double bound in dehydroamino acid infl uences on dramatic limitation of conformational space, not only side-chain but also main-chain [ ] [ ] . according to the peptide's length and neighboring amino acid residues, dehydroamino acid exerts s-shaped, -turn or helical conformation in peptides [ ] [ ] [ ] [ ] [ ] . angiotensin-i converting enzyme (ace) somatic form bears two catalytic domains with two zn metal ions, while the testis form bears remarkable similarity to the aminoacid sequence of ace c-catalytic domain and only one zn metal ion. however, both exhibit their hydrolytic activity to vasoactive peptides such as angiotensin i (angi) and bradykinin (bk), though with different effi cacy. in order to study experimentally the possible interaction and binding events between the ace active site(s) and known ace substrates or inhibitors, we constructed peptide-based catalytic site maquettes (csm). -residue peptides were synthesized through solid-phase peptide synthesis having the aminoacid sequence that correspond to the ace val -ala n-domain segment and to the val -ala c-domain segment and enzyme's active sites were reconstructed through addition of zinc metal ion in solution. the resulting complexes have the metal ion bound in a native-like mode, as manifested by previously reported nmr data. the reconstituted peptides were titrated separately by captopril and angiotensin-i with a molar ratio ace:captopril/angi : and : , respectively. titration was followed by h nmr spectroscopy and spectral changes (chemical shifts, line broadening, etc.) were recorded and analyzed. after the end of angi titration the solution of interacting peptides was titrated with captopril and the titration was followed by h nmr spectroscopy. at the end of each titration d homonuclear h- h tocsy and noesy spectra were recorded. analysis of high-resolution nmr data probes the conformational variation of ace csm and peptide substrates upon interaction, and in the presence of captopril in ace-angi solutions. contemporary development in nanotechnology is fabrication of nanostructured materials using the peptide-based self-assembly. importance of peptide as monomeric components is their capacity to be engineered to mediate spontaneous supramolecular self-assembly and their inherent chemical nature that facilitates chemical and biological recognition process. tubular self-assembled peptide nanostructures are of special interest since these can serve in various applications including 'bottom-up' fabrication of molecular scaffolds, nanoelectromechanical systems and nanomachines. an important discovery by gazit group revealed that the simple dipeptide molecule of phenylalanine which is core recognition motif of the alzheimer's -amyloid polypeptide effi ciently self-assembles into well-ordered nanotubular structure ( ) which could be controlled by relatively simple chemical modifi cations at its termini. based on these observations and our interest in the synthesis of softmaterials for nanotechnology, we have successfully synthesized novel monomeric units of di-peptides with ureido functional group at nterminal of various hydrophobic l-amino acids. these derivatives have been prepared by using solid phase peptide synthesis protocol with cost effective process retaining the chirality of molecule. these ureido dipeptide formed nanostructures with high yield under mild and controlled condition in aqueous media. to fully understand the molecular and supramolecular mechanism guiding the formation of nanostructures we have selected a multidisciplinary approach by combining spectroscopic studies with advanced electron microscopic techniques. the morphology of nanostructures can be controlled by using variety of l-amino acids. these novel ureido di-peptides nanotubes can be used in fabrication of biocompatible peptide-based nanostructures and they will undoubtedly have nanotechnological applications. biomolecules are excellent in hierarchically organizing a characteristic structure by self-assembly. the spontaneous assembly of biomolecules has advanced a bottom-up approach for fabrication of nanostructured materials. we have developed fabrication of controlled nanofi bers through self-assembly of simple and short de novo designedsheet peptides with unique sequences [ , ] . the single straight nanofi ber with high regularity constructed by -sheet fi peptide (sequence: pkfkiiefep) was functionalized with proteins by biotinylated peptides ( ) and using functional anchors that have binding and functional groups. conjugation of self-assembled peptide nanofi bers with biomolecules such as peptides and proteins will expand their potentialities of the nanofi bers for various applications. to identify peptides binding to the self-assembled fi peptide nanofi ber, a phage display combinatorial screening using a random peptide library was performed. after fi ve rounds of biopanning, phage pools with highly specifi c affi nities to fi nanofi bers were screened. as a result of dna sequencing after phage cloning, phage clones were identifi ed. binding affi nities of these clones were quantitatively investigated by an enzyme-linked immunosorbent assay. these clones showed specifi c affi nities to fi nanofi bers, as compared with ff and vi nanofi bers having slightly differences of amino acid sequences. affi nities and specifi cities were dependent on the sequence of each peptide, indicating that different amino acid sequences contributed to peptide interactions with nanofi bers. infrared spectroscopy studies by using chemically-synthesized peptides showed that the peptide binds specifi cally to the fi nanofi ber with structural transitions. owing to their diversity in size and shape, easy access, and biocompatibility, peptides represent versatile units for the construction of h-bonded tubular assemblies and other biomimetic materials with potentially useful applications. so-called peptide nanotubes (pnts) have been obtained through multiple and complementary approaches ( ). originally designed from -peptides made of d-and l-amino acids ( ) , fl at macrocyclic systems forming cylindricalsheet like assemblies have diversifi ed to include oligoamides made from higher amino acid homologs as well as peptide hybrids (e.g. ,peptides). tubular sheet-like assemblies however are not restricted to oligoamides. the urea group which shares a number of features with the amide linkage, i.e. rigidity, planarity, polarity, and hydrogen bonding capacity is an interesting surrogate. we and other have demonstrated that macrocyclic biotic and abiotic n,n'-linked oligoureas have a unique propensity to self-organize into polar h-bonded nanotubes ( ). partial peptide backbone n-methylation has been introduced as a general strategy to generate truncated stacks (i.e. h-bonded dimers) useful to gain access to the thermodynamics of nanotube formation ( ). herein, we describe biotic macrocyclic amide/urea hybrids with partially nalkylated backbones as new candidates for the formation of h-bonded dimers. a cysteine branched pga (polyglutamic acid) hydrogel is being investigated for therapeutic peptide and protein controlled delivery. entrapment of hgh as a model protein in this hydrogel has been carried out in order to check its later release in different aqueous buffered media and stability. different physico-chemical analysis (by maldi-tof, sec-mals-ri, h-nmr, cd, …) of the released hgh showed no effect on destabilization of the protein. controlled release systems of proteins involve important challenges such as maintaining the protein integrity. researchers need an exhaustive understanding of protein stability issue in drug delivery formulations. common degradative reactions in proteins involve: oxidation, deamidation, stability of disulfi de bonds and aggregation, which may affect secondary and tertiary structure of proteins and thus, their biological activity. the high water content which imbibes the polymer network of hydrogels enables a good accommodation for proteins. polyamino acids have been already studied in some biotechnological applications, however, in spite of their potential, they have barely been taken into account for the development of controlled release microparticles. we show that proteins could be easily loaded into cys cross-linked pga hydrogels. the protein hgh has been loaded and released preserving its physico-chemical integrity. thus, these hydrogels are promising for their use as therapeutic protein delivery systems. totally synthetic collagen-like gels by intermolecular folding of designed peptides our goal is to develop artifi cial collagens that can use as safer and functional biomaterials. here, we report the development of collagenlike gels by means of the intermolecular folding of chemically synthesized peptides. the peptides are disulfi de-linked trimers of collagenous gly-x-y triplet repeats with self-complementary shapes. the self-complementary peptides are able to form elongated triple-helix through spontaneous intermolecular folding. upon cooling of the peptide solutions, hydrogels of peptide supramolecules formed. the gel-sol transition was appeared to be reversible, and the transition temperatures were found to be tunable by the design of the peptides. our strategy for the totally synthetic collagen will offer possibilities for the development of innovative biomaterials. recent advance in biotechnology enables us to fi nd the peptides with affi nity for nonbiological materials and with function of mineralizing inorganic materials. the use of the functional peptides is attracting a growing interest for bottom-up fabrication approaches of nanoscale devise. zinc oxide (zno), a semiconductor with a wide direct band gap, possess unique optical, acoustic, and electronic properties, so that it is one of most widely studied metal oxides for solar cells, ultra violet nanolaser, blue light-emitting diode and so on. this wide variety of applications requires various fabrications of morphologically and functionally distinct zno nanostructures. here, the peptides which are selected from the phage-displayed peptide library with a -mer on the surface, can bind zno particle but not other metal oxides particle, and further, the zno-binding peptides play an important role on the crystal growth of zno in its synthesis. the anti-zno peptide can assist the synthesis of zno nanoparticles from a zn(oh) solution even at ºc, and further, the peptide leads to the self-assembly of synthesized zno particles to fl ower-type morphologies. we describe the biomimetic zno synthesis using the artifi cial peptide with affi nity for zno. the design of nanoparticles suitably functionalized for applications in biology or medicine is an ongoing challenge for both chemists and biologists. nanoparticles such as quantum dots, gold nanoparticles or carbon based-materials offer, in addition to their remarkable physical properties, the possibility to be functionalized by biomolecules, making them suitable for sensing, detection, diagnostic, and/or therapeutic applications. recently, quantum dots have been designed for biological imaging owing to their unique size-dependent fl uorescence properties. however, their plausible toxicity still remains a major concern for in vitro and in vivo applications. nanodiamonds, (nds) are also candidates for biomedical applications considering their intrinsic or induced fl uorescence and biocompatibility. the work will describe: i) the functionalisation and the characterization of nm non-fl uorescent cnds and ii) their use in toxicity and transport studies in living cells after the grafting of a fl uorescent model peptide. we chose to introduce a nonpermeant fl uorescent peptide for the tracking of the nanoparticles on or inside the cells. nanodiamonds (cnds, nm) coated by silanisation or with polyelectrolyte layers have been grafted with a fl uorescent thiolated peptide via a maleimido function, leading to aqueous colloidal suspensions stable for months. for each step of the preparation, the diameter of the nds measured by dynamic light scattering (dls), the zeta potential, the amount of amino groups grafted onto the surface, as well as the stability of the different nds suspensions no cytotoxicity was observed up to hours incubation with cho cells with any of the prepared ( g/ml). their capacity to enter mammalian cells, and their localisation inside cho cells, have been ascertained by confocal microscopy, refl ected light and fl uorescence. hepatitis b virus is one of the most common problem and potential danger of all over the world and also in our country. it is known that the use of proteins and peptide antigens as vaccines has several potential advantages over whole viral or bacterial preparations. to elicit the maximum immunogenic response from synthetic peptide antigens ,it is generally necessary to bind the peptide to carrier protein. moreover, to realize the full potential of synthetic peptide antigens protein-peptide conjugate will have to be used with an adjuvant. we have developed new approches for obtaining highly immunogenic peptide conjugatessynthetic polyelectrolytes (pe) were used for the conjugation with peptide molecules in which pe carry out the carrier and adjuvant roles simultaneously. this concept drive us to create new immunogenic bioconjugates of hepatitis b surface antigenic polypeptides (hbsag) with synthetic pe. in this study we used the region - of the s gene. the synthesis of peptides was performed by explorer pls ® automated microwave synthesis workstation (cem), by using f-moc chemistry. copolymers of acrylic acid with different monomers were used as a pe for the conjugation with polypeptides. pe-peptide conjugates was synthesized by microwave assisted method. composition and structure of bioconjugates werw characterized by hplc, spectrofl uorometry and different spectrophotometric methods. protein-protein and protein-ligand interactions are among the few essential cell processes whose understanding provide us insights into fundamental events of the life cycle. aside from in silico methods, natively folded proteins are an absolute prerequisite in all current biotechnological tools used for the study of these interactions. however, the recently developed ianus peptide array has a potential to evolve in to a protein-free method for detection of protein-protein interaction sites. using this assay, protein-protein interactions could be represented by and investigated as peptide-peptide interactions using peptide pairs immobilized on a solid support.( ) . two main obstacles had to be overcome for successful implementation of this array: (i) the library size and (ii) identifi cation of interacting peptide pairs. if, for example, interactions between two small proteins of only approx. amino acids each should be analyzed, a library of ca. peptide pairs would be needed to cover all possible combinations of overlapping peptides derived from these proteins. although libraries of several thousand peptides were already successfully synthesized, the library size could be reduced drastically if the binding pocket of one protein is already known or if the protein-ligand interactions are studied. up to date, two methods for the identifi cation of interacting peptides in a library have been developed in our group. both methods will be demonstrated in studies of protein-protein and protein-ligand interactions. the role of the non-helical tailpiece in myosin ii assembly the hebrew university of jerusalem, israel self-assembly of macromolecules into large complexes is often mediated by folding of disordered regions. we used peptides to investigate the role of the non-helical tailpiece from non-muscle myosin iic (nm-iic) in its self-assembly process. nm-iic is a motor protein composed of a globular motor domain in the n-terminal region and a coiled-coil rod domain in the c-terminal region, which terminates with a non-helical -residue tailpiece (residues - ) . nm-iic molecules undergo self-assembly into fi laments that are necessary for its contractile activity. the tailpiece has an unfolded character and participates in the assembly process of nm-iic. the n-terminal region of the tailpiece (residues - ) has a net positive charge of + while its c-terminal region (residues - ) has a net negative charge of - . we designed peptides that correspond to the entire tailpiece and to its negative and positive regions. these peptides were used to study interactions with the rod, their structures in the free and bound states, and structural changes they undergo upon binding to residues - of nm-iic rod. fluorescence anisotropy binding studies showed that a peptide corresponding to the positive region of the tailpiece (residues - ) bound the nm-iic rod (residues - ) with an affi nity of m. circular dichroism studies showed that the positively charged peptide became folded upon binding the rod, indicated by a shift of the spectral minimum from nm to nm upon binding. a peptide corresponding to the negative region of the tailpiece (residues - ) did not bind nm-iic (residues - ). based on our results, we suggest that the positive region of the tailpiece is required for ordering and aligning neighboring molecules by electrostatic attraction, leading to fi lament formation. our results provide molecular insight into the role of the structurally disordered tailpiece of nm-iic in the selfassembly process. stefanidakis, michael; karjalainen, katja; pasqualini, renata; arap, wadih; koivunen, erkki md anderson cancer center, united states acute myelogenous leukemias (aml) are characterized with non-random patterns of medullary and extramedullary invasion. we hypothesized that a supramolecular complex, the leukemia cell invadosome, which contains certain integrins, matrix metalloproteinases (mmps) and other as yet unidentifi ed proteins, is essential for tissue invasion and may be central to the phenotypic diversity observed in the clinic. here we show that the specifi c binding of mmp- to leukocyte surface ám integrin is required for pericellular proteolysis and migration of amlderived cells. an effi cient antileukemia effect was obtained by peptide inhibitors that prevented prommp- cell surface binding, transmigration through an endothelial cell layer, and extracellular matrix degradation. notably, the functional protein anchorage between ám integrin and prommp- described in this study does not involve the enzymatic active sites targeted by any of the existing mmp inhibitors. taken together, our results provide a biochemical working defi nition for the human leukemia invadosome. disruption of specifi c protein complexes within this supramolecular target complex may yield a new class of anti-aml drugs with anti-invasion (rather than cytotoxic) attributes. the molecular mechanisms of action of the polycationic lysine homoand heterodendrimers on functional activity of adenylyl cyclase signaling system (ac system) in the myocardium and the brain of rats were studied. the lysine homodendrimers of the third (i), the fourth (ii) and the fi fth (iii) generations, as well as the lysine heterodendrimers of the fi fth generation -[(nh ) (lys-glu) (lys-glu) (lys-glu) (lys-glu) (lys-glu) lys-ala-ala-lys(clac)-ala-nh ] (iv), [(nh ) (lys-ala) (lys-ala) (lys-ala) (lys-ala) (lys-ala) lys-ala-lys(clac)-ala-ala-nh ] (v) and [(nh ) (lys-gly-gly) (lys-gly-gly) (lys-gly-gly) (lys-gly-gly) (lys-gly-gly) lys-gly-gly-lys(clac)-ala-ala-nh ] (vi) stimulated by receptor-independent mechanism the activity of heterotrimeric g proteins, preferably of inhibitory type, and interacted with c-terminal regions of their -subunits. the homodendrimers ii and iii and heterodendrimer v were more effective g protein activators. the treatment of the membranes with pertussis toxin (inactivating g i protein), but not with cholera toxin, led to a decrease of g protein activation effect of the dendrimers. the lysine dendrimers disturbed the functional coupling of the receptors of biogenic amines and peptides hormones with g i proteins and, to a smaller extent, with g s proteins. it was illustrated by the decrease of regulatory effects of the hormones on ac activity and g protein gtp binding and by the decrease of receptor affi nity to agonists in the presence of the lysine dendrimers, as result of receptor-g protein complex dissociation. it was shown that the molecular mechanisms of the action and the g protein selectivity of the polylysine dendrimers are similar to those of mastoparan and melittin, natural toxins of insect venom, which also preferably activate g i/o proteins and inhibit g i/o -coupled signaling. acknowledgements: supported by rfbi (grant - - ) and «russian science support foundation». genetics, bielefeld university, germany dna-protein interactions are a key element in the regulation of cellular processes. transcription factors are able to recognize their cognate dna sequences and regulate the expression of proteins. as a model system, the specifi c dna binding of the transcription factor phob from e. coli is investigated in single molecule experiments. structurally, phob belongs to the family of winged helix-turn-helix proteins. it is composed of a transactivation domain (amino acids - ) and a dna binding domain (amino acids - ). after phosphorylation of the transactivation domain, the protein binds to specifi c dna sequences containing a tgtca consensus sequence. different c-terminally modifi ed protein epitopes representing parts of the dna binding domain of phob were chemically synthesized using microwave assisted solid phase peptide synthesis. the binding contributions of these molecules are compared to the complete dna binding domain ( - ). this protein was purifi ed using intein mediated protein splicing, an additional cysteine was ligated to the protein by intein mediated ligation. performing single molecule force spectroscopy experiments, kinetic off-rates were obtained, and sequence specifi c dna-binding of both peptide and protein was proven in competition experiments. alanine scans of strategic residues revealed the contributions of single amino acid residues for peptides and proteins. structural investigations of the peptides, the proteins and dna/protein complexes were performed using circular dichroism measurements. this method revealed structural differences of the peptides, proteins and dna upon complex formation. furthermore, the protein/dna or peptide/dna interaction was determined by surface plasmon resonance experiments and electrophoretic mobility shift assays. acknowledgment: this project was supported by dfg (sfb ). reversed-phase chromatography is important in the development of well-characterized peptide pharmaceuticals. several factors infl uence sensitivity, resolution, and retention for lc and lc-ms applications: bonding chemistry, pore size, particle size, column confi guration, and solvent composition. by decreasing the particle size of hplc packings, column effi ciency is increased. we demonstrate the use of short -mm length hplc columns packed with . m, Å c silica for ultrafast, one-minute peptide analysis with moderate backpressures (< psi) using a conventional hplc system. for more complex peptide samples, such as protein digests, on conventional hplc, a novel column confi guration packed with . m, Å c is described for -to- min. separations that traditionally take to minutes. we also discuss fast two-minute separations on an ultra-high pressure system using a variety of . m, small pore columns with unique selectivity. "unknown-genome" proteomics-based identifi cation of a new nadp-epimerase/dehydratase from desulf. phosphitoxidans by inverted-pcr, edman-sequencing and high resolution mass spectrometry protein identifi cation in proteomics is generally based on the availability of genomic data. using amenable databases, identifi cation in "bottom-up" proteomics is often straightforward but is highly complex in the absence of genome data, which typically requires "de novo"-identifi cation approaches. we present here a new approach for identifi cation of proteins from a bacterial strain, desulfotignum phosphitoxidans, with unknown genomic background, using a combination of (i), inverted pcr of degenerate primers derived from n-terminal edman sequencing, and (ii) high resolution maldi-fticr mass spectrometric peptide mass fi ngerprint-proteomics of expressed proteins. desulfotignum phosphitoxidans is an anaerobic sulfatereducing bacterium from marine sediment in which phosphite oxidation was found crucial for energy metabolism. culturing under different growth conditions provided specifi cally expressed proteins with molecular masses of ca. kda in the presence of phosphate, which were subjected to d-gel electrophoretical separation for soluble and membrane fractions using pdquest comparative analysis. n-terminal sequences of the proteins, determined by edman analysis were used for inverted pcr of degenerate primers, and provided a series of orf candidates, one of which coded for a putative nad-dependent dehydratase. in a complementary approach, protein spots from the d-gels were excised, digested with lys-c protease, and digestion mixtures analysed by maldi-fticr-ms. the accurate peptide masses unequivocally matched to identify a new nad-dependant epimerase/ dehydratase. the detailed functional characterization of the new protein, and further development and application of this approach to proteome analysis with unknown genomic background, are currently in progress. based on specially treated large pore silica and enhanced with a proprietary bonding process, vydac ms reversed-phase (rp) hplc columns offer superior performance for peptides and proteins. the deamidation of human growth hormone (hgh) has been monitored for many years by rp using vydac columns. the vydac ms c column provides the best overall performance characteristics (recovery, resolution, and peak symmetry) for the common important assay of hgh and desamido hgh. although hydrophobic membrane proteins are particularly diffi cult to separate, the vydac ms c column provides better separation and recovery (up to % higher vs. other leading columns) for a reptilian reovirus p protein and myristolyated form, a component of a potentially new vaccine delivery system. separation of the trypsin digest of fetuin, a glycoprotein, exhibits improved selectivity for peptide mapping on a vydac ms c column compared to other c columns, revealing some peaks otherwise not seen. the improved selectivity for peptides on the vydac ms columns results in better primary structure defi nition and easier identifi cation of degradation products and other protein characteristics. hemoglobin peptides in mammalian tissues: facts or artifacts?yatskin, oleg; karelin, andrei; ivanov, vadim shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, russian federation comparative rp-hplc and ms analysis of peptide composition of rat brain, heart, lung and spleen acidic extracts was performed. the extracts were prepared from snap-frozen organs both in the presence and in absence of protease inhibitors ( - m pepstatin a, - m pmsf, mm edta) which stabilize the peptide composition of tissue acidic homogenates. the absence of inhibitors led to appearance of novel peptides and did not affect the concentration of predominant peptide components detected in samples prepared with protease inhibitors. therefore, the latter were considered as present in the tissues prior to extraction, i.e. as endogenous. the majority of predominant peptide components were found common in the studied tissues. we believe that the presence of predominant (> pmol/g) peptide components in the protease-inactivated tissue extracts can be reliably extrapolated to their in vivo presence in the tissues. the endogeneity of minor (< pmol/g) components requires a separate consideration. generally, the results of peptidomic studies strongly depend on sample preparation procedures involved, making diffi cult the straightforward comparison of the results obtained by different research groups. urine is a promising source of endogenous peptide biomarkers, because the protein concentration is -fold lower than in plasma but the peptide concentration is equal. urine contains small metabolites that interfere with peptide analysis and urine fl ow varies between subjects and within a day. these variations pose a challenge to urine sample preparation, which, in case of serum, has been shown to be critical for reproducibility. we aim to optimize the enrichment method for urine peptides, to characterize these and to fi nd potential biomarkers. all methods resulted in good peptide recovery if the peptide concentration was normal and metabolite content low. slightly different peptides were obtained with different methods. the df preferentially recovered hydrophilic peptides, while sec-spe recovered high molecular weight peptides better than the df-method. sec-spe provided the best peptide enrichment especially for samples with high metabolite concentration. normalization was critical especially with very dilute samples. without normalization the number of peptides detected was highly dependent on urine concentration. normalization against specifi c gravity proved the most reproducible results. we have identifi ed several peptides found in all samples and some differences between cancer and control samples. validation of these differences is currently under way. jalili, pegah ; ball, haydn sigma-aldrich, united states; in order to improve the detection of phosphorylated peptides/proteins, we developed a novel protocol that involves the chemical derivatization of phosphate groups with a chemically engineered biotinylated-tag (biotintag), possessing three functional domains; a biotin group for binding to avidin, a base-labile -carboxy fl uorenyl methoxy-carbonyl ( -carboxy fmoc) group, and a nucleophilic sulfhydryl moiety on the side-chain of cysteine. using this approach, the derivatized, enzymatically digested peptides were selectively separated from unrelated sequences and impurities on immobilized avidin. unlike previously published phosphopeptide enrichment procedures, this approach upon treatment with mild base, liberates a covalently bound gly-cys analog of the peptide(s) of interest, exhibiting improved rp-hplc retention and ms ionization properties compared to the precursor phosphopeptide sequence. the results obtained for a model peptide akt- and ovalbumin protein digest, demonstrated that the method is highly specifi c and allows selective enrichment of phosphorylated peptides at low concentrations of femtomoles/ l. in search of physiological substrates of brain prolyl oligopeptidase. prolyl oligopeptidase (pop) is a serine protease, which cleaves small peptides at the carboxyl side of an internal proline residue. substance p, arginine-vasopressin, thyroliberin and gonadoliberin are proposed physiological substrates of this protease. pop has been implicated in a variety of brain processes including learning, memory, and mood regulation, as well as in pathologies such as neurodegeneration, hypertension and mood disorders. however, there is no defi nite information about the physiological substrates of pop in brain. we have determined previously that some pop inhibitors, when administrated orally or intraperitoneal, are able to cross the blood-brain barrier and inhibit pop ex vivo. in this work also inhibition duration according to the doses were determined. using this system we studied the natural occurring peptide profi le in brain tissues from inhibited animals and compare it with the profi le in control tissue. we were able to design a method in which the background peptide level was importantly decreased which allowed us to identify, with high degree of confi dence, the peptides which were changed in rat hypothalamus upon pop inhibitor treatment. conclusions about the physiological substrates of pop are discussed. [ , ] . in the present work, we further studied the dependence of peptide production by the cells on their functional state. using rat hepatocytes as a model, we compared peptide sets generated by: ( ) primary hepatocytes in a differentiated state; ( ) dedifferentiated primary hepatocytes with fi broblast-like phenotype; ( ) rat hepatoma cells. peptides were extracted from the cellular lysates and supernatants by solid-phase extraction and then separated by rp-hplc. peaks were analyzed with maldi-tof and/or maldi-tof/tof. acknowledgements: this work was supported by ras presidium grant "molecular and cellular biology"; russian president grant "scientifi c schools" # . . ; rfbr grant # - - -a. triostin a is a naturally occurring quinoxaline depsipeptide antibiotic originally isolated from streptomyces s- - . with its two quinoxaline moieties it bis-intercalates into dna in a gc selective manner. triostin a contains n-methylated l-valine and l-cystein. the unmethylated analog tandem binds at selectively due to a different hydrogen bonding pattern. substitution of the disulfi de bridge of tandem by an azobenzene moiety leads to photoswitchable analogs. their photoswitchability was investigated and quantifi ed using uv, cd and nmr spectroscopy as well as hplc. in order to determine the three-dimensional structure, conformational analysis by nmr spectroscopy in combination with molecular dynamics calculations was carried out. in this process, distance restraints were obtained from nmr spectra measured in dmso-d and applied in distance geometry/simulated annealing followed by molecular dynamics calculations. as it is possible to distinguish between cis-and transisomer in nmr spectra due to the difference in the chemical shifts, structures of both isomers can be investigated. dna binding studies were carried out using an optical tweezers setup with -dna. furthermore, uv melting curves were recorded for the tandem derivatives in complex with dna. cd spectroscopy was applied to observe changes in dna structure upon formation of the complex. in addition to structural investigations, the antibiotic activity of the tandem derivatives was investigated in minimal inhibition concentration assays against gram-positive bacillus subtilis. acknowledgment: this work was supported by the dfg (sfb ). k. gaus gratefully acknowledges the fi nancial support (phd grant) by the international nrw graduate school of bioinformatics and genome research. cyclic peptide nanotubes are formed by self-assembly of cyclic peptides with alternating l-and d-amino acid. however, peptide nanotube selfassembly phenomenon had not yet been made cleared. intermolecular electrostatic interaction is one of the important driving forces to assemble cyclic peptides to peptide nanotubes. here in, a series of cycilc hexapeptides, cyclo(-l-lys-gly-) , cyclo(-l-glu-gly-) , cyclo(-l-lys-d-ala-) , cyclo(-d-glu-l-ala-) , cyclo(-l-trp-d-lys-) , and cyclo(-l-trp-d-glu-) , were designed and synthesized by solid-phase peptide synthesis using fmoc strategy. turbidity study revealed that the abilities of self-assembly formations of the : mixtures of cyclo(-l-trp-d-lys-) and cyclo(-l-trp-d-glu-) in aqueous solution are extremely higher than the abilities of each individual positively or negatively charged cyclic peptides. transmission electron microscope (tem) experiments showed that mixture of cyclo(-lys-xxx-) and cyclo(-glu-xxx-) formed fi brous structure. tem images indicated that, the diameters of the nanotubes were approximately - nm. diameter of similar cyclic peptides was found to be nm approx. these results also supported that the bundle formation of peptide nanotube. conformations of monomer cyclic hexapeptides were calculated based on nmr observation. estimated formation of peptide nanotube is consisted with the bundle structure. koèevar, nina; obermajer, nataša; kreft, samo faculty of pharmacy, slovenia therapeutic proteins offer a promising potential as effective drug compounds. however, proteins are generally poorly transported across biological membranes due to hydrophilicity. fatty acylation with reactive fatty acid derivatives represents one of the basic methods for increasing the proteins' hydrophobicity and improving membrane permeability. key: cord- -jqm hxps authors: nan title: abstract date: - - journal: eur biophys j doi: . /s - - - sha: doc_id: cord_uid: jqm hxps nan standard proteomics techniques are unable to describe the stoichiometry, subunit interactions and organisation of assemblies since many are heterogeneous, present at low cellular abundance and frequently difficult to isolate. we have combined two existing methodologies to tackle these challenges: tandem affinity purification (tap) and nanoflow esi-ms. we use methods designed to maintain non-covalent complexes within the mass spectrometer to provide definitive evidence of interacting subunits based on the masses of complexes and subcomplexes generated by perturbation both in solution and gas phases. structural models will be presented for three oligomeric protein complexes of unknown structure: the yeast exosome and the human u snrnp and eif complexes. these models will then be examined within the context of their function. recent developments in mass spectrometry have added a further dimension to our studies of protein complexes: that of their collision cross-section. using ion mobility mass spectrometry we have been able to add spatial restraints to our models validating our models with measurements of collision cross-sections. very recently we have had a considerable breakthrough which has enabled us to preserve intact membrane complexes in the gas phase . this enables us to establish lipid and nucleotide binding and to define the stoichiometry and post translational modifications within the intact transmembrane regions of a number of complexes. in vivo molecular sensing: fluorescence beyond labeling f. beltram scuola normale superiore, i- pisa, italy fluorescent molecules are powerful reporter tools that have much extended the impact of optical microscopy, particularly thanks to the flexibility of genetically encoded tags. detection can now target single molecules even in the complex environment of intact live cells offering unprecedented insight on biological processes in real time in live cells and tissues. fluorescent labels, however, can do more that this. our increased ability to tailor molecule and, more in general, nanosystem properties allows us to design, produce and exploit intelligent tags that can actually analyze the cellular environment. today multifunctional nanosystems can be produced that provide a signal dependent on the value of a specific biochemical parameter. importantly these nanosystems can target specific subcellular domains and have the ability to be used also in the case of live organisms. recent results will be discussed that highlight the impact of nanobiotechnology in this context with a particular emphasis given to methods suitable for in vivo studies that can be transferred to the biomedical world. far-field optical nanoscopy s. w. hell max planck institute, göttingen, germany the resolution of a far-field optical microscope is usually limited to d = λ/ ( n sin α) > nm, with n sin α denoting the numerical aperture of the lens and λ the wavelength of light. we will discuss lens-based fluorescence microscopy concepts that feature a resolving power on the nanoscale. all these concepts share a common basis: exploiting selected (pairs of) states and transitions of the fluorescent marker to neutralize the limiting role of diffraction. specifically, the fluorophore is switched on and off, that is, between a bright and a dark state, to detect the emission of adjacent features sequentially in time. the first viable concept of this kind was stimulated emission depletion (sted) microscopy in which the fluorescence ability of the dye is switched off by stimulated emission. in the sted microscope, the extent of the region in which the molecule is able to fluoresce follows d ≈ λ/ n sin α + i/i s , meaning that fluorophores that are further away than d can be separated. i is the intensity that drives a fluorophore from the bright fluorescent state to the dark ground state by stimulated emission. i s depends (inversely) on the lifetime of the states. for i/i s → ∞, it follows that d → , meaning that the resolution can be molecular). altogether, far-field optical 'nanoscopy' is a fascinating development in optics with high relevance to the many areas of sciences, in particular the life sciences. since it has already been a key to answering important questions in biology, and owing to its simplicity and commercial availability, we expect far-field fluorescence 'nanoscopes' to enter most cell biology and many nanoscience laboratories in the near future. grabbing the cat by the tail: discrete steps by a dna packaging motor and the inter-subunit coordination in a ring-atpase c. bustamante, j. moffitt university of california, berkeley, california, u.s.a. as part of their infection cycle, many viruses must package their newly replicated genomes inside a protein capsid. bacteriophage ϕ packages its . mm long double-stranded dna into a nm dia. x nm high capsid using a multimeric ring motor that belongs to the asce (additional strand, conserved e) superfamily of atpases. a number of fundamental questions remain as to the coordination of the various subunits in these multimeric rings. the portal motor in bacteriophage phi is ideal to investigate these questions and is a remarkable machine that must overcome entropic, electrostatic, and dna bending energies to package its genome to near-crystalline density inside the capsid. using optical tweezers, we find that this motor can work against loads of up to ∼ piconewtons on average, making it one of the strongest molecular motors ever reported. we establish the force-velocity relationship of the motor. interestingly, the packaging rate decreases as the prohead fills, indicating that an internal pressure builds up due to dna compression attaining the value of ∼ megapascals at the end of the packaging. we show that the chemical energy of atp is converted into mechanical work during phosphate release. using ultra-high resolution optical tweezers, we determined the step size of the motor and established the coordination of the polymerases around the ring. we propose a comprehensive model of the operation of this motor. watching proteins function in real time via timeresolved x-ray diffraction and solution scattering p. a. anfinrud laboratory of chemical physics/niddk, nih, bethesda, maryland, u.s.a. to generate a deeper understanding into the relations between protein structure, dynamics, and function, we have developed x-ray methods capable of probing changes in protein structure on time scales as short as ps. this infrastructure was first developed on the id b time-resolved x-ray beamline at the european synchrotron and radiation facility, and more recently at the id b biocars beamline at the advanced photon source. in studies of ligand-binding heme proteins, a picosecond laser pulse first photolyzes co from the heme, then a suitably delayed picosecond x-ray pulse passes through the protein and the scattered x-rays are imaged on a d detector. when the sample is a protein crystal, this "pump-probe" approach recovers time-resolved diffraction "snapshots" whose corresponding electron density maps can be stitched together into movies that unveil the correlated protein motions that accompany and/or mediate ligand migration within the hydrophobic interior of the protein. when the sample is a protein solution, we recover timeresolved small-and wide-angle x-ray scattering patterns that are sensitive to changes in the size, shape, and structure of the protein. scattering studies of proteins in solution unveil structural dynamics without the constraints imposed by crystal contacts; thus, these scattering "fingerprints" complement results obtained from diffraction studies. this research was supported in part by the intramural research program of the nih, niddk dc-sign is a trans-membrane protein expressed on antigen presenting cells and recognizes pathogens like hiv- , hepatitis c virus and ebola. by electron microscopy and near-field optical nanoscopy, we demonstrated that at the plasma membrane dc-sign is organized in well-defined nanodomains of - nm in diameter. intensity-size correlation analysis revealed remarkable heterogeneity in the nanodomains molecular packing density. we constructed and characterized several dc-sign mutated forms lacking specific molecular domains. by immunogold labeling and spatial point pattern analysis, we show that the extracellular neck domain is essential for dc-sign nanoclustering. finally, we present a model that describes the probability of a cell to have a certain number of receptors joining the contact site in the initial encounter with an external object. monte carlo simulations subsequently define the parameters that are determinant in the object-cell encounter. our results show that receptor nanoclustering is of particular importance for binding objects of sizes comparable to the nanocluster size, indicating that the nanoscale spatial organization of dc-sign is optimized for binding to virus-sized objects. imaging of mobile stable lipid rafts in the live cell plasma membrane m. brameshuber , j. weghuber , v. ruprecht , h. stockinger , g. j. schuetz johannes kepler university linz, austria, medical university of vienna, austria the organization of the cellular plasma membrane at a nanoscopic length scale is believed to affect the association of distinct sets of membrane proteins for the regulation of multiple signaling pathways. based on in vitro results, conflicting models have been proposed which postulate the existence of stable or highly dynamic platforms of membrane lipids and proteins. here we directly imaged and further characterized lipid rafts in the plasma membrane of living cho cells by single molecule tirf microscopy. using a novel recording scheme for "thinning out clusters while conserving stoichiometry of labeling" , molecular homo-association of gpi-anchored mgfp was detected at • c and ascribed to specific enrichment in lipid platforms. the mobile mgfp-gpi homo-associates were found to be stable on a seconds timescale and dissolved after cholesterol depletion. having confirmed the association of mgfp-gpi to stable membrane rafts, we attempted to use an externally applied marker to test this hypothesis. we used bodipy-gm , a probe that was recently reported to be enriched in the liquid-ordered phase of plasma membrane vesicles. when applied to cho cells at different surface staining, we found that also bodipy-gm homo-associated in a cholesterol-dependent manner, thus providing further evidence for the existence of membrane rafts. [ ] appl phys lett , ( ) . synapsin knock-out mice as an in vitro model of human epilepsy studied with multi-electrode arrays d. f. boido, p. farisello, p. baldelli, f. benfenati department of neuroscience and brain technologies, italian institute of technology, genova, italy mutant mice lacking synapsins (syn), a family of synaptic vesicles (sv) proteins implicated in the regulation of neurotransmitter release and synapse formation, are epileptic. the attacks appear after the third month of age and severity increases with age. several mutations of syn genes have been found in families of patients with epilepsy. we used micro-electrode arrays (meas) to study spontaneous and chemically evoked epileptiform activities in cortico-hippocampal brain slices obtained from wild-type (wt) and synko mice. -months old synko mice show sporadic ictal (ic) events in the entorhinal cortex. a potassium channel blocker, aminopyridine ( ap), elicits ic and inter-ictal (i-ic) events in both wt and synko slices. in the hippocampus of young synko ( -days old) mice, ap induces i-ic events at higher frequencies than in wt mice. also the frequency of ic events, mainly observed in the cortex, is higher in synko. the analysis of adult ( -year old) mice, revealed a clear age-related aggravation, which paralleled the increase in the severity of the epileptic phenotype observed in vivo. many slices from adult synko mice showed an ic event, while wt slices were refractory at this age to experience ic activity. synko mice are useful to study how neuronal network hyperexcitabilty due to mutations in sv proteins leads to the development of epileptiform activity. meas proved themselves to be useful tools to characterize the epileptic signals foci and patterns of propagation. high electron mobility transistor (hemt) structures were used to bridge the gap between the analysis of biological reactions and biophysical characterization. the combination of nanotechnological measurement approaches with biological reactions provides new possibilities for living cell examinations after exposure to ionizing radiation and basically during the irradiation experiments itself. in this transdisciplinary approach experimental data and handling of biological material enables the identification and specification of systems properties of biological responses to ionizing radiation at different hierarchical levels. gan/algan-heterostructures form a hemt with a gate very sensitive to ph-value changes and potential changes in general. to record cell membrane potentials and ion fluxes during and after irradiation experiments living cells are cultivated on the functionalised biocompatible chip surface. here, we present results of x-ray stimulated cell responses grown on gan-chip surfaces. we recorded transistor signal changes of . µa within s caused by an irradiated cell monolayer. to measure cell potentials, not only after irradiation experiments but also during the irradiation itself expands the examination restrictions in an enormous way. measuring diffusion by spatial-cross-correlation e. gratton, m. a. digman laboratory for fluorescence dynamics, university of california, irvine, u.s.a. fluorescence correlation spectroscopy (fcs) has emerged as a very powerful method to study the motions of proteins both in the interior and exterior of the cell. it provides information at the single molecular level by averaging the behavior of many molecules thus achieving very good statistics. single particle tracking (spt) is also a highly sensitive technique to measure particle movement. however, the fcs method suffers in spatial resolution while the spt technique only allows for the tracking of isolated molecules. here we propose a change of paradigm in which using spatial pair cross-correlation functions we can overcome this limitation. our method measures the time a particle takes to go from one location to another by correlating the intensity fluctuations at specific points on a grid independently on how many particles are in the imaging field. therefore we can trace the average path of the particles. for example, our method could be used to detect when a protein passes the nuclear barrier and the location of the passage. this information cannot be obtained with the frap (fluorescence recovery after photobleaching) technique or the image correlation spectroscopy method. the interaction of the bax c-terminal domain with membranes j. c. gomez-fernandez, s. sanchez-bautista, a. perez-lara, s. corbalan-garcía departamento de bioquimica y biologia molecular. universidad de murcia, murcia, spain the c-terminal domain of the pro-apoptotic protein bax (bax-c) acts as a membrane anchor during the translocation to the membrane of this protein leading to programmed cell death. we have used static and mas-nmr techniques to show that the interaction of bax-c with membranes is modulated by the presence of a negatively charged phosphatidylglycerol. the width of the resonance peaks were considerably more increased by bax-c, in the presence of phosphatidylglycerol. bax-c substantially decreased the t relaxation times of phosphatidylglycerol and those of phosphatidylcholine when mixtured with phosphatidylglycerol but they were not decreased when phosphatidylcholine was the only phospholipid present in the membrane. c-mas-nmr showed that t values were decreased when bax-c was incorporated and, when phosphatidylglycerol was also present, the decrease in t affected considerably more to some carbons in the polar region. these results indicate that bax-c interacts differently with the polar part of the membrane depending on whether phosphatidylglycerol is present or not, suggesting that an electrostatic interaction of bax-c with the membrane determines the membrane disposition of this domain. fluorescence spectroscopy showed that the trp residues of bax-c were located in a microenvironment more hydrophobic when phosphatidylglycerol was present. h. g. franquelim , l. m. s. loura , n. santos , m. castanho instituto de medicina molecular, univ. lisboa, portugal, faculdade de farmácia, univ. coimbra, portugal since the efficacy of hiv fusion inhibitors was previously reported to be related to an ability to interact with membranes, we studied the interaction of the hiv fusion inhibitor sifuvirtide, a aa negatively charged peptide, with lipid vesicles. since this peptide has aromatic residues, fluorescence spectroscopy techniques were used with no need for attached probes. results showed no significant interaction with both zwitterionic fluid phase and cholesterol-enriched membranes; however extensive partition to fluid phase cationic membranes were observed. in the dppc gel phase, however, an adsorption at the surface of these membranes was detected by using a differential quenching approach with lipophilic probes, as well as by fret. moreover, the interaction with gel phase membranes seems to be specific towards pc vesicles, since no significant interaction was retrieved for membranes composed by shingomyelin and ceramide. besides fluorescence, atomic force microscopy and zeta-potential were used to further investigate this issue. our results show a selectivity and specificity of the peptide towards rigid domains, where most of the receptors are found, and help explain the importance of the interaction with membranes in the improved efficacy of sifuvirtide compared to other fusion inhibitors, by providing a local increased concentration of the peptide near the fusion site on both cellular and viral membranes. the study of molecular dynamics at the single-molecule level with fluorescence far-field optics offers new detailed insights into scientific problems, especially in living cells. unfortunately, the resolution of common far-field techniques is limited to about nm in the lateral direction by diffraction. in recent years, several concepts such as stimulated emission depletion microscopy (sted) have been successfully applied to overcome the diffraction barrier by exploiting the photophysical properties of fluorescent labels. we present the combination of high resolution sted microscopy with different fluorescence fluctuation techniques providing the unique ability to study molecular dynamics with high spatial (< nm) and temporal resolution (< ms) in living cells. using fluorescence correlation spectroscopy (fcs) and general single-molecule analysis, we were able to explore single-molecule dynamics in up to -fold reduced focal volumes on two-dimensional samples such as lipid membranes with excellent signal-to-noise ratios. special attention is drawn to inhomogeneous lipid diffusion on the plasma membrane of living cells . by extending the available spatial scale of standard single-molecule fluorescence far-field spectroscopy techniques, our experiments outline a new way of approaching scientific problems. modulation of the properties of membrane microdomains as a control mechanism in cellular physiology p. o'shea cell biophysics group, institute of biophysics, imaging & optical science, school of biology, university of nottingham, nottingham ng rd, u.k. this presentation will outline the molecular-physical rationale of how membrane microdomains may modulate the behaviour of membrane receptor systems as a controlling mechanism in cell signaling. a number of external factors that modulate these properties will be indicated that have a bearing on controlling cellular behaviour. some of key questions will be considered such as the factors that control the assembly and disassembly of microdomains, the size and numberdensity of the microdomains and the lifetime that they exist within the membrane. the technical challenges that these questions identify will also be outlined with some possible solutions. throughout this presentation, correlations will be made between theory and experiment as well as between model membrane systems and real cellular systems. structural and dynamic properties of caveolin- and - fragments at the membrane interface c. le lan , j. gallay , m. vincent , j.-m. neumann , b. de foresta , n. jamin cea, ibitecs, sb sm, & ura cnrs , gif-sur-yvette, france, ibbmc, université paris-sud, umr -cnrs, ifr , orsay, france caveolins are major protein components of caveolae, microdomains of the plasma membrane involved in a large number of biological functions, including signal transduction, cholesterol homeostasis and transport. the consensus topological model of caveolin- includes a small central intramembrane region ( - ) flanked by two cytosolic amphiphilic domains ( - and - ) which probably constitute in-plane membrane anchors. we investigated the interaction of the cav- ( - ) juxta-membrane segment with various membrane mimics, using fluorescence, cd and nmr. this segment partitioned better in dpc and in dm/anionic lipids micelles than in dm micelles and this partitioning was coupled with the formation of an amphipathic α-helix. this amphipathic helix was located in an average shallow position, in the polar head group region of the dpc micelle, as shown by fluorescence data and intermolecular noes, with the aromatic doublet w -f probably pointing towards the inside of the micelle on average. the peptide encompassing the homologous sequence of cav- was also localized to the dpc micelle polar head group region, in which it adopted a more stable helical conformation than cav- ( - ) . these data brings experimental support for the role of this segment as an interfacial membrane anchor. resveratrol (trans- , ', -trihydroxystilbene) , a phytoalexin present in grapes and its analogue piceatannol (trans- , , ', '-tetrahydroxystilbene) are biologically active compounds and possess potential chemopreventive and anticancer properties. the activity of resveratrol and piceatannol can be mediated by membrane effects since structure of lipid membrane domains may play an important role in cell signalling pathways. drugs interactions with dmpc bilayers was investigated using a combination of esr spectroscopy and differential scanning calorimetry. spin probes used in epr experiment were located in different part of lipid bilayer. study was performed at temperatures below and above phase transition temperature (t m ). epr spectra were simulated and displayed with ghost condensation method. the values of ϑ and ϕ (the main and asymmetry cone angles of wobbling spin probe, respectively) were taken for free rotational space parameter (Ω) calculation. the decrease of Ω values was observed in the presence of both compounds and the effect was more pronounced in lipid gel phase. order parameter and correlation time were also determined and presented in form of ghost patterns. using this approach the differential influence of studied compounds on membrane heterogenity was revealed. separating hydrostatic pressure from cellular strain: development of an in vitro model system r. sulley , j. whatmore , c. p. winlove , a. shore , j. tooke , r. ellis , k. gooding peninsula medical school, school of physics, university of exeter, uk endothelial cells (ecs) line blood vessels & are constantly subjected to haemodynamic and mechanical stresses and strains. these stimuli are known to influence ecs, modifying their morphology, intracellular signalling & gene expression. most reported systems exposing ec to mechanical forces in vitro alter pressure & strain simultaneously, making it impossible to distinguish the two potentially independent stimuli. this distinction is particularly relevant when examining the interaction of haemodynamic forces on microvascular ecs, which are exposed to low hydrostatic pressure but significant strains. this research aims to create an in vitro system that can independently examine the effects of pressure and strain, over a range experienced by ecs in the microvasculature. human ecs are seeded ( x ˆ cells/cmˆ ) on to the inner surface of compliant mm diameter tubing. which is mounted on a perfusion rig inside a sealed, fluid-filled chamber. a continuous sinusoidal cyclical strain of - % is created by a pump attached to the external chamber. lumenal pressure is generated using two hydrostatic pressure heads. validation experiments show that pressure & substrate strain can be independently varied & controlled over a physiological range. the system is now being used to investigate the effects of pathophysiological haemodynamic abnormalities on ec function. membrane potential dynamics of living cells in response to femtosecond laser irradiation n. i. smith , j. ando , k. fujita , s. kawata photonics advanced research center, osaka university, suita, osaka - , japan, dept of applied physics, osaka university, suita, osaka - , japan the ultrashort pulsed near-infrared femtosecond laser has had a large impact in biomedical research fields and in microscopy, where it has enabled new imaging methodologies. at high intensities, the focused beam of a femtosecond laser has been used to irradiate specific locations inside a cell, often beneath the cell membrane, exploiting the inherent penetration and localized absorption that comes from the multiphoton absorption physics. this has been applied in photobleaching, photouncaging, laser surgery and other experiments where the light is used not merely for observation but is instead an integral tool to interact with the dynamics of cells, to probe and perturb the cell condition. in this talk i will discuss biological and mechanical effects that can be generated by short exposures to femtosecond laser irradiation, such as calcium waves, membrane hyperpolarization, and cell contraction. this talk will concentrate on the changes in membrane potential that can occur when the cell is subjected to focused femtosecond laser beams. both depolarization and hyperpolarization of the membrane potential could be evoked, depending on the laser parameters and on the position of the laser focus. these results have implications for the use of laser beams in microscopy, optical gene transfection, and laser nanosurgery. in a recent study we showed that the melting behavior of supported lipid bilayers (slbs) on mica can be influenced by the solution ionic strength and by the slb preparation temperature [ ] . by changing these parameters we could control the coupling between the two bilayer leaflets obtaining a coupled or decoupled melting behavior. thus, we could provide evidence that the slb model system is also suited for the study of lipid/protein interactions which had been questioned in the past. then we investigated the mutual interactions between the membrane lipids (pope:popg : ) and the kcsa potassium ion channel by studying kcsa proteins reconstituted in slbs. in particular, we studied the melting behavior of the slb and the ion channel distribution relative to the different membrane phases by temperature controlled atomic force microscopy (afm). by decreasing the temperature we found that the proteins underwent diffusion so to be excluded from the growing solid ordered regions. further, the ion channels tended to accumulate at the domain boundaries or they aggregated in the liquid disordered phase. both effects have been suggested to affect protein function. when we started from a low temperature at which the membrane was mainly in the solid ordered phase the membrane melting processes started in the vicinity of the included ion channels. we report fluorescence recovery after photobleaching (frap) measurements performed at variable spot radius for t -egfp-hmop receptors on sh-sy y neuroblastoma cells in the presence of ligands. two different agonists, damgo and morphine, caused markedly different changes to receptor diffusion as compared to the basal state. like receptors in the absence of ligand, receptors bound to morphine exhibited diffusion confined to joint semi-permeable domains, but with smaller domain size and diffusion coefficient. this effect was inhibited by pertussis toxin, suggesting that this dynamic behaviour is associated with early steps of signaling. in the presence of damgo, half of the receptors displayed free long-range diffusion and the other half were confined to smaller isolated domains. hypertonic sucrose buffer suppressed this effect which we attribute to receptor entry into clathrin-coated pits. it is likely that the observation of distinct receptor dynamics in the presence of damgo and morphine involves the agonist-selective phosphorylation of the receptor. the alteration of the physiological transcriptional program is one of the constant features of cancer cells. however, the characterization of the chromatin changes at single-gene level requires going beyond the diffraction limit affecting conventional fluorescence microscopy. the ability of molecular biology techniques to obtain a detailed view of the chromatin status at a sub-promoter resolution has to pay instead the price of averaging over a cell population. we report here the application of an approach based on high-resolution cytometry, chromatin immuno-precipitation and transcriptional profiling (dna microarray) for the characterization of the transcriptional and chromatin changes induced by the oncogenic transcription factor pml/rarα. the presentation will focus on the imaging protocol employed to observe the effects on the chromatin status and the extent of the deregulation induced on transcriptional activity in acute promyelocytic leukemia cells. this multiple-approach examination provides a further step towards the comprehension of the hierarchy of chromatin modifications leading to the establishment of a malignant transcriptional program. dna is rigid negatively charged polymer and in solution exists in extended conformation. i n vivo, volume occupied by dna must be reduced to fit to tiny space of cell nucleus. to condense dna, dna-dna electrostatic repulsion must be cut off that is achieved by interaction with cationic ligands. in binding to dna, oligocations compete with salt cations (k + , na + , mg + ). description of salt dependence of oligocation-induced dna condensation is still lacking. we studied dna condensation by model oligocations, ε-oligo(l-lysines), with variation of charge from + to + . combination of light scattering, uv-monitored precipitation assay and isothermal titration calorimetry allowed covering wide range of dna (c dna ) and salt (c kcl ) concentrations. salt dependence of dna condensation efficiency of the ligand, ec (ligand concentration at the transition midpoint) displays two regimes: salt-independent at low c kcl and salt-dependent at higher c kcl (steep increase of ec with c kcl ). simple formula describing ec as function of ligand charge, c dna and dissociation constant of ligand-dna complex (k d ), was proposed. in the salt-independent regime ec is defined by c dna . salt-dependence of ec is rooted in the variation of k d with c kcl earlier described in ligand-dna binding studies. importance of our findings for description of chromatin is discussed. interaction between proteins from linker region of nucleosome in presence/absence of dna in solution i. b. kipenko , e. v. chikhirzhina , a. m. polyanichko faculty of physics, saint-petersburg state university, russia, laboratory of cell biochemistry, institute of cytology, ras, saint-petersburg, russia interactions in the linker region of the nucleosome play a key role in the structural organization of the chromatin. the most fascinating and least understood is the interplay between non-histone chromatin protein hmgb and a linker histone h . it is known that both h and hmgb bind the linker region of the dna in vivo. however it is still a matter of debate as to whether these proteins assist each other or compete upon binding. the main attention in this work is paid to the investigation of the interactions between the hmgb and h proteins in physiological environment. using circular dichroism (cd) spectroscopy we have studied the interactions between hmgb and h at various hmgb /h ratios (r). it has been shown that there is a cddetectable interaction between the proteins h and hmgb at r < . we have demonstrated that the interaction between these proteins results in changes of their secondary structure. cd indicates that the structural impact of the unordered fragments decreases while the net α-helicity of the proteins increases upon the interaction. we have also shown that large higher order structures are formed in solution. in this work we have also discussed the dna-binding properties of the hmgb and h proteins. the work was supported by a rfbr grant ( - - ) and the government of staint-petersburg. t. u. rodionova , a. m. polyanichko , v. i. vorob'ev faculty of physics, saint-petersburg state university, russia, institute of cytology of the russian academy of sciences, saint-petersburg, russia hmgb is a nonhistone chromosomal protein. data regarding the structure of the hmgb-proteins obtained so far are rather different. thermodynamic experiments reveal predominant α-helical structure of the proteins only at temperatures below + • c, i.e. under physiological conditions they are mainly disordered. despite a lot of experimental data biological role of hmgb still remains unclear. it is believed that the proteins perform structural functions in chromatin and participate in various regulatory processes in cell. using circular dichroism spectroscopy and dna melting analysis we have shown that hmgb changes its structure upon binding to dna. it was shown that at room temperature only about % of amino acid residues form α-helices, while in dna-hmgb complex the degree of the α-helicity of the protein increases to approximately %. based on the data obtained we estimate the size of hmgb binding site as - b.p. we have also demonstrated that despite of strong dna-bending properties of hmgb its binding to dna results in increase of the double helix termostability. the authors are grateful for the financial support from the russian foundation for basic research (grants - - , - - ) and the government of saint-petersburg. structural organization of supramolecular complexes of dna with chromosomal proteins hmgb and h a. m. polyanichko , h. wieser faculty of physics, saint-petersburg state university, russia, dept. of chemistry, university of calgary, canada a combination of uv and ir absorption and circular dichroism spectroscopy together with atomic force microscopy was applied to investigate the structure and formation of large supramolecular dna-protein complexes. this combination of techniques was used to overcome limitations of uv-cd spectroscopy due to considerable light scattering in such solutions. based on the analysis of ftir and uv circular dichroism spectra and afm data the interaction of dna with highmobility group non-histone chromatin protein hmgb and linker histone h was studied. it is believed, that hmgb-domain proteins perform both structural and regulatory functions in chromatin. however, the particular mechanisms of it functioning remain unclear/ our data show that histone h facilitated binding of hmgb to dna by interacting with the sugar-phosphate backbone and binding of asp\glu amino acid residues of hmgb . acting together, hmgb and h stimulated the assemblage of supramolecular dna-protein structures. the organization of the ternary complexes is modulated by the interactions between hmgb and h molecules. the dna-proteins interactions in the presence of metal ions were different, causing prominent dna compaction and formation of large intermolecular complexes. the work was supported by rfbr (grant - - ). biophysical properties and mechanisms of phage dna ejection t. mdzinarashvili , m. khvedelidze , a. ivanova , t. partskhaladze , n. shengelia i. javakhishvili tbilisi state university, tbilisi, georgia, institute of molecular biology and biophysics, tbilisi, georgia to determine the requirements for phage adsorption on bacterial cell and for the realizing resources of following dna ejection thermodynamic and hydrodynamic methods were employed. the temperature, bacterial membrane fragments and receptors had been chosen as such external factors. the phages with short and long tail, both contractile and noncontractile have been studied. our viscometric studies of the phage dna ejection induced by receptor by the example of t phage and its receptor fhua have shown that the minimum protein-to-phage ratio necessary for complete dna release is to . the viscometric study of ddvi phage dna ejection induced by membrane fragments obtained from its host cells has shown that the environmental conditions play significant role in ejection process. both methods show that the thermally induced phage dna ejection for all investigated by us phages have shown that this process is nonenthalpic. finally from our experimental results we conclude that the start of the dna ejection process from the phage particle occurs without additional energy from either a physical or chemical source. we thank gnsf for the financial support. nitroxides induce apoptosis through caspase- activation and collapse of mitochondrial potential k. matczak , a. koceva-chyla , k. gwozdzinski , z. jozwiak department of thermobiology, department of molecular biophysics, university of lodz, lodz, poland nitroxides are new class of antioxidants that have been proved to show high reactivity toward free radicals. they act as superoxide dismutase mimics dismutating superoxide anions, but can also exert pro-oxidative properties. in view of their possible dual activity nitroxides could be of great importance in medicine. we have investigated pro-apoptotic activity of pyrroline and pyrrolidine nitroxides pirolid (pd) and pirolin (pl) in human breast cancer cells. in cancer, it is the failure of malignant cells to undergo apoptosis that is crucial. using microplate fluorescence methods, we estimated kinetics of changes in mitochondrial transmembrane potential and caspase- activity in breast cancer cells mcf- treated with pirolin or pirolid. these features are connected with induction of apoptosis in some type of cancer cells. we observed steady-state increase in caspase- activity up to h of postincubation that was followed by a decrease in the enzyme activity at h. caspase- activation was considerably greater in cells treated with pirolid. both nitroxides also caused notable decrease in mitochondrial transmembrane potential, which suggest that they can induce apoptosis in breast cancer cells through mitochondrial pathway. o. chernyavskiy , l. vannucci , p. bianchini , f. difato , l. kubínová dept. of biomathematics, inst. of physiology, as cr, prague, czech republic, dept. of immunology, inst. of microbiology, as cr, prague, czech republic, lambs, dept. of physics, university of genoa, italy, dept. of neuroscience and brain technologies, the italian institute of technology, genoa, italy the second harmonic generation (shg) imaging along with confocal laser scanning microscopy in reflectance mode can be applied to imaging unstained tissues in vivo, so it can be considered as a fast and non-invasive tool for in vivo studies. murine b f melanoma cells after subcutaneous inoculation in syngeneic mice were let to develop into tumor up to - mm in diameter. microscopic images were taken before and after microwave hyperthermia treatment (mwht). the microscopic images were acquired by -photon imaging in reflectance mode, shg imaging and -photon imaging of tissue autofluorescence. the evaluation of changes in the images after mwht of the tumor demonstrated changes in the architecture and organization in both the tumor capsule and tumor mass. the presented study was supported by the academy of sciences of the czech republic (grant iaa , institutional research concepts no.av z , av z ), and ministry of education, youth and sports of the czech republic (research program lc ). intracellular delivery and fate of peptide-capped gold nanoparticles: towards cellular biosensors y. cesbron , v. sée , p. free , p. nativo , d. g. spiller , m. r. h. white , m. brust , b. lounis , r. lévy liverpool institute for nanoscale science, engineering and technology, liverpool, uk, université bordeaux i / cnrs, bordeaux, france gold nanoparticles (nps) have extraordinary optical properties that make them very attractive single molecule labels. although understanding their dynamic interactions with biomolecules, living cells and organisms is a prerequisite for their use as in situ sensors or actuators. while recent research has provided indications on the effect of size, shape, and surface properties of nps on their internalization by living cells, the biochemical fate of nps after internalization has been essentially unknown. here we show that peptide-capped gold nps enter mammalian cells by endocytosis. we demonstrate that the peptide layer is subsequently degraded within the endosomal compartments through peptide cleavage by the ubiquitous endosomal protease cathepsin l. preservation of the peptide layer integrity and cytosolic delivery of nps can be achieved by a combination of cathepsin inhibition and endosome disruption. this is demonstrated using a combination of distance-dependant fluorescence unquenching and photothermal heterodyne imaging. these results prove the potential of peptide-capped gold nps as cellular biosensors. current efforts focus on in-vivo labeling of nps, nanoparticle-based real-time sensing of enzyme activity in living cells, and the development of photothermal microscopy for single nanoparticle imaging in living cells. towards intravital two photon microscopy study of lymphocytes mobility in lymphonodes m. caccia , l. sironi , m. collini , i. zanoni , t. gorletta , m. di gioia , g. francesca dipartimento di fisica, università di milano bicocca, italy, dipartimento di biotecnologie e bioscienze, università di milano bicocca, italy during the last years the edge between optical fluorescence based microscopy and the world of bio-medical research has become thinner and thinner and two photon laser scanning microscopy (tplsm) is one of the most powerful tool for immunological and medical research. one of the most limiting step in intravital microscopy is the preparation of the animal model and the number of animals to be sacrificed to get good statistics. alternative routes must be searched. we employ tplsm to explanted lymphnodes. two photon excitation and a non descanned detection mode allow to increase, respectively, the excitation and detection efficiency while the explanted organs are kept very close to the condition they experience in live animals by means of an home-made temperature controlled box surrounding the microscope and a system for the flux of physiological fluids. experiments performed on explanted lymphonodes, kept under constant flux of co -o saturated buffers and at o c, agree with literature for what concern t-cell homing and motility. this seems to confirm that t-cells behavior in explanted organs mantained in physiological conditions is very similar to that observed in live animals. we then believe that our tplsm microscope would allow to study cell behaviors in in-vivo with high efficiency and a little technical effort. a generalized quantitative frap method with no restriction on the size of the photobleached area heterochromatin protein in dna damage response -recruitment or dissociation from repair sites? j. w. dobrucki division of cell biophysics, faculty of biochem. biophys. and biotechnol., jagiellonian university, kraków, poland we studied recruitment of dna repair proteins to damage sites in live cells, by microscopy approaches, using a new method of inflicting local, sublethal damage in nuclei of live cells. oxidative damage, which was inflicted by exciting dna-intercalated ethidium with focused green light, triggered recruitment of base excision repair enzymes. surprisingly, an epigenetic regulator, heterochromatin protein (hp ) (zarebski et al., ) was recruited to damage as well. hp is a constitutive component of heterochromatin, and plays an important role in transcriptional repression and regulation of euchromatic genes, however it was not known to be required for repair of oxidative damage. the finding of hp recruitment is particularly puzzling, since in another study hp was shown to dissociate from chromatin as a result of dna damage (ayoub et al. ) . technical aspects of live cell imaging that may explain these contradictory results will be discussed. in this presentation, we report a method for determining both the presence and the stoichiometry of protein complexes at pixel resolution and apply it to disassembling focal adhesions. the method is derived from fluorescence fluctuation methods that have single molecule sensitivity and is based on our previously described n&b (number and brightness) method that measures the number and brightness (aggregation state) of fluorescent molecules in every pixel of a confocal microscope image. the new method exploits the correlation of fluorescence amplitude fluctuations for two colors and detects the presence of molecular complexes and their stoichiometry. while the original n&b method was developed for one color, i.e., a single molecular species, the new method, ccn&b, extends the analysis to two colors and introduces the concept of cross-variance. this method is similar in concept to the two-color pch analysis. however, the covariancebased ccn&b method also generates pixel resolution maps of protein complexes and can be used on commercial confocal microscopes. the method is highly sensitive and has relatively high temporal resolution. we have applied this method to adhesion complexes in cells. in addition of their structural role, to link the extracellular substratum to actin filaments, they also serve as signaling centers that regulate many cellular processes including their own assembly and turnover, migration, gene expression, apoptosis, and proliferation. spatio-temporal analysis of membrane lipid remodeling during phagocytosis s. de keijzer , d. kilic , c. g. figdor , s. grinstein , a. cambi department of tumor immunology, radboud university of nijmegen, nijmegen, the netherlands, department of biochemistry, university of toronto, toronto, canada the constant threat posed by pathogens and cell debris is tackled by phagocytosis, the process through which cells engulf and destroy dangerous material. the signaling, targeting and trafficking during phagocytosis is dependent on cytoskeleton rearrangements and membrane remodeling. it is becoming increasingly evident that lipids play an important role and can affect the phagocytic response. they assemble microdomains which can act as signaling platforms and confer charge and curvature to the membrane surface promoting electrostatic attraction and retention of proteins. little is known about the mechanism(s) regulating membrane lipid remodeling during phagocytosis. here we used fluorescently labeled biosensors based on k-ras and h-ras proteins to obtain spatio-temporal information on phagosomal membrane lipid remodeling during fcreceptormediated phagocytosis. the results show that cell activation by cytokines modulates the kinetics of anionic lipids thus affecting the membrane charge and the recruitment of cytosolic proteins to the phagosomal membrane. our data emphasize the fundamental role of lipids in the generation and transduction of signals in phagocytosis, and we believe this can be extrapolated to many important processes in a cell. fluorescence correlation spectroscopy (fcs) is a useful technique for characterizing the mobility and concentration of fluorescent molecules both in vitro and in vivo. we utilize two-photon fcs to characterize the concentration and mobility of fluorescent molecules within living cells of bacillus subtilis. autocorrelation functions were measured in bacteria expressing green fluorescent protein (gfp) under the lac promoter in both nutrient rich and nutrient poor culture medium. although considerable heterogeneity was evident from cell to cell, on average, both intracellular concentration and mobility were found to be dependent upon culture medium. we also investigated bacteria expressing gfp under control of native promoters for involved in the regulation of the carbon metabolic cycle in bacillus subtilis. the gfp concentration, which should be related to promoter activity, was investigated for single cells and cell populations under different metabolic conditions. some photobleaching was observed during the course of the measurements as a decrease in the average fluorescence intensity. this is due to the small size of the bacteria (∼ fl) and low basal expression levels of gfp (∼ nm) in the absence of iptg. methods to take this into account during data analysis are discussed. a. fassio , s. congia , p. baldelli , f. benfenati dimes, university of genoa, genoa, italy, dnbt , iit, genoa, italy several mutations have been discovered in the synapsin i (syn) gene in families with epilepsy, but the mechanism inducing the epileptic phenotype is unknown. syn is a protein associated with synaptic vesicles (svs) that control sv trafficking and neurotransmitter release. the syn mutations subject of this study are a non sense (ns- ) and of two missense (ms- , ms- ) . syn knockout (ko) hippocampal neurons were transfected with the either wild type (wt) or mutated syns. all syns presented a common punctate pattern of expression at the level of axonal arborizations but, while ns- syn targeted to synapses as wt-syn, ms- and ms- syns reached the presynaptic terminal less efficiently. we next set up a live cell imaging experiment using synaptophysin-phluorin and evaluated the effects of ns- , ms- and ms- syns on the size of sv pools. restoring wt-syn in syn i ko terminals led to a increase of all sv pools. restoring ms- and ms- syns resulted in a phenotype not significatively different from syn i ko background whereas restoring ns- syn caused a decrease of all sv pools as compared either with wt-syn or syn i ko background. these data suggest an alteration in the subcellular distribution and function of syn in patients carrying ms- e ms- mutation and a more severe effect on synaptic activity in patient carrying ns- mutation. imaging dynamics of dc-sign at the plasma membrane of hiv- -stimulated dendritic cells o understanding how viruses interact with host receptors is essential for developing new antiviral strategies. dendritic cells (dcs) can efficiently capture and take up hiv- through multiple attachment factors, such as the c-type lectin dc-sign. however, the initial interactions between hiv- and this receptor on dcs are not fully understood yet. in this work, we have used single-molecule epi-tirf microscopy in combination with fluorescently labelled hiv- virus like particles (vlps) and dc-sign-specific antibodies to image the dynamic interaction between hiv- and dc-sign nanoclusters at the plasma membrane of dcs. by tracking individual trajectories of dc-sign on the cell surface of living dcs we have found heterogeneity in the modes of motion of dc-sign: some clusters are immobile whereas others move very quickly, with a few ones showing a directed motion on the cell membrane. to investigate how such motion might be correlated to dc-sign function as virus attachment factor, we have developed glass platforms functionalized with hiv- vlps to locally stimulate living dcs. these virus platforms have allowed us to measure dc-sign diffusion rates and motion modes over the cell surface of stimulated dcs and represent a powerful tool for studying the dynamic interactions between hiv- and the dc membrane. a. esposito , t. tiffert , j. mauritz , s. schlachter , j. n. skepper , v. l. lew , c. f. kaminski dept. of chemical engineering and biotechnology, univ. of cambridge, u.k., dept. of physiology, development and neuroscience, univ. of cambridge, u.k. plasmodium falciparum (pf ) causes the most lethal form of malaria in humans. early research exposed two paradoxes: ) during its intraerythrocytic cycle pf permeabilizes the host cell so much that a comparably permeabilized healthy red blood cell (rbc) would lyse prematurely, and ) pf digests far more hemoglobin than needed for its metabolism. a model of the homeostasis of a pf infected rbc suggested a common explanation of both puzzles: excess hemoglobin digestion is required to reduce the colloidosmotic pressure within the host cell thus ensuring its osmotic stability to the end of the pf asexual cycle. we investigated these predictions with direct measurements of [hb] and volumes of parasitized rbcs. reliable volume and morphological data was obtained by confocal microscopy and quantitative surface reconstruction. furthermore, we developed a new fret-based method to measure hemoglobin molecular crowding by exploiting the reduction in fluorescence lifetime of a donor fluorophore loaded in the rbc cytosol. fret imaging techniques are powerful tools for probing the biophysics of living cells. these tools provided a first validation of the colloidosmotic hypothesis and a deeper understanding of the homeostasis of the intraerythrocytic stage of pf. hiv- assembly and release occur at the plasma membrane of infected cells and are driven by the gag polyprotein. using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated assembly of fluorescently labeled hiv- at the plasma membrane of living cells with high time resolution. gag assembled into discrete clusters corresponding to single virions. after their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over - hours. using a photoconvertible fluorescent protein, we determined that assembly was nucleated by membrane bound gag molecules, while both membrane-bound and cytosol derived gag polyproteins contributed to the growing bud. assembly kinetics were rapid and three phases are observed. in phase i, the number of gag molecules at a budding site increases following a saturating exponential with a rate constant of ∼ x − s − . hence, gag assembly is complete in ∼ s. in phase ii, a plateau in fluorescence intensity is observed with no exchange of gag protein. the fluorescence intensity decays in phase iii. this decay, in some cases, corresponds to the release of a virion. the time scale from the onset of assembly to release of extracellular particles was measured to be ∼ , +/- s. fast d chromatin dynamics studied in living yeasts using a novel lab on chip technology h. hajjoul , m. dilhan , i. lassadi , k. bystricky , a. bancaud laas-cnrs, université de toulouse, france, lbme, cnrs umr , toulouse, france we present a novel lab-on-chip technology for d particle tracking yeast abased on v-shaped mirrors, which are used to observe fluorescent specimens from multiple vantage points, providing stereo-images that can be recombined for d reconstruction. our technology is based on v-shaped mirrors, which are fabricated by wet etching of silicon wafers, and used as optical and fluidic components. after rigorous optical optimization of the device in vitro, the lab-on-chip is applied to study chromatin dynamics in vivo using budding yeasts as a model system. yeasts cells are visualized using gfp fused to the associated repressor protein. we confirm earlier observations that telomeric sequences, i.e. located close to chromosome ends, accumulate at the nuclear periphery, whereas genes found midway along chromosome arms are mostly present in the nuclear lumen. the dynamics of these sequences is followed in d with an unpreceded temporal resolution of ms, showing that chromosome dynamics is non linear in the short time regime and switches to a linear regime at larger timescales. notably, this behavior is reminiscent of universal responses observed in polymer solutions, and is related to the confinement and the structure of chromosomes. this technique shows a great potential for studying dynamic processes in small living organisms. retrovirus induced remodeling of the host cell actin architecture m. gladnikoff, i. rousso department of structural biology, weizmann institute of science, rehovot, israel retrovirus budding is a key step in the virus replication cycle. yet, despite substantial progress in the structural and biochemical characterization of retroviral budding, the underlying physical mechanism remains poorly understood, primarily due to technical limitations preventing visualization of bud formation in real time. using atomic force-, fluorescence-and transmission electron microscopy we find that both hiv and moloney murine leukemia virus (mlv) remodel the actin cytoskeleton of their host cells and utilize the forces it generates to drive their assembly and budding. highly dynamic actin-filamentous structures which varied in size over the duration of budding appeared to emanate from the assembled virion. these actin structures assemble simultaneously or immediately after the beginning of budding, and disappear as soon as the nascent virus is released from the cell membrane. analysis of sections of cryo-preserved virus infected cells by tem reveals similar actin filaments structures emerging from every nascent virus. substitution of the nucleocapsid domain implicated in actin binding by a leucine-zipper domain resulted in budding of virus-like particles that was not accompanied by remodeling of the cell's cytoskeleton. notably, budding of viruses carrying the modified nucleocapsid domains was an order of magnitude slower than that of the wild type. the results of this study show that retroviruses utilize the cell cytoskeleton to expedite their assembly and budding. t. gensch institute of structural biology and biophysics (isb- ) cellular biophysics, research centre jülich, germany the determination of ion concentrations in cells -in particular in neurons -is very important for understanding cell function and life. ca + is an ubiquitous messenger in almost all cell types, cl − has several roles, e.g. plays an important role in some neuronal signaling pathways including olfaction, nociception and vision. fluorescence lifetime imaging (flim) is of advantage over intensity based fluorescence microscopy, when comparisons between micro-domains of one cell or between different cells of one cell type are performed. several (organic chromophores and fluorescent protein based) ca + -and cl − -sensors have been tested in culture cells with respect to their applicability in flim studies. the ca + -and cl − concentration in rodent olfactory sensory neurons, dorsal root ganglion neurons and neurons of the retina is investigated by time-resolved flim with two-photon excitation. viscosity is one of the main factors which influence diffusion in condensed media. in a cell viscosity can play a role in several diffusion mediated processes, such as drug delivery and signalling. previously, alterations in viscosity in cells and organs have been linked to malfunction; however, mapping viscosity on a single-cell scale remains a challenge. we have imaged viscosity inside individual cells using novel fluorescent probes, called molecular rotors, in which the speed of rotation about a sterically hindered bond is viscosity-dependent. , this approach enabled us to demonstrate that viscosity distribution in a cell is highly heterogeneous and that the local microviscosity in hydrophobic cell domains can be up to × higher than that of water. we have also shown that the intracellular viscosity increases dramatically during light activated cancer treatment, called photodynamic therapy (pdt). we have demonstrated the effect of such viscosity increase on intracellular reactions by directly monitoring the rates of formation and decay of a short lived toxic intermediate, crucial in pdt, called singlet molecular oxygen, in light perturbed cells. characean algae, close relatives of higher plants, represent a convenient model for studying the effect of propagating electrical signals, action potentials (ap) on photosynthesis. illuminated chara corallina cells produce coordinated spatial patterns of chlorophyll fluorescence (chl fl) and extracellular ph. photosynthesis is higher in the cell regions adjacent to acid zones compared to alkaline zones. under physiological conditions, the electrically induced ap differentially and reversibly suppresses photosynthesis in the alkaline and acid regions. in this work we examined the effects of an artificial psi acceptor, methyl viologen (mv), on chl fl with imaging-pam technique. mv is a divalent cation and poorly permeates through biological membranes. the presence in the medium of mv had no effect on fl as well as on p + absorbance signals until the application of a single excitatory stimulus. once an ap was generated in the presence of mv, it induced irreversible inhibition of native (nadpdependent) electron flow and a strong non-photochemical fl quenching all over the cell. this indicates that ap redirects electron flow from the main pathway to the artificial acceptor. we concluded that ap generation opens access for permeation of mv from the medium to the chloroplast stroma across two membrane barriers, plasmalemma and chloroplast inner membrane. we suggested that mv might enter chara cell via plasmalemma ca + -channels activated during ap. evaluation of cell damage after photodynamic and sonodynamic treatment h. kolarova, k. tomankova, s. binder, p. kolar, r. bajgar, m. strnad department of medical biophysics, faculty of medicine and dentistry, palacky university, hnevotinska , olomouc, czech republic photodynamic therapy (pdt) and sonodynamic therapy (sdt) is a new, combined therapy for treating cancer. the basis of the therapy is to administer a small amount of photosensitizer and sonosensitizer, which are selectively taken up by cancer cells, and then expose the body to light and ultrasound to activate these sensitizers. when the sensitizers absorb light of an appropriate wavelength, it may cause their excitation with subsequent energy transfer to oxygen; the oxygen then becomes highly reactive in cancer cells and produces reactive oxygen species (ros). the resulting damage to organelles within malignant cells leads to tumor ablation. sdt uses an agent that is sensitive to ultrasound, allowing deeper penetration and destruction of abnormal cells. changes in human melanoma cells were evaluated using fluorescence microscope and atomic force microscopy.we focused on obtaining pictures of the topography and pictures involving elastic properties of cell surface. the production of ros was investigated with the molecular probe cm-h dcfda, and morphological changes in cells were evaluated using fluorescence microscope. the quantitative ros production changes in relation to phthalocyanine concentration, irradiation doses and ultrasound intensity were measured by a fluororeader. activity correlation imaging: visualizing function and structure of neuronal populations s. junek , t.-w. chen , m. alevra , d. schild department of neurophysiology and cellular biophysics, university of göttingen, germany, dfg research center for molecular physiology of the brain (cmpb), university of göttingen, germany understanding a neuronal network relies on knowledge about both the function and the structure of its neurons. while he simultaneous observation of hundreds of neurons is possible by densely staining brain tissue with functional dyes, the low contrast of these stainings does not allow the identification of neuronal processes from the raw fluorescent images. however, as neurons are known to exhibit complex temporal patterns of activity, fluctuations in signal intensity over time could be exploited to generate contrast in densely stained tissue. we demonstrate that the uniform calcium signals within individual neurons of the olfactory bulb can be exploited to visualize the morphology and projection patterns of these cells in tissue slices. as different neurons exhibit distinct time patterns, it is possible to generate a highcontrast multi-color visualization of the network's active neurons, solely based on the specificity of temporal fluctuations of a single-color calcium dye. it is thus possible to use other spectral channels for additional labelling, for example using cell-type or protein specific markers. the ability to map function and structure of neuronal populations online opens up a number of intriguing applications, such as selecting cells or cell pairs with certain functional or projection profiles for targeted recordings, ablation or stimulation. -live cell imaging - investigation of the dynamics of redox elements in live cells by using fluorescence ratio microscopy g. maulucci , g. pani , v. labate , m. mele , e. panieri , m. papi , g. arcovito , t. galeotti , m. de spirito istituto di fisica, università cattolica s. cuore, roma, italy, istituto di patologia generale, università cattolica s. cuore, roma, italy oxidative stress or signaling events can affect cellular redox environment, which act simultaneously as regulator and indicator of key cellular functions in both physiological and pathological settings. by using a redox-sensitive protein (rxyfp), employed ratiometrically, it is possible to generate high resolution redox maps of cells. the spatial distributions of oxidized and reduced elements have been discriminated in human embryonic kidney cells by the deconvolution of the histograms of redox maps. by transfecting cell with glutaredoxin v (grx-v), a significant shift towards more reduced state with respect to that recovered from non-transfected cells is observed. despite such large differences, a common behaviour in the spatial distribution of reduced and oxidized couples can still be observed: oxidized population shows a pronounced localization on the cell borders, near the plasma membrane, while reduced population appears as a collection of well separated spots homogeneuosly distributed thoroughly the inner part of the cell with a mean dimension of um. furthermore we observe that the role of grx-v consists in causing a shift towards reduced values of the highly reduced region, while leaving unaltered the redox-balance of the intracellular side of the plasma membrane. fluorescence resonant energy transfer (fret) is a popular approach to studying molecular interactions. fluorescence lifetime imaging (flim) provides a robust read-out of fret but has largely been restricted to fixed cells owing to relatively long acquisition times. we present a highspeed wide-field multidimensional fluorescence microscope for flim fret in live cells on second timescales for high content analysis and studying fast dynamics. the system uses a nipkow disc for optical sectioning and a time-gated image intensifier for wide-field flim. a spectrally selected supercontinuum excitation source facilitates versatile realtime flim of live cells transfected with fluorescent proteins. for cell signalling studies we have developed a multiplexed fret approach. ras activation at the plasma membrane is demonstrated using flim to read out tagrfp-raf rbd interaction with mplum-h-ras. simultaneously, a spectral ratiometric read-out is used for a second fret (cfp/yfp cameleon) probe to monitor the downstream calcium flux. to further develop multiplexed fret as a tool for imaging multiple components of cell signalling networks, we are working to include polarisation-resolved imaging for steady-state and time resolved fluorescence anisotropy measurements. monitoring gene expression via novel nucleic acid and delivery methods k. lymperopoulos , c. spassova , a. seefeld , h. s. parekh , d. p. herten bioquant institute,heidelberg university, germany, school of pharmacy, university of queensland, australia application of single-molecule and high-resolution fluorescence methods to monitor gene expression in living cells increase the demand on novel probes and delivery methods.they require fluorophores with high photostability and quantum yield and highly-efficient delivery methods that ensure the minimum interference with cell processes such as metabolism and signal transduction. we used a novel class of dendrimers with varying generations and branching factors and different number of positive charges due to different moieties and functional groups.these different properties were tested for their efficiency to transfect eukaryotic cells with fluorescent oligonucleotides (odns).different parameters (temperature,concentration of dendrimers, ratio of dendrimers and odns) were evaluated and optimised.we utilised these established optimal conditions to deliver a modified concept of smartprobes to mammalian cells targeting mrnas involved in signal pathways.we tested different mrna targets and we optimised the fluorescence signal by varying a range of parameters,namely the fluorescent label and the intrinsic properties of the smartprobe (length of the loop and stem, conformation and number of guanosines).in the near future,we plan to use these probes for monitoring gene expression levels using diffusion imaging microscopy. we present a novel near-field scanning microwave microscope (smm) capable of providing surface impedance measurements of samples with nanometric resolution. the instrument is the integration of a microwave vector network analyzer (vna) and a scanning probe microscope (afm/stm). a key point is that our software, controlling and synchronizing both the instruments, creates simultaneously images of the sample at several frequency points. this can be used to extract several features of the sample depending on the frequency. moreover, close frequencies show the same features, added to random noise. exploiting this redundancy of information, we have achieved remarkable results. we have been working on the optimization of this system for biological applications, to detect functional characteristics of cells generating a variation of their dielectric properties. this instrument offers the possibility of performing local impedance measurements on a single live cell and, if correctly calibrated, it provides also quantitative information (e.g. absolute measurements of membrane permittivity). the system was demonstrated to work on saccharomyces cerevisiae. a better model for a full test of the potentialities of the new technique is given by excitable cells, characterized by a greater variability of dielectric properties. a challenging objective could be directly imaging ion channels. hiv- to efficiently complete a replication cycle has to integrate its genome into the host cellular dna.after hiv- enters target cells,neosynthesized viral dna forms along with other proteins the pre-integration complex (pic).pics are then transported into the nucleus where integration,catalyzed by the viral integrase,takes place.hiv- viral particles engineered to incorporate integrase fused to egfp have proven effective to study pics within nuclei of infected cells.in this study we report the live imaging analysis of nuclear pic dynamics obtained by time-lapse microscopy.intranuclear trajectories of in-egfp-labeled pic were collected in three dimensions and examined by both mean squared displacement (msd) and cage diameter (cd) analysis.in cd the maximum distances measured between two positions occupied by a pic in a time window of minutes were calculated while in our msd analysis -minute long trajectory segments were considered.remarkably,msd revealed the presence of an underlying active transport mechanism.to test the possible role of actin filaments,pic nuclear trafficking was analyzed in cells treated with latrunculin b (actin polymerization inhibitor).preliminary results suggest that the disruption of actin function impairs the active nuclear movement of pics. second harmonic generation microscopy reveals sarcomere contractile dynamics of cardiomyocytes n. prent , c. a. greenhalgh , r. o. cisek , j. aus der au , s. elmore , j. h. van beek , j. squier , v. barzda department of physics and institute for optical sciences, university of toronto, toronto, canada, department of physics, colorado school of mines, golden, usa, department of molecular cell physiology, vrije universiteit, amsterdam, netherlands cardiomyocytes, like other striated muscles, exhibit strong inherent second-order nonlinear optical properties that make them exemplar for live cell dynamic studies with second harmonic generation (shg) microscopy. laser scanning shg microscopy with an incident wavelength around µm, enables fast imaging for extended periods of time with negligible tissue damage. strong shg signal originates from the anisotropic (a-) bands which are comprised of regularly arranged myosin molecules, while actin molecules, the main constituent of the isotropic (i-) bands, produce negligible shg. consequently, the alternating bands along the myofibril are clearly visualized, therefore, enabling the determination of individual sarcomere lengths and the study of sarcomere length dynamics during macro-scale contractions. shg intensity is shown to positively correlate to sarcomere length, which leads to the development of real-time inherent force sensors for in vivo myocytes. rich sarcomere dynamics can be observed during myocyte contraction, which can be used for medical diagnostic purpose of muscular degenerative diseases. imaging cellular communication in insulin secretion ex vivo and in vivo d. w. piston vanderbilt university, nashville, tennessee, u.s.a. the islet of langerhans is the functional unit responsible for glucose-stimulated insulin secretion (gsis), and thus plays a key role in blood glucose homeostasis. the importance of the islet is demonstrated by the proven ability of islet transplants to reverse type i diabetes pathologies in human patients. we are interested in understanding the multicellular mechanisms of islet function, and their role in the regulation of blood glucose under normal and pathological conditions. in many ways, the islet appears to function as a syncytium, which exhibits synchronous behavior of membrane action potentials, ca + oscillations, and pulsatile insulin secretion across all β-cells in the islet. in other ways, the islet works as individual cells, especially in the regulation of gene transcription. using our unique quantitative optical imaging methods and novel microfluidic devices, the dynamics of these molecular mechanisms can be followed quantitatively in living cells within intact islets. these investigations utilize transgenic and tissue-specific knock-out mouse models with demonstrated phenotypes, as well as traditional biochemical and molecular biological approaches. do retinal rod outer segments carry out oxydative phosphorylation? i. panfoli , p. bianchini , d. calzia , s. ravera , a. morelli , a. diaspro biology dept., university of genova, italy, lambs-microscobio res.centre, university of genova, italy, neuroscience and brain technology dept., i.i.t. genova, italy visual transduction in vertebrate retinal rod outer segments (os) is very energy demanding. however, atp supply in os, that are devoid of mitochondria, is still puzzling. by a proteomic analysis we identified in purified bovine os disks, proteins involved in vision, as well as the respiratory chain complexes i to iv and f f o -atp synthase, whose activity was comparable to that of mitochondria and sensitive to specific inhibitors. rhodamine fluorescence quenching experiments showed the presence of a proton potential difference across disks. disks consumed oxygen. confocal laser scanning and transmission electron microscopy showed that cytochrome c oxydase and atp synthase are localized on disks. mitochondrial vital dyes stained os ex vivo, and disks. rhodopsin and mitotracker fluorescence co-localized in os. data, suggestive of an aerobic metabolism in os, point to the existence of "mitochondrial inner membrane-like membranes" ectopically producing atp through oxidative phosphorylation, with respect to mitochondria. new scenarios open on the patho-physiology of many retinal diseases associated to mitochondrial dysfunction. -live cell imaging -abstracts unmixing using lifetime-excitation multidimensional confocal fluorescence microscopy data s. schlachter , s. schwedler , a. esposito , a. d. elder , g. s. kaminski schierle , c. f. kaminski cambridge university, u.k., universität bielefeld, germany fluorescence imaging provides a powerful tool to probe biological systems, enabling the high resolution investigation of the localization, interaction and biochemical modification of biomolecules. multidimensional imaging, in particular, is a growing application of confocal and other forms of fluorescence microscopy. it seeks to measure not only fluorescence intensity in three spatial dimensions, but also other features of fluorescence emission, such as lifetime and fluorescence spectra. although multi-dimensional fluorescence microscopy has been demonstrated before, little attention has so far been paid to the problem of data interpretation and representation when dealing with such large datasets. we present here an instrument capable of recording fluorescence lifetime and excitation data by combining tcspc for lifetime determination and a supercontinuum excitation source for extracting excitation spectra. these technologies permit the acquisition of d and d images with lifetime and excitation spectrum information at every pixel. we demonstrate a method whereby the multidimensional datasets acquired with this instrument are processed to yield biologically relevant parameters, (e.g. unmix fluorophores in a multiply labelled sample). our method relies on a global analysis approach and uses ab plots for displaying multidimensional data. we demonstrate the instrument & processing method on dye and multiply labelled biological samples. advanced neuroimaging with diffractive optical elements p. saggau baylor college of medicine, houston, texas, usa studying neural systems is complicated by the large number and small size of its cellular elements. the traditional way of exploring neuronal function by electrical monitoring with micropipettes is increasingly replaced by molecular imaging. fluorescent molecules allow optical monitoring of neuronal signaling with cellular and often subcellular resolution. in addition to monitoring activity, analyzing the functional properties of individual neurons or neuronal populations requires their controlled activation. optical approaches are well-suited, including molecular photolysis of inert precursors and light activation of engineered proteins that control ionic membrane currents. to fully utilize optical techniques for exploring neural systems requires more than adequate spatial resolution to distinguish neuronal elements and sufficient temporal resolution to induce and/or monitor neuronal signaling. because of the non-linear and non-stationary nature of the studied system it is necessary to access many neuronal sites simultaneously. in modern imaging, wide-field illumination and imageformation are often replaced by patterned excitation and nonimaging photon collection. in many instants, diffractive illumination schemes can substitute for conventional reflective or refractive designs. in particular, the availability of programmable diffractive optical elements has made this an attractive alternative, since they permit highly versatile microscope designs and support a significant increase in the overall spatio-temporal resolution. i will present an advanced imaging approach using diffractive optical elements to analyze structure and function of live neurons in brain slices and intact cortex. efficient evaluation of fret in image cytometry with acceptor photobleaching and ratiometric methods j. roszik, d. lisboa, j. szöllősi, g. vereb department of biophysics and cell biology, university of debrecen, debrecen, hungary fluorescence resonance energy transfer (fret) is a powerful technique that can be applied to study nanoscale intra-and intermolecular events and interactions of molecules in situ in biological systems. a robust, easy to use, self-controlled fret method, independent of donor and acceptor concentration and stochiometry, is acceptor photobleaching fret, which requires only simple image mathematics. another approach with more complicated calculations is the intensitybased ratiometric method, which is not based on destroying the acceptor fluorophores, making it applicable for following molecular interactions in live cells. as the need for using fret in image cytometry for evaluating molecular interactions increases, we have undertaken to develop softwares for these methods involving the calculation and usage of all correction factors needed to obtain reliable energy transfer efficiencies. correction possibilities of the acceptor photobleaching method include unwanted photobleaching of the donor, fluorescent photoproduct of the acceptor after photobleaching, cross-talk of unbleached acceptor into the donor channel and partial photobleaching of the acceptor. in the case of intensity-based ratiometric fret, we can correct for all channel cross-talks and calibrate the fret efficiency calculations. both programs provide registration and semiautomatic processing, and they are freely available. the pendrin (slc a ) gene is responsible, when mutated, for the pendred syndrome, a recessive disorder characterized by sensorineural hearing loss often accompanied by thyroid disfunctions. pendrin is an anion exchanger. the way it works and its role in different tissues, owing to the lack of known isoforms, is matter of wide research and debate. we focused on a still unexplored pendrin function, that is most important in the inner ear: cellular volume control and cl − fluxes regulation. we used hek cells over-expressing wild type pendrin or a mutated isoform together with the eyfp. we challenged cells with hypo-osmolar solutions and followed their volume variations in time. taking advantage of the confocal optical sectioning we independently measured cell volume and fluorescence intensity. in this way, given the dependence of eyfp fluorescence from [cl − ] and ph (measured with snarf ) we could estimate at the same time cl − fluxes, volume and ph variations. the contemporary measurements of the three variables, not yet reported in living cells, allowed to assess the role of pendrin in volume regulation and evidenced its participation to cl − fluxes as compared to the mutated isoform or controls. particle tracking inside the cell largely benefits of the ability to spatially and temporally mark specific structures to follow their "signalling" over a "dark" background as made possible since the advent of the photo-activatable markers. in terms of spatial confinement of the photo-activation process, the use of multiphoton excitation provides several favourable aspects compared to single photon confocal microscopy in photomarking biological structures to be tracked: the confined excitation volumes, of the order of magnitude of subfemtoliter, due to the non-linear requirements provide a unique control of the excitation and consequently photoactivation in the d space. in this context photoactivation experiments can be used to assess quantitative information about the binding kinetics of a macromolecule expressed in different cellular compartments. in this work we extended to photoactivation procedures and models originally developed for the quantitative analysis of frap experiments and we evaluated, for different proteins of medical interest (rac-pagfp), the diffusive behaviour in the cytoplasm and the binding kinetics at the large endosomes. the results are compared with standard photobleaching experiments, in order to evidence the gained sensitivity obtained with photo-activatable proteins. motile plaques involved in stress fiber assembly revealed by high-speed spm for living cells k. tamura, t. mizutani, h. haga, k. kawabata division of biological sciences, graduate school of science, hokkaido university, kita-ku, sapporo, japan stress fibers, which are contractile actin cables aligned in a highly-ordered manner, are important for cell migration, mechanical support of plasma membranes and extracellular matrix organization. although stress fibers are dynamically disassembled and assembled again in cells, it is poorly understood how actin filaments are organized into cables with highly-ordered alignment for stress fiber assembly. for investigation of actin cytoskeletal dynamics during actin cable formation, we performed time-lapse observation of actin cytoskeleton in lamella of living fibroblasts by using scanning probe microscopy (spm). high-speed spm observation revealed that motile plaques defined front-side ends of new actin cables and that preexisting mesh-form actin networks were remodeled into new actin cables. directional order analysis of movement of plaques and pharmacological experiments clarified that plaques were driven by myosin-ii-based retrograde actin flow, indicating that plaques are associated with actin cytoskeleton. immunofluorescence experiments showed that plaques were localized on foci of vinculin, a component of cell-substratum adhesions, suggesting that plaques can bind to extracellular substrata via vinculin. based on these results, we propose a model for actin cable formation that motile plaques initiate remodeling of preexisting actin networks into actin cables aligned in a direction of actin flow by associating with extracellular substrata. abscisic acid (aba) is a plant hormone regulating fundamental physiological functions in plants, such as response to abiotic stress. recently, aba was shown to be produced and released by human granulocytes, by insulin-producing rat insulinoma cells and by human and murine pancreatic β cells. aba autocrinally stimulates the functional activities specific for each cell type through a receptor-operated signal transduction pathway, sequentially involving a pertussis toxin (ptx)-sensitive receptor/g-protein complex, cyclic amp, cd -produced cyclic adp-ribose and intracellular calcium. here, the aba receptor on human granulocytes and on rat insulinoma cells is identified as the lanthionine synthetase c-like protein lancl . co-expression of lancl and cd in the human hela cell line reproduces the aba-signaling pathway. the ptx-sensitive g protein coupled to lancl is identified as g i by transfection of cd + /lancl + hela with a chimeric g protein (gα q/i ). identification of the mammalian aba receptor will enable the screening of synthetic aba antagonists as prospective new anti-inflammatory and anti-diabetic agents. investigation of post-thaw damage of s.cerevisiae yeasts using fluorescent dyes -dab and -dab t. s. dyubko , i. a. buriak , v. d. zinchenko , i. f. kovalenko state scientific institution "institute for single crystals", national acadey of sciences of ukraine, kharkov, ukraine, institute for problems of cryobiology and cryomedicine, national academy of sciences of ukraine, kharkov, ukraine we investigate applicability of the fluorescent probes -dab and -dab available from seta biomedicals (www.setabiomedicals.com) to study the post-thaw damage of saccharomyces cerevisiae yeast cells. the leaving cells stained with these dyes have bright fluorescence in the yellow and red region, respectively. however, the freezing followed by post-thawing causes cell damage, which results in a change of fluorescence intensity. in the native living cells the dyes are preferably localized in the cell membranes and organelles which are highly fluorescent. partially damaged cells have even brighter fluorescence as compared to the living cells. however, their cell ultra-structure is not well-distinguished in the fluorescence mode. ultimately destroyed cells are almost non-fluorescent: the cell membranes are not visible in the fluorescent mode and their organelles are only weakly fluorescent. the number of intensively fluorescing cells with partially destroyed membranes, which were obtained by freezing to - • c, is lower compared to those frozen at - • c. -dab and -dab enable not only to distinguish damaged and undamaged cells, but also allow quantitative estimation of the extent of damage in membranes by cryogenic effects. the thylakoid membrane is a structured network in higher plants which is organised into stacked granal thylakoids that are interconnected by single 'stromal' lamellae. we have studied the mobility of a resident protein of the stromal lamellae, hcf , part of the tat protein translocase. hcf -green fluorescent protein fusion (gfp) was targeted into thylakoids and studied using photobleaching approaches. we show that small regions fail to recover significant levels of hcf -gfp fluorescence within minutes after photobleaching. autofluorescence from the photosystem ii light-harvesting complex (lhcii) in granal stacks likewise fails to recover over this time scale. although the thylakoid membrane is a single continuous entity, these data show that both hcf -gfp and lhcii are constrained within this network. since the hcf homologue, tatb, is highly mobile in the escherichia coli plasma membrane, we believe that the stromal lamellae take the form of distinct domains that are effectively constrained by boundaries within the thylakoid network. cytomechanical modifications induced by drugloaded carriers uptake by breast cancer cells the novel opportunities offered by the nanotechnologies have attracted great interest in the development of novel biomaterials for targeted drug delivery in cancer research. the desired features of pharmaceutical drug delivery for intravenous administration are their small size, biodegradability, high drug content, prolonged circulation in the blood and the ability to target required areas. in this work we have compared efficacy of different type of carriers having complementary properties for pharmaceutical delivery in cancer therapy. drug nano-colloids encapsulated by combination of layer by layer (lbl) techniques and ultrasonication, phytochemical encapsulated artificial oleosomes and drug-loading clay/carbon nanotubes have been used for uptake into breast cancer cells. analysis of viscoelastic response of neoplastic cells induced by cargo-carriers uptake has been carried out by a combination of high resolution optical and scanning force microscopy techniques. furthermore, the effects of drug-reservoir carriers on the cytoskeleton (re)organisation of neoplastic cells were further investigated by confocal microscopy using different fluorescent probes. mapping diffusion by raster image correlation spectroscopy (rics) with analog detection time lapse and flow cytometry data integrated in a common cell cycle proliferation model p. ubezio, v. colombo, m. lupi, f. falcetta istituto di ricerche farmacologiche "mario negri", milano, italy we present a cell cycle simulation tool, connecting the basic proliferation process to the cell population data obtained by time lapse and flow cytometry. the computer program is a general framework within which cell cycle progression can be interactively modeled at the desired level of complexity, including g /s/g m cell cycle phases with variable duration, g phase, cell subpopulations belonging to different generations or differentiation stages, distinct block and cell death parameters for each phase. in this way, we achieved a detailed rendering of cell proliferation of normal or tumor cell populations, additionally including cytostatic and cytotoxic effects of treatments. the program gives as output simulated measures, reproducing those obtained by absolute cell counting, growth inhibition tests, flow cytometric dna histograms and cell cycle percentages, pulse continuous-labeling studies, time-lapse intermitotic times and generation-wise analyses. each technique provides a piece of information related to the underlying proliferation process, catching only some of the phenomena in play, and the measures often cannot be univocally interpreted. we used the cell cycle simulator to fit together time lapse and flow cytometry time courses measures, to achieve a detailed reconstruction of the cell cycle proliferation of an ovarian cancer cell line in vitro after x-ray exposure. -live cell imaging - detection of functional modes in protein dynamics j. s. hub , b. l. de groot deptartment of cell & molecular biology, uppsala university, sweden, computational biomolecular dynamics group, max-planck-institute for biophysical chemistry, göttingen, germany proteins frequently accomplish their biological function by collective atomic motions. yet the identification of a collective motions related to a specific protein function from, e.g. a molecular dynamics trajectory, is often non-trivial. here, we propose a novel technique termed`functional mode analysis' that aims to detect the collective motion that is directly related to a particular protein function. based on an ensemble of structures, together with an arbitrary`functional quantity' that quantifies the functional state of the protein, the method detects the collective motion that is maximally correlated to the functional quantity. the functional quantity could, e.g., correspond to a geometric, electrostatic, or chemical observable, or any other variable that is relevant to the function of the protein. two different correlation measures are applied. first, the pearson correlation coefficient that measures linear correlation only; and second, the mutual information that can assess any kind of interdependence. detecting the maximally correlated motion allows one to derive a model for the functional state in terms of a single collective coordinate. the new method is illustrated using various biomolecules, including a polyalanine-helix, t lysozyme, trp-cage, and leucine-binding protein. logic estimation of the optimum source neutron energy for bnct of brain tumors boron neutron capture therapy (bnct) is a promising method for treating the highly fatal brain tumor; glioblastoma multiform. it is a binary modality; in which use is made of two components simultaneously; viz. thermal neutrons and boron- . a new concept was adopted for estimating the optimum source neutrons energy appropriate for different circumstances of bnct. four postulations on the optimum source neutrons energy were worked out, almost entirely independent of the rbe values of the different dose components. four corresponding conditions on the optimum source neutrons energy were deduced. an energy escalation study was carried out investigating different source neutron energies, between . ev and . mev. mcnp b monte carlo neutron transport code was utilized to study the behavior of these neutrons in the brain. the deduced four conditions were applied to the results. a source neutron energy range of few electron volts (ev) to about kev was estimated to be optimum for bnct of brain tumors located at different depths in brain. the results were discussed. v. l. cruz, j. ramos, j. martinez-salazar instituto de estructura de la materia. csic cannabinoid receptors are an important class of g protein coupled receptors. in particular cb and cb have received considerable interest because they mediate a variety of physiological responses in the central nervous and immune systems. their tertiary structure is still unknown. experimental information suggests the presence of both, an active and an inactive conformation. cb and cb share a common structural framework consisting in a seven transmembrane α-helix bundle connected by three extracellular and three intracellular loops. however, the knowledge of structural differences between both receptors may serve to design new ligands to activate/deactivate selectively only one of those receptors. in the present research, we report a multi-nanosecond molecular dynamics simulation of both receptors in solution, starting from a structure obtained by homology modelling using the x-ray determined bovine rhodopsin protein. we look for differences in the behaviour of cb and cb during the simulation process to shed light about those structural features which can be important for ligand selectivity. in this sense, we observed that cb tend to present a more flexible and opened helix bundle than cb . thus, it is expected than the former receptors would present less steric hindrance for ligand binding. we expect these results will be useful to design more selective ligands. self-assembly and equilibration of bola-lipids membranes studied by molecular dynamics m. bulacu, s. j. marrink groningen university, the netherlands bola-lipids consist of two monopolar, twin-tailed lipids that are held together by chemical linkage between one or both ends of the tails from one lipid and the corresponding ends from the other one. membranes formed by these lipids or by their mixtures with monopolar lipids are known to have additional mechanical stability while retaining membrane fluidity. this is traditionally attributed to the fact that, in the membrane phase, the bola-lipids have predominantly spanning configuration (the two polar heads are positioned at opposite membrane-water interfaces) in detriment to looping configuration (both head groups are located in the same membrane-water interface). we perform molecular dynamics simulations, using the coarse grained martini forcefield. we start with self-assembly simulations of bola-lipids followed by bilayer equilibration. an artificial pore is created in the membrane that significantly increases the flip-flop mobility of the lipids and hasten equilibration. the membrane properties are characterized (area per lipid, thickness, order parameter, pressure profile) with emphasis on the spanning/looping ratio. our study can help designing new artificial membranes, with higher stability under extreme conditions. -multiscale simulation - evidence for proton shuffling in a thioredoxinlike protein during catalysis d. narzi , s. w. siu , c. u. stirnimann , j. p. grimshaw , r. glockshuber , g. capitani , r. a. böckmann theoretical and computational membrane biology, center for bioinformatics, saarland university, germany, institute of molecular biology and biophysics, eth zürich, switzerland, structural and computational unit, embl, heidelberg, germany, paul scherrer institute, villigen psi, switzerland proteins of the thioredoxin (trx) superfamily catalyze disulfide-bond formation, reduction and isomerization in substrate proteins both in prokaryotic and in eukaryotic cells. all members of the trx family with thioldisulfide oxidoreductase activity contain the characteristic cys-x-x-cys motif in their active site. here, using poisson-boltzmann-based protonation-state calculations based on -ns molecular dynamics simulations, we investigated the catalytic mechanism of dsbl, the most oxidizing protein known to date. we observed several correlated transitions in the protonation states of the buried active-site cysteine and a neighboring lysine coupled to the exposure of the active-site thiolate. these results support the view of an internal proton shuffling mechanism during oxidation crucial for the uptake of two electrons from the substrate protein. intramolecular disulfide-bond formation is probably steered by the conformational switch facilitating interaction with the active-site thiolate. a consistent catalytic mechanism for dsbl, probably conferrable to other proteins of the same class, is presented. our results suggest a functional role of hydration entropy of active-site groups . the role of water in zn(ii)-abeta( - ) complexes beta-amyloid (aβ) peptides are the main component of amyloid fibrils detected in the brain of alzheimer patients. fibrils display an abnormal content of cu and zn ions whose binding to aβ-peptides has been recently studied by x-ray absorption spectroscopy [ ] and interpreted in terms of ab initio simulations. in order to perform such simulations it is of the utmost importance to find a compromise between the need of having a realistic description of the actual physical system and the difficulty of dealing with too many atoms and electrons. what is usually done is removing solvent (water molecules), thus studying the system in the so called "gasphase". in this work we investigate the relevance of water in the zn-aβ − coordination mode. relying on a combination of classical [ ] and quantum chemistry [ ] methods we find a significant difference in the zn coordination geometry depending on whether water is present or not. this information is exploited in building a full model system for subsequent car-parrinello simulations where two aβ peptides in water are in interaction in the presence of zn. [ although experiments which can directly probe the mechanical properties of proteins have only been performed recently, it is clear that the complex folds of polypeptide backbones lead to very heterogeneous mechanical behavior. this heterogeneity is likely to play an important role in protein function and interaction and it would be useful to be able to predict what mechanical (and dynamical) properties will result from a given structure. we have been using coarse-grain elastic network models to investigate this question and have found that these models are able to link specific mechanical properties to a number of functional features including enzymatic active sites, folding nuclei and changes in behavior due to point mutations. these models are also adapted to looking at complex formation between proteins and how specific recognition is achieved, while being fast enough to be applied to very large numbers of interactions. refinement of protein model structures using biasing potential replica exchange simulations s. kannan, m. zacharias school of engineering and science, jacobs university bremen, d- bremen, germany comparative protein modeling of a target protein based on sequence similarity to a protein with known structure is widely used to provide structural models of proteins. frequently, the quality of the target-template sequence alignment is non-uniform along the sequence: parts can be modeled with a high confidence, whereas other parts differ strongly from the template. in principle, molecular dynamics (md) simulations can be used to refine protein model structures but it is limited by the currently accessible simulation time scales. we have used a recently developed biasing potential replica exchange (bp-rex) md method (kannan, s. zacharias, m. proteins , , - ) to refine homology modeled protein structure at atomic resolution including explicit solvent. in standard rex-md simulations several replicas of a system are run in parallel at different temperatures allowing exchanges at preset time intervals. in a bp-rexmd simulation replicas are controlled by various levels of a biasing potential to reduce the energy barriers associated with peptide backbone dihedral transitions. the method requires much fewer replicas for efficient sampling compared with standard temperature rexmd. bp-rexmd simulations on several test cases starting from decoy structures deviating significantly from the native structure resulted in final structures in much closer agreement with experiment compared to conventional md simulations. m. s. p. sansom, p. j. stansfeld, c. l. wee, k. balali-mood department of biochemistry, university of oxford, u.k. coarse-grained molecular dynamics simulations may be used to probe the interactions of membrane proteins with bilayers and their component lipids on an extended (∼ µs) timescale ( ) . conversion to atomistic resolution allows more detailed protein/lipid interactions to be examined. this multiscale approach will be examined via three examples: (i) interactions of a small ion channel toxin (vstx ) we present a new competitive approach for the treatment of biomolecular flexibility to provide an alternative to the limitations of current methodologies such as molecular dynamics and normal mode analysis. this method, called static mode method, is based on the "induced-fit" concept and is aimed at mapping the intrinsic deformations of a biomolecule subject to any external excitations: direct mono or multi-site contact, electrical etc... the algorithm allows obtaining a set of deformations, each one corresponding to a specific interaction on a specific molecular site, in terms of force constants contained in the energy model. such a process can be used to explore the properties of single molecular intrinsic flexibility, as well as to predict molecular docking or molecule/surface interactions. from a modelling point of view, the interaction problem can be expressed in terms of reactive sites between the interacting entities, the molecular deformations being extracted from the pre-calculated static modes of each separated ones. the first applications of our method have focused on the intrinsic flexibility of biomolecules like nucleic acids and proteins. more recently, this new methodology allowed us to investigate the folding of the region - of the amyloid β-peptide, via the docking of a zinc ion on the reactive sites of the molecule. conformational study on a myelin basic protein fragment: molecular dynamics simulations in membrane e. polverini , g. harauz dipartimento di fisica, università di parma, parma, italy, department of molecular and cellular biology, university of guelph, guelph, ontario, canada myelin basic protein (mbp) is a multifunctional protein of the central nervous system whose principal role is in maintaining the compactness and integrity of the myelin sheath, the multilamellar membrane wrapped around nerve axons. however, mbp also interacts with other proteins such as cytoskeletal and signalling proteins, adapting its structure to the different roles. mbp is a candidate autoantigen in the human demyelinating disease multiple sclerosis. this study investigated at atomic detail the conformation of a highly conserved central fragment of mbp, constisting of two consecutive regions with different relevant functionalities. the first one is associated with the membrane and comprises the primary immunodominant epitope in multiple sclerosis; the second one was predicted to be a ligand for sh -domains of signalling proteins. molecular dynamics simulations were perfomed in the presence of dodecylphosphocholine micelle, starting from a structure extrapolated from experimental data (harauz and libich, curr. protein pept. sci., ). the results confirm the experimetal hypothesis, showing, in the micelle, a stable alpha-helix anchored to the membrane for the first region and, for the proline-rich second one, a poly-proline type ii helix pointing outwards, ready to interact with the signalling proteins. multiresolution modelling of drug and hormone permeability through a lipid bilayer traditional atomic-level (al) modelling of biomembranes is time-consuming, and hence limited in the range of systems and phenomena that can be simulated. to alleviate this problem, we designed a coarse-grain (cg) representation where each lipid molecule, in reality consisting of more than atoms, is modelled with only cg sites. our cg technique proves two orders of magnitude less demanding of computational resources than traditional al methodology. a unique feature of our approach is that the cg potentials are directly compatible with standard al models, thus facilitating the simulation of multiresolution systems, where the "chemically-sensitive" components (e.g., the solutes in membrane permeation studies) are modelled atomistically, while the surrounding environment is coarse-grained. in this contribution, we present a summary of our multiscale methodology, together with its application to the permeation of large molecules -drugs and steroid hormones. the calculated permeabilities are compared to the available experimental measurements and al simulation data. molecularlevel insights regarding the permeation mechanism are obtained and rationalised. -multiscale simulation - the processes of life involve a variety of events that occur on different scales, ranging from a fewÅ/ps of the triggering steps of the biochemical reactions, up to their macroscopic effects in cells and organs. intermediate steps involve the structural rearrangement of the bio-molecules (∼nm/ - ns), their aggregation, folding (∼ nm-µm/ µs-ms), internal cell diffusion and dynamics (µm-mm/ms-hours), evidently requiring a multi-scale modeling approach. here the multi-scale approaches are first briefly illustrated with a particular attention to the issue of matching the different resolutions, which is essential to achieve a coherent descripition. the focus is then fixed on the coarse grained (cg) models, typically spanning the nm-µm and µs-ms scale, and in particular on their minimalist -simplest and computationally cheapest -versions [ , ] . a parameterization strategy that combines accuracy and predictive power within these models is presented here and applications are shown to relevant cases including the proteins involved in the hiv replication, the green fluorescent proteins and examples of macromolecular complexes. [ ] controlled degradation of collagen is an important process in tissue remodeling and wound healing. collagenase cleaves fibrillar collagens about three quarters of the distance from the amino-terminus. even though the determination of the cleavage site and the collagenase structure took place decades ago, the mode of action of collagenase on collagen is not clear. to understand the mechanism of collagenase activity on collagen, the structure, stability and dynamics of collagen, its conformation around the cleavage site and the possibilities of conformational rearrangements between the two domains in collagenase was explored using md simulation. the results of principal component (pca) and normal mode (nm) analysis of the collagen and collagenase suggests that the c-terminal domain of collagenase recognizes the collagen, and then the n-terminal catalytic domain undergoes rearrangement on the substrate (with the help of linker regions). the sda software for carrying out brownian dynamics simulations of protein-protein diffusional association with the help of biochemical constraints is being used to predict the mode of interaction of collagen and collagenase. different conformations obtained from pca and nm analysis are being used for the docking. the predicted structures of the complex will help us to understand how collagenase recognizes and binds collagen specifically. acknowledgment: daad, germany, csir-srf india, and the klaus tschira foundation. membrane aoration by antimicrobial peptides combining atomistic and coarse-grained descriptions d. sengupta, a. j. rzepiela, n. goga, s. j. marrink university of groningen, the netherlands antimicrobial peptides (amps) comprise a large family of peptides that include small cationic peptides, such as magainins, which permeabilize lipid membranes. previous atomistic level simulations of magainin-h peptides show that they act by forming toroidal transmembrane pores. however, due to the atomistic level of description, these simulations were necessarily limited to small system sizes and sub-microsecond time scales. here, we study the long-time relaxation properties of these pores by evolving the systems using a coarse-grain (cg) description. the disordered nature and the topology of the atomistic pores are maintained at the cg level. the peptides sample different orientations but at any given time, only a few peptides insert into the pore. key states observed at the cg level are subsequently back-transformed to the atomistic level using a resolutionexchange protocol. the configurations sampled at the cg level are stable in the atomistic simulation. the effect of helicity on pore stability is investigated at the cg level and we find that partial helicity is required to form stable pores. we also show that the current cg scheme can be used to study spontaneous poration by magainin-h peptides. over-all, our simulations provide a multi-scale view of a fundamental biophysical membrane process involving a complex interplay between peptides and lipids. the varkud satellite (vs) ribozyme is the largest of the nucleolytic ribozymes, and the only one for which there is no crystal structure. we have determined the overall architecture of the complete vs ribozyme using small-angle x-ray scattering in solution. the substrate stem-loop docks into the tertiary fold of the ribozyme, allowing an intimate loop-loop interaction to occur. this brings two key nucleobases a and g into close proximity of the scissile phosphate, and we believe that these nucleobases are involved in general acid-base catalysis of the phosphoryl transfer reactions. this is supported by functional group substitution, and the ph dependence of the reaction rate for the natural and modified rna. although possessing totally different folds, the functional elements of the vs and hairpin ribozymes are topologically and mechanistically very similar if not identical. this has probably arisen by convergent evolution. other nucleolytic ribozymes have diversified to employ hydrated metal ions and even small molecules to participate in genera acid-base catalysis. by contrast, the larger ribozymes seem to have adopted a different, metalloenzyme, catalytic strategy. why these different groups have evolved different catalytic chemistries is an interesting challenge to our understanding of biocatalysis. comparison of dna and sirna binding and nuclease protection by non-viral vectors for gene delivery j. lam, k. witt, a. j. mason, l. kudsiova, m. j. lawrence pharmaceutical science division, king's college london, uk the biggest obstacle for the success of gene therapy is delivery. with the discovery of rnai, the use of sirna to regulate gene expression as potential therapy has attracted much attention recently. in contrast to dna, sirna delivery does not require nuclear entry. with one less barrier to overcome, the delivery of sirna seems to be easier. it is frequently assumed that comparable delivery strategy could be used for both dna and sirna. in fact the two types of nucleic acids are fundamentally different with distinct properties, which impact their delivery strategies. in the present study it was found that most non-viral delivery vectors including polymers, peptides and lipids were generally more efficient in binding with dna than sirna as shown in gel retardation assay. the inferior sirna binding is possibly due to the rigid structure of sirna, resulting in weaker electrostatic interaction with the cationic vectors. surprisingly, it was observed that all the vectors studied offered better nuclease protection for sirna than dna despite poorer sirna binding. either the nuclease protection for sirna is easier to achieve due to its small size, or the gel retardation assay did not truly reflect the binding efficiency as the weaker sirna complexes may dissociate during electrophoresis. not only the delivery strategy, the results between dna and sirna study must also be carefully adapted and interpreted. single molecule studies of spliceosomal rnas u and u z. guo, d. rueda department of chemistry, wayne state university, detroit, michigan, u.s.a. splicing is an essential step in the maturation reaction of mrna, in which intervening introns from exons. the spliceosome is a dynamic assembly of small nuclear rnas and > proteins that catalyzes splicing. u and u are the only snrnas strictly required for splicing. major conformational changes are expected to take place during spliceosomal assembly and catalysis. we have developed a single-molecule fluorescence assay to study the structural dynamics of a protein-free u -u complex from yeast. our previous data have revealed a -step large amplitude conformational change of the u -u complex. the st step is a mg + -induced conformational change where helix iii and the u -isl are in close proximity in low mg + and separated in high mg + . the nd step corresponds to the formation of the highly conserved helix ib. we now examine the role of the highly conserved bases in the folding dynamics of the u -u complex. the data show that u and the acagag loop play an important role in stabilizing the interaction between helix iii and the u -isl. we hypothesize that u flips out of the u -isl and interacts with the acagag loop to bring them in close proximity. our results raise the interesting possibility that this interaction plays an important role in bringing the ' splice site and the branched a into close proximity of u , which may be critical for catalysis. a structure-based approach for targeting hiv- genomic rna dimerization initiation site s. freisz, s. bernacchi, p. dumas, e. ennifar ibmc -cnrs, strasbourg, france all retroviral genomes consist in two homologous single stranded rnas. hiv- dimerization initiation site (dis) is a conserved hairpin in the ' non-coding region of the genomic rna and essential for viral infectivity. the dis initiates genome dimerization by forming a kissing-loop complex, further stabilized into an extended duplex upon interaction with the ncp nucleocapsid protein. x-ray structures of the dis kissing-loop and extended duplex revealed similarities with the bacterial s ribosomal rna a-site, which is the target of aminoglycoside antibiotics. as a result, aminoglycosides also bind the hiv- dis as shown by our x-ray structures of the dis kissingloop bound to aminoglycosides. using fluorescence, uvspectroscopy and microcalorimetry, we further characterized hiv- dis/aminoglycosides interactions. we found that the affinity of aminoglycosides for the dis was higher than for their natural target, the s a site. they strongly stabilize the dis kissing-loop, blocking its conversion into the duplex form. finally, we also solved x-ray structures of the dis duplex bound to aminoglycosides, revealing an important conformational change following drug binding. these structures show that it is possible to target the hiv- dis dimer before and after the ncp -assisted rna maturation with the same molecule. altogether, our studies create the basis for a rationally driven design of novel potential drugs targeting the hiv- genome. the core protein of hepatitis c virus is a multifunctional protein of aa, consisting of a hydrophilic n-terminal domain with three basic domains (bd -bd ) responsible for the interactions with rna and a hydrophobic c-terminal domain. the n-terminal domain exhibits nucleic acid chaperone properties similar to those of the nucleocapsid protein from hiv. here, we characterized the mechanism of the chaperone properties of a peptide e corresponding to the bd and bd clusters of the n-terminal domain. to this end, we monitored the promotion by this peptide of the annealing of dtar, the dna analogue of the transactivation response element to its complementary sequence, ctar dna, taken as models. the annealing involves two second-order kinetic components that are activated by at least three orders of magnitude by peptide e. this activation was correlated with the ability of peptide e to destabilize the lower half of dtar stem. using, ctar and dtar mutants, the two kinetic components were assigned to two pathways which are connected with the fast annealing of the terminal bases of ctar to dtar and slow extended duplex formation, limited kinetically by the nucleation of central segments of ctar and dtar stems. structure and conformational dynamics of a unique dead box helicase m. rudolph , m. linden , r. hartmann , d. klostermeier hoffmann-la roche, basel, switzerland, biozentrum, univ. of basel, switzerland, univ. of marburg, germany dead box helicases couple atp hydrolysis to rna structural rearrangements. t. thermophilus hera consists of a helicase core and a c-terminal extension (cte) with a putative rnase p motif. crystal structures show that the cte is bipartite, forming a highly flexible dimerization motif with a novel fold and an additional rna-binding module. we provide a first glimpse on the orientation of an rna-binding domain outside the helicase core. in a structure-based model for the complete hera dimer, the rna-binding sites of the helicase cores face each other, allowing for inter-subunit communication. the plasticity of the dimerization motif allows for drastic changes in the juxtaposition of the helicase cores within the dimer. in single molecule fret experiments we identified fragments of the s rrna and rnase p rna as substrates for hera. both substrates switch the hera core to the closed conformation and stimulate the intrinsic atpase activity. rna binding is mediated by the cte, but does not require the putative rnase p motif. atp-dependent unwinding of a short helix in s rrna suggests a specific role for hera in ribosome assembly, in analogy to the e. coli and b. subtilis helicases dbpa and yxin. in addition, the specificity of hera for rnase p rna may be required for rnase p rna folding or rnase p assembly. simultaneous action of two hera subunits on the same rna molecule may be important for efficient rna remodeling in vivo. structural basis for the encapsidation process of turnip yellow mosaic virus m. petersen , j. hansen , s. s. wijmenga nucleic acid center, department of physics and chemistry, university of southern denmark, odense, denmark, physical chemistry/biophysical chemistry, radboud university, nijmegen, the netherlands formation of hairpins with internal loops with c c and c a mismatches in the ' untranslated region is a common characteristic among the plant viruses belonging to the tymovirus genus. turnip yellow mosaic virus possesses two such hairpins, hp and hp . hp , and in particular the presence of the c c and c a mismatches in its internal loop, is important for initiation of the encapsidation of the viral genome. the encapsidation occurs under acidic conditions at the neck of invaginations of the chloroplast membrane. we have now determined the d structures of revertants involved in the evolutionary pathway using nmr spectroscopy. these structures reveal how the grooves in the hairpin become increasingly positively charged in successive generations of evolution. the similarity between the ccca mutant and the wild-type hairpin (hp ) is striking and explains why this mutant gives rise to a viable virus. in addition, a characteristic tilt of the backbone is observed upon occurrence of a protonated c c base pair. both the positively charged major groove and the kink in the rna backbone appear to be crucial for interactions with the viral capsid. at neutral ph, the structure of hp resembles the watson-crick base paired mutant which explains why encapsidation only occurs under acidic conditions. a. percot , j. vergne , m.-c. maurel , s. lecomte ladir, umr , thiais, france, lbeam, upmc, paris, france, cbmn, umr , pessac, france the existence of "rna world" as an early step in the history of life increases the interest for the characterization of these biomolecules. the studied hairpin ribozyme is a self-cleaving/ligating motif found in the minus strand of the satellite rna associated with tobacco ringspot virus. surface-enhanced raman spectroscopy (sers) was successfully used to detect sub-picomole quantities of nucleic acids. sers takes advantage of the strongly increased raman signals generated by local field enhancement near metallic (typically au and ag) nanostructures. sers spectra of dna or rna are strongly dominated by stockes modes of adenine. through an interaction of adenyl residues with silver colloid, adenyl raman signal is times increased compared to raman scattering. in controlled conditions, sers signal is proportional to the amount of free residues adsorbed on the metal surface. upon rna cleavage, residues are unpaired and free to interact with metal. in the present study, we proposed to follow the cleavage reaction of hairpin ribozyme (hpr ) using the sers signal of the liberate adenyl residues. as the sers signal is proportional to the adenyl residues, reactivity of hr was monitored by measuring the raman intensity of the fragment liberated during the cleavage of hairpin. the results obtained were compared with the electrophoresis method performed on the same sample and similar results were obtained. -rna world - eur biophys j ( ) the development of arrays for biomolecular recognition for a broad range of applications in biomedical diagnostics is receiving a constantly increasing attention. the design of efficient protein biochips, however, requires the optimization of protein immobilization protocols for improving the device sensitivity. moreover, innovative platforms for in-vitro detection in highly diluted, small volume samples need to be developed, for which standard micro-fabrication techniques are not suitable. therefore, the development of nano-scale platforms for protein and antibody detection is essential. we report here on a novel approach for the fabrication of multiple protein nanosensors using atomic force microscopy based nanografting and dna-directed immobilization (ddi), which takes advantage of the specific watson-crick hybridization of oligonucleotide-modified proteins to surfacebound complementary oligomers. using nanografting, single-stranded dna nano-structures with well defined local environments were fabricated on a flat surface. dna-protein conjugates were then anchored on the engineered binding sites by ddi in a single chemical operation and detected by the corresponding topographic height increase of the relevant patches. immunological assay were used to demonstrate the biochemical functionality of the immobilized proteins, proving the specificity of biomolecular recognition of our nanodevices, in the micro-molar to pico-molar concentration range. dna accessible surface area and indirect protein-dna recognition: study by bioinformatical approach o. p. boryskina , m. y. tkachenko , a. v. shestopalova , m. y. tolstorukov institute of radiophysics and electronics nas of ukraine, harvard-partners center for genetics and genomics, usa revealing the mechanisms of protein-dna recognition is essential for understanding the regulation of many cellular processes. there is growing evidence that recognition through sequence-specific contacts (direct readout) can be enhanced by recognition via dna sequence-dependent deformability (indirect readout). the role of changes in dna accessible surface area (asa) in distorted dna configurations in complexes with proteins is not fully understood yet, even though such changes and related changes in polarity of dna surface are among key factors of indirect readout. to fill this gap we have developed a publicly-available internet database of protein-dna complexes, which integrates the data on dna conformational parameters with information on asa of dna atoms in the minor and major grooves, protna-asa. the database has been used to analyze the effect of changes in dna backbone configuration on asa of dna atoms in major and minor grooves. we observe that sugar puckering and conformation of torsion angle γ affect the accessibility of dna atoms in both grooves to a noticeable extent. we also report sequence specific preferences of the nucleotides for structural domains of γ. these results can shed new light on the mechanisms of indirect protein-dna recognition. a. arakelyan biology dpt., yerevan state university, yerevan, armenia the influence of ligand, irreversibly binding with dna, on the isotherm of adsorption of ligand binding with dna reversibly has been modeled theoretically. the isotherm of adsorption of etbr on dna in presence of cis-ddpt has been considered at low concentrations of cis-ddpt and etbr.. the isotherm of adsorption of etbr on dna has linear form in scatchard coordinates at low degrees of occupation. the comparison with experimental isotherm permits to estimate the parameters of etbr binding with dna. with taking into account the pseudo-ring structures formation with partially molten regions we consider two types of binding regions, linear and ring at low degrees of occupation. the isotherm of adsorption of etbr on dna and also variation of the number of bounded etbr molecules are calculated with taking into account these two types of binding regions. it was shown that at low concentrations cis-ddp, bounding with dna, changes the isotherm of ligands adsorption. the linear in scathcard coordinates isotherm of adsorption transforms into non-linear isotherm. the degree of transformation depends on the fraction of dna in the ring regions, on the ratio between number of etbr binding cites in the ring and linear regions, and also on the binding constants for these regions. it was shown that low concentrations of cis-ddp affect the dependence of dispersion on the concentration of ligands, changing both the magnitude of maximum and its position. tunable nanoconfinement structures for dna manipulation e. angeli, l. repetto, g. firpo, c. boragno, u. valbusa nanomed labs, advanced biotechnology center and physics department, university of genoa, italy nanostructures, such as nanochannels [ ] or nanoslits [ ] , have been successfully used to confine and stretch dna molecules, offering interesting opportunities of investigation on conformational changes induced by confinement, physical and biological properties, etc. the integration of these nanostructures on lab-on-chip systems has shown their great potential for applications such as dna sieving or single molecule manipulation [ ] , [ ] . arrays of nanochannels fabricated, using a focused ion beam (fib) on a silicon master, are replicated using elastomeric materials, such as poly(dimethylsiloxane) (pdms), and soft-lithography techniques. the cross-section of these flexible polymeric nanoconfinement structures can be reversibly and dynamically tuned, in order to vary biomolecules transport characteristics and confinement conditions of trapped dna molecules. moreover, these nanochannels, with tunable cross-section, are used to study and exploit the sieving mechanism of "entropic recoil" [ ] for the separation of long dna chains. [ ] mannion jt, et al., ( ) biophys. j., , : - . [ ] jo k, et al., ( ) pnas, , : - . [ ] fu j, et al., ( ) nat. nanotechnol., : - [ ] huh d, et al., ( nat. mater., : - . the nucleocapsid protein ncp of hiv- is characterized by two conserved zinc fingers and plays crucial roles in the virus, through its binding to nucleic acids. ncp is required for efficient proviral dna synthesis, by promoting the initiation of reverse transcription and the two obligatory strand transfers. using fluorescence techniques as well as fcs and nmr, we investigated the chaperone properties of ncp on the primer binding sites (pbs) and transactivation response elements (tar) sequences involved in the two obligatory strand transfers. we showed that ncp binds mainly to the (-)pbs loop, which results in an extension of the loop and a destabilization of the upper base pair of the stem. these structural changes chaperone a kissing complex with the complementary (+)pbs loop and its further conversion into an extended duplex. in contrast, the ncp -promoted annealing of ctar-tar results from the destabilization of the bottom of ctar stem, which favors the invasion of the tar stem. by developing new fluorescence methodologies to site-specifically characterize these interactions, we further showed that ncp slows down the ps to ns conformational fluctuations of its nucleic acid targets and freezes the local mobility of the bases contacted by the zinc fingers. exploring dna orientation in flow e. l. gilroy, m. r. hicks, a. rodger department of chemistry, university of warwick, uk dna is one of the most important biomolecules. in order to undertake its biological role the dna needs to fold and unfold for which its structure and flexibility are crucially important. we have studied the characteristics of dna in flow to probe its structure. flow aligned linear dichroism (ld) is a technique that uses light polarised parallel and perpendicular to an orientated sample. it can be used to measure how aligned a sample is, and the orientation of any interacting molecules. to create this alignment, the sample is placed between two concentric cylinders where one is spun to create a shear flow. the longer and more rigid the molecule is the better the orientation and the signal. as the sample is in flow there are other factors than need to be considered when using ld. these include viscosity and temperature. there are many methods to measure the viscosity of a sample of dna at varying temperatures. these include viscometer, rheometer and dynamic light scattering. the effects temperature has on viscosity and the sample itself need also to be considered. the findings of all these studies have been reported and show the significance that viscosity and temperature have on dna ld measurements. possible applications of using ld to study dna have also been discussed, showing the importance of the use of ld and in the results shown. a.-m. florescu, m. joyeux laboratoire de spectrométrie physique, université joseph fourier grenoble , france we present a dynamical model for simulating non-specific dna protein interactions, which is based on the "beadspring" model for dna with elastic, bending and debye-hückel electrostatic interactions, and where the protein interacts with the dna chain through electrostatic and excluded-volume forces. we study the properties of this model using a brownian dynamics algorithm that takes hydrodynamic interactions into account and obtain results that partially agree with experiments and predictions of kinetic models. for example, we show that the protein samples dna by a combination of three-dimensional diffusion in the solvent and one-dimensional sliding along the dna chain. this model evidences the presence, in a certain range of values of the effective protein charge, of facilitated diffusion, i.e. a combination of the two types of diffusion that leads to faster than -dimensional diffusion sampling of dna. moreover, the analysis of single sliding events shows that the number of base pairs visited during sliding is comparable to those deduced from single molecule experiments. in contrast to kinetic models, which predict an increase of the number of different base pairs visited by the protein proportional to the square root of time, our model however suggests that this number increases linearly with time until it reaches a value that is close to the total number of dna base pairs in the cell (published in j. chem. phys. , ( )). use of md simulation to identify the critical radiation-induced lesions of a dna binding protein n. chalabi, s. goffinont, n. garnier, d. genest, orléans cedex , france a key step in the regulation of gene expression, dna structuring and dna repair is the binding of some proteins to specific dna sequences. we have shown previously that ionizing radiation destabilizes such dna-protein complexes mainly through damage to the protein. for the typical lactose operator -repressor system we have shown by fluorescence measurement and mass spectrometry that upon irradiation, all the four tyrosine residues of the dna binding domain (called headpiece) are oxidized into , dihydroxyphenylalanine (dopa). a circular dichroism study revealed a global conformational change and the destabilization of the headpiece. to decide which lesion is critical for the induction of these effects, a molecular dynamics simulation study was undertaken in parallel with a site-directed mutagenesis one. each tyrosine residue of the headpiece was replaced by another amino-acid that mimics the damaged tyrosine. the most common amino-acid used in site-directed mutagenesis being alanine, we have replaced one tyrosine ( , , or ) in the nmr-based structure of the headpiece from pdb databank ( lqc) by an alanine. the conformational stability of each tyr→ala mutant has been studied by molecular dynamics simulation (md) and compared to that of the native sequence and of the different tyr→dopa mutants. the mammalian high mobility group protein at-hook (hmga ) is a nuclear protein associated with mensenchymal cell development and differentiation. disruption of its normal expression pattern is directly linked to oncogenesis and obesity. our laboratory has utilized a variety of biochemical and biophysical methods to investigate the molecular mechanisms of hmga recognizing at-rich dna. using a pcr-based selex procedure, we discovered that hmga is a sequence-specific dna-binding protein and recognizes the following two sequences: '-atattcgcgawwatt- ' and 'atattgcgcawwatt- ', where w is a or t. using a double-label emsa assay, we found that hmga binds to at-rich dna as a monomer. using isothermal-titrationcalorimetry, we demonstrated that hmga binds to at-rich dna with very high binding affinity whereby the binding of hmga to a-tracts is entropy-driven and to alternate at sequences is enthalpy-driven. this is a typical example of enthalpy-entropy compensation in which the hydration difference between hmga -dna complexes is a main reason for the compensation. interestingly, the binding of hmga to different at-rich sequences is accompanied with a large negative heat capacity change indicating an important role of solvent displacement and charge-charge interaction in the linked folding/binding processes. partition of gibb´s free energy of drug-dna complexation we report a computation methodology, which leads to the ability to partition the gibb's free energy for the complexation reaction of aromatic drug molecules with dna. using this approach it is now possible to calculate the absolute values of the energy contributions of various physical factors to the dna binding process, whose summation gives a value that is reasonably close to the experimentallymeasured gibb's free energy of binding. application of the methodology to binding of various aromatic drugs with dna provides an answer to the question 'what forces are the main contributors to the stabilization of aromatic ligand-dna complexes' ? we report the effects of khz ultrasound irradiation of double-stranded dna solutions under conditions of transient cavitation. a new method was developed for studying these effects which is based on combination of two procedures: ultrasound irradiation of the solutions of double-stranded dna fragments and subsequent analysis of the high resolution denaturing gel electrophoresis data. statistical treatment of the data on the mobility of '-end-labelled restriction fragments with known sequence allowed us to discover the phenomenon of sequence specific ultrasonic cleavage of dna. our analysis results in the following conclusions: ) all steps with '-ward cytosine [ '-d(cpn)- '] have significantly higher cleavage rates than others; the intensity of cleavage diminishes in the order cpg > cpa ≈ cpt > cpc; ) the cleavage rates of all steps depend on the type of flanking base pairs; ) the cleavage rates of the complementary base pair steps are not identical. thus, subtle sequence specific conformational and physical-chemical variations modulate the reaction of sugar-phosphate single bonds on the ultrasound exposure. a theory of the mechanisms on the simultaneous binding of two aromatic drugs to dna it has long been recognized that certain combinations of dna-binding aromatic drugs, x +y, lead to synergistic biological effects. considering x as a basic ligand and y as an added ligand, the change of the integral biological response of x in the presence of y has been interpreted in terms of two mechanisms: the interceptor and protector action of y on x. this mechanisms have been characterized by two criteria, r d and a d , reflecting the removal of x from dna by y (biophys. chem., , vol. , pages - ) . in this work we develop the theory of the interceptor-protector action of a mixture of two biologically-active dna-binding aromatic drugs. the theory is based on solution of a general system of mass balance equations in the three-component system x -y -dna with respect to the two factors, r d and a d . the outcome is a set of expressions enabling estimation of the change in biological response of x on addition of y as a function of equilibrium parameters under different restrictions. the results are in good agreement with known in vitro data on caffeine+antibiotic action in leukemia cell lines. the influence of mn + ions on the structure of natural calf thymus dna was studied by raman spectroscopy. measurements were done at room temperature and ph . ± . , in the presence of the physiological concentration of mm na + ions, and in the presence of mn + concentrations that varied between and mm. no condensation of dna was observed. dna backbone conformational changes were not detected in the whole concentration range of mn + ions as judging from the raman spectra. no evidence for dna melting was identified. binding of manganese(ii) ions to the charged phosphate groups of dna, stabilizing the double helical structure, is indicated in the spectra. as judged from the marker band of dc near cm − , altered nucleoside conformations in dc residues are supposed to occur, in the mn + concentration range of - mm. binding of divalent ions to n of guanine and, possibly, in a lesser extent to adenine was observed as judging from the raman marker bands at , , and cm − . toward rapid dna sequencing -ab initio study of nucleotide sandwiched between au( ) plates c. morari, d. bogdan, i. turcu national institute for research and development of isotopic and molecular technologies, cluj-napoca, romania recently a new technique for dna sequencing based on measurement of transversal conductive properties of a single strained dna molecule has been proposed. such a method would allow single-base resolution by measuring the electrical current perpendicular to the dna backbone. until now, it is still not clear if the electrical signals obtained for the four nucleotide can be clearly distinguished by a hypothetical experimental setup. several factors -like the influence of the pentose group or the presence of water -may influence quite strongly the electrical signatures of the four bases. therefore, in order to obtain a working device, the understanding and detailed description of the conduction mechanisms through dna bases connected to a metallic electrode is essential. the goal of theoretical studies in this field is to describe the electric signatures of dna bases from a theoretical point of view. our study is focused on the detailed description of the electronic structure of dna's base pairs "squeezed" between two au plates. while such a geometrical model closely mimic the sequencing devices proposed in the literature, it allows us to compute meaningful physical properties such as density of states, charge transfer and orbital localizationby using "ab initio" methods. the results allow us to give qualitative prediction over the potential use of such a device in the dna's sequencing technology. multinuclear platinum complexes represent a new class of anticancer agents, distinct in dna binding and antitumor activity from their mononuclear counterparts. bbr as a first representative of this class has undergone phase ii clinical trials for treatment of ovarian and lung cancers. the structure of this trinuclear pt drug consists of two trans-ptcl(nh ) units bridged by a trans- the main lesions formed by multinuclear pt complexes in dna are long-range intra-and interstrand cross-links (cls) bridging two guanines separated by up to four base pairs. since interstrand cls can prevent dna strand separation interfering with critical cellular events they represent a serious obstacle in cell survival. in order to contribute to understanding the biological effects of dna interstrand cls of bbr , we analyzed the effect of the single, site-specific , -interstrand cl formed by this metallodrug between two guanine bases on opposite strands in the '- ' and '- ' direction on the thermal stability and energetics of short dna duplexes. the results demonstrate that , -interstrand cls of bbr in both '- ' and '- ' directions exist as two distinct conformers that are not interconvertible and affect thermodynamic stability of the dna differently. side-by-side and end-to-end attraction of doublestranded dna c. maffeo , b. luan , a. aksimentiev university of illinois at urbana-champaign, urbana, usa, ibm watson research center, yorktown heights, usa genomic dna is densely packed inside cell nuclei and viral capsids. such close packing suggests that electrostatic repulsion between negatively charged dna in the condensed states is balanced by counterion-induced attraction. several theoretical models have been proposed to explain dna attraction, however, specific microscopic mechanisms are not known. here, we report all-atom molecular dynamics simulations of the effective force between double-stranded dna in side-by-side and end-to-end orientations. in the side-by-side orientation, the dna molecules were found to form a bond state in the presence of magnesium ions. in the bond state, the dna molecules contact each other via their negatively charged phosphate groups, bridged by magnesium ions. the maximum attractive force in the side-by-side orientation is about pn per dna turn. in the end-to-end orientation, a strong ( > pn) attractive force was observed at short (< . nm) end-to-end distances regardless of the electrolyte concentration. the presence of a phosphate group at the 'ends of the fragments was found to direct dna end-to-end self-assembly and produce bound states resembling a continuous dna molecule. the computed potentials of the mean force suggest that the end-to-end attraction, rather than being mediated by counterions, is likely caused by hydrophobic and van der waals interactions between terminal nucleobases of the fragments. doxorubicin (dox) is an anticancer antibiotics with a four-membered ring system containing an anthraquinone chromophore, and an aminoglycoside. it has good anticancer activity against a wide spectrum of tumors and is one of the most extensively used antitumor chemotherapeutic compounds currently in clinical use. interestingly, conversion of dox to pyrrolinodoxorubicin analog (p-dox) exhibits - times higher toxic effects in human breast cancer cell line (nagy, a. et al., pnas, ( ) . molecular mechanisms responsible for this enormously enhanced cytotoxicity have not been entirely clarified. there is good evidence that a key component of the mechanism of action of dox is its intercalation into dna and the formation of dox-dna adducts. therefore, we have examined in detail, using the methods of molecular biophysics, dna interactions of p-dox in cell-free media and compared these results with the same studies focused on the parental dox. we find distinct differences between dna interactions of dox and p-dox and suggest that these differences are responsible at least in part for different biological effects of these two anticancer drugs. design of a microfluidic devices for the detection of oligonucléotides by sers designing fast and efficient analytical tools allowing the detection of free dna or rna at very low concentration within small volumes, without specific molecular labeling, remains a major issue of significance to perform diagnostic or to detect pathogen agents. our strategy is to use surface-enhanced raman scattering (sers) to probe selectively the spectral signature of each base in polynucleotides. sers takes advantage of the strongly increased raman signals of species when adsorbed on adapted silver colloids. we already demonstrated that adenyl raman signal of pa in presence of silver colloids is times enhanced compared to bulk signal. we plan to use nanoliter droplets of uniformed size that form spontaneously in microchannels when two immiscible fluid streams merge. these tiny droplets are almost ideal reactors as they create homogeneous control condition. we will design an optimized channels network platform that result in droplets production hosting both nucleotides and silver colloids: internal fluids recirculation provide fast and efficient mixing, favoring base adsorption on silver nanoparticles. sers will be used to determine the chemical composition of the droplets. nicks represent the most common damage in dna which occurs naturally in living cells. structural properties of nicked dna fragments have been an object of numerous studies due to its special role in reparation processes. here we report experimental results covering ultrasound irradiation of nicked dna solutions. several single-stranded nicks were produced into one strand of dsdna fragments by the nicking enzyme bst i. we have quantitatively estimated the ultrasonic cleavage rates in nicked dna fragments with known sequences using the polyacrylamide gel electrophoresis. computer analysis of the cleavage pattern in the '-end labeled and primarily intact strand reveal cleavage enhancement in the regions of about b. p. up and down the nicks which were initially produced into complementary strand. the intensity of cleavage near the nicks is (in average) about times higher than cleavage in the same sites of the intact dsdna fragments. at the same time, the cleavage rates in positions beyond the regions of the nick markedly grow weak even comparing to the sequence-specific cleavage of intact double-stranded dna fragments. thus, the presence of the nick serves as an expressive structural indignation, which exceeds modulation of the structure caused by the base-pair sequence and is capable of absorbing mechanical stresses applied to the nearby sites of the molecule. comparing the native and an irradiated lactose repressor-operateur complex by md simulation g. naudin, n. garnier, d. genest, rue charles sadron, orléans cedex , france the function of the e. coli lactose operon requires the binding of a protein, the tetrameric repressor, to a specific dna sequence, the operator. the formation of this complex involves the interaction of at least one protein dimer with the operator sequence. this occurs via the dna-binding domains (called headpieces) of the two constitutive monomers. we have previously shown that upon irradiation with gamma rays the complex is destabilised mainly because the repressor losses its dna binding ability. radiation-induced lesions were identified that may be responsible for this deleterious effect: all tyrosine residues of the headpieces are oxidized into , -dihydroxyphenylalanine (dopa). in order to unravel the mechanisms leading to the observed destabilization of the operator-repressor complex, we compare by md simulations two complexes: -the native complex formed by a dimer of two headpieces and a fragment of dna with the operator sequence and -the damaged complex in which all tyrosines are replaced by dopa. analysis of these trajectories shows a loss of stability and binding energy as well as changes in the structure of the damaged complex with respect to the native one. by comparing precisely the evolution of the two complexes we can explain how the oxidation of the tyrosine residues of the headpieces into dopa may trigger the destabilization of the complex. local conformation of confined dna studied using emission polarization anisotropy in nanochannels with dimensions smaller than the dna radius of gyration, dna will extend along the channel. we investigate long dna confined in nanochannels with dimensions down to * nm, using fluorescence microscopy. studies of the statics and dynamics of the dna extension or position in such confinements as a function of e.g. dna contour length, degree and shape of confinement as well as ionic strength has yielded new insight into the physical properties of dna with relevance for applications in genomics and fundamental understanding of dna packaging in e.g. viruses. our work extends the field by not only studying the location of the emitting dyes along a confined dna molecule but also monitoring the polarization of the emission. we use intercalating dyes whose emission is polarized perpendicular to the dna extension axis, and by measuring the emission polarized parallel and perpendicular to the extension axis of the stretched dna, information on the local spatial distribution of the dna backbone can be obtained. we will discuss results in shallow ( nm) and deep ( nm) channels and describe how the technique can be used to investigate non uniform stretching of a single dna molecule. raman spectroscopy of dna modified by new antitumor nonclassical platinum complexes o. vrana, v. kohoutkova, v. brabec institute of biophysics as cr, královopolská , cz- brno, czech republic platinum anticancer agents (cisplatin, carboplatin, oxaliplatin) are in widespread clinical use especially against testicular, ovarian and head and neck carcinomas. there is a large body of experimental evidence that dna is the critical target for the cytostatic activity of cisplatin. platinum complexes form several types of adducts, which occur in dna with a different frequency and differently distort the conformation of dna. clinically ineffective trans isomer of cisplatin (transplatin) also covalently binds to dna bases. the trans geometry in dichloridoplatinum(ii) complexes was activated by various ways. the replacement of at least one amine ligand by planar amine ligand represents example of such activation. raman spectroscopy is powerful technique for examining both structural and thermodynamic properties of nucleic acids in solution. interactions of cis-and trans-pt(ii) complexes having nonplanar heterocyclic amine ligand such as piperidine, piperazine and -picoline with dna have been investigated by laser raman spectroscopy. raman difference spectra reveal that the pt(ii) complexes induce great structural changes in b-dna and indicate disordering of b-dna backbone, reduction in base stacking and base pairing and specific metal interaction with acceptor sites on purine residues. acknowledgement: this work was supported by grant as cr, iqs . a. v. vargiu , a. magistrato , p. carloni , p. ruggerone cnr-infm-slacs and physics dept., university of cagliari, cagliari, italy, cnr-infm-democritos and sissa/isas, trieste, italy, iit and sissa/isas, trieste, italy the minor groove of dna is the target of several anticancer agents, which interfere with replication and translocation processes, leading to cell death. the molecular recognition event is a key step to achieve detailed knowledge of the interactions behind selectivity and affinity of a ligand towards a particular nucleic acids sequence. recognition is a multiroute process which can involve many steps before the formation of the most stable adduct. in particular, many studies have pointed out the importance of events like sliding along the groove and dissociation (which is a relevant step in the translocation among different sequences) for the affinity and the specificity of minor groove binders. despite this, no studies on the dynamics of this event were reported in the literature. in this contribution i present our recent work on the subject. umbrella sampling and metadynamics were used in the framework of classical md to characterize mechanisms andfree energy profiles of molecular recognition routes by the antitumoral agents anthramycin, duocarmycin and distamycin. our results are in very good agreement with the available experimental data, and provide insights on the influence of factors like size, charge and flexibility on the molecular recognition process. amplification of oligonucleotide sequence recognition using bioresponsive hydrogels s. tierney, b. t. stokke biophysics and medical technology, dept. of physics, the norwegian university of science and technology, ntnu, no- trondheim, norway we describe development and characterization of oligonucleotide functionalized hydrogels for amplifying the molecular recognition signal occurring on hybridization between dioligonucleotides. the µm radius hemispherical hydrogels were integrated on a high resolution interferometric fiberoptic readout platform supporting determination changes in the optical length of the hydrogel with nanometer resolution. the hydrogels were designed with hybridized dioligonucleotides grafted to the polymer network as network junctions in addition to the covalent crosslinks or oligonucleotides grafted to the network chains. the probe oligonucleotide destabilizing the junction point by displacement hybridization yielded an optical signal about five times as large as for binding within the hydrogel design with a comblike grafted oligonucleotide. the optical signal was found to be dependent on the concentration of the probe, the sequence and matching length between the probe and sensing oligonucleotide. concentration sensitivity applied as specific label-free detection of oligonucleotide is estimated to be in the nanomolar region. the current design support detection in excess of x sequences. amplification of the molecular recognition employing the developed oligonucleotide imprinted hydrogel for label-free sensing of probe oligonucleotide sequences or taking advantage of the oligonucleotide sequence designed as aptamers for determination of other types of molecules are discussed. tracing t-cells by paramagentic nanoparticles in the brain of the rat model of als d. bataveljic , g. vanhoute , g. bacic , p. andjus inst. for physiology and biochemistry, univ. of belgrade, serbia, bio imaging lab, univ. of antwerpen, belgium, school of physical chemistry, univ. of belgrade, serbia amyotrophic lateral sclerosis (als) is a devastating neurological disorder affecting upper and lower motoneurons. since immune disbalance is known to be an important manifestation of the disease we were particularly interested in following the labeled immune cells in the familial als rat model, hsod- g a . a t -or t -weighted mri protocol was used with a mini surface coil placed over the skull of the anesthetized animal in a . t wide bore magnet. in order to compare this approach to standard high field small animal imaging a . t mri system was also used. there was a congruence of images of lesions in the brainstem at the two field strengths. it was confirmed with gd-dtpa contrast that the blood brain barrier (bbb) is compromised at the interbrain level. in order to study immune cell infiltration rats were i.v. injected with magnetically labeled antibodies against helper cd + or cytolytic cd + killer t cells. by combined t , t and t * weighted imaging cd + lymphocyte infiltration was observed in the brainstem-midbrain region while the cd + cells were more confined to the brainstem region. comparison of mri of labelled cd + vs. cd + lymphocytes reveals the relevant cellular inflammatory mechanism in als. the appearance of the mri signal from the latter t cell type points to the mechanism of bbb disruption as suggested from a recent study on the role of cd + t cells in a model of multiple sclerosis. emodin uptake study in u- mg cells using optically trapped surface-enhanced raman scattering probes s. balint , s. rao , p. miskovsky , d. petrov icfo -the institute of photonic sciences, barcelona, spain, department of biophysics, university of p.j.Šafárik, košice, slovakia emodin ( , , -trihydroxy- -methylantraquinone) is a photosensitizing pigment present in plants of herbal laxatives. emodin inhibits nuclear transcription factor-κb activation and induces free radical production in human mononuclear cells resulting in its antiviral and anti-cancer activity. the uptake and distribution of emodin inside the u- mg cell line is studied by combining optical tweezers and surfaceenhanced raman spectroscopy (sers). sers greatly enhances the spectrum of an otherwise weakly scattering material which is achieved by attaching nano-sized silver colloids to micron-sized dielectric beads. the distribution of emodin in the cell is studied by simultaneously trapping and exciting the sers bead and scanning it across the membrane while recording the emitted light. secondly, the beads are statically placed inside the cell and excited at certain intervals in order to track the migration of emodin through the membrane. the results give new insight in to the metabolic pathways of emodin and demonstrate a new imaging and detection technique that is fast and less invasive than current standards. acknowledgement: miin fis - (spain), fundació cellex barcelona, nadacia spp (slovakia), apvv- - (slovakia) amphotericin b (amb) is a polyene antibiotic that has been widely used for treatment of systemic fungal infections. the main mechanism of biological mode of action of amb is considered to be associated with formation of ionic membrane pores or channels in the lipid membranes. the aim of this work was to study the influence of the k + and na + ions on the aggregation process of amb in aqueous medium. the analysis of electronic absorption and fluorescence spectra of amb shows that the increasing k + concentration have influence on the level of aggregation of the drug much more than the same amount of na + ions. this effect is especially noticed at neutral ph values. the rls technique was used to study aggregation of amb in solution, in the environment of the k + and na + ions. the application of this technique makes it possible to study the electronically coupled chromophores, especially molecular aggregates. the results of the atr-ftir and raman spectroscopic studies also support this conclusion. these results provide a better understanding of the interaction between k + and na + ions and antibiotic which has not been previously considered to be significant for biological action of amb. g-quadruplexes: combining theory with experimental spectroscopic methods guanine-rich oligonucleotides can form unique tetrameric structures with four coplanar guanine bases, known as gquadruplex motifs. the g-quadruplexes have been found in vivo in the terminal parts of telomeres and other genomic regions. ligands, specifically binding to the g-quadruplex regions inhibit telomerase activity and thus can play an important role in the cancer therapy. most recently, the potential use of these structures has been tested in biosensors and nanotechnology industry. in the present work we use the infrared spectroscopy, including the relatively novel technique of the vibrational circular dichroism (vcd), in a combination with molecular dynamics and quantum chemistry computational methods to investigate the structure and spectroscopic response of the quadruplexes formed by the d(g) and selfassociated dgmp. we obtained a good agreement between the computed and experimental spectra, confirming that the proposed geometrical models are realistic. the vcd technique appears especially convenient for the studies as it can detect the liquid-crystalline phases of the g-quadruplexes by an anomalous enhancement of the signal. j. bogdanovic pristov, a. mitrovic, k. radotic, i. spasojevic institute for multidisciplinary research, belgrade, serbia fructose, due to its high antioxidative capacity, represents a significant component of non-enzymatic defense of some plants against cold-provoked oxidative stress. in the present study, we have investigated role of fructose in seasonal adaptation of picea omorika (pančić) purkinye to cold. this endemic coniferous species is exposed to subfreezing temperatures that range from - to - • c during the autumn/winter and high temperatures exceeding • c during the summer. characteristic epr signal of free or weakly bound mn + was used as an indicator of oxidative status of needles, since coldrelated oxidative damage leads to mn + release from photosystem ii. it was observed that prooxidative conditions developed in the autumn, at the beginning of cold season, which corresponded to significant increase of fructose level. total sod, as well as mnsod activity also rose significantly higher in the autumn. observed activation of antioxidative system (non-enzymatic and enzymatic) led to adaptation of needles to cold, as oxidative status during winter was decreased and similar to the status of needles in cold-free seasons. calyx of held: sted nanoscopy of a glutamatergic terminal p. bingen , t. m. staudt , c. kempf , h. horstmann , j. engelhardt , t. kuner , s. w. hell german cancer research center / bioquant, heidelberg, germany, max planck institute for biophysical chemistry, göttingen, germany, institute for anatomy and cell biology, university of heidelberg, heidelberg, germany the calyx of held, a large glutamatergic synaptic terminal in the auditory brainstem circuit has been increasingly employed to study presynaptic mechanisms of neurotransmission in the central nervous system. a highly detailed model of the morphology and distribution of cytoskeleton, synapsin, synaptic vesicles, calcium sensors, mitochondria, the presynaptic membrane and its active zones is derived by colocalization analysis of these different key elements of synaptic transmission in the rat brain. the various cellular components are visualized with subdiffraction resolution by stimulated emission depletion (sted) microscopy. imaging individual structural elements exhibit a focal plane resolution of < nm inside µm thick tissue sections. three-dimensional shim and pem to study collagen arrangement and crimping pattern p. bianchini , m. franchi , l. leonardi , a. diaspro lambs-ifom microscobio research centre and department of physics, university of genova, genova, italy., italian institute of technology (iit), genova, italy, department of human anatomical sciences and physiopathology of the locomotorapparatus, university of bologna, italy ligaments have been generally described as multifascicular structures with collagen fibre bundles cross-connecting to each other or running straight and parallel with crimps. a different collagen array and crimping pattern in different ligaments may reflect a different mechanical role. aim of this study was to relate the d collagen arrangement and crimping pattern by backward and forward second harmonic imaging microscopy (shim) and -photon excitation microscopy ( pem). shim on a laser-scanning system is a powerful and unique tool for high-resolution, high-contrast, three-dimensional studies of tissue architecture. although it is a coherent process the multiple scattering through the tissue give us the capability to acquire signal in both backward and forward direction [ ] . shim and pem were combined in a dual-mode nonlinear microscopy to find out collagen fibre arrangement and crimping pattern. both polarization dependence and differences between forward and backward signals allowed to yield information on local structure [ ] . currently used tcspc flim systems are characterised by high counting efficiency, high time resolution, and multiwavelength capability. the systems are, however, restricted to count rates on the order of a few mhz. in the majority of applications, such as fret or autofluorescence, the photostability of the samples limits the count rate to much lower values. the limitation of the count rate is therefore no problem. however, if flim is used for ion concentration measurements or imaging of chlorophyll in plants the available count rates can exceed the counting capability of a single tcspc channel. we therefore developed a flim system that uses eight fully parallel tcspc channels. by using a polychromator for spectral dispersion and a multichannel pmt for detection we obtain multi-spectral flim data at a rate of several frames per second. we will demonstrate the application of the system to dyamic changes of the fluorescence lifetime of chlorophyll in living plants. -imaging and spectroscopy - scattering effects on non linear imaging of thick biological samples f. cella , z. lavagnino , a. diaspro lambs-ifom, microscobio research center, university of genoa, italy, iit, italian institute of technology, genoa, italy non linear optical scanning microscopy became a useful tool for living tissue imaging. biological tissues are highly scattering media and this leads to an exponential attenuation of the excitation intensity as the light travels into the sample. while performing imaging of biological scattering tissues in two photon excitation ( pe) regime, the localization of the maximum pe intensity was found to shift closer to the surface and the imaging depth limit appears strongly limited by near surface fluorescence . in this work we computed illumination and photobleaching intensity distribution in order to characterize the effects induced by scattering. simulations of pe illumination and photobleaching intensity profiles have been performed for different scattering coefficients and at different focus depth. furthermore imaging of fluorescent immobile sample (polyelectrolyte gel) allowed to perform an experimental test on thick turbid media. results confirm that under these conditions no photobleaching effects due to scattering occur close to the surface. [ ]ying et al., appl. opt. , ( ) . [ ] theer p. and denk w, j. opt. soc. am. a. ( ) [ ]mazza d. et al., appl. opt. ( ) . biospectroscopic probes for real time measurement of hydrogen-deuterium exchange p. carmona , m. molina instituto de estructura de la materia (csic), madrid, spain, escuela universitaria deÓptica, madrid, spain isotopic exchange has long been used for the analysis of biomolecular structure and dynamics. hydrogen-deuterium exchange rates depend on ph, temperature and biomolecular environment. this is due to hydrogen bonding, low solvent accessibility, and steric blocking. time resolved measurement of hydrogen-deuterium exchange for subsequent d correlation spectroscopic analysis can, then, be very useful to obtain structural information from the said viewpoint. we have developed a microdialysis quartz cell for use in conjunction with raman spectroscopy to investigate hydrogen-isotope exchange reactions of biomolecules. the system requires only µl volumes of the initial substrate and perturbing effluent solutions. we have obtained a d o efflux rate of k d = . ± . min − with the greatest mwco ( kda) used here, which involves that an exchange rate of . min − is the limiting rate that could be resolved with the said cell system. analogous results have been obtained using an infrared biospectroscopic microdialysis probe. the use of the method described here has the advantage of avoiding sample dilution (and subsequent signal loss) involved in the known stop flow methods. acknowledgements: the authors gratefully acknowledge financial support from the spanish ministerio de ciencia e innovación (project ctq - /bqu). polarized transient absorption to resolve electron transfer between tryptophans in dna photolyase photoactivation of dna photolyase comprises electron transfer through the chain fadh • -w -w -w . photo-excited fadh • abstracts an electron from the tryptophan residue w in ∼ ps (monitored by transient absorption spectroscopy). the subsequent electron transfer steps (from w to w •+ and from w to w •+ ) are difficult to resolve experimentally, because electron transfer between chemically identical species does not give rise to net absorption changes. to overcome this difficulty, we make use of the fact that polarized excitation (pulse laser) induces a preferential axis (that of the excited flavin transition) in the system (photoselection), and that w and w form different angles with that axis (known from the crystal structure). thus, polarized detection should allow distinguishing between them. using polarized "classical" transient absorption on a nanosecond time scale and the pump-probe technique on a picosecond scale, we demonstrate the feasibility of the method and provide evidence that electron transfer from w to w •+ is faster than the ps time constant of the initial electron transfer from w to excited fadh • . synchrotron based fourier transform infrared (sr-ftir) microspectroscopy was applied to investigate apoptotic death of u- mg cells induced by the photosensitizer hypericin (hyp), in using different transport systems (hyp alone vs. hyp/ldl complexes) and incubation protocols. the differences between ir spectra of non-treated and hyp treated cells are mainly manifested in the positions of amide i and amide ii vibrational bands of proteins. these vibrational shifts are attributed to the protein structure changes from dominantly alfa-helix, in the non-treated cells, to beta-sheets and random coil structures, which prevail h and h after photodynamic treatment, respectively. the observed conformational changes of proteins can be explained as the consequences of the processes leading to apoptosis as was verified by flow cytometry experiments. the results confirm suggestion that ir spectroscopy can be successfully applied for the detection of early apoptotic processes. a. k. de, d. goswami indian institute of technology kanpur, india molecular fluorescence has been an indispensable tool in modern day optical imaging. one of the state-of the-art challenges in fluorescence microscopy is having better depth resolution as embodied by the confocal and multi-photon laser-scanning microscopic techniques. however, each technique bears its own limitation in having sufficient out-of-focus signal for the former while the low non-linear photon absorption cross-section for the latter. for confocal microscopy using one-photon excitation, we have shown how the clever choice of pulsed illumination instead of continuous-wave excitation leads to a gigantic enhancement in fluorescence that also has immediate applications in microscopy. moreover, single-photon illumination with ultrafast pulses leads to a novel way of achieving axial resolution along with numerous other advantageous applications e.g. reduced photo-bleaching of the chromophore. on the other hand we have thrown new insight demonstrating that the use of mode-locked laser pulses in multi-photon microscopy induces severe solvent-induced photo-thermal damage and prescribed methods to get rid of it. besides, the use of pulse pair excitation in multi-photon microscopy leads to probe and control the dynamics of fluorophores which has crucial role in selective excitation of fluorophores from quantum control perspectives. all these cutting edge research works will be presented in addition to our recent work on application of laser pulse shaping in multi-photon microscopy. interkingdom signalling in pseudomonas aeruginosa b. davis , r. jenson , p. williams , p. o'shea institute of biophysics, imaging and optical science, university of nottingham, u.k., institute of infection, immunity and inflamation, university of nottingham, u.k. quorum sensing is the process through which some bacterial species coordinate cell-cell communication. pseudomonas aeruginosa; the pathogen responsible for over % of chronic lung infections and the leading cause of mortality in cystic fibrosis patients, expresses two major classes of quorum sensing molecules characterized as n-acyl homoserine lactones and -alkyl- -quinolones. these compounds, in addition to their signalling roles have also been found to possess virulent properties, not only against competing species of bacteria such as staphylococcus aureus but also eukaryotic cells such as t-lymphocytes. the process of bacterial quorum signalling molecules influencing eukaryotic cell activity is termed 'interkingdom signalling'. to date it has been suggested that the quorum sensing molecules outlined above act on eukaryotic cells through interactions with an as yet unidentified plasma membrane or cytosolic receptors. this project is directed towards developing an understanding of how these compounds elicit eukaryotic response through a combination of membrane based interactions at physiologically relevant concentrations. particular emphasis is placed on studies of ligand binding with membrane microdomains in and the consequent downstream signalling are also considered. this work is significant as it will not only lead to a better understanding of pseudomonas infection, but may also lead to the discovery of new classes of agents for the treatment of infective diseases. a xas study of the sulphur environment in human neuromelanin and its synthetic analogues neuromelanin is a complex molecule accumulating in the catecholaminergic neurons that undergo a degenerative process in parkinson's disease. it was shown to play an either protective or toxic role depending on whether it is present in the intraneuronal or extraneuronal milieu. in the present study x-ray absorption spectroscopy is employed to investigate the sulphur binding mode in natural human neuromelanin, synthetic neuromelanins and in certain structurally known model compounds, namely cysteine and trichochrome c. based on comparative fits of human and synthetic neuromelanin spectra in terms of those of model compounds, the occurrence of both cysteine-and trichochrome-like sulphur coordination modes is recognized and the relative abundance of these two types of structural arrangement is determined. data on the amount of cysteine-and trichochrome-like sulphur measured in this way indicate that among the synthetic neuromelanins those produced by enzymatic oxidation are the most similar ones to natural neuromelanin. c. cremer kirchhoff-institute for physics, university of heidelberg, germany here we report on "spectral precision distance/position determination microscopy (spdm) with physically modifiable fluorochromes (spdm phymod ) to analyse the spatial distribution of single nuclear proteins and dna sequences at the macromolecular optical resolution level. like other methods of "spectrally assigned localization microscopy" (salm), spdm phymod is based on labelling 'point like' objects (single molecules) with different spectral signatures, spectrally selective registration and high precision localization monitoring by far field fluorescence microscopy. the intranuclear spatial location of single molecules was determined up to a density up to ca. molecules/µm of the same type, and distances down to - nm were nanoscopically resolved. quantum dots (qds) are semiconductor nanoparticles with increasing application as fluorescent markers in biology.we investigated structure of the cell walls of different species complexed with cdse qds using fluorescence microscopy, fluorescence spectroscopy and ftir techniques. in the experiments we used the cell walls isolated from three distinct plant species: arabidopsis thaliana, acer sp. and picea omorika. we studied both unlabeled and cdse-labeled cell walls. fluorescence spectroscopy and microscopy were used for detection of qds alone or complexed to the cell walls. emission spectra were deconvolved using the nelder-mead algorithm in matlab . . we calculated approximate probability distribution (apd) for positions of spectral component maxima. there was certain difference between unlabeled cell walls and those complexed with qds. the ftir spectra also show some difference between the complexed and pure cell walls. the results show that structure was changed, but not significantly in reaction with cdse qds. these results are promising in context of use of qds as labels in cell wall studies. the characterization of the complex of cell wall structure with qds is a part of the study of nanoparticles application in investigations of plant materials. modulating the response of single neurons and neuronal networks with biophysical stimuli f. difato, a. maccione, l. berdondini, f. benfenati, a. blau italian institute of technology, department of neuroscience and brain technologies, genoa, italy during differentiation, cell processes create connections with other cells to form tissue capable of performing complex tasks. biophysical constraints provide necessary inputs for cellular organization in living organism . to better understand how biophysical conditions influence tissue development, it is necessary to bridge the gap between experiments on single cells and complex tissues , . to achieve this goal we pair optical tweezers with electrophysiology measurements . by adopting neuronal networks as a biological model, neuronal signal transmission can be recorded either by patchclamp electrophysiology or microelectrode arrays (meas). dissociated neurons will be cultured on meas to record neuronal network activity at different sites of the network while applying spatio-temporally defined biophysical stimuli to individual neurons. a. diaspro , k. cortese , p. bianchini , c. gagliani , c. tacchetti iit -italian institute of technology, morego, genova, italy, microscobio, university of genoa, italy correlative light/electron microscopy (clem) is becoming increasingly frequent in molecular and cellular biophysics. we successfully applied the method to analyze the d structure of rough and smooth russell bodies used as model systems. the major advantages of this approach are the following: (i) the ability to correlate several hundreds of events at the same time, (ii) the possibilitˆto perform d correlation, (iii) the potential to immunolabel both endogenous and recombinantly expressed proteins at the same time and (iv) the effective combination of the high data analysis capability of flm with the high precisionaccuracy of transmission electron microscopy in a clem hybrid morphometry analysis. we have identified and optimized critical steps in sample preparation, defined routines for sample analysis and retracing of regions of interest, developed software for semi/fully automatic d reconstruction and defined preliminary conditions for an hybrid light/electron microscopy morphometry approach. the relevance of the presented approach lies in two important key elements, namely: the development of optical nanoscopy methods and the potentiality for exploring different correlative frameworks like optical nanoscopy vs. optical microscopy adding scanning force microscopy techniques. multidrug resistance is a well known phenomenon which limits effectiveness in treating malignancy with chemotherapy by modifying the internalization and/or externalization flow of the drugs through the cancerous cells. combined chemotherapies, such as mvac, are therefore currently used in bladder cancer treatment. however, about % of patients do not respond this chemotherapy because of inherent or acquired drug resistance. we developed a non invasive predicative test on urinary cells to estimate the chemotherapy effectiveness before treatment, based on the fluorescence emission of mvac. we first studied the mvac photophysical properties in solution and using five cell lines: a drug sensitive cancer cell line mgh-u s, its multidrug resistant subline mgh-u r, a not tumorigenic cell line sv-huc- , its tumorigenic counterpart mc-sv-huc t- and a cell line from transitional cell carcinoma t . the results revealed a penetration and localization of the drug depending of the cell line type, allowing us to find a specific fluorescence signature for the identification of mvac resistant cells. similar data have been obtained for cytospined fixed culture cells and patients urinary cells. -imaging and spectroscopy -abstracts multilayered photoresist system as a ghost model for biological samples in confocal microscopy. l. ferrari, f. cilloco, f. r. bertani, s. selci istituto dei sistemi complessi cnr rome we have realized a bilayer photoresist system as a model to perform optical characterization in the vis-ir range of multilayered complex biological specimen. our goal is to obtain structural and spectroscopic reference parameters from the identification of reflectance pattern features within a novel project granted by miur (skintarget, ideas firb). our model is composed by a nm thick layer (shipley photoresist ) with a refractive index n= . - . , wavelength dependent, and a nm tick hsq (hydrogen silsesquioxane) layer with a refractive index n around . , both spin-coated over a glass substrate. we present the analysis of reflectance pattern that can be obtained by a confocal laser reflective system as compared to the expected values as coming from analytical calculations, using a matrix approach, or a microscopic electromagnetic finite element analysis. interfacial roughness and consequent optical scattering are analyzed using a neural network approach. translational biophysics: multimode imaging for preclinical and clinical applications d. l. farkas cedars-sinai medical center, los angeles, usa our focus is where light and patient meet, and improvements yielding better outcomes. surgery is moving towards minimally invasive intervention, where biophotonics represents a major area of hope and growth. the translation of useful laboratory-derived knowledge into clinical practice has been hampered by the difficulty of detecting, characterizing and monitoring molecules and cells in the human body, especially dynamically. advanced bi ophotonic imaging is best suited for studying such entities, but has been lagging in clinical acceptance, in spite of major advances, and a clear need for the kind of resolution (spatial, temporal, spectral) and specificity that it alone can offer. biophysics-based new strategies are needed to address this challenge. the development of biophysical methods for translational medicine will be reviewed, with emphasis on our recent advances. our approach is a multimode one -combining methods to achieve early, quantitative detection of abnormalities. with imaging fulfilling its dual role of better describing anatomy and physiology, intrasurgical histopathologyequivalent molecular and cellular imaging is achievable in vivo, as is a closer spatio-temporal connection between imaging and intervention. some application areas to be covered: cancer (early detection by spectral reflectance/autofluorescence; progression quantitation by oct; nano-and targeted chemotherapy assessment in vivo); neurobiology (imaging fast calcium transients and alzheimer's plaques); hyperspectral mie scattering imaging for in situ displasia; design and use of an advanced multimode imaging endoscope with in vivo delineation of hirschsprung's disease for better intervention; monitoring of stem cell fate in vivo. immobilization of liposomes in a sol-gel matrix: a fluorescence confocal microscopy study r. esquembre , s. n. pinto , j. a. poveda , m. prieto , c. r. mateo ibmc, universidad miguel hernández, elche, spain, cqfm, instituto superior técnico, lisbon, portugal immobilization of liposomes shows interesting applications in protein biology, membrane biophysics and biosensor technology. previous fluorescence spectroscopy works revealed that the entrappement of zwitterionic phospholipid liposomes in a silica sol-gel matrix alters the thermodynamic properties as well as the fluidity of the lipid bilayer. interactions between the polar head of phospholipids and the porous surface of the host matrix could be responsible of such behaviour. in order to get more insight into this possibility we have immobilized, for the first time, giant unilamelar vesicles (guvs) and the shape and size of these structures as well as the possible existence of lipid domains have been visualized through fluorescence confocal microscopy. this technique allows for direct observation of the effect of encapsulation on an individual liposome, in contrast to the averaged information given by macroscopic spectroscopic techniques. liposomes composed of pure popc or dopc as well as mixtures dopc/dppc were labelled with the fluorescent probes bodipy, rhd-pe and rhd-dope. preliminary results shows that only the smallest guvs ( - µm) survive to the encapsulation process but often with a slight loss of its sphericity, probably due to pressures suffered during the matrix gelation. however no change in the gel/fluid phase proportion has been observed for immobilized dopc/dppc guvs, regarding to solution. solvent fluctuations play a key role in controlling protein motions and functions. here, we have studied how the reaction catalyzed by the light-activated enzyme protochlorophyllide oxidoreductase (por) couple with solvent dynamics (g. durin et al, biophysical j. ( ) , . to simultaneously monitor the catalytic cycle of the enzyme and the solvent dynamics, we designed temperature-dependent uv-visible microspectrophotometry experiments, using flash-cooled nano-droplets of por. the temperature-dependant formation of the first two intermediates in the por reaction were measured, together with the solvent glass transition temperature (t g ) and the build-up of crystalline ice. we find that formation of the first intermediate occurs below t g and is not affected by solvent dynamics, whereas formation of the second intermediate occurs above t g and is influenced by solvent dynamics. these results suggest that internal protein motions drive the first step of the por reaction whereas solvent slaved motions control the second step. we propose that the concept of solvent slaving applies to complex enzymes such as por. amphotericin b (amb) is one of the main polyene antibiotics widely used to treat deep-seated fungal infections. the mechanism of biological action of amb is most probably directly related to the ability of the drug to form hydrophilic pores in the membrane core, thus affecting physiological transport of ions. the effects of amb-cu + complexation are demonstrated by the electronic absorption and fluorescence spectra. the absorption spectra of amb in water (ph= ) after the injection of water solution of copper(ii)sulfate display a complex structure with hypsochromic-and bathochromic-shifted bands indicative of formation of molecular aggregates of the drug. formation of the electronically coupled chromophores of amb, especially aggregates, was analyzed at different cu + concentrations by the rls (resonance light scattering) technique. intensity of the fluorescence emission spectrum (characteristic of the dimeric form of amb) decreases after the amb-cu + complex formation. this effect of the formation of the amb aggregated structures by amb-cu + are different from the spontaneous molecular aggregation process, as deduced from the spectroscopic analysis. morfo-functional asymmetry of the olfactory receptors of the honeybee apis mellifera l e. frasnelli , g. anfora , f. trona , f. tessarolo , r. antolini , g. vallortigara cimec, centre for mind/brain sciences, university of trento, italy, iasma research and innovation center, fondazione e. mach, s. michele all´adige (tn), italy, biophysics and biosignals lab., dept. of physics, university of trento, italy lateralization, i.e. the different functional specialisation of the left and right side of the brain, has been documented in many vertebrate and, recently, invertebrates species. in the honeybee apis mellifera l. olfactory memory seems to involve at first the use of the right antenna. the present study investigated physiological and anatomical differences between left and right antennae of honeybees. electroantennographic responses (eag) were recorded from the left and right antennae of honeybees to linalool, a floral volatile compound, and isoamyl acetate, an alarm pheromone, at doses (from , to µg). the number of sensilla on the left and right antennae was recorded by scanning electron microscopy (sem). each antenna segment, from insects, was observed from different viewpoints in order to image the whole antenna surface and compute the number of sensilla. the tested compounds induced higher eag responses on the right than on the left antenna at every dose. sem showed that the placoidea olfactory sensilla were slightly more abundant on the right antenna surface than on the left one. results suggest an asymmetry in the peripheral odour perception mechanism in the honeybee a. mellifera. super-resolution imaging of dna through single molecule switching of intercalating cyanine dyes c. flors, c. n. j. ravarani, d. t. f. dryden school of chemistry, university of edinburgh, u.k. a growing trend in far-field super resolution fluorescence microscopy involves the replacement of photoactivatable fluorophores by common dyes such as cy, atto or alexa [ ] . it has been shown that these dyes can blink in useful timescales for single-molecule based imaging by adding suitable buffers. this strategy greatly simplifies the sample preparation and imaging scheme, enabling its application to a wider range of biological systems. we have explored if a similar approach might be useful to study dna topology using intercalating cyanine dyes such as yoyo- . there are two main advantages of this approach: i) dna labelling with intercalating dyes is straightforward, and ii) the free dye in solution is essentially non-fluorescent, greatly reducing the fluorescence background. we show that yoyo- can blink in the absence of oxygen and in the presence of cysteamine, which allows its application to nanoscale imaging. we exemplify its use by imaging λ-dna and a puc plasmid. we also explore the compatibility of several intercalating dyes with biological systems such as enzymes or cells. our results suggest that dna intercalating dyes are a promising option for fluorescence super-resolution studies of dna topology. seeing more in total internal reflection fluorescence (tirf) microscopy r. fiolka, a. stemmer nanotechnology group, eth zürich, zürich, switzerland total internal reflection fluorescence (tirf) microscopy is an effective widefield imaging tool that selectively excites a very thin sample layer within the evanescent excitation field at the glass-water interface. lateral resolution of standard tirf microscopy is limited to approx. nm using green emission, which can be insufficient for a large class of biological investigations. additionally, the evanescent excitation field is prone to light scattering, creating out of focus blur in the final image. we present several techniques that address these mentioned shortcomings of standard tirf microscopy. using evanescent standing waves, the lateral resolution in tirf microscopy can be extended by a factor of . , reaching nm. we further show techniques to reduce the blur induced by light scattering of the evanescent field. finally, we demonstrate optical sub-sectioning capabilities in tirf microscopy by acquiring several images with different penetration depth of the evanescent field and applying suitable post processing algorithms. thereby the obtainable z-resolution exceeds the classical limit of widefield microscopy, and object structures lying within the evanescent field can be reconstructed. the natural photosentizer hypericine (hyp) exhibits potent properties for tumor diagnosis and photodynamic therapy. evidences of hyp release from ldls prior to passive diffusion within cells are addressed in this study. fluorescent properties of hyp have been used for dynamic studies of its interaction with low-density lipoproteins (ldls) and u glioma cells. subsequent non-specific staining of intracellular membranes compartment were observed by mean of colocalization fluorescent imaging studies. it was shown, that monomers of hyp are only redistributive forms. increasing of hyp concentration leads to the formation of non-fluorescent aggregates within ldls as well as within the u cells, and can preclude its photosensitizing activities. in all experiments, hydrophobic character of the molecules appears as the driving force of its redistribution process. acknowledgment: this work was supported by the slovak res. and dev. agency contracts no. lpp- - . we also kindly thank the synchrotron soleil for using the detection system of the disco beamline. ledgf/p switches from a dynamic to a tight chromatin interaction upon binding to hiv- integrase j. hendrix , z. debyser , y. engelborghs laboratory of biomolecular dynamics, katholieke universiteit leuven, belgium, laboratory of molecular virology and gene therapy, katholieke universiteit leuven, belgium human transcriptional co-activator ledgf/p is hijacked by hiv- integrase (in) during the replication of hiv. little is still known about the molecular complex of these two proteins in the living cell. in this work we first studied the cellular chromatin interaction of egfp-tagged ledgf/p with tunable-focus fluorescence correlation spectroscopy (tf-fcs) and show that ledgf/p is in equilibrium between a free brownian motion and a very slow movement on the chromatin. being dependent on the size of the laser focus, this slow movement represents a continuous associationdissociation-reassociation process that is governed by diffusion. concentration-dependent continuous photobleaching measurements (cp) furthermore revealed the existence of high-affinity chromatin binding sites. next, we co-expressed mrfp-tagged in and confirmed its intracellular interaction with ledgf/p by fluorescence cross-correlation spectroscopy (fccs). interestingly, cp and fluorescence recovery after photobleaching (frap) indicated that the affinity of this complex for chromatin is exceptionally high. by twophoton fluorescence lifetime imaging ( p-flim) we verified if the cellular stoichiometry was altered when the proteins were expressed together. we believe that this work is useful for the understanding and targeting of hiv-replication. biophysical identification of orf from clavulanic acid biosynthesis cluster as a cyp l. s. goto , c. o. hokka , j. f. lima , o. r. nascimento , a. p. u. araújo grupo de engenharia bioquímica, ufscar, br, grupo de biofísica molecular "sérgio mascarenhas", usp, br streptomyces clavuligerus produces the clinically important β-lactamase inhibitor clavulanic acid. biosynthesis related genes reside in three gene clusters, one of these, named clavulanic acid gene cluster, includes most of the known clavulanic acid biosynthetic enzymes. the penultimate step along clavulanic acid biosynthesis remains unclear. required transformation involves at least two events: oxidative deamination and double epimerization of ( s , s )-clavaminic acid into ( r, r)-clavaldehyde. downstream the known part of clavulanic acid cluster lays orf , a putative gene encoding a tentative cytochrome p -like protein which knockout has been proven deleterious to clavulanic acid biosynthesis. should such protein exist, it would be candidate to fulfill the clavulanic acid pathway missing step. in this work, orf encoded protein is characterized aiming to place it as a real p . for this task, molecular cloning and recombinant expression of orf were accomplished. purified protein was submitted to spectroscopic measurements such as circular dichroism and electron paramagnetic resonance which indicate p features, including catalytic relevant heme iron redox states and homolytic peroxide scission mechanisms. further, peroxide reaction adducts were characterized by spin trapping. this work is supported by fapesp. muscle structure and gabaergic innervations in the limbs of barnacle cyprid l. gallus , s. ferrando , c. gambardella , a. diaspro , p. bianchini , p. ramoino , v. piazza , g. tagliafierro libiom, dibio, università di genova, italy, ifom-lambs/microscobio, difi, università di genova, italy, dipteris, università di genova, italy, ismar cnr, venezia, veneto, italy balanus amphitrite is a sessile crustacean that settles at the larval stage of cyprid. in this stage we studied the gabaergic innervation of limb striated muscular fibers, by immunohistochemistry. the second harmonic generation (shg) and -photon excitation ( pe) microscopy were used to set out the muscle structure and its relationship with nerve terminal. sections were observed at a multimodal nonlinear microscope composed by the leica clsm. the laser system used is a ti:sapphire chameleon-ultra (coherent inc, santa clara, ca, usa), tunable between nm and nm and characterized by a pulse width of fs delivered at a repetition rate of mhz by means of a homebuilt set-up (bianchini p. and diaspro a. j biophotonics : - ). the z-stacks were performed in order to obtain the -dimensional distribution of the muscular fibers. in the posterior ganglion gad immunoreactive (ir) motor neurons were arranged in clusters near the emergence of the limb nerves. gaba and gababr ir neuromuscular junctions (nj) were localized in the limb muscle fibers; vgat ir cells surrounded each limb muscles. these results suggest that gaba plays a key role in the regulation of limbs movement. the shg was very useful to outline the relationship between nerve terminals and limbs muscle fibers. giant unilamellar vesicles (guvs) are very useful model membrane systems to study many aspects of lipid-lipid and lipid protein interactions, particularly employing fluorescence microscopy related techniques (bagatolli ) . the use of this model system can be particularly useful to study aspects related with the lateral structure of bacterial membranes using the aforementioned approach (i.e. fluorescence microscopy). bacterial cells have a size close to the resolution limit of optical microscopy and details about the organization of their membranes are not easy to achieve using such technique. recently a new electroformation method to prepare guvs composed of compositionally complex mixtures under physiological conditions was developed in our laboratory (montes ). in the present work we further extended this electroformation method to prepare guvs composed of bacterial lipid extracts and lipopolysaccharide (lps). in our experiments we used e.coli lipid extract to prepare small vesicles containing various lps species, from smooth strains and rough mutants. suvs were used as starting point to electroform guvs using various types of buffers with high ionic strength. the successfully obtained bacterial-guvs were used to study the interaction of these membranes with known lps-binging proteins (lung surfactant protein d, sp-d) and peptides (polymyxin b). our results remark the usefulness of this particular bilayer models to perform studies mimicking bacterial membranes. mechanical properties of polymeric membranes probed by afm m. kocun , i. mey , w. müller , m. maskos , a. janshoff georg-august universität, institute for physical chemistry, göttingen, germany, johannes gutenberg universität, institute for physical chemistry, mainz, germany many biological cell functions are dependent on the mechanical properties of the membrane. polymeric membranes that mimic native cell membranes are valuable research tools which can be used to better understand the physics of biological membranes. we have investigated free standing artificial membranes prepared from polybutadiene-b-polyethylene oxide (pb-b-peo). the membranes were prepared from vesicles ruptured on porous silicon substrates. these polymeric membranes were studied by confocal laser scanning microscopy (clsm) and atomic force microscopy (afm). force indentation curves were performed on the membranes and theoretical models were used to extract elasticity constants from the results. the study of polymeric membranes can give insight to the function of biological membranes, furthermore polymeric membranes can be used to create new hybrid systems by incorporating biological (lipids, proteins), artificial (polymers, dyes) and inorganic (nanoparticles) components. site-directed spin labeling study of the lightharvesting complex cp the topology of the long n-terminal domain of the photosynthetic light-harvesting complex cp was studied using electron spin resonance (esr). cp is a minor antenna complex of the photosystem ii (psii), a multisubunit protein complex. wild-type cp protein containing a single cysteine at position and nine single cysteine mutants were produced, allowing to label different parts of the domain with a nitroxide spin label. in all cases the apoproteins were either solubilized in detergent or they were reconstituted with their native pigments (holoproteins) in vitro. the spin label esr spectra were analyzed in terms of a multi-component spectral simulation approach, based on hybrid evolutionary optimization and solution condensation. these results permit to trace the structural organization of the long n-terminal domain of cp . we took the crystal structure of light-harvesting complex ii (lhcii), major antenna complex of psii available in pdb as a starting point and constructed a model for cp based on esr data. present opportunities and future developments in soft x-ray transmission and emission microscopy b. kaulich, a. gianoncelli, v. babin, m. kiskinova elettra -sincrotrone trieste, s.s. km . in area science park, i- trieste-basovizza, italy soft x-ray transmission and emission imaging and spectromicroscopy are bridging the gap to other microscopy techniques in terms of lateral resolution, penetration depth and chemical sensitivity. the novel soft x-ray spectromicroscopy approach of the twinmic instrument at the elettra synchrotron radiation facility is combining several imaging modes for morphology characterization, such as scanning, projection and full-field imaging with several contrast techniques including brightfield, darkfield, differential phase and interference contrast at sub- nm lateral resolution. complementary chemical information is provided by chemical imaging and micro-spectroscopy using the photon-in/photon-out xray absorption and x-ray fluorescence. unique for twinmic is the low-energy x-ray fluorescence setup operated at - ev photon energies, which allows simultaneous analysis of the morphology, and the distribution of light elements on cellular and sub-cellular level. in the presentation the principles of the methods used in the twinmic instruments the performance and potentials of the instrument will be demonstrated by several examples of applications in the field of human, animal and plant biology, biophysics and chemistry, physiology and genetics. the potential impact of microscopy techniques using free-electron x-ray lasers on biophysics will be illustrated by the diproi project at fermi@elettra. -imaging and spectroscopy - fluorescence anisotropy and afm used as tools to characterized porin´s reconstituion in luv´s s. c. lopes, i. sousa, p. eaton, p. gameiro requimte, faculdade de ciências, universidade do porto, rua do campo alegre, - porto, portugal a major requirement to perform structural studies with membrane proteins is to define efficient reconstitution protocols that assure, not only, a high incorporation degree in preformed liposomes, but also a protein directionality and topology that mimics its in vivo conditions. for this kind of studies, protein reconstitution in membranes systems via a detergent-mediated pathway is usually successfully adopted, since detergents are generally used in the initial isolation and purification of membrane proteins. in this study we report the reconstitution of ompf in preformed dmpc and e. coli liposomes using two different techniques for detergent removal: ( ) exclusion chromatography and ( ) incubation with detergent adsorbing beads. the incorporation degree was determined by bicinchoninic acid assay and fluorescence anisotropy was used to determine ompf effect on the structural order of membrane lipids. these results show that protein insertion in membranes depends both on the technique used to remove detergent and on the lipids used to prepare the liposomes. moreover more anisotropy and atomic force microscopy studies will allow a better characterization of bacterial model system membranes. the wavelength dependence of the luminescence for different sized aunp by -photon clsm k. li, m. schneider pharmaceutical nanotechnology, saarland university, campus a , d- saarbrücken, germany gold nanoparticles (aunp) with different sizes can exhibit original luminescent properties if excited with pulsed nearinfrared laser light which makes them a suitable object to be detected in biological tissues. the main obstacle is to distinguish between the object of interest (emitted light) and the autofluorescence from the sample which limits the scope of application of aunp. therefore, our aim is to characterize the luminescent properties of such nanoparticles regarding their excitation and emission. in this study, the excitation and emission spectra of aunp with different sizes below nm in an excitation range of nm to nm were investigated. our study shows the emission spectra curves of aunp are broadband spectrum and vary with the changing of excitation wavelength from nm to nm. the results also suggest the minimum laser power necessary to trap the aunp depends on particle size and excitation light wavelength and a maximum power above which the particles are destroyed. in all the experiment above, to avoid the simultaneous effects from slide and cover slip is a must. rocking, tumbling, and sliding: real-time nanomotion of a membrane-bound virus p. kukura , h. ewers , c. mueller , a. renn , a. helenius , v. sandoghdar laboratory of physical chemistry, eth zurich, ch- zurich, switzerland, institute of biochemistry, eth zurich, ch- zurich, switzerland the interaction of a virus with its receptors in the plasma membrane is decisive for its infection of cells. optical studies have revealed that after binding, virus particles move laterally on the membrane, but the complexity of the cellular environment and the drawbacks of fluorescence microscopy have prevented access to the molecular dynamics of early virus-host couplings. here, we examine a model system, in which single simian viruses (sv ) interact with their gm ganglioside receptors in supported lipid bilayers. we employed scattering interferometry and single molecule fluorescence localization to visualize the vectorial rotational motion of virions. at low receptor concentration, we observed sliding and tumbling of single virions during rapid lateral diffusion. in contrast, at increased receptor concentration the virions repeatedly underwent periods of standstill, reminiscent of their behavior prior to endocytosis. by an unprecedented combination of millisecond time and nanometer spatial resolutions, we revealed that during these immobile periods, the virions rock back and forth among nanoscopic spots separated by nm. our insights, together with the structure of the viral capsid, suggest aggregation of receptors in nanodomains and recurrent swap of binding between receptor molecules and neighboring viral protein pentamers. herein, we have developed different membrane probes binding spontaneously to the outer plasma membrane leaflet and showing sensitivity to membrane properties such as surface charge and phase state. the first two types of probes were based on -hydroxyflavone fluorophore. one of them showed a high sensitivity to the surface charge and to the phase state of lipid bilayers, while the other was only sensitive to the surface charge. the third type, which was based on nile red fluorophore, was sensitive only to the phase state. surprisingly, the probes that were sensitive only to the surface charge did not respond to apoptosis, while the other two types of probes showed significant spectroscopic response to it. moreover, the latter exhibited response to cholesterol depletion, which was similar to that observed on apoptosis. thus, according to our data, the intact living cells present a remarkable fraction of the cholesterol-rich domains, while apoptotic loss of the transmembrane asymmetry decreases it dramatically. these probes represent a new tool for quantification of surface charge and cholesterol-rich domains in cell membranes. -imaging and spectroscopy - due to its porosity and unique optical properties porous silicon (psi) is an attractive template to develop biomaterials and biosensors. porous silicon microcavity (psimc) structures were prepared then functionalized for covalent protein attachment of glucose oxidase (gox) or solubilized bacteriorhodopsin (br). functionalization and protein infiltration was monitored by specular reflectometry sensitive to change in refractive index, when a molecule is attached to the large internal surface of psi. protein infiltration into the porous scaffold was confirmed by edx spectroscopy and the structures were imaged by biphoton microscopy. second harmonic generation and enhanced two-photon excited fluorescence emission from porous silicon was observed when resonantly exciting the structures. in addition, when the microcavities were infiltrated with gox or br, the proteins acted as a very efficient internal two-photon-excited fluorescence emitter, hence protein infiltration enabled the in-depth visualization of the porous structure by taking advantage of the optical sectioning capacity inherent to the non linear optical microscopy technique. patterning of bio-molecules: methods for characterization of neuron-substrate interfaces we investigated how to use and improve micro printing techniques to obtain molecules patterns on cell culture substrates. with micro contact printing (µcp), we generated geometrically defined depositions of poly-d-lysine (pdl) using poly-dimethylsiloxane stamps and optimized neuronal culturing conditions. rat and mice hippocampal neurons grown on those patterns showed to be alive and functional. we then applied µcp to study axonal development by combining cell culture assays with atomic force microscopy (afm) to investigate in more detail the molecule deposition on the surface and to measure morphological changes in the growth cone (gc) during the early phases of neurite development. we found distinct shapes of the gcs depending on whether they were growing on l adhesion molecule patterned by indirect-µcp or on pdl coated surfaces. we also attempted to transfer such patterns on multi electrode arrays in order to constrain neuronal cell bodies on the electrode area and to improve electrophysiological recordings from neuronal networks. other patterning techniques were therefore explored using a nano-drop printing system. patterned surfaces were analyzed with afm and scanning electron microscopy to combine different approaches aimed to the improvement and characterization of the printing techniques. insights on the mechanism of electron transfer in complex i a. l. maniero , c. bergamini , m. bortolus , r. fato , s. leoni , g. lenaz università degli studi di padova, padova, italy, università degli studi di bologna, bologna, italy complex i (nadh dehydrogenase) plays a central role in cellular energy production, transferring two electrons from nadh through a series of iron-sulfur clusters (fes) to ubiquinone (coenzyme q); the electron transfer is coupled to the translocation of protons across the membrane. the fes center n is the last acceptor in the electron-transfer chain, but the mechanism through which the enzyme couples the e − reduction of the fes centers to the e − reduction of ubiquinone (q→sq→qh ) is unclear [ ] . in our experiments, submitochondrial particles were treated with different inhibitors, in the absence or in the presence of different quinone analogues, and nadh addition initiated the electron transfer. we assess by epr (electron paramagnetic resonance) spectroscopy the relative abundance of the reduced n center and of the semiquinone radical, and coupled epr data to enzymatic activity assays and to fluorescence measurements on the effect of inhibitors on reactive oxygen species (ros) production. we identify two different classes of inhibitors showing different effects on ros production. moreover the redox state of the complex has shown to depend both on the inhibitors and on the quinone analogues. a possible mechanism of the electron transfer, that can explain the experimental findings, will be presented. conventional fluorescence microscopy suffers from a resolution limit imposed by the diffraction of light. stimulated emission depletion (sted) microscopy overcomes this resolution barrier without being limited by the wavelength by taking advantage of the photophysics of the observed sample into the image formation process, and has proven to be a powerful approach for exploring relevant biological issues. the outstanding resolution of sted microscopy is achieved by drastically minimizing the spatial extent of the focal region from which fluorescent molecules can emit signal in the sample. so far, mainly complex and relatively expensive lasers systems providing pulsed beams have been used to inhibit the fluorescence in the outer area of the focal region. here we report on the development of a new setup based on a fast beam scanning confocal microscope using compact turn-key and inexpensive continuous wave lasers. the great potential of this simple configuration is demonstrated for a selection of commonly used fluorescent markers. characterization of hepatitis b antigen particles by atomic force microscopy each year, over one million people die from hepatitis b virusrelated chronic liver disease, including cirrhosis and hepatocellular carcinoma. the major surface antigen of hepatitis b virus (hbsag) is a cysteine-rich, lipid-bound protein with amino acids. recombinant hbsag produced in yeast can self-assemble into -nm immunogenic spherical particles that are used in licensed hepatitis b vaccines (protein/lipid ratio is / in mass). hbsag size and shape have been mainly investigated by transmission electron microscopy after negative staining of the particles. however, under these conditions, no details of the particle surface can be obtained because of the shadowing effect due to the uranium salts. here we describe new structural insights of hbsag particles using atomic force microscopy (afm) performed under physiological conditions. we applied atomic force microscopy to define structural details of the surface organization with a resolution in the nanometer range. as expected, the diameter of hbsag particles is , ± , nm in average. the surface of these particles clearly shows the presence of protuberances that most probably correspond to proteins. indeed, reduction and alkylation induces the disappearance of the protuberances. the number of the protuberances estimated from afm micrographs is about per spherical particles. a biophysical study of equine herpesvirus- entry into cells g. mckenzie , p. o'shea , j. kydd , c. rauch school of veterinary medicine and science, university of nottingham, uk, institute of biophysics, imaging and optical science, university of nottingham, uk equine herpesvirus- (ehv- ) can cause respiratory disease in young horses, varying degrees of paralysis and abortion during the later stages of pregnancy. furthermore, there has been a recent increase in the number of outbreaks involving paralysis of thoroughbred horses in training. virus disseminates rapidly after initial infection via cell associated viraemia. controlling this may prove crucial in combating the pathogenicity of the disease. we have investigated the cellular interactions of ehv- . initially, binding of ehv- to fluorescein phosphatidylethanolamine (fpe)-labelled phospholipid vesicles of various compositions was examined. a variety of microscopy techniques were then employed to study the events surrounding the binding of virus to equine peripheral blood mononuclear cell (pbmc) membranes. confocal microscopy images have highlighted possible colocalisation of ehv- with membrane 'rafts'. total internal reflection fluorescent microscopy was then used to identify viral binding at the membrane with high contrast images, able to observe single virus particles. establishing the viral entry pathway into pbmcs would allow drugs that target this process to be employed, reducing clinical viraemia. the interpretation of in-vivo binding rate measurements allows inferring the molecular interactions that regulate cellular functions. fluorescence recovery after photobleaching (frap) is a widely used tool to quantify binding reactions in vivo. however the lack of "golden standards" for these measurements requires the measured binding rates to be validated with other techniques. we present a new fluorescence correlation spectroscopy (fcs) method to measure the in vivo bound fractions and residence times for molecules that interact with an immobile substrate. we apply this method to measure binding of mutants of the transcription factor vbp (vitellogenin binding protein) to the dna. comparison of fcs with frap results in comparable estimates of the measured diffusion constants and bound fractions. however, fcs provides an estimate of the vbp residence time at the dna, while frap does not. this limitation in the analysis of vbp is due to the larger photobleaching volume used in frap, if compared to the observation volume of an fcs experiment. in sum, we present a method to measure binding rates with fcs. substantial agreement of this method with frap is shown. however, further validation on tightly bound molecules will be necessary to assess if frap and fcs agree in the measurement of residence times. -imaging and spectroscopy - we have investigated the changes in the mechanical properties of the zona pellucida (zp), a multilayer glycoprotein coat that surrounds mammalian eggs, that occur after the maturation and fertilization process of the bovine oocyte by using atomic force spectroscopy. the response of the zp to mechanical stress has been recovered according to a modified hertz model. zp of immature oocyte's shows a pure elastic behaviour. mature and fertilized oocyte's zps evidence, instead, a transition from a purely elastic behaviour, which occurs when low stress forces are applied, towards a plastic behaviour has been observed. the high critical force necessary to induce deformations, that well supports the non-covalent long interactions lifetimes of polymers, increase after the cortical reaction. afm images show that oocytes' zp surface appear to be composed mainly of a dense, random meshwork of nonuniformly arranged fibril bundles more wrinkled surface characterize marure oocytes with respecto to immature and fertilized oocytes from a mechanical point of view, the transition of the mature zp membrane toward fertilized zp, through the hardening process, consists in the recovery of the elasticity of the immature zp, while maintaining a plastic transition that, however, occurs with a much higher force with respect to that required in mature zp. dynamical behavior of ribose and deoxyribose supercooled water solutions s. e. pagnotta , s. cerveny , a. alegria , j. colmenero centro de fisica de materiales, centro mixto csic-upv/ehu, san sebastián, spain, departamento de fisica de materiales, universidad del pais vasco (upv/ehu), facultad de quimica, san sebastián, spain, donostia international physics center, san sebastián, spain ribose and '-deoxyribose are probably the most widespread monosaccharides in nature. they can be extensively found in ribonucleic acid (rna) and '-deoxyribonucleic acid (dna), respectively, where they form, together with a nitrogenous base and a phosphate group, a peculiar building-block structure called nucleotide. in the present work, the relaxation dynamic of ribose and deoxy-ribose water solutions at different concentrations has been studied by broadband dielectric spectroscopy and differential scanning calorimetry in the temperature range of - k. two relaxation processes are observed for all the hydration levels; the slower (process i) is related to the relaxation of the whole solution whereas the faster one (process ii) is associated with the reorientation of water molecules in the mixture. as for other polymeric water solutions, dielectric data for process ii indicate the existence of a critical water concentration above which water mobility is less restricted. moreover, according with these results, atr-ftir measurements of the same sugar solutions showed an increment in the intensity of the oh stratching sub-band close to cm − as water content increases. the regulation of the formation of cytoskeletal protein complexes by actin-binding proteins m. nyitrai, a. vig, t. kupi, z. ujfalusi, s. barkó, g. hild university of pécs, faculty of medicine, department of biophysics, pécs, hungary in living cells various groups of proteins are associated to supramolecular actin filament structures, often in a nucleation factor dependent manner. for example, actin structures associated with formins can bind tropomyosin and profilin, while those polymerised by the nucleation of the arp / complex bind cofilin and myosin i. the molecular mechanisms underlying the regulation of the formation of these protein complexes is still ambiguous. we have shown recently that formins can bind the actin filaments and change their conformational state. subsequent binding of other actin-binding proteins, such as tropomyosin and myosin, can reverse these changes. it appears that the reversal effect assumes that the actin-binding protein binds the filaments in a well-defined and specific binding site. the altered conformational state of the actin filaments observed after the binding of these proteins provides a possible explanation for the modified affinity of the filaments for other-actin binding proteins. based on the results available so far we assume that the affinities are modified differently by different nucleation factors, and the conformational changes introduced to actin by actin nucleation factors can serve as the molecular bases for the regulation of the formation of actin based intracellular protein complexes. experiments are currently in progress to test and further corroborate the existence of such regulatory mechanisms in living cells. correlation between sub-cellular distribution of photoactive drug hypericin (hyp), determined by applied delivery systems (hyp vs hyp/ldl), and mode of the cell death is addressed in this study. co-localization of hyp with mitochondria, lysosomes and golgi apparatus in u- mg glioma cells was determined by confocal laser scanning microscopy in using organelle specific fluorescent dyes as well as by time resolved fret experiments. flow cytometry experiments were realized to study a photodynamic effect of hyp ( nm/ jcm − ) on cells. significant differences in the proportional representation of live, apoptotic and/or necrotic cells were observed for different types of delivery systems of hyp hours after hyp ( x − m) photoactivation. conclusions: i) sub-cellular distribution of hyp depends on using delivery systems, ii) the mode of cell death depends more on concentration of hyp inside cells, than on different type of delivery systems (for non selective wide-field cell illumination), iii) fluorescence lifetime is sensitive parameter to study sub-cellular distribution of hyp. novel time-resolved spectroscopic methods have been used to investigate the interactions between a fluorescently-labelled mutant of the peptide melittin and supported lipid bilayers, formed by self-assembly at a silica-water interface via vesicle deposition. time-resolved evanescent wave-induced fluorescence spectroscopy (trewifs) is a surface-selective technique in which the evanescent field from a pulsed laser source is used to photoexcite fluorescent species at an interface. the resulting fluorescence decay kinetics, measured using time-correlated single-photon counting, report on the micro-domains experienced by those fluorescent species at an interface. extending trewifs to time-resolved evanescent wave-induced fluorescence anisotropy measurements (ew-trams) provides dynamic rotational information of a fluorescent species, reporting on its mobility at an interface. presented here are trewifs and ew-trams data obtained from the fluorescence of an alexa -labelled melittin mutant interacting with a dipalmitoylphosphocholine bilayer at room temperature, physiological ph and ionic strength. the results provide new insights into the conformation, location and motion of cytolytic peptides interacting with cell membranes. optical tweezers are used to controllably apply forces to red blood cells and the resulting chemical and structural changes are monitored using raman spectroscopy. the forces are applied in vivo and mimic that which the cell undergoes mechanically as it passes through vessels and smaller capillaries. the first result presented will be spectroscopic evidence of a transition between the oxygenation and deoxygenation states of hemoglobin that is caused by the stretching of the red blood cell. the transition is due to mechanically induced enhancements of hemoglobin-membrane and hemoglobin neighbor-neighbor interactions. the latter, lesser known effect is further studied by modeling the electrostatic binding of two of the protein structures using molecular dynamics methods. secondly, polarized raman spectroscopy is utilized to study the packing and ordering of the hemoglobin proteins in the red blood cell as it is stretched. depolarization ratios for a number of heme group modes change, indicating that the applied force additionally packs and orders the proteins inside the cell which further demonstrates the role of cell deformation in the oxygen transport kinetics. acknowledgements: this work was supported by miin fis - (spain) and fundació cellex barcelona fret microscopy is widely used to study protein-protein interactions in living or fixed specimens. currently, most commonly used visible fluorescent proteins for live-cell fret studies are the cerulean and venus variants of the cyan and yellow fluorescent proteins. even though this fret pair appears to be ideal for monitoring protein-protein interactions, the most commonly used fixed laser wavelengths do not excite cerulean at peak absorption. recently, we characterized an ideal donor, the monomeric teal fluorescent protein (mtfp), which is excitable using the commonly available ( ) opt. june/july, ). we used teal as a donor for various red fluorescent proteins as acceptors including tdtomato, mko , morange , mtagrfp, mkate. we have employed a "fret standard" genetic construct to minimize variability in separation distances and positioning of the fused donor and acceptor fps. using spectral fret imaging and fluorescence lifetime measurements in living cells expressing the fused proteins, we have characterized both sensitized acceptor emission and the change in the donor lifetime distribution as a result of quenching for each of the fused fp pairings. our results indicate that some red fps are better acceptors than others in terms of quenching the teal donor and sensitizing the emission of the acceptor indicating a fret event. s. m. perepelytsya, s. n. volkov bogolyubov institute for theoretical physics, nasu, -b metrologichna str., kiev, , ukraine stability of the dna double helix is determined by na + counterions, neutralizing the negatively charged phosphate groups of the macromolecule backbone. but under spatial conditions they may be replaced by much heavier ions, for example cs + . to determine the influence of heavy counterions on internal dynamics of the double helix the conformational vibrations of na-and cs-dna are studied. for this purpose the model of conformational vibrations of dna with counterions is used [perepelytsya s.m., volkov s.n., eur.phys.j. e. , , ] . as the result the frequencies and amplitudes of vibrations for b -dna with na + and cs + counerions are calculated. the frequencies of internal modes of the double helix are about , , and cm − . the frequencies of ion-phosphate modes are about and cm − for na-and cs-dna respectively. the calculated amplitudes of vibrations show that light counterions not disturb the dna internal dynamics, but heavy counterions make move all structure elements of the double helix. using the valence-optic approach the intensities of the dna vibrations in raman spectra are calculated. the calculations show that the ion-phosphate mode in cs-dna spectra is prominent, in contrast to na-dna spectra, where it has very low intensity. obtained results describe the intensity increase of the band cm − in cs-dna spectra that was observed in [bulavin l.a., et al., arxiv: . v ]. s. soria , f. quercioli , r. mercatelli , f. bianco , i. cacciari , g. righini centro studi e ricerche "e. fermi", p. del viminale , rome, italy, ifac-cnr, istituto di fisica applicata "n. carrara", via madonna del piano , sesto fiorentino (fi), italy, isc-cnr, istituto dei sistemi complessi, via madonna del piano , sesto fiorentino (fi), italy we report on the application of a simple white light source based on the supercontinuum generation from commercial photonic crystal fibres to confocal fluorescence microscopy and fluorescence lifetime imaging (flim) microscopy. the coherent white light can be tuned by varying the wavelength and intensity of the pump, a ti:sapphire laser. there are several advantages jn the use of sc sources: spatially coherent white radiation, tuning ranges of approximately nm, high brightness, a robust compact system (potentially all-fibre) and relatively low cost. being pulsed, sc sources are suitable for flim. we have used this system for measuring foerster resonance energy transfer (fret) in order to study interactions between ions channels and proteins of membrane within lives cells resolving the quaternary structure of plague f capsular antigen a. soliakov , j. r. harris , m. r. hicks , a. rodger , r. woody , a. watkinson , j. h. lakey cell and molecular biosciences inst., newcastle univ., uk, chemistry dept., univ. of warwick, coventry, uk, biochemistry and molecular biology dept., colorado state univ., usa, pharmathene uk, billingham, cleveland, uk most gram-negative pathogens express multi-subunit fibres on their surfaces that mediate host cell attachment, biofilm formation, invasion of host defenses and protection against phagocytosis. here we have studied capsular antigen fraction (or caf ), secreted through the conserved chaperone/usher pathway by the plague agent yersinia pestis [ ] . caf is highly immunogenic and is used in a recombinant subunit vaccine against plague. recent immunological studies indicated vaccines containing polymeric caf have higher protective efficacy than vaccines containing its monomeric variant. this difference in protective efficacy was attributed to the quaternary structure. however the quaternary structure of caf has not been characterized [ ] . in our study we have used transmission electron microscopy of negatively stained specimen and linear dichroism spectroscopy to determine the quaternary structure of recombinant caf . whereas electron microscopy revealed morphology of caf fibres bound to the surface of a carbon coated grid, linear dichroism gave the orientation of subunits in flow aligned caf fibres in solution. our results show recombinant caf comprises extended linear fibres and circularized fibres, both of which have high degree of conformational freedom. despite the vast application of lambert-beer law in biology, this empirical law cannot accurately describe the light absorption process of molecules with a long-lived excited state. this family of molecules includes most fluorescent molecules, which are very important in biology. lambert-beer law is in fact only valid at very low intensity of incident light and very low concentration of chromophore. if the system doesn't meet these conditions, it falls into a nonlinear regime when it is affected by a phenomenon which we call "dynamic photobleaching": the depletion of chromophores from the first layers, due to their transition to the excited state, leads to a sub-exponential propagation of light in the medium [ ] . this phenomenon leads to the necessity of a new formula for the light absorption dynamics which depends on the lifetime of the excited state of the chromophore. the predictions of the theory were successful in describing the absorption dynamics of azobenzene [ ] , but now they have been tested also on a biologically relevant molecule like chlorophyll. the results indicate that the absorbance is affected by the intensity of the incident light, and it is therefore a non reliable way of determining, for example, the concentration of the molecule. annular pupil filter to improve spatial highfrequency signal to noise ratio in linear and non linear microscopy e. ronzitti , v. caorsi , a. diaspro lambs, microscobio, department of physics, university of genoa, genoa, italy, semm, ifom-ieo, university of milan, milan, italy, neuroscience and brain technologies department, iit, genoa, italy shot-noise significantly affects and deteriorates the imaging capabilities of typical two-photon excitation and confocal laser scanning microscope, especially in biological applications where the detected signal can be remarkable slight [ ] . in particular, shot-noise substantially influences the spatial high-frequency range inducing a remarkable reduction of the optical transfer bandwidth. the insertion of an annular filter on the microscope objective lens in the illumination light pathway is here proposed to retrieve the high frequencies information loss [ ] .the electromagnetic interference effect induced by the filter insertion, gives a redistribution of the optical transfer function. in particular, the microscope frequency response in filter scheme exhibits an enhancement of signal to noise ratio at the high frequencies able to recover the high frequencies hampered by shot-noise [ ] . the goal of biomarker studies is to develop simple noninvasive tests that identify disease states. focus is beginning to shift from identification of individual biomarkers to identification of biomarker panels comprising multiple targets of different molecular species. there are, however, no current technologies available that allow for a comprehensive and simultaneous analysis of the expression levels of multiple cellular components, i.e. proteins and rna. surface plasmon resonance (spr) polaritons are surface electromagnetic waves that propagate in a direction parallel to the interface between a metal surface and an external medium e.g., liquid. these oscillations are very sensitive to any change of this boundary, a phenomenon that has been exploited to facilitate label-free, real-time detection of biological interactions, e.g. protein binding interactions. we are utilising the power of spr to develop technologies that facilitate diagnostic procedures for complex diseases such as alzheimer's disease and chronic obstructive pulmonary disease, through identification and detection of patterns of biomolecules indicative of disease. our approach will facilitate better disease characterisation, improve early detection strategies and aid drug discovery. surface generated fluorescence detection by supercritical angle confocal microscopy d. verdes, m. rabe, s. seeger institute of physical chemistry, university of zürich, winterthurerstrasse , zürich, switzerland a two channel confocal microscope for surface and simultaneously in solution fluorescence detection is presented. the microscope's core element is a parabolic mirror objective that collects the fluorescence above the critical angles for total internal reflection (tirf) of the water/glass interface. an aspheric lens incorporated into the parabolic mirror is used for diffraction limited focusing and collecting the fluorescence at low angles with respect to the optical axis. this low angles excitation approach is technically straightforward and gives an advantage over high numerical objectives that require very high angles for tirf illumination. by separating the collection of the fluorescence into supercritical and subcritical angles, two detection volumes highly differing in their axial resolution are generated at the interface. the surface selectivity of the detection volume is obtained on the basis of the dipole emission profile near a dielectric interface. its angular distribution is highly anisotropic and consists of a superposition of traveling and evanescent waves, which both are detected using the parabolic mirror objective. unlike with objective tirf microscopy, the parabolic mirror objective achieves easily diffraction limited excitation/detection volume at the water/glass interface. the objective optical performance is shown by measuring the actin cytoskeleton of cultured cells, fret energy transfer within adsorbed clustered proteins and single molecules detection. differential polarization laser scanning microscopy. anisotropy in biological samples g. steinbach , i. pomozi , g. garab biological research center, hungarian academy of sciences, szeged, hungary, pi vision bt., budapest, hungary differential polarization (dp) spectroscopy provides unique information on the anisotropic organization of biological samples [tinoco et al. ann rev biophys biophys chem : ]. however, anisotropy is often a microscopic property. the dp-lsm, constructed in our laboratory enables us to image the main dp quantities: linear and circular dichroisms (ld&cd), also in fluorescence detection ( . further applications include: periodic structure of isolated amyloids, anisotropy variations in cell walls related to drought resistance, and strong anisotropy of 'artificial chlorosomes', nanorods of synthetic porphyrins. dp-lsm might thus represent a novel tool in the better understanding of highly organized molecular macroassemblies. t. m. staudt , p. bingen , c. kempf , h. horstmann , j. engelhardt , t. kuner , s. w. hell german cancer research center/ bioquant, heidelberg, germany, max planck institute for biophysical chemistry, göttingen, germany, institute for anatomy and cell biology, university of heidelberg, heidelberg, germany the calyx of held, a large glutamatergic synaptic terminal in the auditory brainstem circuit has been increasingly employed to study presynaptic mechanisms of neurotransmission in the central nervous system. a highly detailed model of the morphology and distribution of cytoskeleton, synapsin, synaptic vesicles, calcium sensors, mitochondria, the presynaptic membrane and its active zones is derived by colocalization analysis of these different key elements of synaptic transmission in the rat brain. the various cellular components are visualized with subdiffraction resolution by stimulated emission depletion (sted) microscopy. imaging individual structural elements exhibit a focal plane resolution of < nm inside µm thick tissue sections. -imaging and spectroscopy - r. worch, t. weidemann, p. schwille biotec, biophysics group, technische universität dresden, germany interleukin- (il- ), a small soluble protein, is a principal regulatory cytokine, playing an important role during the maturation and clonal expansion of antigen specific b-cells in mammals. at the plasma membrane, il- is recognized by a receptor that consists of two single spanning transmembrane proteins: a high affinity il- r alpha chain, and a low affinity il- r gamma chain. it is still controversial by which molecular mechanism the signaling complex is fully activated: dimerization of chains, large conformational change upon il- binding or a combination of both. moreover, the influence of the lipid environment in which the activation takes place is poorly characterized. to address these issues we aim to reconstitute the receptor component in artificial membrane systems to study the various mutual interactions by means of fluorescence-based techniques, mainly fluorescence correlation spectroscopy. due to reduced background in a chemically defined system this may provide details not yet accessible in the living cell. k. wicker, s. sindbert, r. heintzmann randall division of cell and molecular biophysics, king's college london, se ul london, u.k. fluorescence confocal microscopy, an indispensable tool of modern biology, allows the imaging of live fluorescent specimen with high lateral as well as axial resolution. through the introduction of a sufficiently small confocal pinhole, the lateral resolution can be enhanced compared to that of a wide field microscope. however, this gain in resolution comes at the cost of a decrease in detection efficiency, as light blocked by the pinhole is lost. we present a method for improving the lateral resolution (extending work of sandeau et al. [ ] ) and detection efficiency of scanning microscopes by adding an interferometer with image inversion in one of its arms to the detection pathway [ ] . this surpasses the lateral resolution achievable in a conventional confocal microscope (closed pinhole) while increasing the detection efficiency substantially. point spread function measurements for a uz-interferometer (uzi) are shown. the light in this setup follows threedimensional u-and z-shaped paths and relies on reflections off planar surfaces only in order to achieve image inversion. we achieved an interference contrast of % for white light, and excellent agreement with theoretical predictions. g. vicidomini, a. schönle, j. keller, r. schmidt, c. von middendorff, s. w. hell max planck institute for biophysical chemistry, department of nanobiophotonics, göttingen, germany fluorescence far-field microscopy is a indispensable tool in modern life science. however, the resolution of its standard variants is limited by diffraction to ∼ nm in the focal plane and ∼ nm along the optical axis. to overcome this limit, a new class of super-resolution microscopy techniques has been designed. one example is pi microscopy. to obtain isotropic resolution, pi uses interference of the wave fronts from two opposing lenses. the point spread function (psf) of pi system is characterized by multiple maxima/sidelobes, which replicate the object in the image. therefore image restoration is mandatory to render unambiguous imaging. the situation is further complicated because the positions and the relative heights of the multiple maxima/sidelobes of the psf depend on the phase difference (pd) between the two wave fronts at the focus. if the refractive index of the sample varies in space, this pd becomes a function of position and pi image formation process looses its shift invariance. the pd function (pdf) may not even be known a-priori and must then be estimated from the image, leading to a blind image restoration problem. here, we propose a maximum a-posteriori based method to solve the problem. we either assumed a mathematical model for the pdf that depends on a small number of parameters or allowed for an arbitrary pdf but introduced a smoothing constraint. designer multicomponent lipoplexes have recently emerged as especially promising transfection candidates, since they are from to times more efficient than binary complexes usually employed for gene delivery purposes [ ] [ ] [ ] . here, we show, for the first time, that after internalization binary complexes of lower transfection potency remain in compact perinuclear endosomes, while multicomponent systems have intrinsic endosomal rupture properties that allow plasmid dna to escape from endosomes with extremely high efficiency [ ] . endosomal rupture results in an extraordinarily homogeneous distribution of unbound plasmid dna throughout the cytoplasm and in the nucleus [ ] . fluorescence spectroscopy and stopped-flow technique were utilized for the study of the kinetics of incorporation of hypericin (hyp), a natural photosensitizing pigment, into lowdensity lipoproteins (ldl). triphasic kinetics of hyp association with ldl was observed when solutions of hyp and ldl were mixed together. the most rapid phase of hyp incorporation is completed within tens of msec, while the slowest one lasts - min. the most of hyp molecules are incorporated into ldl in the slowest phase. the kinetics of the incorporation of hyp into ldl particles pre-loaded with hyp were also investigated. the observed decrease of the lifetime and total intensity of hyp fluorescence with the increase of the incubation time of hyp with hyp/ldl complex is a sign of the formation of aggregates and the dynamic quenching of singlet excitation state of hyp inside ldl. to study the kinetics of a transfer of hyp molecules between ldl particles, the time evolution of the stopped-flow and time-resolved fluorescence experiments were investigated after the mixing of the complex hyp/ldl= : with appropriate amounts of free ldl. for each final hyp/ldl ratio the increase of the lifetime and total intensity of hyp fluorescence was observed. the half-time of this process is similar to that one of the slowest phase of hyp incorporation into free ldl. a. bosca , r. magrassi , g. firpo , l. repetto , c. boragno , u. valbusa italian institute of technology, genova, italy, nanomed labs (difi-cba), genova, italy the technique of choice to measure the electrophysiological activity of neuronal cells is the so called patch-clamp method because of its precision and sensitivity; this procedure could play a major role also in the investigation of the behavior of a biological neuronal network and so represents an important tool for understanding its functionality and for screening the effects of drugs and compounds on it. the final aim of this project is the development of a planar patch clamp device suited to measure simultaneously the electrical activity of cultured neurons associated in a network. this device will be made of polymeric disposable material and will include a microfluidic perfusion system. in order to create a smooth micro sized pore in a thin polymeric membrane we exploited the prototyping capabilities of focused ion beam etching and we extended the air moulding technique by combining it with soft moulding, obtaining micro structured substrates with the requested features. we also subjected our substrates to a chemical treatment capable of rendering its surface stable and hydrophilic and we verified that it makes them suitable for neuron culturing. use of lipoamino acids for nasal delivery of therapeutic proteins c. bijani , s. sice , j. elezgaray , c. degert , o. broussaud , e. j. dufourc physica pharma, pessac, france, umr cbmn, cnrs-université bordeaux , iecb, pessac, france most of the therapeutic proteins are clinically administered through an intravenous injection several times a day / week. because the repeated injections are not convenient and cause pain in patients, an alternative route of administration is desirable such as oral or nasal. unfortunately, proteins are easily degraded by proteolytic enzymes in the gastrointestinal tract and, therefore, have a low bioavailability when administered via oral route. physica pharma has gained experience in forming sprayable solutions combining lipoamino acids (laa) and small therapeutic organic compounds with the aim to improve their intranasal absorption. in the present work we investigated the ability of laa to complex large molecule such as human erythropoietin, human growth hormone and salmon calcitonin in order to form easily sprayable colloids. these three proteins are used respectively to treat anaemia, growth problem and hypercalcemy. circular dichroism and dynamic light scattering were used to further characterize the laa-protein colloids. a specific molar ratio of laa versus protein was found for wich the proteins keep their secondary structure and have an overall isotropic size slightly increased. molecular dynamics show that proteins are indeed coated with laa. such a complex is shown to pass very easily through a culture of nasal cells growth at confluence. in this study we describe two systems based on soft matter designed for the drug delivery and for the replacement of synovial fluid in osteoarticular pathologies. (i) a new class of temperature sensitive hydrogels pva/poly(ma m nipaam n ) shaped as microspheres obtained with a water-in-water emulsion photocrosslinking reaction. microparticles of pva/poly(ma m nipaam n ) with m:n theoretical molar ratios equal to : ; : , : have been studied in terms of average size and responsiveness to temperature characterized by confocal laser scanning microscopy (clsm), dynamic light scattering (dls) and differential scanning microcalorimetry (dsc). (ii) a physical network based on hyaluronic acid with a small extent (degree of substitution: %) of hydrophobic moiety grafted on the backbone, hyadd , has been characterized in order to account for the influence of thermal treatment on the stability of the hydrogel. dynamic light scattering (dls) and small angle neutron scattering (sans) have been used for dynamic-structural characterization of hyadd hydrogels. diffusion of macromolecular probes has been studied by fluorescence recovery after photobleaching (frap) to study the mesoscopic texture of the hydrogel and molecular dynamic (md) simulations were used to approach the time evolution of the physical junction points and of chains clusters. multi-scale estimation of water soluble diffusivity in polysaccharide gels g. eisele , s. bertini , d. paganini , l. piazza ronzoni institute, milan, italy, distam, university of milan, italy diffusion properties in gels play important role in food and biotechnological applications. an attractive goal is to design gels in such a way that the active molecules are delivered by the material according to specific release sequences. the transport of macromolecules within polymeric gels depends: on the obstruction effects of the surrounding gel strands, on the molecular interactions between the gel and the solute, on the interactions between the solute molecules themselves and the interactions between the solute and the solvent. physical polysaccharide gels were evaluated in this work to probe diffusion over both microscopic and macroscopic distance scales. physical gels from agar and starch were investigated by high and low resolution nmr techniques in order to characterize their structures. obstruction effects of the surrounding gel strands were considered by studying diffusion of glucose. local diffusion, due to brownian motion, was quantified by low resolution nmr spectroscopy. the fickian diffusion coefficient was measured by modelling experimental concentration-distance curves obtained by means of a two-compartment diffusion-cell. diffusion coefficients depend on the viscoelastic properties of the gel matrix and on water-polysaccharide interactions. j. l. cuellar, g. koehler, m. fischlechner, e. donath medizinische institut für physik und biophysik, leipzig, germany rather than being just pathogens, from the view point of biotechnology and materials science viruses can be regarded as nanocontainers in which nucleic acids have been enclosed in a protective self assembled protein cage. in some viruses an additional lipid-protein envelope wraps the capsid shell. the capsid is constituted of several copies of one single protein subunit or just a few, arranged in a regular fashion showing icosahedral symmetry. the mechanical properties of the cage are important for understanding the infection mechanism involving the release of the encaged genome. by means of atomic force microscopy, it becomes possible to probe such material properties by nanoindentations of single viral particles. it is very interesting to learn how strong or brittle a virus can be. here we have studied the mechanical properties of empty rubella virus particles (rlps) due response of external applied forces. we found that rlps are extremely soft comparable to that of some rubber materials. a peculiarity of the rubella virus is that the capsid is considerably smaller than the surrounding shell filling only a fraction of the lumen provided by the envelope. the envelope in rubella has a distinguishable response on the material properties of the virus. deformation and fracture of the capsid requires comparatively larger forces. our results indicate that the ph is a major factor influencing on rubella particle material properties. this can be related to the infection mechanism. pentavalent antimony (pa) (glucantime, sanofi-aventis or glaxo) are the mainstream agents of choice for leishmaniasis treatment. in therapeutic doses the pa treatment has cardiac side effects like electrocardiographic (ecg) alterations, that include qt segment prolongation, t wave flattening or inversion, inversion of st segment, p, r and t waves amplitude reductions, torsade de pointes arrhythmias and sudden death by cardiac arrest. the objective of this study was to characterize the arrhythmogenic potential of pa. we used guinea-pig to assess the chronic effects of pa therapeutic dose on corrected qt interval, qt dispersion, ventricular action potential (ap) amplitude and duration at % and % of maximal repolarization and survival rate. guinea-pig received daily mg/kg pa or saline for days. eight lead ecg were recorded before and in the last treatment day. at the end the animals were killed and the left ventricle papillary muscles excised for ap recording with the intracellular microelectrode technique. our results of chronic pa treatment showed significant increase of qrs complex duration, qt interval duration, qt dispersion and incidence of t wave flattening or inversion and arrhythmias. the ap analysis demonstrated prolongation at % duration. the treatment was lethal in % of the animals. we concluded that pa is a proarrhythmic drug that upon chronic use may causes arrhythmias and mortality by disturbances in the ventricular repolarization process. m. m. khvedelidze , t. j. mdzinarashvili , t. partskhaladze , n. nafee , m. schneider ivane javakhishvili tbilisi state university, tbilisi, georgia, institute of molecular biology and biological physics, tbilisi, georgia, biopharmaceutics and pharmaceutical technology, saarbrücken, germany, pharmaceutical nanotechnology, saarbrücken, germany we have studied the thermodynamical properties of chitosan-coated nanoparticles (cnp) and non-coated nanoparticles (np) and have gained some insights about plga nanoparticles' properties using supersensitive differential microcalorimetry. the experiments show that in a wide ph interval the changes in transition temperature did not take place. it was shown that such nanoparticles could be used in acidic surrounding for drug transfer. stability and their other properties are less depended on either the particles were in bidistilled or deionized water, or the suspension of particles were located in buffer. to determine the interaction of plga nanoparticles with dna. in the case of dna presence in cnp solution the calorimetric experiments show that the heat absorption peak is constricted, what biophysically means that interaction between them takes place. for more exact determination the contribution of cnp in spectrum, we have compared the spectra of pure dna with the spectrum of the same concentration dna plus cnp. the optimal ratio for dna loading onto the cnp was found to be : . protein-based "epoxy-like" physical hydrogels for stem cell transplantation s. c. heilshorn, c. wong po foo, j. s. lee materials science and engineering, stanford univ., u.s.a. stem cell transplantation has emerged as a promising therapy for multiple injuries and diseases; however, cell survival after transplantation is often poor and unpredictable. we hypothesize that co-injection of stem cells encapsulated within an optimized physical hydrogel will enhance viability. whereas, current physical hydrogels require a shift in environmental conditions (e.g., ph, temperature) to initiate the sol-gel phase transition during encapsulation, our newly designed molecular-recognition gels do not require environmental triggers. instead, these "physical epoxy-like" gels consist of two components that undergo a sol-gel transition upon mixing due to specific hydrogen bonding. the gel viscoelasticity is predictably tuned through precise variation in the molecular-level design of the two components, created using recombinant protein techniques. the design of the two components is based on simple polymer physics considerations and utilizes bio-mimimcry. adult neural stem cells or mesenchymal stem cells are encapsulated within these gels with high viability at constant physiological conditions. the gels promote the growth and differentiation of neural progenitors into neuronal phenotypes, which adopt a d-branched morphology. the gels are further optimized for use in the central nervous system by tethering neuroprotective peptides to the gel through molecular-recognition sites. these peptides are released-on-demand by cells through the action of proteolytic enzymes. we consider the integration of the portal protein of the bacteriophage virus φ into lipid bilayers of giant unilamellar vesicles (guvs) membranes with the aim of constructing a functional cargo-device able to transport dna and later translocate it outwards. our nano-engineering plan consists of growing guvs from bilayer membranes built up from proteoliposomes previously prepared by extrusion. we have designed two alternative chemical routes for integrating the portal protein in the lipid bilayer, the first considering the native protein and the second a mutant modified with a hydrophobic belt made of histidine-tags. in our contribution we will present details on the different nano-engineering strategies and experimental evidence about the integration of the portal protein in the membrane with an orientation adequate to allow for functional dna translocation. p.-h. guelluy , m.-p. fontaine-aupart , m. hoebeke biomedical spectroscopy, ulg, belgium, laboratory of molecular photophysics, univ. of paris-sud, france ppme is a second generation photosensitizer (ps), and a promising candidate for photodynamic therapy (pdt) treatment. we have previously demonstrated that ppme can be easily and efficiently encapsulated in dmpc liposomes, used as ps-vector. we therefore compared the photophysical and photochemical properties of free and encapsulated ppme incubated with human carcinoma cells. absorption and fluorescence microspectroscopy as well as flim analysis allow evaluating the aggregation state of ppme inside the different cellular organelles and the extracellular medium. confocal microscopy established undoubtedly the colocalization of ppme, by robust probabilistic exclusion method, within mitochondrion (central siege of apoptosis). after ps activation ( h and h), the balance apoptosis-necrosis was double-estimated by facs device and fluorescence confocal microscopy. quantification of hydroxyl radical was purchased by spin trapping-esr spectroscopy and quenching technique. all these techniques required peculiar settings because of the fluorescence activity of ppme. these results allow to ascertain that the vectorization of ppme affords a better cellular penetration and a monomeric state of ppme. the presence of ppme inside mitochondrion orientated the cellular death in apoptosis h following ps-activation. but necrosis is the major actor h after treatment. active substrates to study mechanotransduction j. le digabel, p. hersen, b. ladoux laboratoire matière et systèmes complexes, université paris diderot -cnrs, paris, france cellular processes imply an important coordination of interactions with the extracellular medium. accumulating evidences demonstrate that cell functions can be modulated by physical factors such as the mechanical forces acting on the cells and the extracellular matrix, as well as the topography or rigidity of the matrix. these extracellular signals can be sensed by mechanosensors on the cell surface or in the cell interior to induce various cell responses. we have developed an original approach based on micro-fabricated substrates of polydimethylsiloxane (pdms) to study cell migration. we used a closely spaced array of flexible micropillars (diameter∼ ?m) to map the forces exerted by cells on their substrates. in this case, the micropillars act as passive force sensors. here, we propose to analyze the cell response to an external applied stress by a well-controlled actuation of the substrate. to do so, we used magnetic pillars. such substrates allow us to modify dynamic adhesion conditions of cells and to better understand the coupling phenomena between mechanical sensing and biochemical activity of a living cell. using polyacrylamide hydrogels doped with ferromagnetic iron oxide particles or ferrofluids, we can make magnetic pillars with diameters of to microns while a magnetic field can be locally applied with a magnetic needle. such a technique will be helpful to study the mechanical response of cells to an external force or to local changes in their microenvironment. glucose scavenging activities of pamam dendrimers m. labieniec , t. gabryelak , c. watala university of lodz, deptartment of general biophisics, lodz, poland, glycation is a spontaneous, non-enzymatic modification of biomacromolecules with hexoses, mainly glucose. in terms of its pathophysiological relevance, it targets predominantly proteins, but also nucleotides and phospholipids, and is of major importance to both physiology (ageing) and pathology (diseases with a metabolic background, like diabetes). earlier we demonstrated in vivo that the administration of pamam g to diabetic rats resulted in a significantly reduced blood glucose, as well as the early (hba c ) and late (ages) protein glycation products. in this study we investigated the ability of pamam g ( surface nh groups) and g ( nh ) to inhibit glycation of proteins in plasma, and a model protein -bsa. pamam g and g competed chemically with protein nh for the binding of glucose, and hampered protein glycation. in a high-glucose medium they underwent an excessive glycation themselves. this modification was more effective in pamam g , in which surface nh were more mobile and accessible. pamam modification with glucose rendered these dendrimers less polycationic in aqueous solutions. pamams neither affected bsa conformation nor formed stable complexes with a protein. we conclude that pamams are very effective glucose scavengers. thus, even less toxic pamams of lower generations, like g , may appear useful in further medical applications as the agents attenuating the detrimental effects of sever hyperglycaemia on biomacromolecules. selective drug delivery and novel drug approaches by polyelectrolytes s. krol cbm-cluster in biomedicine, area science park, s.s. , . km; trieste, italy the use of polyelectrolytes in the past was mainly related to targeted drug delivery and nanoparticle preparations for medical applications. but rarely the polyelectrolytes were investigated for the own features as a drug. in our present work we use a special physical feature of cancer cell membranes as a target for a specific polycation. we found that the polycation is selectively up-taken by cancer cells (leukemia, hepatocarcinoma, cancer stem cells) while normal cells remain unaffected. another interesting application of polyelectrolytes are in form of a multilayer coating on the surface of nanogold particles. for this topic, two new approaches were developed for a drug delivery through the blood brain barrier. in the first case, high amounts of creatine were bound to the gold particles and delivered as protective agents for ischemic stroke (viota et al., j colloid interface sci. , ( ): - ). in the second case coated multifunctional gold particles were prepared as a drug for neurodegenerative disease on the basis of protein aggregates. the use of gold as core for coated nanoparticles offers the possibility to study our systems as "theranostics", system which are modified to recognize selectively diseased cells and carry the moieties to treat or destroy the malfunctioning cells. possible treatments can be local hyperthermal therapy by using the particles as amplifier and enhancer or photodynamic therapy with gold as a drug. m. koneracka , v. zavisova , m. muckova , p. kopcansky , a. jurikova , n. tomasovicova , g. lancz , m. timko , m. fabian institute of experimental physics, slovak academy of sciences, košice, slovakia, hameln rds a.s., modra, slovakia, institute of geotechnics, slovak academy of sciences, košice, slovakia the aim of this study was to develop biodegradable and biocompatible paclitaxel loaded magnetic plga nanospheres (nps) suitable for biomedical applications. biodegradable poly(d,l-lactic-co-glycolic acid) (plga) was used as a capsulation material and the magnetic fluid was used as a magnetic carrier. incorporation of magnetic particles and drug in the plga polymer matrix was confirmed by infrared spectroscopy (ftir) and differential scanning calorimetry (dsc). the release of the drug from the prepared nps to the surroundings under the different conditions was studied also. the prepared magnetic plga nps with encapsulated paclitaxel (spherical shape, size - nm) have good stability in the presence of high nacl concentration at • c, the toxicity of prepared samples declared times higher value of lethal dose ld in comparison with pure paclitaxel (ld = mg/kg) and showed the significant response to external magnetic field which is useful from the point of view to achieve pharmaceutically acceptable drug delivery systems for tumour treatment. success of human gene therapy depends upon the development of delivery vehicles or vectors, which can selectively deliver therapeutic genes to target cells with efficiency and safety. many cationic polymers have been used to condense dna by electrostatic interaction into small particles (polyplex), for protecting the dna from degradation and enhancing uptake via endocytosis. polyethylenimine (pei) appears to be one of the most advanced delivery system that can condense dna efficiently forming pei-dna polyplex complex. the physicochemical properties of different molecular weights of pei, such as condensation ability, buffer capacity, time kinetics, ftir and surface charges of the pei-dna complexes may be important factors to obtain a higher transfection efficiency of the polycation vectors. our intent in this study was to characterize pei-dna complexes to see whether these physicochemical properties have any influence on their disposition characteristics and cellular uptake process. we found that pei-dna complexes, obtained by the k pei at n/p ratio > , were more stable in the presence of tissue culture medium & serum, and did not dissociate in nacl easily. key words: polyethylenimine, polyplex, polycations, dna, transfection, gene delivery. effect of nanopatterned substrate on neuronal growth cones activity in the last years an increasing interest has been address to explore, at the level of the single cell, the physical interaction between neurons and micro-and nano-patterned surfaces which mimic the biological environment and can induce specific biological behavior. a consistent number of substrates have been tested (nano-grooves, pillars, gap nanowires) but the effect of nano-topographical features at subcellular level e.g. branching and pathfinding of growth cones(gc)is still unexplored. using nanoimprinting lithography we fabricated gratings on glass with grooves of variable pitch, depth and width all in the nm range. embryonic stem cell derived neurons were seeded on nanostructured and on flat control glasses. we investigate gc morphology with nm resolution by afm. a significant effect on sub-cellular architecture was observed.on nanopatterned substrates % of gc were branched with a large number of long and thin filopodia(average length and height . µm and nm)while on control only %of gc were branched with an higher percentage of long and thick filopodia (average length and height , µm and nm). on the contrary we did not observe a significant influence of the nanopatterning on the alignment and elongation of neurites. in both cases the distribution of angles between axon and filopodia showed a preferential direction at • . in conclusion, the tested nanopatterns do not influence the neurite directions but do enhance the gc morphology and explorative activities. growth enhancement and adhesion control of pc on micropatterned ns-tio thin film c. lenardi , a. v. singh , p. milani c.i.ma.i.na., dip. di scienze molecolari applicate ai biosistemi, università di milano, italy, c.i.ma.i.na., semm, european school of molecular medicine, fondazione ifom, milano, italy, c.i.ma.i.na., dip. di fisica, università di milano, italy cluster assembled nanostructured titanium dioxide (ns-tio ) has been explored as novel substrate for in vitro cell culture. in this work, we report micropatterned ns-tio thin film as putative microdevice for neuron culture and growth. in additions, we show a simple scheme of molecular patterning of bovine serum albumin (bsa) as cell anti-adherent substrate complementary to ns-tio micropattern which favors a selective spatially confined adhesion of neurons. bsa was drop coated and physisorbed over glass coverslip covered with a thin conducting layer of indium tin oxide (ito) and then pmma was spin coated over it using standard protocols. later, using combinations of e-beam lithography and pulsed microplasma cluster source (pmcs), a thin layer of ns-tio was deposited over micropatterned pmma. further, lift off process enabled us to generate complementary micropatterns of hydrophobic bsa (cell repellent) and hydrophilic ns-tio (cell adhesive). cell culture studies have confirmed that pc cells like to grow on ns-tio substrate and not on bsa layer. the technique offers a novel approach for neuronal cell assay applications. gene delivery with chitosan: influence of chain length on intracellular trafficking and dissociation s. lélu , c. d. l. davies , n. reitan , s. p. strand department of physics, ntnu, trondheim, norway, department of biotechnology, ntnu, trondheim, norway chitosan, a cationic polysaccharide presenting low cytotoxicity, is a promising nonviral gene delivery vector. chitosan complexes dna into nanoparticles, and the complexation and cell transfection efficacy are function of the chitosan/dna ratio and of the intrinsic properties of chitosan, i.e. degree of polymerization dpn, charge density, and molecular structure. nanoparticles formed by shorter chitosan (dpn - ) mediated higher transgene expression than nanoparticles based on longer chitosans. the purpose of this work is to relate the dpn of linear chitosan with the cellular uptake, intracellular trafficking and dissociation of chitosan-pdna complexes, measured by confocal laser scanning microscopy and fluorescence correlation spectroscopy (fcs). cells were incubated with oligomers (dpn and ) either free or complexed with plasmid-dna. both chitosan oligomers seem to penetrate cell nucleus in association with or free from pdna. hours after incubation, accumulation of oligomers in cell nucleus is similar for free and complexed dpn , whereas it increases for complexed dpn compared to free, indicating a possible delayed dissociation of the complexes based on dpn in the nucleus and suggesting a dissociation of pdna-dpn in cytoplasm. these data are consistent with previous studies which suggested that longer chitosan chains led to tighter complexes, and hence to a delayed dissociation process and lower transfection efficiencies compared to shorter chitosans. material surface properties greatly influence dna purification and pcr yield in microsystems c. potrich , l. lunelli , l. marocchi , l. pasquardini , g. guella , d. vozzi , l. vanzetti , p. gasparini , c. pederzolli fbk, trento, italy, univ.trento, italy, univ.trieste, italy modern microchip platforms integrate dna purification, target amplification by pcr and dna detection in a single device. combination of these processes minimizes sample loss and contamination problems as well as reduces analysis time and costs. different strategies are available to perform dna extraction on a chip. here we exploited amino-coated materials as a tool for specific binding of dna through the electrostatic interaction between amine groups and nucleic acids. we analyzed the ability of different treated substrates to selectively absorb/desorb the genomic dna with the aim to purify dna from unwanted components. amino-coated substrates were characterized by afm, xps, fluorescence microscopy and absorption spectroscopy to define the surface chemical and morphological properties. the distribution of the dna adsorbed on materials was homogeneous and the eluted dna was tested for pcr. same materials were analyzed for their compatibility with pcr and the use of different enzymes and reagents or proper surface treatments was employed. we established the best conditions for dna amplification in silicon/pyrex microdevices depending on the type and the fabrication method used and on the quality of reagents more than on the passivation treatment or increment in standard taq polymerase concentration. nanoporous alumina fabricated using unconventional acids for enhanced biomolecular physisorption n. patra, m. salerno, r. losso, r. cingolani italian institute of technology, genova, italy in the last decades the available porecell sizes of porous alumina have been extended, but operating conditions to obtain some pore diameters still have to be optimized, particularly below the nm limit. in this range the use of biocompatible porous alumina thin films could find applications in biosensors, after functionalization by appropriate physisorbed biomolecules. while pore ordering and film growth rate are mainly influenced by the electrical parameters of voltage and current, respectively, the typical pore size primarily depends on the electrolyte. in the search for alternative anodization conditions, we have investigated several acids that have never been used before, namely gallic, lactic, propionic, and glycolic acid. the anodizations were carried out in galvanostatic conditions at fixed concentration and durations, by varying the current. atomic force microscopy was used to test the oxidized surface morphology. in particular, lactic and propionic acids demonstrated feasible. lactic acid gave best results for ∼ ma/cm current density, corresponding to roughly constant ∼ v potential, with resulting pore diameter in the nm range, whereas propionic acid performed best for ∼ ma/cm , corresponding to ∼ v, resulting in nm pore diameter. both kind of films looked lightgray, different from the yellowish oxalic acid porous alumina, which can be a hint of lower ion contamination. chitosan-arabic gum nanoparticles as potential vehicles for peptide delivery l. moutinho, s. rocha, m. coelho, m. c. pereira lepae, chemical engineering department, faculty of engineering, university of porto, portugal chitosan (cs) -arabic gum (ga) nanoparticles were produced by an ion-ion interaction process, using different weight ratios of polysaccharides. according to zeta potential (zp) measurements, particles are ionically stable. zp values ranged from to mv for cs:ga ratios of : . and : . , respectively. cs:ga : yielded uncharged nanoparticles and aggregates, showing that, at this ratio, the negative charges of ga neutralize the positive charges of cs. the particle diameters ranged from to nm, as measured by dynamic light scattering (dls), and displayed a slight tendency for decreasing as the ga concentration increased. transmission electron microscopy (tem) images showed numerous spherical nanoparticles. peptides were encapsulated within the nanoparticles, by mixing them with chitosan solution prior to adding ga. this system can protect short peptides from rapid metabolization, prolonging their blood half-life. physical characterization of nanocarriers for drug delivery s. motta , y. gerelli , g. sandri , e. ricci , p. brocca dept. of chem, biochem and biotech for medicine -university of milan, italy, dept. of physics -university of parma, italy, dept. of pharmaceutical chem -university of pavia, italy nanoparticles used as nanocarriers for pharmaceuticals can improve solubility and bioavailability of problematic drugs and protect labile or toxic molecules. fundamental parameters like drug encapsulation efficiency and release kinetics are tuned by the physico-chemical features of the drug/carrier complex. a challenging aspect of the pharmaceuticallyoriented issues resides in the relatively low number of molecules allowed to build up nanosystems with different properties and morphology, according to the specific drug and to the intended therapy. we have studied: ) soybeanlecithine/chitosan nanoparticles for progesterone and tamoxifen delivery; ) solid-lipid nanoparticles (compritol ato, poloxamer , tween and chitosan) for cyclosporine-a delivery via ophthalmic formulation. both void carriers and drug-loaded nanoparticles have been studied, to understand how the structure of the carriers can be modified by the active molecules. several non-invasive physical techniques have been used to achieve a detailed knowledge of the systems: a) dynamic light scattering for the dimensional distribution of the nanocarriers, b) zeta potential to determine surface charge, c) cryo-tem for morphological analysis d) x-ray scattering (in small angle configuration) and dsc to access information on the inner structure. . a. schollier , a. halperin , m. sferrazza , g. fragneto institut laue-langevin, grenoble, france, universite libre de bruxelles, belgium, cnrs-ujf grenoble, france protein adsorption on surfaces is responsible of several unwanted effects in technological and pharmaceutical applications: fouling of contact lenses, clotting in blood containing devices, inflammation around artificial organs for instance. those phenomena can be repressed with certain types of polymer brushes, in particular peg (polyethylene glycol) brushes, and a better understanding of the mechanism of adsorption could lead to improvements in the design of biomaterials. we have developed a theory describing this mechanism and carried out measurements to confirm the theoretical predictions [langmuir , , ] . three cases are predicted: primary adsorption at the grafting surface, secondary adsorption at the outer edge of the brush, and ternary adsorption within the brush itself. we prepared samples with different grafting densities and different degrees of polymerization and used different protein size and concentrations. neutron reflectivity experiments, able to determine the structure and composition of material interfaces with a fraction of nanometer resolution, were carried out and, by using deuterated proteins (anti-freeze protein, dihydrofolate reductase and myoglobin) on different peg compositions and grafting densities, it was possible to locate the proteins in the brush and to distinguish between the different kinds of adsorption : primary adsorption is dominant for short brushes and ternary adsorption for long brushes. effect of powder air polishing on nanocomposite dental materials measured by atomic force microscopy m. salerno , g. derchi , a. m. genovesi , l. giacomelli italian institute of technology, genova, italy, istituto stomatologico tirreno, lido di camaiore, italy in dentistry, airpolishing with glycine and bicarbonate powders is widely used to remove accumulated plaque. however, the resulting teeth and gingival surface roughness, which is a risk factor for further plaque accumulation, has to be considered. in this in vitro study the effect of the above mentioned technique on the surface of a novel nanocomposite dental material is evaluated by means of atomic force microscopy (afm). square specimens ( . × . cm size) were airpolished either with glycine or bicarbonate, using different application times ( , , sec) and distances ( , , mm) . four specimens were evaluated for each timedistance combination, and checked versus untreated specimens (controls). the specimens were imaged with tappingmode afm at two different scan sizes ( × and × µm ) in two different regions for each sample. the surface roughness was measured as the rms of the feature heights. for the sec mm group airpolishing resulted in an increased surface roughness as compared to the controls; however, glycine was associated with a lower roughness than bicarbonate (glycine: ± nm; bicarbonate: ± nm; controls: ± nm; p< . for treated specimens vs controls, p< . for glycine vs bicarbonate, anova test). similar results were obtained for all the other timedistance combinations. the work comprises the design of polysaccharide based nanoparticles for drug delivery. chitosan/arabic gum and arabic gum/maltodextrin nanoparticles were prepared by polyelectrolyte complexation and spray drying [ ] , respectively. dynamic light scattering characterization established that arabic gum/maltodextrin nanoparticles are more polydisperse (diameters ranging from to nm) than chitosan/arabic gum particles (diameters of approximately nm). scanning electron microscopy measurements demonstrated that the particles are spherical and have a smooth surface. the systems are highly stable to forces up to nn as observed by atomic force microscopy. due to their nature they are hydrophilic and biodegradable. the nanoparticles were used to entrap short peptide sequences and antioxidants and were proved to be efficient in maintaining the biological activity of the molecules. kyotorphin (ktp) was found in in bovine brain. despite revealing remarkable analgesic properties, analgesia was only induced after central delivery. this limited ability to cross the blood-brain barrier (bbb) combined with the unknown mechanism of action largely confines its pharmacological use. to surpass these problems, we designed new ktp derivatives: ktp-rc and ktp-rc-lipogen. biophysical studies were carried out using fluorescence methodologies to characterize the peptides' interaction with biomembrane model systems. partition coefficient quantification showed a clear preference of the derivatives towards fluid zwitterionic and anionic membranes. moreover, a relationship between anionic lipid percentage and in-depth insertion in membrane was established. additionally, studies with fluorescent probe di- -anepps revealed different membrane-interaction profiles of morphine and ktp-derivatives, suggesting distinct actions between them. the analgesic efficacy of the compounds was studied in vivo after systemic administration in models of acute and tonic pain. unlike ktp, both ktp-rc and ktp-rc-lipogen displayed high efficacy from doses as low as . and . mg/ g, respectively, indicating bbb crossing. the observed correlation between higher partition/insertion in the membrane and enhanced analgesic action proved the biophysical rationale to be a powerful strategy for early screening in cns drug development. metabolic syndrome (ms) is now regarded as a major risk factor for cardiovascular disease. as prooxidant/antioxidant balance is disturbed in the course of this disorder, it is possible that melatonin may exhibit protective effect against oxidative damage of blood cells. ms was diagnosed according to international diabetes federation definition ( ) . the investigated group consisted of patients before and after the melatonin supplementation ( mg per day for two months) and was compared to control group of healthy individuals on normal diet. our experiments show that erythrocytes from patient group exhibit significantly higher tbars and total cholesterol levels whereas the protein thiol concentration, na + k + atpase and glutathione peroxidase activities were decreased in comparison to those of healthy volunteers. after two month melatonin supplementation, tbars and cholesterol concentrations significantly decreased, whereas the na + k + atpase, catalase and glutathione peroxidase activities increased. the glutathione concentration was also higher. these results show that melatonin supplementation has a protective effect on erythrocytes of ms patients. action of ferritin, a nanoparticle model, on ros formation and glutamate uptake in synaptosomes t. waseem, a. alekseenko, s. fedorovich institute of biophysics and cell engineering, minsk, republic of belarus nanoparticles are currently used in medicine as agents for targeted drug delivery and imaging. however it has been demonstrated that nanoparticles induce neurodegeneration in vivo and kill neurons in vitro. the cellular and molecular bases of this phenomenon are still unclear. we have used the protein ferritin as a nanoparticle model. ferritin contains iron particles (fe + ) with size nm and a protein shell. we investigated how ferritin influences uptake and release of [ c]glutamate and free radical formation as monitored by fluorescent dye dcfda in rat brain synaptosomes. we found that even a high concentration of ferritin ( µg/ml) did not induce spontaneous [ c]glutamate release. in contrast the same concentration of this protein inhibited [ c]glutamate uptake two fold. furthermore ferritin induced intrasynaptosomal ros (reactive oxygen species) formation in a dose-dependent manner. this process was insensitive to µm dpi, an inhibitor of nadph oxidase and to µm cccp, a mitochondrial uncoupler. these results indicate that iron-based nanoparticles can cause ros synthesis and decrease glutamate uptake, potentially leading to neurodegeneration. functionalized carbon nanotubes (cf-cnts) have a promising future as vectors for drug delivery and for neuronal cell growth and communications . it has been demonstrated that cnts can be employed in drug delivery systems. the toxicological properties of cnts result strictly correlated with nanotube solubility . in this study we focused our attention on multi-walled cnts (mwcnts) because they are easy to manipulate and we covalently functionalize them in different ways to increase their solubility.. we grew different derivatives of a dendrimer on mwcnts surface with positive charges at the periphery, in order to study the different solubilisation properties and correlate them to the biological activity in gene therapy . slowdown of - β-peptide aggregation by addition of two synthetic biocompatible polymers p. sciortino , r. carrotta , g. cavallaro , d. bulone , p. l. san biagio ibf-cnr, palermo, italy, ctf dept., university of palermo, palermo, italy fibril deposit formation of amyloid β-protein (aβ) in the brain is a hallmark of alzheimer disease (ad). fibrils formation is triggered by molecular conformational changes and protein-protein interactions involving partially unfolded regions of different aβ peptide molecules. increasing evidence suggests that toxicity is linked to diffusible aβ oligomers, which have been found in soluble brain extracts of ad patients, rather than to the insoluble fibres. new therapeutical approach, based on searching molecules capable of regulating the peptide aggregation, is currently developing. here, we study the effects on the aggregation of aβ - peptide of two different synthetic polymers with structure similar to that of a protein (α,β-polyasparthylhydrazide -pahy and α,β-polyasparthylhydrazide-polyethyleneglycol -pahy-peg). static and dynamic light scattering measurements showed that the aggregation kinetic is slowed down by the presence of both polymers. optical microscopy revealed the presence of aggregates of different dimension in all samples. transmission electron microscopy allowed to establish that all aggregates are made of fibers, as confirmed by fluorescence spectroscopy measurements on thioflavine binding. a. smorodchenko , d. sittner , a. rupprecht , j. goyn , a. seiler , a. u. bräuer , e. e. pohl institute of cell biology and neurobiology, charité-universitaetsmedizin, berlin, germany, german federal institute for risk assessment, zebet, berlin, germany ucp is a member of the mitochondrial anion transporter family and one of the three ucps (ucp , ucp and ucp ) associated with the nervous system. in our previous work we have shown that ucp appears in brain at embryonic day (e ). we hypothesized that the ucp expression can be related to neuronal differentiation. to prove this idea we now investigated protein and mrna levels in two different systems: (i) mouse embryonic stem cells of line d (mesc) and (ii) brains from pre-and postnatal mice. ucp was not present in undifferentiated mesc. during differentiation of mesc in neurons the expression of neuronal marker map and ucp started at the same time -as early as on day in culture -and were increasing simultaneously. (ii) the comparative analysis of gene transcripts prepared from whole embryos, brains and different brain regions (neocortex, hippocampus, cerebellum) demonstrated that levels of ucp mrna were increasing from e till e , reached an expression peak between e and postnatal day (p ) and remained constant in adult animals. in contrast, expression of ucp was increasing permanently until birth, whereas ucp expression was invariant in time. our results suggest that ucp contributes to specific neuronal functions. the plant hormone abscisic acid stimulates the proliferation of human hemopoietic progenitors through the second messenger cyclic adp-ribose s. scarfì , c. fresia , c. ferraris , s. bruzzone , f. fruscione , c. usai , m. magnone , m. podestà , l. sturla , l. guida , g. damonte , a. salis , a. de flora , e. zocchi advanced biotechnology center, genova, italy, dept of experimental medicine, biochemistry section, and cebr, university of genova, italy;, institute of biophysics, cnr, genova, italy, stem cell center, s. martino hospital, genova, italy abscisic acid (aba) is a hormone involved in pivotal physiological functions in higher plants. recently, aba was demonstrated to be produced by human granulocytes, β pancreatic cells and mesenchymal stem cells (msc) and to stimulate cell-specific functions. here we show that aba expands human hemopoietic progenitors in vitro, through a cadpr-mediated increase of the intracellular calcium concentration. incubation of cd + cells with micromolar aba also induces transcriptional effects, which include nf-κb nuclear translocation and transcription of cytokines encoding genes. stimulated human msc produce and release aba at concentrations sufficient to exert growth-stimulatory effects on co-cultured cd + cells, as demonstrated by the inhibition of colony growth in the presence of an anti-aba monoclonal antibody. these results provide a remarkable example of conservation of a stress-hormone from plants to humans and identify aba as a new hemopoietic growth factor involved in the cross-talk between hp and msc. t. golovanova, g. b. belostotskaya sechenov institute of evolutionary physiology and biochemistry, russian academy of sciences, saint petersburg, russia a subpopulation of small cells (volume ± µm ) have been found in the neonatal cardiac myocytes culture whereas the same parameter of the remaining cells was ± µm on the st day. in contrast to main part of hypetrofied cardiomyocytes, the small cells were able to proliferate, form colonies and differentiate spontaneously into cardiac myocytes in the culture. on the th day there were slight, slow ( - beats/min), arrhythmic contractions in the centre of colonies. pulsing cells were not united by common contraction and had individual beating profile. on the - th days, the colonies displayed comprehensive contractile activity with the pulsation rate - beats/min and reached beats/min on the d - th day and beats/min on the - th days in the culture. it has been shown that receptors of the surface membrane and sarcoplasmic reticulum of colony cells gradually mature. by estimating the ca + transition under the specific agonists action: acetylcholine, kcl and caffeine, we have detected the activity of basic structural and functional elements of excitation-contraction coupling in contractile cells inside colonies. the small cells ability to proliferate and differentiate under the influence of near mature cardiomyocytes allows us to put forward a hypothesis, that they belong to a category of resident stem cells. changes in the gating properties of na + channels were studied in es-derived neural stem (ns) cells during in vitro neuronal differentiation with the use of the whole-cell and cell-attached variants of patch-clamp technique. ns cells represent a novel stem cell population remaining stable and highly neurogenic over multiple passages.voltage-clamp recordings during neuronal differentiation of ns cells indicated significant changes in the key properties of na + currents. a voltage-gated and tetrodotoxine-sensitive na + current,absent under self-renewal conditions, was first recorded following application of differentiative agents. current density increased with time of exposure to differentiating conditions. whole-cell and single channel analysis revealed that the observed increase in current density was due at least in part to changes in steady-state activation and inactivation properties. namely, half activation potential shifted from - mv to - mv, while half-inactivation potential shifted from - mv to - mv. furthermore, a contribution to the increase in na + current density could also be given by an enhancement in channel expression, as suggested by an augmentation in the number of single channels per patch area, with increasing neuronal differentiation. interestingly, those changes in na + channel activity well correlate with the capability of ns cells to generate action potentials during in vitro neuronal differentiation. a. viale, g. bonizzi, a. cicalese, f. de franco, c. pasi, s. pece, a. orleth, p. di fiore, p. pelicci european institute of oncology, milan, italy we are characterizing the biological differences between normal and transformed scs. scs are defined by their ability to generate more scs, termed self-renewal, and to produce cells that differentiate (asymmetric cell division). scs, however, possess the ability to expand in number (i.e. during development, in adulthood after injury/disease); this increase is not accounted by asymmetric divisions, in which only one daughter cell maintains sc identity. recent findings in invertebrates indicate that scs can also generate daughter cells destined to acquire the same fate (symmetric cell division). on the other hand, sc quiescence is critical to maintain tissue homeostasis after injury. here we show increased symmetric divisions of cscs in breast tumors (due to inactivation of the p tumor suppressor) and dependency of leukemia development on quiescent leukemia scs (due to transcriptional up-regulation of the cell cycle inhibitor p by leukemia-associated fusion proteins). we suggest that sc asymmetric divisions function as a mechanism of tumor suppression, that sc quiescence is critical to the maintenance of the transformed clone and that symmetric divisions of scs permit their geometric expansion. a. uccelli department of neurosciences ophthalmology and genetics, genoa, italy stem cells are considered as a possible source of cells for tissue repair. in this scenario damaged tissues will be reconstructed by newly formed cells with the result of a recovery of lost functions. thus, the rationale for utilizing stem cells for the treatment of neurological diseases such as multiple sclerosis (ms) stemmed from the idea that they differentiate in neural cells regenerating the damaged tissues at the basis of irreversibile disability. however, while multipotential embryonic stem cells may provide in the future an optimal source of cells competent for myelin and axons repair in ms, their use is still far from being exploitable in a clinical setting. moreover, it is widely accepted that adult stem cells may in vitro transdifferentiate in neural cells but recent experimental data have challenged the biological importance of this event in vivo. nevertheless, several experimental studies have provided new evidences supporting the use of adult stem cells derived form different human tissues for the treatment of ms. in the cases of mesenchymal stem cells and neural stem cells current experimental data support an unexpected therapeutic plasticity mediated by diverse paracrine mechanisms including neuroprotection, induction of local neurogenesis and modulation of the immune response. therefore, current experimental and clinical studies support the use of stem cells for the treatment of neurological diseases such as ms. unravelling the structure of the water splitting site of photosynthesis and implications for mechanism of catalysis j. barber imperial college london, u.k. photosystem ii (psii) is a multi-subunit membrane protein complex which catalyses the oxidation of water to molecular oxygen and reducing equivalents. the reaction occurs at a catalytic centre composed of mn ions and a ca ion, is thermodynamically demanding and generates highly oxidised species. unavoidable side reactions cause detrimental effects on the protein environment leading to the rapid turnover of the reaction centre d protein. to understand the mechanisms of water oxidation and d turnover structural information is required. initially the positioning of various protein subunits and their transmembrane helices were determined by electron microscopy. more recently a refined structure of the cyanobacterial psii unit has been elucidated by x-ray crystallography giving details of specific environments of the redox active cofactors. the implications of these structural studies will be discussed in relation to the unique facets of psii function, particularly the water splitting reaction. importantly this new knowledge is providing a blue print for the design of photochemical catalysts which can mimic the photosynthetic water splitting reaction and thus give hope that new technologies will emerge to provide humankind with a sustainable energy supply. photochemically controlled molecular devices and machines v. balzani department of chemistry "g. ciamician", university of bologna, italy the macroscopic concepts of a device and a machine can be extended to the molecular level. molecular-level devices and machines operate via electronic and/or nuclear rearrangements and, like macroscopic devices and machines, they need energy to operate and signals to communicate with the operator. the extension of the concepts of a device and a machine to the molecular level is of interest not only for basic research, but also for the growth of nanoscience and the development of nanotechnology. if a molecular device or machine has to work by inputs of chemical energy, it will need addition of fresh reactants ("fuel") at any step of its working cycle, with the concomitant formation of waste products that compromise the operation. currently there is an increasing interest in the use of light to power molecular devices and machines. the lecture will illustrate examples of recent achievements [ , , ] , which include molecular wires, switches, plug-socket systems, extension cables, antennas, and light powered nanomotors. biophysics laboratory, institute of botany, azerbaijan national academy of sciences, baku, azerbaijan it is well known that fast component of induction curves of millisecond delayed chlorophyll fluorescence (ms-df) originates via radiative recombination of reaction center with product on the donor side of psii. in view of our previous data (j. photochemistry and photobiology b: biology , ) it was shown that partners for radiative recombination of p q a − localized as a hole on the donor side of psii. depending on ph this hole might be on the camn -cluster or on y z and their recombination with p q a − can be monitored by the ms-df fast component. for analysis of site of damages, photoinhibition of psii particles from spinach at different ph was monitored by ms-df. during photoinhibition of psii ( µmol photons m − s − ) the fast component of ms-df was shown to be more stable at ph . and ratio of the fast component to the steady-state level of ms-df was approximately constant, while at ph . the fast component essentially decreased, the steady-state level increased and ratio of these components rapidly went down. it is possible concluded that strong light damaged of recombination reaction with in the presence ppbq -artificial electron acceptor -the protection of psii from photoinhibition has been observed only at acidic condition. stress factors such as heavy metals, strong light and low temperature were investigated by measurement of transient pictures of ms-df in intact plants, isolated chloroplasts and pure psii particles. the heavy metals action on psii was investigated on wheat seedlings. the targets for toxic action of al, mn and co ions were found to be a q a -q b acceptor side of psii. the damage site for cd + may be partners for recombination with p + -depend on of medium ph -either y z or camn -cluster. investigated ions (mn + , al + , cd + and co + ) have lead to reduction of chlorophyllprotein complexes pigment fund and cd + and co + destroyed also an apoproteins, especially of psii. the photoinhibition of psii was investigated in intact leaves of barley and maize seedlings at low and normal temperature. a very sharp reduction of intensity of ms-df second fast component, possibly y z * p ÿq a − radiative recombination of maize seedlings was observed even after short illumination by strong light at • c. ph dependence photoinhibition of chloroplasts and pure psii particles have shown that strong light damaged of camn -cluster in great degree than y z . hydrogenases are considered as potential energy sources. in particular, the ability of the green alga c . reinhardtii to reduce protons to hydrogen gas upon illumination by means of a [fefe]-hydrogenase is a phenomenon of great scientific interest, as it would need only light and water to generate energy. however, the catalytic activity is strongly inhibited by the o produced during photosynthesis; furthermore, the protein is expressed at very low levels, only in conditions of strict anaerobiosis. this mutually exclusive nature of o and h photoproduction represents a crucial problem in the development of h bio-production. the study of the structurefunction relationship of [fefe] hydrogenases, which would help to clarify the molecular mechanisms underlying both h production and o sensitivity, requires the characterization of purified native and modified proteins, which can be obtained by site-directed mutagenesis. we expressed the algal hydrogenase in the cyanobacterium synechocystis sp. pcc , which holds a bidirectional [nife]-hydrogenase with a well known maturation system ( ) . we obtained two constructs to stably transform synechocystis, enabling it to express the c. reinhardtii hydrogenase in an active form. this suggests that the [nife]-hydrogenase maturation pathway is able to drive the biosynthesis of functional [fefe] enzymes. these data open new perspectives about the indispensable presence of hyde, hydf and hydg auxiliary proteins ( , ) to obtain a correctly folded [fefe]-hydrogenase. the a low energy monomeric chlorophyll of cp to investigate the role of low energy spectral forms in energy transfer among chlorophyll protein complexes, we characterized one of the redmost chlorophylls of psii antenna complexes, the a of cp . pigment stoichiometry of the wild type (cp wt) and mutant lacking the a binding residue (cp a ) reconstituted complexes confirmed the loss of a chlorophyll a. to exclude the possibility of a marked excitonic interaction of the a with other chlorophylls, we quantified the decrease in dipole strength after mutagenesis as a function of the frequency and performed a second derivative analysis of the difference absorption spectrum cp wt minus cp a . these data, along with circular dichroism spectra of both complexes, support the idea that the a is a monomer in cp , in disagreement with recent suggestion of involvement in an excitonic dimer (mozzo et al., bba ) . chla a reorganization energy and inhomogeneous broadening were then determined through a thermal broadening analysis in the - k temperature range. these parameters allowed us to calculate the förster overlap integral of the homogeneously broadened a bandshape, a fundamental factor for energy transfer rate estimations between chromophores. a comparison with foi of chlorophyll in solution suggests that chla a may connect cp complex to psii core favoring energy transfer from cp towards other inner antenna low energy chlorophylls. in oxygenic photosynthetic organisms, reaction centre complexes catalyze electron transport from water to co using light energy absorbed by tetrapyrrole pigments bound to so called "antenna proteins". a major challenge in performing this type of photosynthesis consists on the difficulty of operating "one electron" transport through a multi-step pathway in the chloroplast environment which is the source of oxygen of biosphere. in fact, synthesis of ros, mainly singlet oxygen and superoxide anion and consequent photoinhibition is an intrinsically unavoidable consequence of photosynthesis that must be prevented and/or controlled in order to avoid photodestruction and death. mechanisms involved include chlorophyll (chl) triplet quenching, ros scavenging and controlled heat dissipation of excess chl singlet excited states. the latter process has been studied for over years with little success in elucidating its mechanism. reverse genomics and ultrafast-spectroscopy led to the proposal that the transient formation of carotenoid radical cations, followed by charge recombination might be the underlying mechanism of energy dissipation while three proteins belonging to the lhc superfamily could be the site hosting the reaction. probing the dark cycle to reveal regulatory networks controlling photosynthetic efficiency the food, fibre and fuel needs of an ever-increasing population are one of the challenges facing current society. higher plant photosynthetic efficiency is ∼ % whereas the theoretical maximum is thought to be ∼ - % giving potential for great improvements. it is not however clear where in the photosynthetic process the additional losses are occurring. we are adopting a systems biology approach to reveal the regulatory networks that control photosynthetic efficiency, the challenge being to make quantitative measurements of the input parameters to models of the network of photosynthetic reactions, and also to identify missing physical parameters and processes. the aim being to understand how they interact with other key physiological pathways and what impact they hold over photosynthetic efficiency. we report initial results in this poster from experiments using conventional and novel proteomic methods. e.g. an optical technique based on a non-linear dir method is being developed for proteomic and metabolomic analysis. unlike many conventional proteomic methods, which provide information on the relative levels of various proteins and metabolites, the viability of the dir method as a quantitative, sensitive and high throughput proteomics platform has recently been demonstrated. in collaboration with klug this project will employ dir as a both a proteomic and metabolomic tool. t. morosinotto , p. arnoux , g. saga , g. m. giacometti , r. bassi , d. pignol dip. di biologia, padova, italy, cea, cadarache, france, dip. di biotecnologie, verona, italy violaxanthin de-epoxidase (vde) is the enzyme responsible for zeaxanthin (z) production. the synthesis of this carotenoid in plants exposed to high light conditions is an important photoprotection mechanism, enhancing excess energy dissipation and reactive oxygen species scavenging. the inactive enzyme is normally soluble but, upon activation by low ph, it binds to the thylakoids membrane, where its substrate is found. we present here the first structural data on this enzyme obtained at both acidic and neutral ph. at neutral ph, vde is monomeric with its active site occluded in the lipocalin barrel. upon acidification, the barrel opens up resulting in a functional dimerization of the enzyme. the channel linking the two active sites of the dimer can harbour the entire carotenoid substrate and thus allow the parallel de-epoxidation of the two violaxanthin β-ionone rings, making vde an elegant example of the adaptation of an asymmetric enzyme to its symmetric substrate. structural data opened the possibility to investigate deeper this enzyme and further work allowed the identification of its active site, the protein domains responsible of its membrane association as well as key residues involved in the ph dependent conformational change. hydrogen is considered the fuel of the future if produced from sun light-driven water splitting. a hydrogen economy based on genetically modified organisms, immobilized enzymes or biomimetic synthetic catalysts requires a profound knowledge of the structure and function of the respective enzymes in nature. light-induced water splitting is performed by a tetranuclear manganese cluster located in photosystem (ps) ii of oxygenic photosynthesis. the protons generated by ps ii can be converted to molecular hydrogen by the enzyme hydrogenase, which is for example found in green algae and cyanobacteria. [nife]-and [fefe]-hydrogenases are the two main classes of this enzyme. they contain bridged binuclear transition metal cores, which are tuned by a special ligand environment to efficiently convert protons to hydrogen -or vice versa -via a heterolytic mechanism. rhodobacter sphaeroides strain a- isolated from mineral springs in armenia produces h with high rate in anaerobic conditions under light. in our previous work the inhibition of h production by n,n' -dicyclohexylcarbodiimide (dccd), the f f -atpase inhibitor, was shown, and dccd-inhibited atpase activity was determined. therefore it is possible to admit a role of this atpase in h production by r. sphaeroides; otherwise dccd inhibition of hydrogenase is not ruled out. in order to examine the mediatory role of proton-motive force (pmf) or proton atpase in this process transmembrane electrical potential (∆Ψ) and ∆ph are determined and the atpase activity is studied in r. sphaeroides grown under light. pmf was determined under anaerobic conditions in the dark. at ph . the ∆Ψ was of - mv and the reversed ∆ph was + mv, resulting in the pmf of - mv. but ∆Ψ was not affected by dccd. moreover, adenine nucleotide phosphates (anp) content is essential for cell functioning and may result with atpase activity. the percentage of atp was calculated from the total quantity of anp in whole cells, it was . %. a relatively high concentration of adp (∼ %) and amp (∼ %) and accordingly low energetic charge were noted; these might be indicative for the atpase activity, a further study is required. relationship between h production and atpase activity by rh. sphaeroides is suggested; possible mechanisms are discussed. identification of the sites of chlorophyll triplet quenching in relation to the structure of lhc-ii from higher plants. evidence the chl a molecules involved in the triplet-triplet energy transfer to the central luteins in trimeric lhc-ii are identified by time-resolved and pulse epr techniques. the concept of spin conservation during triplet-triplet energy transfer is exploited. the sites with the highest probability to form triplet states, which are quenched by the central luteins, are chl and chl . unquenched chl triplet states are also produced by photo-excitation in the lhc-ii complex. putative sites of these triplet states are chl , chl , chla and chl , since they do not contribute to the formation of the observed carotenoid triplet states. fluorescence lifetime spectrum of the plant photosystem ii core complex the photosystem ii kinetic model (diffusion or trap-limited) is still much debated. there is discussion about whether energy transfer from the core antenna (cp and cp ) to the reaction center complex (d -d -cyt b ) is rate-limiting (transfer to trap-limited). this study investigates this problem in isolated core particles by exploiting the different optical properties of the core antenna and the reaction center complex near nm, due to p and an isoenergetic pheophytin. this was used as a marker feature for the reaction center complex. if the transfer to the trap-limited model were correct, assuming excited-state thermalization, the specific reaction center fluorescence decay lifetime should be shorter near nm, where there is reaction center complex specificity, than at the other emission wavelengths. such a selective reaction center feature was not observed in fluorescence decay measurements. at the experimental resolution used here, we conclude that the traplimited energy transfer to the reaction center could, at the most, be % limiting. thus, the transfer to the trap-limited model is not supported. photosynthetic apparatus response under heat stress n. pshybytko , i. divak , l. kabashnikova , e. lysenko institute of biophysics and cell engineering, national academy of sciences of belarus, belarus, institute of plant physiology, russian academy of sciences, russia the mechanisms of psii thermoinactivation and adaptation under high temperature impact and participation of hydrogen peroxide at these processes were studied. the twice suppression of oxygen evolving activity of thylakoids with simultaneous decrease in d protein content and the release of extrinsic kda polypeptide from woc after - min heating at o c were registered. using inhibitor analysis it was shown that thermoinduced degradation of d protein after min heating occurred by proteases. the participation of ftsh protease in thermoinduced d protein degradation was observed. level of transcription of psba gene in chloroplast was raised after min heating and was decreased through h. the content of hydrogen peroxide was increased three times after min of heating and was decreased to normal level through h and was raised after . h again. it is interesting that level of peroxidation lipids products was increased after . h heating only. received data indicated that hydrogen peroxide is signal molecular at the photosynthetic apparatus under heat stress. during heating the inactivation of woc and d protein is occurred. as result the h o is generated. hydrogen peroxide as signal molecule activates transcription of psba. turnover of psii is occurred. more long heating induces degradation of proteins and lipids and h o represents as the destructive agent. a. nurisso , m. a. morando , f. j. cañada , j. j. barbero , a. imberty cermav-cnrs, grenoble, france, centro de investigaciones biológicas, madrid, spain the mechanism of symbiosis between legume plants and rhizobial bacteria is a relevant topic of interest since it is at the basis of the nitrogen fixation process. this mechanism is strictly related to the production of lipochitooligosaccharides, nod factors (nf), which allow the bacterial invasion into the legume host roots. in medicago truncatula, the recognition of nf from the symbiont sinorhizobium meliloti requires the nfp gene, able to encode a lysm-motif receptor-like kinase. the extracellular part of this protein is characterized by three lysm motifs: only one of them seems to be involved in the nf recognition. herein, we report an in silico investigation of different structural aspects of this process. first of all, the conformational behaviors of a new generation of nf analogues were elucidated by mds simulation. then, a homology model of the lysm domain from m. truncatula was proposed and compared with a model of lysm domain from pisum sativum: docking calculations of natural nf identified a common binding site in which the carbohydrate portion is the main responsible of the binding. both studies provide support for the idea that the carbohydrate part of nf plays a key role in the interaction with lysm domains, while the lipid moiety modulates ligand specificity probably interacting with a potential second receptor. interaction between frutalin with biomembrane models and its correlation with cell adhesion property frutalin is a homotetrameric lectin d-galactose (d-gal) binding that activates natural killer cells in vitro and leukocyte migration in vivo, being a potent lymphocyte stimulator. in this study we investigated the interaction of frutalin with different phospho/glyco-lipids using langmuir monolayers as biomembrane mimetic system. the results attest the specificity of the protein for the carbohydrate d-gal when attached to the biomembrane model. the adsorption kinetics for frutalin to the mixed monolayers containing glycolipids showed that the interaction depends on the presence of charged groups and on the position of the d-gal on the polar head of the glycolipid. using brewster angle microscopy (bam), we investigated the morphology of the interface for the binary mixtures containing galcer, where small domains were formed at high lipid packing, suggesting that frutalin can induce the formation of likelipid rafts domains in vitro. the results obtained with the membrane models were associated with those from fibroblast adhesion induction. the cell adhesion promoted by frutalin is in accordance with the results observed in langmuir monolayers, which probes the specificity of the interaction between the lectin and d-gal on cell-membrane surfaces. based on these results, frutalin can be considered as a promise biotechnological tool to actuate in tissue engineering regeneration. supported by fapesp, cnpq and capes aggregation and glycation processes of proteins are of peculiar interest for several scientific fields. serum albumins are widely studied proteins for their ability to self-assemble in aggregates and also to undergo to non enzymatic glycosylation in cases of diabetes. in this work we report a study on thermal aggregation of glycated bovine serum albumin (bsa) prepared with different concentrations of glucose at ph . . increasing concentration of sugar modulates the effect of different glycation levels on the protein aggregation. fluorescence spectroscopy, ftir absorption, static and dynamic light scattering are used to follow the time evolution of the aggregation process and of protein conformational changes. conformational changes of secondary and tertiary structures are measured by ftir absorption; the kinetics of amide i, amide ii and amide ii' bands are monitored. the kinetics of tryptophans fluorescence give complementary information on the tertiary structure changes and on the polarity modification of the fluorophores environment. the aggregates growth is studied by dynamic light scattering measurements and rayleigh scattering peak. the results show that the partial unfolding of the protein is not affected by the glycation, while the presence of increasing amounts of glycated molecules progressively inhibits the aggregates formation. solvent occupancy analysis in ligand structure prediction: the case of galectin- s. di lella, m. a. martí, d. a. estrin inquimae-conicet, universidad de buenos aires, argentina formation of protein ligand complexes is a fundamental phenomenum in biochemistry. during the process, significant solvent reorganization is produced along the contact surface. using md simulations in explicit solvent combined with statistical mechanics analysis, thermodynamic properties of water molecules around proteins can be computed and analyzed in a comparative view. based on this idea, we developed a set of analysis tools to link solvation with ligand binding in a key carbohydrate binding protein, human galectin- (hgal- ). specifically, we defined water sites (ws) in terms of the thermodynamic properties of water molecules strongly bound to protein surfaces. we then succesfully extended the analysis of the role of the surface associated water molecules in the ligand binding and recognition process to many other carbohydrate binding proteins. our results show that the probability of finding water molecules inside the ws, p(v), with respect to the bulk density is directly correlated to the likeliness of finding an oxhydril group of the ligand in the protein-ligand complex. this information can be used to predict possible complex structures when unavailable, and suggest addition of ohcontaining functional groups to displace water from high p(v) ws to enhance drug, specially glycomimetic-drugs, protein affinity and/or specificity. glyconanoparticles: nano-biomaterials for application in biotechnology and biomedicine s. penadés laboratory of glyconanotecnology, cicbiomagune -ciber-bbn, san sebastian, spain to study and intervene in carbohydrate interactions our laboratory has developed a chemical strategy (glyconanotechnology) to produce sugar functionalized gold nanoclusters with multivalent carbohydrate display (glyconanoparticles, gnps). by combining these tools with biophysical and analytic surface techniques (itc, afm, tem, spr) we have demonstrated and evaluated ca + -mediated carbohydratecarbohydrate interactions involved in cell adhesion processes. the gnp system complements other currently available multivalent systems incorporating carbohydrates and presents some advantages as: ) exceptionally small core size; ) high stability in water and physiological buffers; and ) multivalency and multifunctionality with control over ligands number. specific gnps have been applied to the inhibition of melanoma metastasis in mice and as inhibitors of hiv-trans infection. magnetic gnps have also been used in cellular labelling and imaging as probes for mri. in this lecture, the glyconanotechnology strategy will be presented and some application will be highlighted. -glycobiophysics - expanded ataxin- induces membrane mechanical instability: evidences from model systems and cells c. canale , s. averaimo , d. pesci , d. paulis , v. fortunati , a. gliozzi , m. mazzanti , c. jodice , a. relini iit, genoa, italy, university of milan, italy, university of tor vergata, rome, italy, university of genoa, italy spinocerebellar ataxia type is a neurodegenerative disorder involving the expansion of a polyglutamine stretch beyond a threshold in the protein ataxin- , with intracellular deposition of amyloid aggregates. ataxin- variants with polyglutamine stretches of pathological length are not associated to disease when the stretch is interrupted by histidines. mounting evidence suggests that ataxin- aggregates interact with the nuclear membrane. to get insight into the mechanisms leading to neurological disorders, we studied model lipid membranes containing an expanded pathological form of ataxin- or expanded normal forms interrupted by histidines. electrical measurements on planar bilayers showed the occurrence of current steps, much larger for the pathological form, indicating the formation of pore-like structures. atomic force microscopy measurements showed that the longer the polyglutamine tract, the smaller was the force required to penetrate the bilayer with the afm tip. the smallest penetration force, and then the strongest membrane destabilization, was observed for membranes containing the expanded pathological form. experiments on cos cells provided evidence that pathological protein aggregates damage the nuclear membrane eventually causing cell autophagy. modification by dopamine adducts links αsynuclein to oxidative stress in parkinson disease m. bisaglia , e. greggio , l. tosatto , f. munari , i. tessari , p. polverino de laureto , s. mammi , m. r. cookson , l. bubacco university of padova, italy, nih, bethesda, md, usa oxidative stress has been proposed to be involved in the pathogenesis of parkinson disease (pd). a plausible source of oxidative stress in nigral dopaminergic neurons is the redox reactions that specifically involve dopamine (da) and produce various toxic molecules, i.e., free radicals and quinone species. α-synuclein (αsyn), a small protein found in lewy bodies characteristic of pd, is also thought to be involved in the pathogenesis of pd. to investigate the possibility of a synergistic role of oxidative stress and αsyn in pd, we analyzed the modulation of da toxicity by αsyn overexpression in dopaminergic human neuroblastoma cells. our results indicate that the increased expression of αsyn enhances the cellular toxicity induced by the accumulation of intracellular da. we then correlated our results with the structural modifications induced by the oxidation products of da on αsyn by studying two potential pathways for the oxidative chemistry associated with da. the first one is the auto-oxidation reaction which leads to the concomitant formation of radical and quinone species. the second is the enzymatic oxidation, mediated by tyrosinase, which leads only to the accumulation of quinones. our data suggest a link between da and αsyn in the progression of pd and provide novel insights on both the mechanisms involved in the oxidative chemistry and the aggregation properties of αsyn. dynamic light scattering aggregation studies of aβ ( - ) peptide s. bertini, r. beretta, d. gaudesi, a. naggi, g. torri ronzoni institute, milan, italy heparan sulfate (hs) proteoglycans play a role in formation of amyloid plaques by facilitating formation of amyloid aggregates. heparins and low-molecular weight heparins which mimic hs sequences could interact with amyloid peptides putatively involved in neurodegenerative processes. the aim of this work is to study the influence of chain length and structure of heparin oligosaccharides on the aggregation of amyloid. to this purpose, the interaction with amyloid peptide aβ − with a number of heparin/heparin oligosaccharides had been investigated using dynamic light scattering (dls) which permits to determine the size and the stability of molecular aggregates in solution. as indexes of aggregation in solution, two parameters were chosen, i.e., the area under the autocorrelation function (af) and the average hydrodynamic ratio (rh). we evaluated the aggregation kinetic of different preparations of aβ and the ph of solubilization. a clear indication was obtained for the trend of aggregation of the peptide aβ − alone and in the presence of oligosaccharides. the size of the aggregate in the presence of the tetrasaccharide is definitely lower than for the peptide alone. the size of the aggregates increases with increasing size of the oligosaccharides. in fact, the octasaccharide promotes further aggregation. it is crucial for pharmaceutical protein formulations to have low aggregate content. using the monoclonal igg antibody rituximab as a model system, we have studied the mechanisms by which antibodies aggregate at physiological ph when incubated at - • c. light scattering showed two coupled stages: an initial fast stage followed by several hours of exponential growth of the scattered intensity. data analysis showed the fast formation of a large species, which subsequently increased in size. the aggregate number density had a maximum suggesting that aggregates increase in size by coagulation. the analysis also predicted the actual underlying increase in aggregate mass to be linear and reach saturation. this was confirmed by size-exclusion chromatography of incubated samples. in an arrhenius plot the activation energy of the first stage was similar to the unfolding energy of the ch domain, suggesting a pivotal role of this domain in the aggregation process. cd and fluorescence showed only minor structural changes in the temperature interval studied. we conclude that coagulation is the main mechanism driving rituximab aggregation and that aggregation is due to small structural changes. -condensed colloidal phase in biology: from proteins crystals to amyloid fibrils -abstracts solid-state nmr structural studies of alzheimer's disease amyloid β( - ) in lipid bilayers j. d. gehman , a. k. mehta , f. separovic school of chemistry, bio institute, university of melbourne, vic, australia, department of chemistry, emory university, dickey dr., atlanta, ga, usa amyloid-β peptides are believed to cause loss of nerve cell function in individuals suffering from alzheimer's disease, where evidence suggests that interaction with the cell membrane correlates strongly with cytotoxicity. previous studies report a range of different but plausible structures, which depend on the molecular environment of the peptide. this sensitivity to sample preparation suggests that the structure relevant to disease is found in lipid bilayer membranes. while model membrane vesicles are too large to be studied by conventional solution nmr, solid-state nmr is one of the few technologies available to study such systems. we present recent measurements which suggest a novel peptide structure in a lipid bilayer environment: (i) rotational-echo double-resonance (redor) distance measurements between selectively enriched c-carbonyl and n-amide positions constrain dihedral angles of intervening residues and suggest that at least part of the aβ( - ) peptide folds into a β-sheet like conformation, in contrast to the helical and coiled structures in previous reports; and (ii) double quantum filtered draws measurements indicate that the extended strand does not assemble into an in-register parallel sheet as reported for amyloid fibrils. a mutation in app gene with dominant-negative effect on amyloidogenesis: a light scattering study e. del favero , g. di fede , f. tagliavini , m. salmona , l. cantù university of milan, segrate, italy, "carlo besta" national neurological institute, milan, italy, istituto di ricerche farmacologiche "mario negri", milan, italy we studied the effect of a single-point mutation (a v) on the aggregative properties of alzheimer's peptides aβ − . by laser light scattering it was possible to follow the early stages of aggregation of aβ − wt and aβ − mut (within hours after sample preparation), when intermediates in the aggregation pathway, rather than the mature insoluble fibrils, are formed. results show that both the kinetics and the extent of aggregation are strongly enhanced by the mutation. on the reverse, coincubation of mutated and wild-type peptides slows down the aggregation process and results in aβ aggregates that are much more unstable against dilution. it is evident that the interaction between mutant and wild-type aβ − interferes with nucleation or nucleation-dependent polymerization, hindering amyloidogenesis. these results account for the highly amyloidogenic effect of the mutation in vitro, if alone, reversely reduced by the mutated-wild type mix. also a clinical case exists that constitutes the in-vivo evidence of these two opposing effects. this finding may offer grounds for the development of therapeutic strategies, based on modified aβ peptides or peptido-mimetic compounds, for the potential treatment of alzheimer's disease. the collagen i is the major structural protein of the body and it is found organized in a hierarchical manner in the extra-cellular matrix of several tissues (bone, tendon, cornea and sclera, etc..). it is responsible for their specific architecture. it is already known that collagen i is capable of forming cholesteric liquid crystalline phases that once stabilized by a ph increase, lead to collagen matrices that mimic the organization of the organic matrix found in bone [ ] . in the present work, we analyze physico-chemical ways to modulate long range organizations obtained in this liquid state. we study the effect of collagen concentration, ph ( . / . ) and acids (hydrochloride acid / acetic acid) over the liquid crystalline order. in a original approach, correlation of collagen endofluorescence and shg signals has enabled us to quantify cholesteric pitch and phase transition as a function of collagen concentration within a gradient. cholesteric pitch have proved to be very responsive to physico-chemical conditions. indeed, the results allowed us to quantify a i / cholesteric transition as a subsequent transition from cholesteric to a phase hexagonal or colummar. prion diseases are deadly neurodegenerative diseases affecting human and mammalian species. according to the 'protein-only' hypothesis, the key event in the pathogenesis is the conversion of the α-helix-rich monomer (prp c ) into a polymeric β-sheet-rich pathogenic conformer (prp sc ). we have used a combination of biophysical techniques with molecular dynamics simulations (md) to elucidate the molecular mechanisms of prp c unfolding and polymerization. under well established conditions, three β-sheet-rich soluble oligomers were generated from the partial unfolding of the monomer, which were found to form in parallel. to obtain a deeper insight into the molecular events, doublecystein mutants were designed, thus 'locking' different regions of prp. single mutations were also performed, which affected dramatically and selectively the prp oligomerization pathway. furthermore, we have now identified the minimal region that leads to the same oligomerization profile as the full-length prp, namely, h h . the existence of at least three distinct oligomerization pathways and the effect of single mutations reveal the conformational diversity of prp and a possible relationship with prion strain phenomena. the identification of domains involved in the conversion process may lead to a better understanding of the effect of mutations or gene polymorphism on the evolution of prion pathology. investigating toxic protein nanoclusters and their interactions with living cells s. nag, b. sahoo, a. bandyopadhyay, c. muralidharan, r. abhyankar, s. gurav, s. maiti tata institute of fundamental research, homi bhabha road, colaba, mumbai , india amyloid protein aggregation is responsible for many neurodegenerative diseases, such as alzheimer's and parkinson's. it appears that quasi-stable small soluble aggregates are the key to amyloid toxicity. though amyloid aggregation appears to be a nucleation mediated process, simple nucleation theory is unable to explain the stability of these intermediate species. we investigate this issue with fluorescence correlation spectroscopy in solution and on cell membranes. our initial data, varying the ph and the ionic strength of the solution, show that a charged colloid model can explain the overall stability of these species. in addition, this understanding suggests possible ways of modulating the stability of these aggregates in solution. some of these strategies successfully decimate the stable aggregate population, and also reduce the toxicity of amyloid beta to cultured neurons. afm studies of artificial amyloid fibrils and filaments p. mesquida , a. kurtossy , c. macphee department of mechanical engineering, king's college london, london, u.k., school of physics, university of edinburgh, edinburgh, u.k. amyloid fibrils are beta-sheet-rich superstructures of peptides or proteins. although these aggregates have first been found in connection with protein-misfolding diseases, such as alzheimer's or parkinson's disease, there is evidence that the ability to form fibrils is a generic property of any polypeptide rather than a result of specific, disease-related amino-acid sequences. fibrils can easily be formed in vitro from nondisease-related proteins and even from synthetic peptides. these artificial fibrils can thus serve as model systems to investigate the biophysical properties of amyloid fibrils. the system presented here is built from the artificial peptide ttr − , which contains amino-acids and which forms well-defined nanorods of ca nm diameter. we present atomic force microscopy (afm) results of their inner morphology by chemically dissecting the rods into their constituent filaments without completely breaking up the aggregates. in contrast to the stiff rods, their constituent filaments of ca - nm diameter were much more flexible. this shows that the particular way filaments are arranged around each other can massively change the mechanical rigidity of the resulting structures in amyloid fibrils, which could be one cause of the different strains in amyloid diseases. the formation of insulin fibrils is characterized by an initial apparent lag-phase, related to the formation of oligomers, protofibrils and aggregation nuclei. afterwards, the aggregation proceeds via fibril elongation, thickening and/or flocculation, and eventual gelation. here, we focus on the formation of such a gel, made of insulin amyloid fibrils, upon incubation at high temperature and low ph. by light scattering and rheological techniques, we monitor the development of the structural, dynamical and mechanical properties of fibrillar aggregates, up to the dynamic arrest of the sample and to the appearance of a non-ergodic behaviour, which marks the onset of gelation. our experiments were able to reveal the structural details hidden in the apparent lag-phase, displaying the slow fibril nucleation and elongation. this initial stage is followed by the known exponential growth of structures of different sizes. these two kinetic stages of structural growth are mirrored by the kinetics of the viscoelastic properties and, in particular, by the growth of the elastic modulus. our results show that the appearance of a noteworthy elastic network, is associated with the initial fibril nucleation and elongation more than with the formation of large structures, which causes the eventual gelation. capturing the initial events of in-cubo crystallization of membrane proteins c. v. kulkarni , o. ces , s. iwata , r. h. templer chemical biology centre and department of chemistry, imperial college london, united kingdom, division of molecular biosciences, imperial college london, united kingdom, institute für chemie, heinrichstrasse- , university of graz, austria lipid based methods for the crystallization of membrane proteins including in-cubo crystallization are becoming popular in recent times. however, a complete understanding of the basic principles behind these methodologies is still elusive. the crystallization of membrane proteins in the lipid cubic phases involves following major steps: removal of membrane proteins from the native membrane using detergentlike molecules, followed by their insertion and equilibration into a lipid bilayer. the crystallization itself commences with precipitant induced osmotic dehydration which in turn stimulates the nucleation that subsequently leads to a crystal growth. using time-resolved x-ray scattering (saxs) and ultraviolet spectroscopy we have been able to gain new insights into the mechanism behind protein insertion into these complex three dimensional lipid structures including information on the timescales of protein folding relative to crystal growth and the effect of protein insertion on the morphology of the surrounding lipid matrix. several proteins can form amyloid fibrils under given environmental or thermodynamic conditions that affect their native conformation. lysozyme forms amyloid fibrils upon incubation of solutions at acid ph and at about • c for a few days. differential scanning calorimetry experiments confirm that lysozyme is mainly unfolded above • c at ph . we monitored the growth of aggregate size by dynamic light scattering for some days at different temperatures. our results show that the fibrillogenesis is characterized by an initial apparent lag phase and a subsequent growth with quadratic dependence upon time of the scattering intensity. this behaviour recalls a simple kinetic model of nucleation and elongation, with nuclei in equilibrium with monomers. at the end of the incubation at high temperature, we collected atomic force microscopy images which show fibrils with a diameter of a few tens of nanometers and a length of a few microns, characterized by a periodicity along the elongation axis. interestingly, the fibrils morphology exhibits no branching or thickening. this is consistent with the non-exponential growth observed in light scattering experiments. in order to elicit the role of repulsive electrostatic interaction in protein unfolding and self-assembly we extended our study at different ph. in this work we used afm to follow the amyloidogenesis pathway of transthyretin (ttr) to form protofilaments. single-molecule force spectroscopy (smfs) of native ttr and protofilaments were also compared in order to evaluate dynamic and structural differences. we observed that this pathway proceeds through the formation of transient amorphous aggregates, followed by the occurrence of annular oligomers. although implicated in cytoxicity, the role of such oligomers within the amyloidogenesis pathway is poorly understood. we show that the annular species display a tendency to stack, forming tubular-like structures that precede the formation of protofilaments. the protofilament height and pitch resemble those of ttr amyloid reported in previous structural studies. upon solvent exchange, we also observed protofilament disassembly that revealed structures reminiscent of the initial ttr annular oligomers. smfs of protofilaments showed a time-dependent increase in the length of the manipulated structures, suggesting that associations between monomers stabilize with time. force spectra of native ttr and protofilaments contained transitions spaced by ∼ nm, indicative of sequential unfolding of individual β-strands. based on these results a model of ttr protofilament assembly is proposed. ability to undergo amyloid aggregation is affected by protein size c. parrini , h. ramshini , f. chiti , a. relini physics department, university of genoa, italy, biochemical sciences department, university of florence, italy short polypeptide chains, typically between and residues, are generally involved with the conversion from the native state into insoluble fibrillar aggregates. the low prevalence of large proteins is disproportionate with their high occurrence in the human proteome. in order to explore the propensity of large proteins to form amyloid-like fibrils, the -residue hexokinase-b from saccharomyces cerevisiae (yhkb) has been induced to aggregate under two separate conditions, at low ph in the presence of salts and at ph . in the presence of trifluoroethanol. such conditions are among the most promising to form amyloid-like fibrils by normally globular proteins. under both conditions yhkb aggregates very rapidly into species with significant β-sheet structure, as detected by circular dichroism, and a weak thioflavin t and congo red binding. atomic force microscopy revealed globular aggregates eventually clustering into large amorphous aggregates at low ph, while in the presence of trifluoroethanol ribbon-like structures with distinct morphology from typical amyloid fibrils were observed. they had irregular width, no twist, and were connected by thinner fibril segments perpendicular to them. in general, yhkb aggregates displayed an unusual softness, as they were very easily perturbed by the afm tip. these results suggest that inability to form amyloid fibrils may prevent large proteins from being associated with protein deposition diseases. the initial stage of proteins aggregation leading to amyloid fibrils: a saxs study m. g. ortore , f. spinozzi , f. carsughi , t. narayanan , g. irace , s. vilasi , p. mariani dip. saifet, univ. pol., ancona, italy, esrf, grenoble, france, dip. biochim. biofis., ii univ., napoli, italy under some conditions, a protein converts from its soluble form into highly ordered aggregates called amyloid fibrils, which are associated with many human diseases. in order to tackle the prevention and treatment of these diseases, we need to understand the mechanism of the pathological aggregation of proteins. in vitro amyloid fibril formation is preceded by the formation of metastable non fibrillar forms, which are responsible for cytotoxicity underlying neurodegeneration. the molecular mechanisms leading proteins into prefibrillar aggregates are still unclear. we present a saxs study performed at id beamline of esrf on the apomyoglobin mutant w fw f, which at physiological ph, firstly aggregates in prefibrillar forms that are cytotoxic and then forms amyloid fibrils. the first stages of w fw f oligomerization are induced by a ph jump. data show that big changes in w fw f in solution happen in less than ms. singular value decomposition (svd) of the data yields a set of functions, from which all the scattering curves can be reproduced. the major result of our study is the determination of the presence of different oligomers in each step of the process. hence, time-resolved saxs experiments together with the estimation of different oligomers via svd method, can be a new and useful approach to investigate the first stages of amyloidogenesis. -condensed colloidal phase in biology: from proteins crystals to amyloid fibrils - study of structure/toxicity relationship of amyloids by infrared spectroscopy h. p. ta amyloid diseases (alzheimer, parkinson, type ii diabetes, prion) correlate with protein aggregation. all the proteins associated with these pathologies aggregate into amyloid fibrils with a common ß-cross structure. according to many in vitro studies, the toxicity of various amyloids seems to be linked to some intermediates formed during the aggregation pathway and to their interaction with membranes. our aim is to understand the link between structure and toxicity of amyloids by studying their interaction with model membranes. for this, we use as an amyloid model, the prion-forming domain pfd - (wild type wt) of het-s, a prion protein of the fungal p. anserina. when expressed in yeast, wt is not toxic whereas one of these mutants, m is toxic. in vitro, m forms very unusual short amyloid fibers contrary to wt which polymerizes as long fibers. furthermore, atr spectra have shown that m is essentially assembled into mixed parallel and anti-parallel ß-sheets whereas wt displays a predominant parallel organization. we are currently studying the interaction between wt or m with lipid langmuir film by polarized modulation-infrared reflexion absorption spectroscopy (pm-irras). this technique allows determining the secondary structure of proteins and their orientation in the membrane. the study of interactions with different charged phospholipids is following. a. stirpe, m. pantusa, r. bartucci, l. sportelli, r. guzzi dipartimento di fisica, laboratorio di biofisica molecolare & udr cnism, università della calabria, rende (cs), italy an increasing number of experimental studies is demonstrating that the propensity of forming amyloid fibrils is not limited to proteins related to neurodegenerative diseases but it is a more general phenomenon. we present data on human serum albumin (hsa) aggregation induced by a combination of thermal and metal ions effects investigated by optical density, fluorescence and electron paramagnetic resonance (epr). the turbidity experiments as a function of temperature show that the hsa aggregation starts at about • c which is higher than the denaturation temperature (∼ • c). in the presence of copper and zinc metal ions the onset temperature for protein aggregation is markedly reduced as the metal:protein molar ratio is increased from : to : . moreover, the copper is more effective in inducing protein aggregation compared to zinc ion. the hsa aggregation analyzed by tht fluorescence at different incubation temperatures lacks a lag phase and the kinetic traces can be fitted by double exponential functions. the tht fluorescence increase evidences the formation of protein aggregates with fibrillar features. epr experiments for the cu(ii)-hsa complex show the binding of cu(ii) in the protein native state in a square planar coordination with equatorial n atoms, and is not influenced by the heat treatment of the protein. the overall results suggest that hsa aggregation is compatible with a downhill process that does not require the formation of an aggregation nucleus. protein condensation diseases -a colloid physicists viewpoint p. schurtenberger adolphe merkle institute, university of fribourg, ch- marly , switzerland a broad class of diseases, such as cataract, alzheimer's disease and sickle-cell disease, involve protein association phenomena as an essential aspect. the basic element common to all members of this class of molecular condensation diseases is the subtle interplay between protein interactions that produces condensation into dense, frequently insoluble mesoscopic phases. among this class of diseases, cataract is particularly important as the world's leading cause of blindness. this disease is most often the consequence of an uncontrolled aggregation (or phase separation) of the proteins in the eye lens that results in a loss of its transparency. the high concentration protein mixtures present in the eye lens are normally stable and produce a high refractive index that aids the eye in adaptive focusing of light. moreover, the proteins themselves exhibit a rich variety of repulsive interactions, attractive interactions, sizes, phase transitions and self-association. these mixtures also exhibit the pathological aggregation and opacification of the cataract disease that has inspired their study. in my presentation i will illustrate how we can use a combination of small-angle neutron and xray scattering experiments combined with molecular dynamics computer simulations to identify, measure and model the molecular interactions and emergent optical and viscoelastic properties and the phase behavior of the relevant, complex cytoplasmic mixtures. fluorescence microscopy studies of iapp fibrillation at model and cellular membranes d. c. radovan , n. opitz , r. winter department of physical chemistry i -biophysical chemistry, tu dortmund university, dortmund, germany, max-planck institute for molecular physiology, dortmund, germany type diabetes mellitus (t dm) is characterized by islet amyloid deposition and beta cell death, the main culprit being a small a.a. peptide hormone, islet amyloid polypeptide (iapp), which forms fibrils under pathological conditions. we studied the interaction of iapp with giant (guvs) and large (luvs) unilamellar vesicles as well as with ins- e cells. by using confocal / two-photon excitation fluorescence microscopy and guvs, we tested the influence of charge (dopc:dopg : ) and model raft systems, displaying liquid-ordered (l o ) / liquid-disordered (l d ) phase coexistence (such as dopc:dppc:cholesterol : : ), respectively, on the kinetics of iapp fibril formation. a preferential partitioning into the l d phase was observed and fibrils grew along with lipid uptake. fluorescence spectroscopy leakage experiments with carboxyfluorescein-filled luvs and the corresponding tht kinetics of iapp fibril formation were carried out as well. moreover, using the wst- reduction assay and fluorescence microscopy, we could show that the red wine compound resveratrol is a potent inhibitor of iapp fibrillation and its cellular toxicity on ins- e cells, these findings highlighting the potential role of resveratrol in future clinical applications, i.e., in the treatment of t dm. a remarkably high viscosity has been induced in aqueous solutions of lysozyme by the addition of certain structurally related organic solvents, such as tetramethylurea (tmu), dimethylsulfoxide (dmso), dimethylformamide (dmf), and hexamethylphosphortriamide. dmso-induced gelation is observed in samples fulfilling the two following requirements: ( .) lysozyme concentration in excess of mm, and ( .) volume fractions of dmso exceeding . . based on spectroscopic data, the whole process was characterized as consisting of two mutually independent stages. the first involves an extensive transition of the polypeptide backbone, from a predominantly helical to increased random coiled and beta-sheet structures, with the occurrence of non-orthodox protein secondary structures at regions above the solvent critical point. the second stage consists of short-lived interchain contacts leading to an entanglement of the macromolecular system as a whole. here we present a set of scattering and microrheology experiments that investigate both the structural and dynamic properties of the gels under various experimental conditions. studying alpha-synuclein aggregation by fluorescence polarization based kinetics l. tosatto, f. munari, i. tessari, g. de franceschi, m. pivato, p. polverino de laureto, m. bisaglia, l. bubacco università degli studi di padova, padova, italy alpha-synuclein (syn) is linked to parkinson's disease (pd) by two evidences: the accumulation of amyloid fibrils of the protein and the autosomal dominant forms of the disease (a t, a p and e k mutants). protein oligomers seem to be the most toxic species, causing dopaminergic neuronal death, probably disrupting cell membrane (volles & lansbury, ) . the aggregation of syn follows a nucleation dependent mechanism, i. e., aggregation is favoured only after the formation of an oligomer composed of a critical number of monomers (wood et al., ) . as the constitution of the nucleus is a rare event, early aggregation stages are difficult to study. to explore the early stages of the aggregation process a fluorescence polarization (fp) based method (luk et al., ) was applied to study syn oligomerization. fp depends on the size of the fluorescent molecule, therefore it is suitable for the detection of oligomers formation. we measured aggregation kinetic properties under several different conditions. a to mixture of syn and oregon green labelled syn was used to analyze the aggregation behaviour of wild type (wt) and pd mutants. the wt protein shows the fastest aggregation rate. this aggregation process is slowed down by the presence of the chaperone - - eta in wt syn samples. finally, dimers formed by a disulfide bond at the n-terminus or c-terminus of syn, when tested for their aggregation behaviour, show a different propensity to aggregate. a. pardini , r. bizzarri , a. diaspro , p. bianchini , c. usai , p. ramoino , g. checcucci , g. colombetti istituto di biofisica, cnr, italy, a) nest, sns, iit udr, pisa, italy; b) nest, cnr-infm, pisa, italy, univiversity of genova, genova, italy the colored ciliates blepharisma japonicum and fabrea salina show photomotile responses triggered by endogenous pigments of the hypericin family. b. japonicum exists in two forms: the wild one contains red blepharismin; the other one, generated by irradiating the cells with dim visible light, blue blepharismin. b. japonicum shows step-up photophobic response, whereas f. salina, that contains fabrein, shows step-down photophobic responses and positive phototaxis. we showed by confocal microscopy that the pigments are localized not only in pigment granules, but also in the cilia. this fact implies that the structure of the pigment in the cilia differs from that in the cell body, (pigment granules are too big to fit in the axoneme) and suggests that ciliary pigments might play a decisive role in photoreception. fluorescence lifetime imaging microscopy (flim) shows that there is a spatial distribution of lifetimes, which are shorter for the pigment in the cilia. this might indicate a functional role for these pigments. furthermore, lifetimes for f. salina are always longer than those of blepharisma. in order to further characterize the structure of ciliary pigments by means of spectroscopic methods, we have also performed spatially resolved static and time-resolved fluorescence anisotropy measurements. biosensing applications of micro/nano structured silicon several topics related to porous silicon (ps) biosensing properties were carefully considered in this study. ps allows the increasing of the immobilised biomolecule number on its surface and the creation of stable covalent bonds due to its controllable chemistry. beside this, ps is suitable for electrical (conductance, impedance), electrochemical and optical amplification of the detected signal. the experimental results on the fabrication of the ps microstructures such as: (i) protein immobilization and detection using microarray technique; (ii) dna biomolecule detection by impedance or by fluorescence spectroscopy; (iii) very sensitive sers biosensors (raman signal of -mercaptoundecanoic acid); (iv) sensitive element for neurons in nutmix culture are in detail presented. various characterisation techniques have been used, optical and scanning electron microscopy (sem), x-ray diffraction, raman, laser fluorescence and impedance spectroscopy for investigation molecule attachment on the au/ps structures. we have demonstrated that different morphologies of ps as-prepared or coated with gold nanoparticles have an important role in biomolecule/cell detection, due to its large internal surface combined with specific optical properties, being in the same time sensing element/support for immobilization of sensing biomolecules as well as transducer for biochemical interactions. heterotrimeric g-proteins interact with their g-protein coupled receptors (gpcrs) via key binding elements comprising the c-terminal segment of the α-subunit and the two lipid anchors at the α− and γ−subunit. direct information about diffusion and interaction of gpcrs and their g-proteins is mandatory, as these properties will affect the timing of events in the complex signal transduction cascade. in the case of the photoreceptor rhodopsin, receptor packing in the membrane and the related diffusion coefficients are discussed controversially ( , ). by using single particle tracking we show that the encounters of rhodopsin with the fluorescently labeled cterminus of the α-subunit as well as with the holo-g-protein transducin change upon rhodopsin light-activation. our results indicate confined areas of interaction for the c-terminal segment of the α-subunit with inactive rhodopsin disk membranes and less restricted diffusion of the receptor-bound cterminal segment after light-activation. this suggests dynamic short-range order in rhodopsin packing and specific structures for efficient interaction ( ) in vivo, skeletal muscle actively shortens and also resists lengthening, for example when landing after a jump. we study the energy during shortening and during lengthening by measuring the rate of pi release which results from atp hydrolysis. we show here the rate of pi releases in an in vitro protocol that involves muscle stretched followed by muscle shortening. it is hypothesised that the magnitude of deceleration of pi release in the stretch phase is less than the acceleration during the release due the energy input of the motor that is used to apply the length changes. therefore the end point of pi release is similar to isometric conditions. pi release responses to a ramped stretch ( %) followed ms later by a symmetrical release at low velocities ( . l /s − ) were measured in permeabilised fibre bundles of rabbit psoas at • c. laser diffraction and high speed video were also recorded to confirm length change. we show that the rate of pi release drops during the stretch phase, returns to isometric levels during the length hold phase, and finally accelerates during the ramped release. tracking of sarcomere length change using video analysis demonstrated that laser diffraction is unreliable at times during stretches due to lack of uniformity of the sarcomere spacing. microbial rhodopsins: receptors, channels, and pumps from a single design j. l. spudich, e. negri-spudich center for membrane biology, university of texas, houston, texas, u.s.a. the microbial rhodopsin family is comprised of ∼ homologous proteins containing transmembrane helices forming a pocket for the chromophore retinal. most are lightdriven ion pumps ("transport rhodopsins") and others are photosensory receptors ("sensory rhodopsins"). phylogenetic analysis indicates frequent lateral gene transfer of proton pumps among prokaryotic and unicellular eukaryotic species, followed by coupling of the pump's mechanism to the cell's existing signal transduction machinery to create photosensors. this evolutionary path is strongly supported by our studies of sensory rhodopsins in various organisms which demonstrate remarkably diverse signaling mechanisms with diverse transducer partners. the best studied are the phototaxis receptors in haloarchaeal prokaryotes (sri and srii) and in eukaryotic algae (channelrhodopsins). sri and srii transmit signals by protein-protein interaction to control a phosphorylation cascade that modulates motility. channelrhodopsins are light-gated cation channels that depolarize the membrane mediating calcium ion influx into the flagellar axoneme. crystallography and molecular biophysics have begun to clarify how modifications of the same architecture enable the rhodopsins to carry out their distinctly different molecular functions. interconversions of their functions by mutation reveal the elegant simplicity by which evolution uses existing genes to create proteins with novel functions. gold colloids-fluorophore complexes for protein detection assay l. sironi , s. freddi , l. d'alfonso , m. collini , m. caccia , g. tallarida , s. caprioli , g. chirico dipartimento di fisica, università di milano-bicocca, italy, laboratorio nazionale mdm, agrate brianza (mi), italy noble metal nanoparticles (np) are endowed with peculiar optical properties related to the surface plasmon resonances (spr). the interaction of surface plasmons of gold nanoparticles with fluorophores a few nanometers away from the surface modifies their brightness and excited-state lifetime, and this effect can be exploited to obtain nanodevices for proteinprotein recognition. we studied different types of constructs based on gold nps on which derivatives of fluorescein were bound. the interaction of this fluorophore with the gold surface plasmon resonances, mainly occurring through quenching, affects its excited-state lifetime, that is measured by fluorescence burst analysis in standard solutions. the binding of proteins to the gold nps through antigen-antibody recognition further modifies the dye excited-state lifetime. this change can therefore be used to measure the protein concentration. streptavidin-functionalized gold nps of size - nm are used to bind biotin-fluorescein and biotin-antibodies for specific proteins. we have first tested the constructs for bovine serum albumine (bsa) detection. the data reported here indicate that one can measure the concentration of bsa in solution with an apparent limit of detection of ± pm. we have then extended the study to the antitumor protein p -p antibody interaction in standard solution and directly in cellular extracts. calcium transport and phototransduction in isolated rod outer segments g. rispoli dip. biologia ed evoluzione, università di ferrara, italy ca + concentration in photoreceptor rod outer segment (os) strongly affects the generator potential kinetics and light adaptation. light stimuli may produce voltage changes exceeding mv: since the os ca + extrusion is entirely controlled by the na + :ca + ,k + exchanger (nckx), it is important to assess how the nckx ion transport is affected by voltage and intracellular factors. the nkcx regulation was investigated in whole-cell recorded os, using ionic conditions that activated maximally forward and reverse exchange. in all species examined of amphibia and reptilia, the forward (reverse) exchange current increased about linearly for negative (positive) voltages and exhibited outward (inward) rectification for positive (negative) voltages. since hyperpolarization increases ca + extrusion rate, the recovery of the dark level of ca + (and of the generator potential) after light stimuli results accelerated. mg-atp doubled the size of forward and reverse exchange current without modifying their voltage dependence, indicating that mg-atp regulates the number of active exchanger sites and/or the nckx turnover number. ca + jumps achieved via photolysis of caged-ca + produced current transients, possibly originating from electrogenic partial reactions. no monovalent cation substituted for na + at the nckx binding sites, but rb + substituted for k + , while sr + , ba + , mg + substituted for ca + with an apparent permeability ratio of . , . , < . : , respectively. a. pfeifer , k. zikihara , s. tokutomi , j. heberle , t. kottke bielefeld university, bielefeld, germany, osaka prefecture university, sakai, japan the blue light receptor phototropin regulates the growth of plants towards the light. it contains two light-, oxygen-, or voltage-sensitive (lov) domains and a kinase domain. lov domains bind noncovalently flavin mononucleotide (fmn) as chromophore. upon illumination, the triplet excited state of flavin reacts within few microseconds with a nearby cysteine under formation of a photoadduct, which represents the signaling state. in response to adduct formation, a jα helix adjacent to the lov domain dissociates and allows for autophosphorylation by the kinase domain. the mechanism of the photoreaction and the signaling pathway from fmn to the jα helix are still unclear. we have investigated the lov domain of arabidopsis phototropin by microsecond ft-infrared spectroscopy. the difference spectrum recorded at µs provides evidence that the flavin is unprotonated in the triplet excited state. therefore, a previously proposed ionic mechanism of bond formation is disfavored. changes in secondary structure were detected concomitant with adduct formation that relax with a time constant of µs. this early adduct intermediate has not been previously characterized. the final adduct state is formed in milliseconds by further alterations in secondary structure. these findings raise the question of whether the early or late adduct intermediate propagate the signal to the jα helix. -photosensory biophysics - the psychostimulant amphetamine increased no generation measured by epr as well as amino acid release in the rat brain nitric oxide (no) is a novel messenger that modulates many functions of the nervous system. the involvement of no in brain damage was shown mainly by indirect evidence and the data are controversial. the short half-life of no makes its direct detection difficult. we measured no generation using epr spectroscopy based on determination of the amount of paramagnetic mononitrosyl-iron complexes. the aim was to elucidate whether psychostimulant drug amphetamine (amph) modulates formation of no and lipid peroxidation (lpo) products as well as the neurotransmitter release in rat brain. the output of glutamate, aspartate, gaba and acetylcholine (ach) was monitored in striatum by microdialysis with hplc detection. amph produced fold elevation of no generation and lpo formation in brain areas. while amph increased the aspartate, gaba and ach release, the glutamate output was not affected. pretreatment with the neuronal nos inhibitor was highly effective in abating the rise of no and neurotransmitter levels but failed to influence the lpo intensity elicited by amph. the findings suggest that activation of no synthesis is a potent factor in the amph-induced neurotransmitter release. light scattering study of dna over a wide chain length range: comparison with wormlike model p. baeri, m. zimbone dipartimento di fisica e astronomia, università di catania, italy this work reports light scattering measurements on dna in aqueous solutions ( mm nacl , mm edta and mm tris-hcl buffer, ph . ) over a wide range of molecular weights ( - base pairs) and shows that, in the above standard solvent, shorter chains ( < base pairs) behave as a "wormlike chain" and their diffusion coefficient as obtained by dynamic light scattering measurements, confirm the prediction of standard wormlike model, whilst longer chains ( > base pairs) behave in a different manner. dynamic and static light scattering and sem analysis indicate that dna molecules base pairs long, condense into compact structures in our solvent condition. calculations done using a wormlike model are also presented and discussed in comparison both to our experimental data and to other data reported in the literature. a. asandei, t. luchian 'alexandru i. cuza'university, faculty of physics, laboratory of biophysics and medical physics, blvd. carol i no. , iasi, romania amphotericin b (amb) is an antifungal antibiotic which, despite the severe side effects, is still used for the treatment of systemic fungal infections. in this study we investigated the influence of ph upon the selectivity and the transport properties of amb channels inserted in reconstituted, ergosterolcontaining zwitterionic lipid membranes. our electrophysiology experiments carried out on single and multiple amb channels prove that at ph= . these channels are anion selective, whereas at neutral and alkaline ph's (ph= and ph= ) they become cation selective. we attribute this to the ph-dependent ionization state of the carboxyl and amino groups present at the mouth of amb molecules. surprisingly, our data reveal that the single-molecule ionic conductance of amb channels varies in a non-monotonic fashion with ph changes, which we attribute to the ph-dependent variation of the surface and dipole membrane potential. we demonstrate that when added only from one side of the membrane, in symmetrical salt solutions across the membrane and low ph values, amb channels display a strong rectifying behavior, and their insertion is strongly favored when positive potentials are present on the side of their addition. we report a detection method for the redox state of proteins which combines fret-based fluorescence/confocal microscopy on dye-labeled protein with cyclic voltammetry. by using this combined method, electron transfer properties can be revealed from protein to electrode or from redox enzyme to substrate. we applied the fluorescent detection to azurin, a blue copper protein from the bacterium ps. aeruginosa, fluorescently labeled on the n-terminus for monitoring the redox state of the protein. the dye fluorescence is quenched by energy transfer to the copper in oxidized, but not in reduced azurin. fluorescence results demonstrated that cy -labeled wtazurin switched in fluorescence intensity by up to % by varying the applied potential. labeled zinc-azurin was used as a control sample and did not show any fluorescence switching. for single molecule studies wt-azurin was labeled with atto dye and a mixed sam was used, with -hydroxy- octanethiol as a blocking agent to prevent non-specific binding of protein on the surface, and , decanedithiol for the specific covalent binding to the protein. preliminary data show that single-molecule fluorescence switching with the potential is indeed possible. -single molecule biophysics - is there a specific erythrocyte membrane receptor for fibrinogen? an atomic force microscopy approach f. a. carvalho , s. connell , r. a. ariëns , n. c. santos instituto de medicina molecular, univ. lisbon, portugal, university of leeds, u.k. fibrinogen (fg) contributes to erythrocyte (rbc) hyperaggregation by an increase in. rbc-fb protein binding considered to be non-specific. glycoprotein α iib β is a specific integrin receptor for fibrinogen on platelets. we showed that there is a single molecule interaction between fg and an unknown receptor on rbc membrane, with a lower affinity when compared with platelet binding. we evaluated if rbc-fg binding is through an integrin-like receptor or not. interactions between fg and platelet/rbc receptors were studied by force spectroscopy. force curves were performed between fg-functionalized atomic force microscope tips and rbc or platelets. to evaluate if the fg-rbc binding is calciumdependent, similar studies were performed in the presence of ca + or edta. we also carried out studies in the presence of a α iib β inhibitor and of methyl-ß-cyclodextrin (to disrupt lipid rafts by cholesterol depletion). rbc-fg single forces were of - pn and of - pn for platelet-fg binding in presence of calcium. a significant decrease of the platelets-fg force-rupture was obtained in the presence of edta or α iib β inhibitor. significant lower fg-rbc force value was obtained in the presence of mβcd, but not in the presence of edta. conclusion: fg-rbc binding seems not to be calcium-dependent but the existence of cholesterol on rbc membrane is important. nanoscopic and spectroscopic investigation of p -based complexes at single molecule level a. r. bizzarri biophysics and nanoscience centre, cnism, facoltà di scienze, università della tuscia, largo dell'università - viterbo, italy p is a transcription factor that plays a widely recognized role in preventing cancer development in response to dna damage. the tumor suppressor activity of p involves the formation of several complexes whose detection and study, at single molecule level, could be extremely relevant to understand, in detail, the mechanisms governing the cancer defense processes, as well as to develop ultrasensitive biosensors. p -based complexes, are investigated at molecular level, by combining atomic force microscopy (afm), atomic force spectroscopy (afs) and surface enhanced raman spectroscopy (sers) with the support of computational docking. our attention is mainly devoted to study complexes between p and the electron transfer azurin which has been demonstrated to interact with p , by promoting its stabilization. a possible competition between azurin and the cellular oncogene mdm is also investigated. unwinding the dna helix in force clamp condition p. bianco , l. bongini , m. dolfi , l. vincenzo physiolab, dbe, university of florence, italy, university of florence and centro interdipartimentale studio dinamiche complesse, firenze, italy we use a dual-laser optical-tweezers (dlot, smith et al., science, ) to define the highly cooperative conformational transition in the molecule of dna, where the natural b-dna has converted into a new overstretched conformation called s-dna (bensimon et al., phys. rev. lett., ; cluzel et al., science, ) . single molecules of double stranded λ-phage dna (in a solution with mm naci, mm tris-hcl, mm edta, ph . . and • c) are stretched either in length clamp or in force clamp mode. when the dna molecule is stretched in length clamp mode with a ramp lengthening, it shows the previously described highly cooperative overstretching transition at ∼ pn, attributed to unwinding from the b-form to the . times longer s-form. stretching the molecule in force clamp mode with a staircase of force steps at s intervals (step size - pn, rise time - ms) shows, for any given clamped force f in the region of the overstretching transition, different amounts of dna elongation (∆l) with exponential time courses. the analysis of the elongation rates allows to recover all the necessary parameters for an effective two-state model able to reproduce the out-of-equilibrium properties of the system. the results imply an unwinding cooperativity of bps. this value is significantly lower than that obtained assuming force independent rate constants. supported by miur, ente cassa di risparmio di firenze and itb-cnr (milano). c. m. becker , a. benedix , b. l. de groot , a. cafisch , r. a. böckmann saarland university, saarbrücken, germany, mpi for biophysical chemistry, göttingen, germany, university of zürich, zürich, switzerland modifying the stability or the binding behavior of the involved proteins by mutation can influence the activity of cellular processes. for an efficient identification of possible mutation-sites a fast calculation of the free energy of proteins is crucial. here we developed a fast and reliable method (cc/pbsa) [ ] for the prediction of the change in stability of proteins and binding affinity of protein-protein complexes upon mutation. the energy function of cc/pbsa is based on gas phase energies, solvation free energies and entropic contributions. the protein flexibility is taken into account by generating random conformations based on geometrical constraints only applying the concoord [ ] program. we applied cc/pbsa on the tem -blip complex, which is important in bacterial antibiotic resistance. the results of single-and double-point alanine scanning are used to detect hot spots, cooperative effects, and the corresponding energy distribution. cc/pbsa is freely accessible on our web-server: http://ccpbsa.bioinformatik.uni-saarland.de the human recombinase hrad is a key protein for the maintenance of genome integrity and for cancer development. this protein plays a central role is the dna strand exchange occurring during homologous recombination. here we report the polymerization and depolymerization of hrad on duplex dna observed with a new generation of magnetic tweezers, allowing the measurement of dna twist with a resolution of • in real time. at odds with earlier claims, we show that, after initial deposition of a multimeric nucleus, nucleoprotein filament growth occurs by addition of single proteins, involving dna twisting steps of ± • . simple numerical simulations support that this mechanism is an efficient way to minimize nucleoprotein filament defects. this behavior, consisting of different stoichiometry for nucleation and growth phases, may be instrumental in vivo. fast growth would permit efficient continuation of strand exchange by rad alone while the limited nucleation would require additional proteins such as rad , thus keeping this initiation step under the strict control of regulatory pathways. besides, our results combined with earlier structural information, suggest that dna is somewhat less extended ( . versus . Å per bp) and more untwisted ( . versus • per bp) by hrad than by reca, and confirm a stoichiometry of - bp per protein in the hrad -dsdna nucleoprotein filament. biofunctional micropatterned surfaces to study the spatio-temporal organisation of lfa- r. diez-ahedo , d. normanno , c. g. figdor , a. cambi , m. f. garcia-parajo bionanophotonics, ciber-bbn and ibec, barcelona, spain, tumor immunology, nijmegen center for molecular life sciences, the netherlands lymphocyte function associated antigen- (lfa- ) adhesion depends on receptor occupancy and lateral organization on the cell membrane. however, the signals and mechanisms which dynamically reorganize lfa- into high avidity clusters are still a subject of many studies. to obtain deeper insight on the mechanisms that control and regulate lfa- clustering, patterned surfaces of immobilized lfa- ligand areas were fabricated using microcontact printing. the diffusion of lfa- expressed by monocytes stretched over patterned surfaces was followed in time using single molecule tirf microscopy. single lfa- nanocluster trajectories on individual cells showed an increase of immobile lfa- fraction and a slow-down of diffusing lfa- on the ligand areas compared to the non-ligand areas. moreover, single-cluster intensity analysis indicated a reorganization of lfa- nanoclusters in microclusters upon ligand binding. finally, single particle motion analysis of lfa- trajectories in close neighborhood to the ligand areas showed no assisted diffusion of lfa- towards the adhesive regions, consistent with random ligand-encountering and binding. we are currently investigating the effect of cell membrane organizers to regulate the spatio-temporal organization of lfa- . r. diez-ahedo et al, small, in press. optical and electrophysiological detection of single phages across a lipid membrane n. chiaruttini , p. boulanger , m. de frutos , l. letellier , u. bockelmann , v. viasnoff nanobiophysique, espci paristech, cnrs, paris, france, ibbmc, université paris xi, cnrs, orsay, france, lps, université paris xi, cnrs, orsay, france we present an investigation study of the ejection of single t bacteriophages. in vivo studies of dna ejections from the bacteriophage capsid show that the t genome is introduced in the bacterial host in two steps. first % of the genome is ejected then after a pause of a few minutes the rest is internalized. bulk in vitro studies showed that various mutants of t eject their genome in solution following a single or a multistep process. by immobilizing single bacteriophages on a surface and following their ejection by fluorescence microscopy we showed that in all cases the ejection occurs in one step, but some mutants seem to have a subpopulation for which the triggering signal of the ejection is transmitted more slowly to the capsid entrance. we then reconstituted the phage receptor fhua into giant liposomes and followed the ejection of the dna into the liposome by fluorescence. finally we incorporated fhua in a suspended bilayer and followed the infection of the phages through the bilayer both by fluorescence labeling and electrophysiological measurements. we will discuss the influence of the cross membrane potential on the ejection speed of the dna. helixlike pili is a prerequisite of uropathogenic e. coli to adhere to host and withstand urine flow the gram-negative uropathogenic escherichia coli (upec) bacteria, invades the urinary tract region and cause in some cases severe infections, pyelonephritis, if they can withstand the rinsing action of urine and ascend to the kidney, via the bladder and ureters. to mediate adhesion, upec express quaternary surface organelles that are assembled from ∼ identical subunits into a helix-like coil, with a single adhesin located at the tip. it is believed that the single adhesin mediate attachment to host cells while the helix-like structures act as shock absorbers to dampen the irregularly shear forces induced by urine flow. to unravel the biomechanical properties of such quaternary structures, in particular in terms of their force-elongation and kinetic behavior, force-measuring optical tweezers (fmot) have been used. a plethora of different types of pili have been identified in the literature and we show, using fmot, that those dissimilarities might reflect the host environment. for example, we have found differences among pili expressed at diverse environment inside the urinary tract, which imply that pili presumably have evolved to resist specific forces under in vivo conditions. it is thus worth striving for understanding bacterial adhesion in order to figure out alternative to the over-abundance of antibiotics worldwide. single molecules studies have probed protein conformational fluctuations and monitored the oscillating activity of enzymes that remained masked in ensemble measurements. various statistical models have attempted to connect the conformational motions to the fluctuating activity of enzymes and suggested that they adopt inter-converting conformations each one of them exhibiting different catalytic activity. the main drawback of these studies is the non specific interactions that may bias the observed catalytic behavior. we have developed new innovating tools to study the behavior of single enzymes. as a first step we utilized liposomes as scaffold to confine enzymes while keeping them under physiological conditions. keeping them in their native conformation allowed us to monitor the inherent properties of their behavior. as a second step we developed a new model that accurately connects enzyme activity behavior to conformational motions. using this approach we accurately predicted the behavior of single lipases in a highly controlled environment. we modulated the enzyme's conformational mobility and activity by systematically varying its accessibility to liposomes. as we anticipated, that resulted in altering the time the enzyme spends on active conformations.our results provide new insights on interpreting the behavior of enzymes. simulations of single-molecule fret experiments on ribozymes m. hajdziona, a. molski adam mickiewicz university laboratory for dynamics of physicochemical processes, poznan, poland rnazymes are important biological molecules and that can catalyze the cleavage of their own nucleotide chains. this precess is called self splicing [ ] . there are several methods to to study kinetic properties of ribozymes at the singlemolecule level. in this work we focus on fret (förster resonance energy transfer) of single, immobilized molecules. single-molecule fluorescence spectroscopy gives an insight into the behavior of individual molecules rather than the average behavior of an ensemble of molecules, which makes it possible to observe the kinetic heterogeneity. we simulated fret trajectories for single immobilized ribozymes. in our computer simulations we consider two-and three-state kinetic models motivated by actual experiments, published in [ ] and [ ] . we fitted the histograms of on and off times to recover the kinetic parameters of the models. we investigated the bias and standard deviation of the recovered parameters when one or two thresholds are applied to fret trajectories between adjacent states. we found that two thresholds give better parameter estimates than one-threshold analysis. references frap probes the average dynamical properties of a population of fluorescently labeled molecules whereas spt obtains these properties from observations of the trajectories of individual molecules tagged with a colloidal particle (the "bead"). when results of frap are compared with those of spt, frap yields significantly higher diffusion constants than spt. to understand the origin of this difference, we have developed and tested a model system to evaluate the influence of the bead on the dynamics of a diffusing molecule. we use a dsdna tether to attach a bead to a supported lipid bilayer (slb). the dna tether is modified at one end by cholesterol for anchoring to and diffusion in the membrane, and at the other end by biotin for tagging with a bead. with this system, we can vary several parameters: distance between slb and bead, size and nature of the bead, lipid composition of the slb, external force. we can also model the brownian motion of the bead and the hydrodynamic flow around it. we will present results using φ = nm neutravidin-coated latex particles attached to an eggpc slb via a bp to bp dna tether. elucidation of the mechanism of the lambda bacteriophage epigenetic switch l. finzi physics department, emory university, dowman dr, atlanta, ga , usa the lambda bacteriophage epigenetic switch determines the growth lifestyle of the virus after infection of its host (e. coli ). it is now clear that the switch consists of a ∼ . kbplong dna loop mediated by the lambda repressor protein. using tethered particle microscopy (tpm), magnetic tweezers and afm, our laboratory has novel, direct evidence of loop formation and breakdown by the repressor, the first characterization of the thermodynamics and kinetics of the looping reaction and its dependence on repressor non-specific binding and dna supercoiling. these in vitro data provide insight into the different possible nucleoprotein complexes and into the lambda repressor-mediated looping mechanism which leads to predictions for that in vivo. the significance of this work consists not only of the new insight into a paradigmatic epigenetic switch that governs lysogeny vs. lysis, but also the detailed mechanics of regulatory dna loops mediated by proteins bound to multipartite operators and capable of different levels of oligomerization. mechanical force at the molecular level is involved in the action of many enzymes. for example, the phi dna polymerase mechanically unwinds the dna helix as it moves processively along the dna replicating one strand of the dna molecule. using optical tweezers we have developed a single molecule mechanical assay to elucidate the physical mechanism of dna unwinding by the phi dna polymerase as the protein replicates the dna. a single dna hairpin is hold between an optical trap and a mobile surface. as a single polymerase works on the dna hairpin, its replication and unwinding reactions can be measured in real time by measuring the change in extension in the dna polymer, revealing the fluctuations of its rate in response to the dna sequence. moreover, by gradually pulling on the opposite strands of the dna hairpin we can promote the controlled mechanical unwinding of the dna helix and determine the effect of increasing unwinding forces on the polymerization and unwinding rates. the effect of force and dna sequence on the activities of the wild type and an unwinding-deficient polymerase mutant will allow us to determine the inner workings of this molecular motor. single centrosome manipulation reveals its electric charge and associated dynamic structure we demonstrate laser manipulation of individual early drosophila embryo centrosomes in between two microelectrodes to reveal that it is a net negatively-charged organelle with a very low isoelectric region ( . ± . ). from this single-organelle electrophoresis, we infer an effective charge smaller or of the order of electrons, which corresponds to a surface-charge density significantly smaller than that of microtubules. by investigating the centrosome hydrodynamic behavior, we show that its charge has a remarkable influence over its own structure. specifically, we find that the electric field which drains to the centrosome expands its structure to a physiological size a % larger than previous electron microscopy determinations, a self-effect which modulates its structural behavior via environmental ph. this methodology further proves useful to study the action of different environmental conditions, such as the presence of ca + , over the thermally-induced dynamic structure of the centrosome. magnetic contrast neutron reflectivity delivers significant improvements in resolution for membrane structure analysis neutron reflection (nr), with its high resolution and use of contrast variation, is a unique tool to gain information on the orientation and structure of lipid bilayers in the z-axis perpendicular to the surface. to improve the use of nr we have used a highly oriented and stable layer of membrane proteins and lipids made possible by self-assembly upon a gold surface. this also provides a model of the bacterial outer membrane. as the dimensions of the layer can be predicted with accuracy, the system provides a molecular ruler for improvements in methodology and the complexity of the layer structure adds significantly to the modelling challenge. early improvements in resolution were obtained through sample preparation and gold smoothness. further improvement however required clearer discrimination between similar models. this was achieved using the new approach of magnetic contrast variation which uses a magnetic layer to provide two different scattering length densities for oppositely polarised neutrons. during this data collection the sample is unchanged. we show that this approach delivers significant improvements in data analysis and resolution of the protein, lipid and solvent structures. the method will provide a new level of understanding of membrane structure and dynamics. single molecule force spectroscopy and recognition imaging p. hinterdorfer institute for biophysics, johannes kepler university of linz, altenbergerstr. , a- linz, austria in single molecule force spectroscopy, interaction forces of ligand-containing tips with receptors on probe surfaces are quantified. here he attachment of human rhino virus (hrv ) to the cell surface, the first step in infection, was characterized. sequential binding of multiple receptors was evident from recordings of characteristic quantized force spectra. this suggests that multiple receptors bound to the virus in a timely manner. unbinding forces required to detach the virus from the cell membrane increased within a time frame of several ms. the number of receptors involved in virus binding was determined and estimates for on-rate, off-rate, and equilibrium binding constant were obtained. furthermore, we show that accurate free energy values of membrane protein unfolding can be obtained from single molecule force measurements. by applying a statistical theorem developed by jarzynski, we derived equilibrium unfolding free energies of from unfolding force data acquired at different force loading-rates and temperatures. finally, we present a method for the localization of specific binding sites and epitopes with nm positional accuracy. a magnetically driven afm tip containing a ligand covalently bound via a tether molecule is oscillated at a few nm amplitude, during scanning along the surface. in this way, topography and recognition images on membranes and cell surfaces were obtained simultaneously. mapping of the forces acting on biomolecules in cell membranes has spurred the development of effective labels, e.g. organic fluorophores and nanoparticles, to track trajectories of single biomolecules [ ] . standard methods use particular statistical observables, namely the mean square displacement (msd), to extract cues on the underlying dynamics. yet, msd is not an appropriate tool to access force fields and becomes easily a biased estimator in the presence of positioning noise. here, we introduce general inference methods [ ] to fully exploit information hidden in the experimental trajectories, providing sharp estimates for the forces and the diffusion coefficients within membrane microdomains. rapid and reliable convergence of the inference scheme is demonstrated on trajectories generated numerically. the inference method is then applied to infer forces and potentials acting on the receptor of the ε-toxin labelled by lanthanide-ion nanoparticles. results show a constant diffusivity inside a complex force field confining the receptor inside a specific domain. our scheme is applicable to any labelled biomolecule, and results presented here show its general relevance to the issue of membrane compartmentation and protein motion. large scale domain motions are structural rearrangements often adopted by enzymes to achieve their full functionality. understanding the mechanism responsible for such movements by means of computational approaches is of great interest especially in rational drug design, since induced fit or population shift effects are closely related aspects of the problem. unfortunately, the simulation time required to sample these events by conventional techniques such as plain molecular dynamics, is unfeasible. here, the domain motion required by adenosine kinase (ak) to achieve its (pre-)catalytic conformation was studied using well-tempered metadynamics [barducci, a. et al. phys rev lett. ( ) , : ] with path collective variables (pcvs) [branduardi, d. et al. j chem phys. ( ) , : ]. first, a low energy path for the apo form of the enzyme was obtained, and the potential of mean force along the pcvs was reconstructed. then, the large scale movement was simulated in the presence of two inhibitors known to bind to the enzyme in different conformational states. the adopted approach was proven to be successful both to understand the mechanistic features of the ak domain motion and to provide a picture of the population shift effects upon ligand binding. force measurement study of the interaction between s rrna and the ribosomal protein l single molecule force measurements enable to probe the complex structure and folding dynamics of rna molecules and furthermore to investigate the numerous interactions with proteins that affect rna folding in vivo. we use a double optical tweezers setup to study a region of the ribosomal rna s from e.coli and its interaction with the essential ribosomal protein l . the apparatus permits us to probe the dynamics of the rna structure with milisecond time-resolution. first, we pull the rna and show that it mechanically unfolds in several reproducible steps. we use these results in combination with structure prediction tools to evaluate the possible structures of the rna fragment in vitro. the most probable structure exhibits a unique binding site for l . then, force measurements are done on the rna in presence of l . we show that the protein specifically binds the rna and stabilizes it. the binding site is recognized with a resolution of a few bases. finally, two bases are mutated on the rna fragment. in presence of these mutations, in vivo and in vitro binding of l to s rna is abolished. the single molecule approach gives an explanation of this result: the force measurements show that the mutated rna folds differently from the natural one and that it does not bind l . control of the translocation of single dna molecules through alpha-hemolysin nanopores g. maglia, h. bayley department of chemistry, university of oxford, ox ta, oxford, uk the analysis of single nucleic acid molecules by electrophoretic threading through nanopores is under intense investigation as a rapid, low cost platform for dna sequencing. biological nanopores such as staphylococcal alpha-hemolysin (hl) have an added advantage over solid-state nanopores because they can be modified by genetic engineering with atomistic precision. although we showed that all four dna bases can be identified in an immobilized ssdna molecule ( ), the translocation of free dna is too quick to observe single bases. here we show that by a small increase of the net internal charge of the hl nanopore (e.g. by introducing a ring of arginine residues), we have augmented the frequency of dna translocation events through the pore and dramatically lowered the voltage threshold required for dna translocation ( ) . by further increasing the net positive charge of the transmembrane barrel region of the pore (e.g. by introducing extra positive charges) we have also reduced the speed at which dna translocate the pore by more than two orders of magnitude. these experiments provide a means of controlling dna translocation with protein nanopores, which might be translated to solid-state nanopores by using chemical surface modification. the atomic force microscope (afm) is a tool for imaging, measuring and manipulating matter at the nanoscale. single-molecule pulling experiments give information on the thermodynamics and kinetics of biomolecules. the purpose if this work was to develop software to simulate single-molecule pulling experiments and to analyze singlemolecule pulling data. the long term objective is to asses the accuracy and precision of the parameters recovered from single-molecule pulling data.we carried out simulations for two different models [ , , ] , using a wide range of loading rates. from the simulated force-extension curves we extracted the kinetic parameters and compared them with the values used for simulations. the kinetic parameters were the intrinsic rate coefficient (k kramers rate), the location of transition state (x ) and the free energy of activation (∆g). we have found that the loading rates have a small effect on the recovery of the free energy of activation, but have a significant effect on the recovery of the kramers rate. we report the tracking of single myosin v molecules in their natural environment, the cell. myosin v molecules, labeled with quantum dots, are introduced into the cytoplasm of living hela cells and their motion is recorded at the single molecule level with high spatial and temporal resolution. we perform intracellular measurements of key parameters of this molecular transporter: velocity, processivity, step size and dwell time. our experiments bridge the gap between in vitro single molecule assays and the indirect measurements of the motor features deduced from the tracking of organelles in live cells. a mathematical model of neurotransmission at the input stage of the cerebellum t. nieus , s. solinas , l. mapelli , e. d'angelo dept. neuroscience and brain technologies, iit, italian institute of technology, genova, italy, dept. of biomolecular sciences and biotechnology, milan, italy, dept. physiological and pharmacological sciences and cnism, university of pavia, italy the granule cell (gc) of the cerebellum has some peculiar properties compared to other cells of the vertebrate brain. the gc has a small soma (diam= microm) and just a few ( to ) excitatory and inhibitory inputs (e&i). the e&i gc synaptic inputs are formed inside the cerebellar glomerulus, which favors neurotransmitter diffusion between neighboring sites ( ) protracting postsynaptic receptor activation and ( ) causing cross-tall between e&i synapses through presynaptic receptors. neurotransmitter release probability (p) can be regulated by long-term plasticity (ltp and ltd at e synapses) and by gaba b and mglu presynaptic receptors (both at e&i synapses). the p change in turn modifies shortterm plasticity, affecting the first response in a train much more than the followings. to gain insight into the role that p changes might have in computations performed at the cerebellum input stage, we have built detailed biophysical models of the e&i synapses. the model has allowed to investigate glomerular processing of high frequency inputs reaching the cerebellum. optimal synaptic transmission through the mfs inputs resulted when gos inhibited synchronously the gcs. the role of feed-forward inhibition onto synaptic transmission is under investigation. unzipping dna with a nanopore j. muzard, n. chiaruttini, u. bockelmann, v. viasnoff cnrs/espci nanobiophysique, rue vauquelin, paris, france the use of proteinaceous or artificial nanometer size pores has become a promising approach for sensing biomolecules at the single molecule level. it was shown that alphahemolysin, a toxin from staphylococcus aureus, can be employed to sense the translocation of dna strands through a lipid bilayer. the pore dimension allows the electrophoretically driven passage of single stranded dna whereas double stranded structures need to open prior their translocation. we study the unzipping process of the double stranded part of dna both experimentally and theoretically. we show that in a first approximation the duplex opens progressively in a sequence dependent manner. the experimental results can be accounted for by modeling the unzipping process as a free diffusion of the unzipping fork in the energy landscape defined by the sequence of the basepaires. we then discuss the effect of the pore/dna interaction on the translocation process. we further characterize the effects of the pore geometry, showing that the pre-confinement of the dna in the pore vestibule is essential to the unzipping process. we eventually discuss the possibility to use this nanopore approach to sequentially probe rna secondary structures. we report on the interaction forces in the range -- pn determined between pairs of the lectin soybean agglutinin (sba) and a modified porcine submaxillary mucin (tn-psm) using a single-molecule approach. lectins are carbohydrate-binding proteins with biological activities related to i.e. cellular recognition, adhesion, growth and metastasis. here, dynamic force spectroscopy is used to investigate pairs of sba and tn-psm with the aim to understand the mechanisms of binding and cross-linking of multivalent lectins. the unbinding force increased from pn to pn with increasing force loading rate and the lifetime of the complex in the absence of applied force was . - . s. published kinetic parameters describing the rate of dissociation of other sugar lectin interactions are in the range . x − - . x − s. the long lifetime of the sba -tnpsm complex is compatible with a previously proposed "bind and jump" mechanism. this mechanism has also been suggested for lectins binding to multivalent carbohydrates and globular glycoprotein. the bind and jump mechanism is also similar to that observed for binding of proteins to dna, and suggest a common conserved binding mechanism of ligands to the two biopolymers and possibly between ligands and all biopolymers, as recently suggested. the rate of topoisomerase ii activity correlates with persistence length of dna q. shao , l. finzi , d. dunlap dept. of physics, emory university, atlanta, georgia, u.s.a., dept. of cell biology, emory univ. med. school, atlanta, georgia, u.s.a. type ii topoisomerases catalyze the transection of one double helical segment by another to modify the topological state of dna. reversible ' linkages to the phosphate-sugar backbones are established on each strand of the "gate" segment to create an opening through which the enzyme drives the "transfer" segment. a recent crystal structure shows that the "gate" segment bends approximately • upon binding to the enzyme. bending a stiff polymer like dna requires considerable energy and could represent the rate limiting step in the catalytic (topological) cycle. using modified deoxyribonucleotides in pcr reactions, more rigid dna fragments have been produced and used as substrates for topoisomerase ii-mediated relaxation of plectonemes introduced in single molecules using magnetic tweezers. before relaxation the persistence lengths were measured by fitting force extension data with the worm-like chain model. topoisomerase ii was found to release mechanically introduced supercoils more slowly in stiffer dna suggesting that dna bending might be a rate limiting step in topoisomerase ii activity. h. seidel , t. klose , h. lilie , c. g. hübner institute of physics, ratzeburger allee , lübeck, germany, institute of biochemistry and biotechnology, kurt-mothes-str. , halle, germany one essential element of a virus is its protein shell, the viral capsid, which encloses the viral genome. the murine polyomavirus is a non-enveloped dna tumor virus with an icosahedral t= d structure. besides the knowledge of the structure, it is of utter importance to understand the process of viral assembly [ ] . the assembly reaction of polyoma vp does not show the typical sigmoidal kinetics in light scattering experiments. the apparent kinetics is of fourth order, which appears rather unrealistic. in order to gain knowledge on capsid composition during assembly beyond ensemble average, we apply methods of single molecule fluorescence, namely fluorescence correlation spectroscopy (fcs), fluorescence-intensity-distribution-analysis (fida), and single-particle-imaging (spi). the molecular brightness and the diffusion time reported by fcs are in agreement with the results from light scattering. fida as well as spi, moreover, point to a polymerization of subunits to complete capsids along the assembly pathway without pronounced intermediates. mixing experiments show that already early in the course of the assembly reaction no exchange of pentamers between capsids occurs, and that the effect of breathing [ ] can be excluded for polyoma vp . genetic information coded in dna provides complete instructions to cells regarding metabolism and proper functioning. a number of endogenous and exogenous sources can damage the genomic dna. unrepaired dna damage can give rise to mutations and may cause cell death. cells have evolved different mechanisms to repair damaged dna and to protect genetic integrity. uracil in dna occurs as a result of incorporation of dump in the place of dtmp during replication and deamination of cytocine, resulting in u:a and u:g base pairs, respectively. uracil-dna glycosylase (ung) is a dna repair protein that searches and removes uracil from dna. ung removes mismatched base by flipping it out from the base stack into the active site. the exact mechanism by which ung searches dna for uracil is unknown. therefore, in our study we use atomic force microscopy (afm) to investigate the interaction between single ung molecules with dna carrying the u:a or u:g mismatches. furthermore, we study the complexation of dna and ung protein. afm images of ung bound to dna reveal the structural changes at the level of single complex, e.g. dna kinking that may occur upon binding of ung to dna. imaging of dnaprotein complexes can provide a a new level of understanding of ung-dna interaction. atomic force microscopy (afm) has been a useful device to visualize cellular and molecular structures at a single-molecule resolution. especially, afm imaging under the trec t m (topography and recognition) mode (recognition imaging) can map a specific protein of interest within an afm image. in this study, we employed an antibody-coupled afm cantilever in recognition imaging, and dissected the structural/functional domains of α actinin- , an actin binding protein that cross-bridges actin filaments and anchors it to integrin via tailin-vinculin-α actinin adaptor-interaction. a use of monoclonal anti-α actinin- antibody enabled us to map its epitope in the amino-terminal actin-binding domain within a single molecule afm image of α actinin- . counting fluorescent molecules by photonantibunching h. ta, m. schwering, d. p. herten bioquant, heidelberg university, germany information on the stoichiometry of labelled biomolecules is highly desirable. a method called photo-antibunching has successfully been used to determine the number of fluorescence emitters in multichromophoric systems. a statistical model to estimate the number of fluorescent molecules in a confocal observation volume is established based on photonantibunching in time-correlated single-photon counting (tcspc) scheme with avalanche photon diodes (apds) for detection of individual photons. numerical simulations based on realistic experimental conditions show that the model is able to estimate the number of molecules with moderate errors. experiments on immobilized double-strand dna oligonucleotides with photon stabilizing agents show promising results even when the number of molecules is ∼ . the proposed method allows us to monitor labelled single molecules and in the near future we plan to implement it in complex biological systems (cell extracts/live cells). single molecule afm force spectroscopy and steered molecular dynamics of contactin- protein j. w. strzelecki, k. mikulska, a. balter, w. nowak institute of physics, nicolaus copernicus university, grudziadzka , - torun, poland contactin- (cntn ) is an axonal cell adhesion protein that contains six igc and four fniii domains and is responsible for creating neural outgrowths. recent research shows that mutation of cntn gene may be responsible for autism, while its absence in transgenic mice results in lack of smell sense. we use a homemade afm single molecule puller and steered molecular dynamics simulation to test its elastic properties through force spectroscopy. stretching experiments show unfolding of fniii domains while igc domains stay coiled as they are stabilized by disulfide bonds. unfolding of contactin- molecule appears to play role of buffer that helps to protect neural contacts from damage when neural cells are subjected to shock or tumor. single molecule studies of the thermophilic bacillus ps f -atpase have revealed kinetic and structural information that cannot be discerned using other methods, including the presence of and degree physical substeps (yasuda et al. ) and the order and kinetics of chemical substeps. we are interested in using single molecule techniques to observe the effects of mgi mutations on enzyme kinetics and torque production in f from the yeast saccharomyces cerevisiae. using a high speed imaging camera, we have captured the rotation of wild-type and mutant forms of yeast f -atpase. rotation data for the wild-type and preliminary data for some mgi strains will be presented. we show for the first time that at saturating atp, wild-type yeast f rotates approximately four times faster than the thermophilic f . kinetic and substepping behaviour in yeast appears to be similar to that observed in bacterial f . -single molecule biophysics - biophysical characterization of a dna gquadruplex formed in the promoter of human mef d gene w. zhou , l. ying molecular medicine, national heart and lung institute, imperial college london, london, sw az, uk, chemical biology center, imperial college london, sw az, uk g-quadruplex is believed to be involved in many crucial biological processes, such as the gene regulation and the maintenance of human telomere. mef d, a member of mef (myocyte enhancer factor- ) family of transcription factors, regulates the response of heart to cardiac stress signals. we found that a g-rich sequence starting at - bp of mef d promoter can form a very stable intramolecular g-quadruplex. here we present a detailed biophysical characterization of this quadruplex. we also found that this quadruplex is more stable than its duplex form under physiological conditions by cd melting and single molecular fret. we observed subpopulations in smfret measurements possibly due to different conformations of the quadruplex. we characterized its unfolding process by monitoring the change in fret when it hybridizes to its c-rich complimentary strand. finally, we proposed several possible structures of this quadruplex based on the smfret and fluorescence dms footprinting results. this research is supported by national heart and lung institute foundation. by stretching a polymer in solution using single molecule techniques it is possible to infer about its physical properties. afm stretching experiments allow for a full characterization of the elasto-mechanical properties of the sample under study. in the presented work, single molecule afm force spectroscopy experiments were used to determine mechanical properties of a peptide obtained from exon (ex ) of the human elastin gene. elastin is a protein with important mechanical properties and, in particular, it shows quasi ideal elastic behavior associated to the presence of many hydrophobic unstructured domains (such as ex ) into the protein structure. ex coded polymer was used as a starting point to obtain bio-materials with specialized elastomechanical functions. in particular, a mutated polypeptide based on the ex sequence was synthesisized with the aim of obtaining a new polymer with the same mechanical and physical properties of the native molecule but with increased aggregation properties, induced by a cross-linking reaction. afm stretching experiments were used to verify the mechanical properties of the engineered proteins at a single molecule level. the obtained results allowed not only to address this question, but also to give some insight into the first aggregation steps of the polymer towards the formation of reticulated structures. luminescent lanthanide-ion doped nanoparticles (nps) are attractive single-biomolecule labels. they are synthesized directly in water, are highly photostable, and display narrow emission without intermittency. we coupled y . eu . vo nps to ε-toxins produced by clostridium perfringens, which bind to a specific receptor on mdck cells. single-molecule tracking experiments using these labels produce long ( min) uninterrupted trajectories with temporal resolution down to ms or localization precision down to nm. we found that the toxin receptor exhibits confined motion in cell membrane microdomains. to analyze the receptor trajectories, we used a novel approach based on an inference method [ ] . this method fully exploits the information of the ensemble of the trajectory, in contrast to the usual mean square displacement analysis. applying both techniques to recorded trajectories, we highlight the difference in extracted parameters. from the shape of the confining potential, obtained by mapping the forces inside the domain, we can deduce information about the mechanism of confinement. in combination with experiments on cholesterol depletion and cytoskeleton destruction, this technique will shed light into the nature of the membrane micropatterning. background: implantable cardioverter defibrillators (icds) are save-life device for patients at risk of sudden cardiac death, and help cardiac patients to avoid slow beat-rate by means of anti-bradichardia pacing (abp) feature. however, recent literature evidenced the presence of ventricular tachyarrhythmias (vts) immediately following abp, leading to the hypothesis that icd devices might be pro-arrhythmic. aim: study differences between pacing-associated tachyarrhythmias (pat) and other spontaneous vts. signals and methods: spontaneous vt episodes are retrieved from patients with icd. vts are examined and pat episodes, classified. characterization of vts is based on the analysis of seconds immediately preceding vt-onset quantified by: heart cycle (hc-prevt), prevalence of premature ventricular contraction (pvcprev) and prevalence of paced beats (pprev). significant differences are evaluated by student-t or chi-square tests with p< . . results: vt episodes from patients are pat episodes ( %). cardiac activity preceding pats vs. non-pats differs: pat episodes have higher hc-prevt (p< . ), greater pprev (p< . ), and greater pvcprev (p< . ). analysis also shows that pat episode often occur when the paced beat immediately follows a premature contraction. conclusion: the study indicates that new icd generations should avoid abp after premature ventricular contraction. towards mechanistical understanding of adsorption: combining technologies in situ and in real time p. h. bjöörn q-sense ab, västra frolunda, sweden a growing number of researchers in different surface related fields present evidence from more than one analytical technique when detailing their findings. thus a logic and useful development is to combine technologies for simultaneous measurements on a single sensor surface. to develop a biosensor platform such as an assay fast and accurately, mechanistical understanding of why the platform works is essential. quartz crystal microbalance-with dissipation (qcm-d) technology and instrumentation provides an open platform and enable easy and precise quantification of mass, thickness and viscoelastic properties of surface bound molecules. these parameters provide both a reference tool in assay development, but also detecting and identifying your target molecules as the combination of mass and material property info in many cases provide a unique response for a specific molecule. by combining qcm-d with other technologies, a range of useful info is put within easy reach of the researcher. recent advances will be presented where simultaneous real time and in situ measurements using qcm-d together with electrochemistry, ellipsometry and fluorescence microscopy enables manipulation of films as well as quantification of the variation of water content as a result of conformational changes in immobilized molecules. examples will include new data from the formation of protein films, polyelectrolyte multilayers and polymer brushes. intrinsic gravity versus metabolically inert infrastructure and basal metabolic rate in living mass i. r. bhattacharjee , r. kashyap , b. shaptadvipa assam agricultural university, jorhat- , india, international institute of intrinsic gravitation biology (i gb), jorhat- , india 'self gravitation bio' is an emerging concept in biophysics. intrinsic or 'self' gravitational force might exert when mass increases beyond critical level in biological particle pyramid. (http://en.wikipedia.org/wiki/biomechanics of intrinsic gravity) 'metabolically inert infrastructure' (mii) consists of total body mass (body water, dissolved substances, mineral and organic deposits) and serves as storage of nutrients, transport and distribution of these materials. to act independently as living body, mii is suggested also to provide structural support to the organism with density-gradient buoyant force against intrinsic and extrinsic gravitational attraction for the biological mass. it is shown that 'amniotic fluid', 'isotonic saline to ailing patient', 'growth factors', 'cultural medium' and other 'medium matrices' act as counter-gravity mechanism for micro to macro living organisms under center-of-biomass reference frame. controversy over relationship between bmr (basal metabolic rate), rmr (resting metabolic rate), pal (physical activity level), on one hand, and mass of the living organism, on the other, (as described in rubner's / law, kleiber's / law that continued to be contested by many) can be favorably resolved substituting the concept of self gravitation bio, considering 'mass' as synonym of gravitational force under center-of-biomass reference frame. bioenergetics would be an unequal but opposite entity of self-gravity reinforced by extrinsic gravity. platelet arrest on von willebrand factor (vwf) occurs transiently via the platelet receptor gpib. depending on the combination of shear stress and surface density of vwf binding sites the platelets either only adhere, pull tethers or generate microparticles. these processes are influenced by the properties of the platelet membrane and the cytoskeleton. under shear flow conditions these platelet characteristics were examined with reflection interference contrast microscopy and a viscosimeter. in addition we quantified platelet adhesion energy and tether pulling forces. disrupting platelet f-actin had several effects. the tethers were shorter, the membrane contact area was larger, the receptor αiibβ density of the microparticles was depleted and no platelet spreading occurred. breaking up the microtubular system had different implications. the number of severed tethers tripled, the contact area was smaller, microparticle formation was increased and the area of spread platelets was reduced. however changes in the membrane elasticity had no effect on the platelets. our results suggest that platelet attachment and adhesion is not only determined by the platelet adhesion receptors and their cytoplasmic linker proteins, but that cytoskeletal structures have also a crucial influence on how platelets interact with thrombogenic surfaces. gene expression is orchestrated by a host of regulatory proteins that coordinate the transcription of dna to rna. regulatory proteins function by locating specific binding sequences of dna and binding to these sequences to form the transcription initiation complex. in many instances, these regulatory proteins only have several hundred copies that must efficiently locate target sequences on the genome-length dna strand. the non-specific binding of regulatory proteins to random sequences of dna is believed to permit the protein to slide along the dna in a stochastic manner. periodically, a thermal kick or an interaction with another bound protein will disengage the regulatory protein from the dna surface, leading to three-dimensional diffusion. eventually, the protein will reattach to the dna at some new location that is dictated by both the diffusivity of the protein and the dna configuration. cycling through these random events constitutes a search strategy for the target site. we build a reaction-diffusion theory of this search process in order to predict the optimal strategy for target site localization. the statistical behavior of the dna strand acts as a necessary input into the theory, and we consider several governing behaviors for the dna strand. we explore the impact of dna configuration and protein occlusion on target site localization in order to predict how protein expression will vary under different experimental conditions. a. di garbo , s. alloisio , s. chillemi , m. nobile istituto di biofisica cnr, via g. moruzzi , pisa, italy, istituto di biofisica cnr, via de marini , genova, italy in the nervous system of vertebrates there are more glial cells than neurons: from to times, depending on the animal type. glial cells are not able to generate action potentials but, nevertheless they play an important role for the functioning of the different brain's area. the astrocytes are the more abundant cells of the macroglia and through their processes they modulate synaptic activity. in this contribution a biophysical neural network model consisting of a pyramidal neuron, an interneuron and the astrocyte is studied. the corresponding dynamical properties are mainly investigated by using numerical simulations. the results show that the presence of the atp and of the interneuron strongly impacts the neural activity. moreover, it is shown that the fluxes of calcium through the cellular membrane strongly affect the modulation of the neural activity arising from the astrocyte. microscopic origin of the very-low energy vibrational dynamics in proteins g. d'angelo , v. conti nibali , c. crupi , a. paciaroni , u. wanderlingh department of physics, faculty of science, messina university, italy, department of physics, faculty of science, perugia university, italy important functions of biological processes require large atomic rearrangements and conformational fluctuations. for proteins, atoms are mostly displaced along the soft directions identified by the delocalized, low frequency vibrations. the study of very low energy vibrational spectrum of proteins is therefore of particular interest. as a step toward understanding their functional role, we have investigated the low frequency vibrational motions ( . ÷ mev) in different proteins by performing inelastic neutron scattering and low temperature ( . ÷ k) specific heat measurements. for the first time for biological systems, the well-known boson peak found in neutron scattering spectra at ∼ mev is put in relationship with a clear anomaly of the measured specific heat located at around k. this departure from the debye-like behavior further expands the analogy of proteins with glassy systems. quite interestingly, in the very low sub-mev energy range and below ∼ k, we observe an additional anomalous behaviour, which could be ascribed to the existence of twolevel-systems states. increasing the hydration degree, the low energy vibrational region is drastically altered, revealing that the addition or removal of the hydrogen bond network around the protein deeply modifies the interatomic forces, affecting the character of the vibrational modes. a. cupane , m. levantino , m. cammarata , g. schirò department of physical and astronomical sciences, university of palermo, via archirafi , i palermo, italy, european synchrotron radiation facility, grenoble, france our efforts in recent years have been to study in great detail the way hemoglobin works in confined geometries [ ] [ ] [ ] . to this aim we have contributed to the development of a new experimental technique, time-resolved wide-angle x-ray scattering (tr-waxs), that enables one to watch proteins at work in solution [ , ] . a very recent and challenging application of this technique is the study of hemoglobin functioning inside intact red blood cells (rbc). our preliminary results show that, by using laser pulses of about ns, it is possible to photolyse hemoglobin inside rbcs obtaining about % - % photolysis. good structural signals from hemoglobin are obtained, with limited radiation damage to the cells: this opens the possibility of studying the conformational transitions of hemoglobin in its "natural" environment. preliminary results concerning the timing of the r->t quaternary transition inside the rbc and comparison with the behaviour in dilute solution will be discussed. physical review letters and physical review e invite submission of your best work in biological physics. submissions must present significant new results in physics; topics may range from the microscopic to the macroscopic. we will provide information on the different types of articles published in the journals, on authors and referees, and on the review process. for publication in physical review letters, the work should be important and of broad interest. for rapid communications in physical review e, the work should be important for the field. biological physics papers published in physical review are indexed in medline. in an effort to bring important research to the attention of a wider community, the physical review journals have recently begun highlighting important work with viewpoints in the online publication physics. all physical review journals may be accessed through the website http://publish.aps.org/. modeling of fibrin gels using confocal microscopy, light scattering and turbidimetry f. ferri , d. magatti , b. cardinali , a. profumo , m. rocco dipartimento di fisica e matematica, università dell'insubria, como, italy, istituto nazionale per la ricerca sul cancro (ist), genova, italy fibrin gels are biological networks playing a fundamental role in blood coagulation. confocal microscopy of fibrin gels shows a collection of straight fibers, not uniformly distributed in space, connected together at low-order ( ) ( ) branching points. although each fiber is quite straight (mass fractal dimension d m = ), for the overall system d m > . based on the confocal images, we generated threedimensional ( d) synthetic gels made of cylindrical sticks of diameter d, joined together at randomly distributed nodal points. the resulting d network is no more random but ordered on length scales of the order the average fiber length, and exhibits a fractal morphology with d m ∼ . - . . the gel structure is analyzed by means of its d correlation function g d (r)=<n(x) n(x+r)> x , where n(x) is the gel local density. since the gel is isotropic, g d depends on the modulus r=|r| and can be obtained by averaging d correlation functions evaluated at different heights of the gel volume. from this analysis we recover the fiber diameter d (fwhm of g d ), the fractal dimension d m (power-law decay of g d ) and the gel mesh-size ξ (minimum in g d ). furthermore, the d-fourier transform of g d (r) gives the gel power spectrum i(q), which compares quite well with elastic light and multi-wavelength turbidimetry data taken on real gels. a model coupling vibrational and rotational motion for the dna molecule e. drigo filho, j. r. ruggiero, r. a. d. s. silva unesp -sao paulo state university, brazil a largely used mechanical model for vibrational motion of dna has been proposed by peyrard and bishop (pb). in this model, dna is represented by a pair of harmonic chains coupled by a nonlinear potential. the most frequently used nonlinear potential for this purpose is the morse potential. some extensions of the original model are proposed considering, for example, the helicoidal structure of dna. an important objective for this kind of model is to understand the phenomenon of thermal denaturation and, through this, get some knowledge about other processes such as the genetic transcription and drug intercalation. it is possible to obtain from this type of model interesting properties, such as the average stretching between base pairs as a function of the temperature using the transfer integral operator. dynamical properties of this model were also explored and several phenomenological quantities are studied, such as energy localization. in this work, we extend the original pb model by introducing rotational motions for the nucleotides. in this way, both the vibrational and rotational motion for each nucleotide are considered. the stretch of the base pairs is given by the morse potential in the same way as in the original pb model. however, the coupling between the two kinds of motion, rotation and vibration, is obtained through a nonlinear combination of them in the morse potential. a. dobovišek , m. brumen , p.Županović , d. juretić university of maribor, faculty of natural sciences and mathematics, maribor, slovenia, jožef stefan institute, ljubljana, slovenia, faculty of science, university of split, split, croatia mepp is a physical principle, widely used for quantitative explanation of non equilibrium phenomena in physics, chemistry and biology [ ] [ ] [ ] . here, we applied mepp to study two and three state reversible michaelis-menten kinetic schemes of enzymatic reactions. by applying constraints as a diffusional limit for kinetic constants, constant free energy differences between enzymatic states and constant thermodynamic force, we calculated shannon information entropy and entropy production of the entire reaction system as a function of forward rate constants. we found maxima in the net steady-state metabolic flux, total entropy production and shannon entropy for equal values of forward rate constants. moreover, for these values an analytical expression derived gives a relation between substrate and product concentrations at which enzymes operate in the optimal way. in conclusion, we demonstrated that mepp is an appropriate selection principle for evolutionary optimization of enzymes. attractive interaction between like-charged lipid surfaces mediated by spherical nanoparticles with spatially distributed charge is theoretically described by using functional density theory and mc simulations. the spatial distribution of charge within a single nanoparticle is considered by two effective charges at a finite distance. the minimization of the free energy is performed to obtain the equilibrium configuration of the system. both, the rigorous solution of the variational problem and the mc simulations show that orientational ordering of nanoparticles subject to the gradient of the electric field gives rise to an attractive interaction between charged lipid surfaces for high enough charge densities of the interacting surfaces and large enough separations between charges within the nanoparticle. the attraction is explained by orientational ordering of dimeric charges in the electric field which lowers free energy. viral genomes encode for a series of membrane proteins which are embedded or attached to the lipid membrane of the host, or penetrate them during the very first step of viral invasion. we are focussing especially to those of the former which are known to assemble and from channels or pores for small ions or substrates. with these channel forming proteins the virus manipulates the host cell interior for the benefit of its replication. strategies are developed to answer the question about the assembly process of these proteins based on the 'two stage model' and the substrate flux. only few experimental data are available to answer these questions, consequently we stress computational methods to derive the adequate answers. the methods applied are ab inito molecular dynamics (md) simulations based on density functional theory (dft) and conventional md simulations. potential routes for assembly of the three transmembrane domains of a protein from sars-cov will be outlined and compared with computational data from other viral membrane proteins. with a novel pore lining motif suggested for a, the dynamics of ions at selected positions within the putative pore will be assessed. probing the molecular mechanism of antibiotics diffusion through the ompf channel e. hajjar , a. kumar , m. winterhalter , p. ruggerone , m. ceccarelli department of physics, university of cagliari, italy, jacobs university, bremen, germany in gram-negative bacteria, the outer membrane porin-f (ompf) is the preferred entry point of antibiotics. bacteria can resist to antibiotics by altering the expression and the structures of ompf. a key feature in the structure of ompf is the presence of a constriction zone, characterized by both spatial and electrostatics restrictions. to study the process of antibiotics translocation at a molecular scale, we performed molecular dynamic simulations accelerated with the metadynamics algorithm. we studied the diffusion of antibiotics with different structural and chemical properties through ompf wild-type and variants. the free energy surface suggests faster translocation for the cephalosporins compared to the penicillins antibiotics, and also for ompf mutants compared to the wild type. further, the conservation of favored orientation and affinity sites of antibiotics inside the ompf channel reveal which specific interactions govern translocation. the calculated energy barriers and rate determining interactions for translocations compared well with the electrophysiology measurements and liposome swelling assays from our collaborators. this study demonstrates how theory and experiments can be combined to reveal the structural determinants and mechanism of ompf permeation. this will benefit to the design of antibiotics with improved transport properties. m. g. gauthier, j. bechhoefer dept. of physics, simon fraser univ., burnaby, bc, canada dna replication requires two distinct processes: the initiation of pre-licensed replication origins and the propagation of replication forks away from the fired origins. experiments indicate that these origins are triggered over the whole genome at a rate i(t) (the number of initiations per unreplicated length per time) that increases throughout most of the synthesis (s) phase, before rapidly decreasing to zero at the end of the replication process. we propose a simple model for the control of dna replication in which the rate of initiation of replication origins is controlled by the interaction with a population of ratelimiting proteins. we find the time set by reaction-diffusion kinetics for such proteins to find, bind to, and trigger a potential origin. the replication itself is modeled using a formalism resembling that used to study the kinetics of first-order phase transitions. analyzing data from xenopus frog embryos, we find that the initiation rate is reaction limited until nearly the end of replication, when it becomes diffusion limited. initiation of origins and hence i(t) is suppressed when the diffusionlimited search time dominates. we find that, in order to fit the experimental data, the interaction between dna and the rate-limiting protein must be subdiffusive. we also find that using a constant nuclear import of the limiting proteins leads to a more accurate description of the experimental data. the microsoft research -university of trento centre for computational and sisytems biology, trento, italy we propose a new method for inferring the structure of a biochemical network from the time-series of the reactant species. it consists of two parts: the first is the quantification of the correlation between the time-series profiles. correlation in time series data can be used to reveal dependencies between variables and to infer the graph of connectivity among species. the second part consists in the elimination from the connectivity graph of the relationships that have a non-null correlation coefficient, but that are not biochemically plausible. the cutting of false correlations from the graph is performed through the estimation of the parameters ("calibration") of the network. to calibrate the network for detecting null dynamics correlations, we developed the software tool kinfer (knowledge inference). based on a new probabilistic model of the variations in reaction concentrations, kinfer infers the values of the kinetic rate constants, their confidence regions and the level of noise in the input data by maximizing the likelihood to obtain the observed variations given the network model. the a priori knowledge required as input is minimal, as it consists only in the time-series of reactant concentrations. the minimal a priori knowledge and the probabilistic formulation of the calibration method make the accuracy of the predictions strong against experimental, biological and stochastic noise, and allows to use it to cut the null-dynamics edges of the connectivity graph. a. kuzmanic, b. zagrovic mediterranean institute for life sciences, split, croatia root-mean-square deviation (rmsd) is a measure used to give information on the global structure of macromolecules. for example, pairwise rmsd (prmsd) is used to assess similarity of the lowest energy nmr structures or for clustering large ensembles of structures. on the other hand, to obtain information on the local structure of a macromolecule and its dynamics, root-mean-square fluctuation (rmsf) is often used. rmsf can be calculated from md simulations, but also from experimental x-ray b-factors. since prmsd and rmsf report on different features, it is interesting to ask what the relationship between them is. first, we provide a mathematical derivation showing that, given a set of conservative assumptions, the <prmsd> rms is directly related to the <rmsf> rms and, consequently, experimental b-factors. second, we demonstrate this on structures taken from distributed-computing md simulations of the native and unfolded state of villin headpiece. both our analytical and computational results suggest a strong correlation: <rmsf> rms = [(s- )/ s] / <prmsd> rms , where s is the number of compared structures. furthermore, if <prmsd> rms is defined as a generalized radius of gyration in the space of d structures and using rmsd as a measure of distance, the following identity holds: <rmsf> rms = <prmsd> rms. our results provide a basis for determining the level of structural diversity of molecular ensembles, as captured by <prmsd> rms , directly from experiment. charge migration along dna helices may be biologically important because extended electronic states could play a role in the processes of sensing of dna damage and/or dna repair via long-range charge transfer. we measured conductivity and other physical characteristics of several models of natural and diversely damaged molecules of dna. dna polymers were mimicked by various sequences of nucleotide-long double helices with fully watson-crick (wc) paired bases, with several central bases mismatched, and also with chemical modifications that included removal of bases from a few central nucleotides (abasic dna), and neutralization of phosphate charges by their derivatization. the model dnas were investigated by scanning tunneling microscopy, time-resolved thz spectroscopy, raman spectroscopy, circular dichroism, and modeled by molecular dynamics simulations. dna has the highest conductivity in its biologically most relevant double helical form with wc base pairs and negatively charged phosphates equilibrated with counterions. mismatches and all chemical modifications always lower the conductivity. the mechanism of charge transfer is consistent with electron or hole hopping between parallel stacked bases. these observations and data showing that the natural dna has also the most regular double helical form suggest that the continuous base stacking is critical for charge transfer. to control the passage across the bacterial cell wall nature created a large number of "nano"-channels which may act as selective gates for water soluble molecules. here we focus on porins from e. coli which control permeation through interactions with the channel surface. comparing single channel temperature dependent conductance measurements with all atom modeling allow conclusions on the mode of permeation. for example, surprisingly modeling ompf-conductance revealed not only a good agreement with the experiment over a broad range of temperature but also the selectivity for ions. the primary task of porins is to provide facilitated diffusion. we investigated facilitated diffusion of maltooligosaccharide or antibiotics. time resolved conductance measurements allows conclusion on the flux and molecular modeling identifies the limiting interactions with the surface, reveal potential barriers and pathways. exploiting the selectivity of natural or bioengineered channels has promising applications for detecting molecules, characterizing molecular interaction, sequencing dna, protein folding etc. traditionally, studies of diffusion-controlled reaction of biological macromolecules have been made in diluted solutions. however, the high concentration of macromolecules in intracellular environments results into non-specific interactions (macromolecular crowding), which have a great importance on the kinetics and thermodynamics of possible reactions that occur in these systems. in the literature there are studies concerning monte carlo (mc) simulations, giving results that are satisfactory agreement with experimental data, showing, for example, that the protein diffusion in cell cytoplasm is reduced considerably. in addition, there are mc studies about enzymatic reaction, which predict a temporal dependency of the velocity constant in macromolecular crowding. in this work, we try to compare the predicted behavior by mc simulation with the results obtained from the study of the diffusion and reaction of a model protein (alpha-chymotrypsin) using spectroscopic techniques in highly confined media in order to study experimentally the temporal dependence of its diffusion and reaction coefficients. a. paciaroni , a. orecchini , c. petrillo , a. de francesco , f. sacchetti university of perugia, italy, cnr-infm, genova, italy the single-particle and collective dynamical properties of protein hydration water have been studied by neutron scattering experiments in a wide temperature and hydration range. an unprecedented accuracy has been achieved thanks to the availability of a large amount of fully deuterated protein powder and the use of the high-flux spectrometers in and brisp. the protein under investigation was the maltose binding protein (mbp), which is a well-known and widely studied model of biosensor systems. we found that the low-temperature single particle dynamics of mbp hydration water shows clear features that can be traced back to amorphous systems. more in detail, its vibrational density of states is simply described as the superposition of the contributions of low-density and high-density amorphous ice. the quasielastic signal, which appears at the higher temperatures, can be excellently described with a fractional power law which may put in relationship with the peculiarities of fractal systems. quite strikingly, there is a strong similarity, on both the qualitative and quantitative point of view, with the behaviour of hydrated proteins. the collective dynamics of protein hydration water is characterised by the presence of two modes, whose dispersion curves are reminiscent of those of bulk water. however, the relevant damping factors suggest a strong similarity with glassy systems. m. malferrari , f. francia , s. sacquin-mora , g. venturoli università di bologna, bologna, italy, cnrs upr , paris, france the coupling between electron transfer and protein dynamics has been compared in reaction centers (rc) from the wild type (wt) and the carotenoid-less mutant r , by combining brownian dynamics simulations and the kinetic analysis of charge recombination. upon incorporation of the rc into a progressively dehydrated trehalose matrix the electron transfer between the primary photoreduced quinone and the photoxidized donor accelerates progressively and becomes broadly distributed. this behaviour reflects the hindrance of protein relaxation following charge separation and the inhibition of interconversion between conformational substates. in extensively dehydrated matrices the recombination kinetics is two-times faster and three-times more distributed in the wt rc, indicating a larger inhibition of the internal protein dynamics. in line with this findings brownian dynamics simulations reveal a larger rigidity of the carotenoid-containing structure, in which a cluster of residues close to the quinone acceptors is stiffened as compared to the r rc. the in silico and experimental results indicate that the introduction of an internal void in the rc structure has long-range effects on the protein dynamics and that the coupling between the glassy matrix and the rc interior depends markedly on the local mechanical properties of the protein. n. maghelli , v. krstic , n. pavin , f. julicher , i. tolic-norrelykke mpi-cbg, dresden, germany, mpi-pks, dresden, germany in the fission yeast schizosaccharomyces pombe, the nucleus is positioned at the cell center. since the nucleus determines the cell division site, keeping the nucleus at the center is crucial for ensuring symmetrical cell division ( ) . microtubules push against the cell ends and exert force on the nucleus ( ), but how the cell regulates these forces in order to center the nucleus remains unknown. here we tackle this problem by using a combination of live cell imaging, cell manipulations by optical tweezers, and a theoretical model. we show that microtubule pushing forces can center the nucleus because of a larger number of contacts between the microtubules and the proximal cell end than the distal one. moreover, kinesin- motors (klp / ) increase the rate of microtubule catastrophe (transition from growth to shrinkage) in a microtubule length-and contact-dependent manner. thus, the motor behavior results in a longer contact between a microtubule and the proximal than the distal cell end. taken together, our experimental and theoretical results provide a novel centering mechanism, where kinesin- motors increase the efficiency of nuclear centering. electropermeabilization is a commonly used physical method which can induce a transient permeabilization of the cell membrane allowing the entry of therapeutic molecules into the cell and is thus of great interest in the fields of cancer treatment and gene therapy [ ] . however, very little is known about the mechanisms occurring at molecular level. there is clearly some microscopic reorganization of the membrane which is responsible for this change in its transverse transport properties. rather than studying the change of these transport properties, we adopt a simple strategy based on the use of giant unilamellar vesicles and videomicroscopy, as described below. we apply a series of permeabilizing electric pulses to the liposomes, and we observe a size decrease down to a critical radius beyond which their size no longer changes. this decrease in size points to the fact that during the physical processes leading to electropermeabilization, lipids are lost from the vesicles. our results published in [ ] suggest different possible modes for lipid loss, which can be small vesicles, pores, or tubules formation. imaging of brain activation using core techniques as fmri, pet and synchrotron radiation in parallel c. poitry-yamate, g. margaritondo, r. gruetter ecole polytechnique fédérale de lausanne, switzerland functional magnetic resonance imaging (fmri), magnetic resonance spectroscopy (mrs), positron emission tomography (pet) and synchrotron x-ray emission imaging form a highly complementary set of imaging core technologies for studying brain function and energy metabolism. owing to differences in the information each conveys and the temporal and spatial scales on which they measure labeled substances, a single working hypothesis can be tested from different angles to provide a cross-validated, consistent and coherent explanation. the cibm is a unique research facility in europe for advancing our understanding of biomedical processes in health and disease. the housing of a -tesla human magnet, . and . -tesla animal magnets, an animal pet imaging facility and fully-equipped neurochemistry and rf laboratories has enabled us to develop and perform well-targeted experiments around one research theme. in parallel, a longterm collaborative project using synchrotron x-ray transmission and emission imaging at elettra enables us to combine these core technologies towards understanding brain function in vitro and in vivo, from tissue to cells. a brief presentation of these methods will be followed by their application in studying the visual system from man to mouse. p. picone , r. carrotta , d. giacomazza , m. di carlo dip. chimica e tecnologie farmaceutiche, università di palermo, italia, ibf -cnr, palermo, italia, ibim -cnr, palermo, italia diabetes and alzheimer's disease are connected in a way that still is not completely known. diabetes has been implicated as a risk factor for developing alzheimer's disease. some diabetes drugs appear to decrease the cognitive decline associated with alzheimer's disease. it has been recently demonstrated that extracellular injection of insulin is able to protect neurons against a-beta amyloid cell death. one of the proposed theories to explain such an effect is that the hematic glucose levels affect the metabolism of the hippocampus, a part of the brain (associated with memory, emotion and motor skills), which is strongly damaged in alzheimer's patients. the aim of this study is to investigate the effect of insulin on the a-beta induced degeneration and oxidative stress on the neuroblastoma lan cell line. in particular, the present study looks into the role of insulin in the inhibition of abeta specific degenerative apoptotic pathways. preliminary results indicate that insulin dissolved in culture medium in its hexameric form (as tested by absolute scale light scattering) is able to reduce neurodegeneration induced by a-beta amyloid in a dose dependent manner. the link between diabetes and alzheimer's disease may provide new targets for future alzheimer's treatments. moreover, due to the increased incidence of diabetes in western countries, a deeper understanding of such a link is relevant in order to control the escalation in the number of people dealing with dementia. a. pelizzola dipartimento di fisica, politecnico di torino, torino, italy many features of protein folding have been shown to be described by an ising-like model (one-dimensional, with longrange, multispin interactions) whose equilibrium thermodynamics is exactly solvable [ ] [ ] [ ] . we have generalized such a model to the problem of mechanical unfolding. the equilibrium thermodynamics is still exactly solvable, and the characteristic kinetic responses found in force ramp and force clamp experiments are well reproduced [ , ] . unfolding and refolding pathways and intermediates can also be studied, again with good agreement with experiments [ ]. applications to various proteins and rna fragments will be discussed. [ the key role of water in living systems has been widely studied in literature, along with its anomalies, consequence of the extensive three-dimensional hydrogen bonding of water molecules. moreover protein-water interactions take place at protein surface where cell water has been recognized to behave differently from bulky water. the two-states theory of water assumes that water is a mixture of microdomains of different structure and density, the low-density water (ldw) and the high-density water (hdw) domains, and ions partition selectively into ldw or hdw domains. the idea developed in this work was to explore the ordered water structure by measuring delayed luminescence (dl) from salt aqueous solutions in which water structuring is anticipated. it appeared that dl signal from salt solutions is significantly relevant when prevalence of ldw domains is foreseen, with a decay time probability distribution function characterized by a broad maximum in the microsecond range. the obtained results support the ability of dl to reveal the different properties of ldw and hdw domains induced by salt molecules. moreover, the results reveal the existence of clusters, whose characteristics strongly depend on the specific ion effects, of surprisingly long lifetimes not observed till now. this could give new insight into biological water properties. self assembly of patchy particles and dnafunctionalized dendrimers f. sciortino dipartimento di fisica e infm-cnr-soft università 'la sapienza', roma, italy i will report numerical results on the phase behavior of very simple models of patchy particles with the aim of understanding the interplay between phase separation and selfassembly and how the fraction of surface allowing for attractive interactions controls the collective behavior of the system. the case of janus particles, particles characterized by a surface divided evenly into two areas of different chemical composition, will be discussed. i will also discuss the self-assembly of a simple model for four single strands of dna tethered to a central core, and show that the model exhibits a rich phase diagram that includes at least four thermodynamically distinct amorphous phases (polyamorphism) in a one-component system. the dense phases consist of a hierarchy of interpenetrating networks, reminiscent of a woven cloth. peptide dimer motifs in the phospholipid environment -structure, interaction and molecular design p. e. schneggenburger , a. beerlink , t. salditt , u. diederichsen iobc, universität göttingen, germany., irp, universität göttingen, germany. based on recently reported homodimeric peptide pores with a d,l-alternating configuration a novel double helical hairpin-motif of a membrane active gramicidin a analog was designed. [ , ] the cd spectroscopic analyses of the peptide-lipid complexes revealed the structural preservation and elucidated the importance of a zwitterionic interaction of the peptide termini. [ ] the peptide design was enhanced regarding the versatile functionalization with analytical probes as well as molecular recognition moieties like peptide nucleic acids (pnas) to observe the effects of aggregation and specific organization within model lipid membranes even at high peptide-to-lipid ratios. [ ] x-ray reflectivity on lipid bilayer stacks in combination with heavy atom labeling and spectroscopic studies of vesicle systems provides information about the peptide structure and interaction in the native fluid state of the membrane system. [ , ] for this, the fmoc-diiodo-allylglycine building block was created to serve as a novel iodine label pinpointing at a certain position with respect to the membrane normal. we study thermal undulations of giant unilamellar vesicles (guvs) of lipids by flickering spectroscopy. getting values for the mechanical parameters of lipid bilayers requires the experimental fluctuation spectra to be scrutinized in view of the classical helfrich's theory. pure bending modes are revealed unable in predicting the large fluctuations systematically found at high wavevectors. hybrid curvature-dilational modes have been invoked as a more efficient mode of motion in producing high curvatures. a bimodal spectrum of the thermal undulations has been theoretically developed for the shell-like topology. from this new description, two important consequences emerge a priori, the dependence of the fluctuation dynamics on either vesicle size and on bending/compression parameters. for popc and dopc vesicles containing cholesterol the experimental fluctuation spectra are well described by the new spectrum. reconciliation between experiments and theory is achieved when this bimodal spectrum is considered. the new theory opens enormous possibilities for better exploring membrane mechanics in guv models. under normal conditions, platelets circulate in the vascular system having very low interaction with each other and with other cells. the platelets become activated when the biological system is disturbed, for instance by vascular damage in which blood gets in contact with collagen. upon activation, different types of receptors/molecules are exposed on the cell membrane to support adhesion, spreading and aggregation of the platelets onto the damaged vessel. the measurement of altered platelet function is particularly important in cardiovascular diseases such as thrombosis. we are investigating biosensor technologies for the detection of functional properties of platelets. an important study is the specific and non-specific stimulation of platelets in a biosensor cartridge. we will present experimental results on biosensor platelet activation using the platelet-specific membrane markers p-selectin and gp b. multi-joint analysis of locomotion in the first neonatal rats flown in space d. sulica, j. vinersan "carol davila" university of medicine and pharmacy, bucharest, romania the first mammalian neonatal animals in space were the rats flown on the space shuttle endeavor during a -day mission, sts- . the development of locomotion in weightlessness was evaluated using two litters of neonatal rats, launched at postnatal days and . age-and cage-matched animals were used as ground controls. free walking was videotaped from the landing day. although preliminary analysis of walking showed differences in both hindlimb and forelimb joint angles and a hyperextension of the hindlimbs was apparent, the numerical values reached the significance level only for the ankle angle measured at specific moments of the step cycle: foot contact, maximum loading with weight, foot lift and maximum flexion during swing. we report here on the behavior of all the joints during the whole step cycle, by computing the integral of these angles over the step cycle. the results were affected by the differences in the walking speed (the young animals walked faster than the controls), so we scaled the integrals by the step cycle duration. we found that, besides the ankle, the knee was also more extended throughout the whole step cycle in both groups of animals. moreover, all the joints (including the toe and the hip) were affected in the same way (hyperextended), since the differences were still significant when we added together these angles. the animals recovered slowly, with significant differences remaining after days of readaptation. the effect of dextran concentration on red blood cell deposit formation j. strzelecka, b. grzegorzewski department of biophysics, collegium medicum in bydgoszcz, nicolaus copernicus university - bydgoszcz, poland red blood cell (rbc) deposit formation was examined by means of an optical method. blood was obtained from healthy donors and measurements were performed at initial hematocrit %. the intensity of scattered light was measured during sedimentation of rbcs suspended in saline -dextran solutions at different polymer concentrations ( - g/dl). the changes in the intensity of the scattered light manifest rbc aggregate formation, their sedimentation and the process of deposit formation. the deposit formation curve was determined. it is shown that the concentration of dextran affects the deposit formation. an empirical model has been used to describe the experimental data. the parameters of the deposit formation curve as a function of dextran concentration are analyzed. water is essential to life and a major scientific interest lies in a detailed understanding of how it interacts with biological macromolecules in cells. we studied water dynamics in whole cells with neutron scattering [ , , ] . the cellular environment is extremely crowded with macromolecules and water molecules are permanently in close contact to biological interfaces. we measured water dynamics in e. coli and human red blood cells with neutron scattering [ , ] . the data revealed two populations of water in the cells: a major fraction which has dynamical properties similar to those of bulk water (relaxation times ∼ps) and a minor fraction in the order of ∼ % which is interpreted as bound hydration water with significantly slower dynamics (relaxation times > ps). in this contribution we report on investigation of model membrane dynamics by means of quasi elastic and inelastic incoherent neutron scattering and on the effect of membrane inserted pore forming peptide gramicidin. model membrane are realized by highly oriented, hydrated phospholipid bilayer stacks of dmpc ( , -dimyristoyl-snglycero- -phoshatidylcholine) hydrated with d o in excess of solvent condition. the bilayer were supported on mica substrates and prepared at different concentrations of gramicidin, a -residue oligopeptide showing antimicrobial activity by forming pores on the membrane surfaces which allow water and small ions to permeate across the membrane. incoherent qens and ins spectra, measured on in and in spectrometer at ill, allows to obtaining information on the mean dynamics of the hydrogen atoms in the system. moreover, by proper orientations of the membrane plane respect to the scattering wave vector q, we were able to derive information on in plane and out of plane motions of the phospholipids. the using of media products for the creating attracted bioenergetic brain rhythmus advertisement v. i. vlastopulo, v. g. nikolajev str. gen. petrova , app. , odessa, ukraine the technical efficiency of bioenergetical influence of advertisement is present with assistance of consciousness of memory at revision and hearing of advertisement on tv, radio stations, mobile phones, at supermarkets and other places. in a basis of useful model it is put a task to improve the method of creating the attracted advertisement, in which the creation of bioenergetical influence by the oscillation of not less electromagnetic fields or video-images is introduced with their creating as the base on the spatial or flat structure formative macro matrix or matrixes with repeatable structure with the brain α-rhythm frequency of extreme attention and δ-rhythm frequency of meditation. the point of the patent on the device is in the bioenergetical influence increases in addition to the information influence by advertisement of pictures and audio oscillations the bioenergetical influence increases at the consciousness contribution of human memory at the moment of watching or hearing of television, radio stations, working in the internet, music in the supermarkets, banks, clubs, metro and other places. cell adhesion and motility are processes involved in fundamental biological phenomena. they imply multimolecular scaffolds as anchorage points and actin cytoskeleton filaments to build internal stress and eventually crawl onto the substrate. these processes, very dynamic by nature are out of equilibrium. we study cell adhesion on micro-patterned substrates where the introduction of a finite distance between the possible anchorage points of the cell modifies drastically the organization of the cytoskeleton and the anchorage point's distribution. because statistical quantification shows that some shapes are more likely than other, we believe they represent particular organizations of the system which should minimize the energy dissipation. we checked this hypothesis by using the cellular potts model. shapes obtained by simulation are in excellent qualitative agreement with experimental shapes. they depend on phenomenological parameters such as interaction between cells and the extra cellular matrix, a line tension and an elastic modulus. the aim of this work is to link model parameters to physico-chemical properties of cells and to establish phenomenological relations between relevant biochemical regulators controlling the cytoskeleton organization. c. canale, d. ferrera, f. benfenati, l. gasparini the italian institute of technology, genova, italy alzheimer disease (ad) is characterized by cerebral extracellular deposits of β-amyloid (aβ) fibrils. aβ aggregation is a multi-step process involving the formation of various conformational species including soluble intermediate species (i.e. aβ oligomers), protofibrils and fibrils. such aggregates may have various effects on neuronal and glial function and differentially contribute to ad neurodegeneration. aim of this study was to investigate the structural properties of distinct aβ aggregated species and dissect out their effects on neuronal viability. recombinant aβ and aβ peptides were aggregated in vitro in conditions differing by ionic strength, temperature and ph and were analyzed by gel electrophoresis, thioflavin t binding assay and atomic force microscopy (afm). afm analysis was performed using both hydrophilic and hydrophobic substrates, to analyze the full spectrum of structural species. stable low molecular weight oligomers were obtained when aβ was incubated at • c for days in low salt concentration buffer. doughnut-shaped conformational species were detected by afm alongside globular aggregates ( . - . nm height range). acidic ph promoted aggregation of aβ into thioflavin-positive fibrils and protofibrils. protofibrils appeared as beaded chains having a mean height of . ± . nm. effects of aβ on viability of mouse hypppocampal neurons were assessed and correlated with their conformational features. a. bellova , e. bystrenova , m. koneracka , p. kopcansky , f. valle , j. bagelova , f. biscarini , z. gazova institute of experimental physics, slovak academy of sciences, kosice, slovakia, ismn cnr, bologna, italy peptide amyloid aggregation is a hallmark of amyloid diseases including azheimer's disease or type ii diabetes. recent works have addressed the potential of nanoparticles to affect amyloid aggregation. the experimental data are very controversial suggesting that particle characteristics markedly influence the final effect of nanoparticles on the amyloid aggregation (initiation, acceleration or inhibition of amyloid aggregation). we investigate the ability of electrostatically stabilized magnetic nanoparticles of fe o to affect the amyloid aggregation of lysozyme, as a prototype amyloidogenic protein. we have used a combination of spectroscopic (tht fluorescence) and local microscopic techniques (afm). we found, that the ability of magnetic nanoparticles to inhibit formation of amyloid aggregates or destroy pre-formed amyloids exhibit concentrationdependence. the values of inhibition ic and depolymerization dc were determined suggesting that nanoparticles interfere with lysozyme aggregation at stoichiometric concentrations. the observed features make magnetic nanoparticles of potential interest as a therapeutical agent against amyloid diseases. (this work was supported by project of esf and by slovak academy of sciences in frame of cex nanofluid, vega grants , and and eu-strp biodot.). a. bellova , l. balogova , b. chelli , e. bystrenova , f. valle , j. imrich , p. kristian , l. drajna , j. bagelova , f. biscarini , z. gazova institute of experimental physics, sas, kosice, slovakia, faculty of sciences, p. j. safarik university, kosice, slovakia, ismn cnr, bologna, italy numerous diseases have been linked to a common pathogenic process called amyloidosis, whereby proteins or peptide clump together to form amyloid aggregates in the body. an attractive strategy to develop therapies for these diseases seems to be reduction of polypeptide aggregation. we have tested several acridine derivatives characterized by various glycosyl groups for their potential to affect the lysozyme amyloid aggregation in vitro. the ability of glycosyl acridines to interfere with lysozyme aggregation was investigated by tht assay. we found that structure of acridine side chain is factor affecting their anti-aggregation activity significantly. for the most effective compounds the values of ic and dc were obtained. the reduction of protein aggregation was confirmed by afm. to investigate influence of the glycosyl acridines on the cell processes we examined effect of compounds on cell viability. we performed glycosyl acridines characterization by high anti-aggregation activity and low toxicity suggesting their possible application for therapeutical purpose. (this work was supported by project of esf and by slovak academy of sciences in frame of cex nanofluid and vega grants , and and eu-strp biodot.). a. j. beevers, a. m. dixon department of chemistry, university of warwick, uk due to the immense medical importance of proteins which span the membrane of cells, detailed molecular structural information of these systems is essential. practical difficulties in employing high-resolution structural elucidation techniques have resulted in a relative paucity of fully resolved membrane protein structures. therefore a variety of lower-resolution techniques are used to determine structural information of the transmembrane (tm) domains of proteins. one example of such a membrane protein is erbb- , a receptor tyrosine kinase responsible for triggering cell division and which is prone to a mutation in its transmembrane domain resulting in permanent activation and oncogenic effects. we have predicted an interface for the mutated tm domain dimer using site-specific infrared spectroscopy containing a repeating sequence of ile, val and leu . applying the in vivo toxcat assay to the tm domain sequence and to specific mutants of it, confirms this proposed interface whilst another proposed interface is discounted. current studies are focussing on the effect of the tm mutation to the activation of the erbb- receptor and to any possible change in this interface. bacteriophages are complex molecular assemblies which multiplication relies on bacteria infection. the process starts with the binding of the phage on its specific host receptor and the injection of its genome into the host cytoplasm. our work aims to determine the physical mechanisms and forces driving the dna transfer from the phage capsid. the in vitro dna ejection has been analyzed by using light scattering and gel electrophoresis measurements for three phages (t , spp and lambda) belonging to the same family (syphoviridae). our results reveal two forces contributing to drive the dna transfer: the first one is originated from the pressurization due to the strong confinement of dna into the capsid; the second one comes from a pulling mechanism originated by the presence of condensed dna outside the capsid. these two contributions were characterized in in vitro conditions but they likely play a role in the in vivo transfer. the ejection kinetics was also analysed and the characteristic time of the mechanism was studied as a function of the temperature. it appears to follow an arrhenius law, allowing the determination of the activation energy that governs the transfer. the energy values are close for the different phages, suggesting that the mechanism regulating the ejection is common for a given phage family. below these general features, our studies also reveal differences between the three phages. the effect of &beta-amyloid peptide on polymer cushioned membranes s. dante , r. steitz , t. hauss , c. canale , n. a. dencher istituto italiano di tecnologia, genova, italy, bensc, helmholtz center berlin, germany, tu-darmstadt germany beta-amyloid (aβ) is a peptide implicated in the neurodegenerative process characteristic of the alzheimer's disease (ad). to clarify its mechanism of action it is crucial to elucidate the interaction of aβ with the neural membrane. in previous work we demonstrated the capability of aβ to penetrate and perturb stacked lipid bilayers. in this study we considered polymer cushioned lipid bilayers as a model for neural membranes. the polymer cushion is aimed to preserve the membrane natural fluidity; it is obtained depositing charged polyelectrolites layer-by-layer; the lipid membrane is built on the top of the polymer film by fusion of unilamellar vesicles. the floating membranes were kept always in contact to the subphase. the kinetics of adsorption of the lipid double layer at the polymer/water interface was monitored by neutron reflectivity; different experimental conditions to obtain the best surface coverage were exploited. after administration of aβ to the subphase the lipid membrane still adhered to the polymer cushion, but its structure was modified by the interaction with aβ. neutron reflectivity showed a change of the scattering density profile in the direction perpendicular to the membrane plane, suggesting a penetration of aβ inside the double layer. a change in the surface morphology was detected by afm imaging; afm film-rupture experiments showed that aβ weakens the lipid packing. x. cheng , r. pacheco-gomez , a. rodger , h. matthew , d. i. roper university of warwick, u.k., university of birmingham, u.k. ftsz, the ancestral homolog of eukaryotic tubulins, is a gt-pase that assembles into a cytokinetic ring structure (z ring) essential for cell division in prokaryotic cells. the z ring also recruits other proteins (e.g. zapa, ygfe, zipa) to the division site, where they participate in formation of the septum that separates the two daughter cells. we have studied ftsz polymerization and its dynamic behaviour in real time by right angle light scattering. similar to tubulin, ftsz polymerizes into dynamic protofilaments in the presence of gtp; polymer assembly is accompanied by gtp hydrolysis. the kinetics of inorganic phosphate (p i ) released from the gtp hydrolysis have been studied as well, employing a fast and sensitive colourimetric assay. at ph . , approximate % of the p i was released into the media within minutes of gtp addition. the effects of gtp, ph, k + , and mg + were studied in both cases, and the results were used to build up a model for the mechanism of fibre assembly and disassembly. ygfe, a ftsz accessory protein, is identified as a functional zapa orthologue. finally, we have studied the ygfe bundling to ftsz polymers. it strongly promotes ftsz bundling and is an inhibitor of the gtpase activity. many genomes of viruses encode small membrane spanning proteins which are proposed to modify membrane permeability for ions and small molecules. these channel or pore forming proteins are getting into the focus for antiviral therapy since they are essential for some of the viruses. one of the general themes of the mechanism of function of the proteins is to self-assemble to form the functional form. we present a study on the open reading frame (orf) a membrane protein encoded in structural region of human severe acute respiratory syndrome coronavirus (sars-covs). the full length orf a protein is residues long and contains a single transmembrane (tm) domain. full length protein is synthesized using solid phase peptide synthesis and reconstituted into artificial lipid bilayers forms cation-selective ion channels. the bilayer recordings show cation selection channel activity with a major conductance level of around . ps also at elevated temperatures ( . • c). in silico studies with a amino acid tm domain are done to assess conformational space of the monomeric orf a helix. with this monomeric helix homooligomeric helical bundle models are built and embedded in a fully hydrated -palmitoyl- -oleoyl-sn-glycerol- phosphatidylcholine (popc) bilayer. results of both experimental channel recordings and computational modeling show sars orf a to act as a channel forming protein. -biomolecular self-assembly - microtubules are involved in many vital processes. their rigid structure can resist high forces while their intrinsic ability to switch stochastically between growth and shrinkage phases allows them dynamically to reorganise. in cells, a sizeable network of microtubule binding proteins control and regulate microtubule dynamics. alp and dis are members of the dis / xmap family that are major players in s.pombe. the deletion phenotypes of alp and dis are similar, but nonetheless distinct. both are involved in the formation of spindles but alp is also involved in the maintenance of cytosolic microtubules in interphase. the restrictive temperatures of alp -deletion and dis -deletion mutants are different. alp interacts with alp , a potential member of the tacc protein family. i am working to reconstitute alp /dis -dependent microtubule dynamics in vitro, using purified s. pombe tubulin. both alp and dis express well in insect cells and can be readily purified. preliminary data show that both proteins bind tubulin at low salt concentrations and that both influence the dynamics of pig brain microtubules. my goal is a complete functional analysis of alp and dis , individually and in combination, to test candidate molecular mechanisms for alp /dis -catalysis of s. pombe microtubule dynamics. the syphoviridae coliphage t is a well-suited model to study the assembly of large viral capsids. biochemical and biophysical approaches were used to reconstitute in vitro the assembly pathway of its capsid. the t structure was recently solved from cryo-em and image reconstruction. its icosahedral capsid (t = ) is built from the major head protein (pb , copies) forming both the pentons and hexons and from the portal protein (pb , copies) located at one vertex. its assembly proceeds by steps. pb and pb first assemble into a precursor structure called prohead i, which is converted to prohead ii by proteolysis of pb and pb by a head maturation protease. packaging of the kbp dsdna is then driven through the portal pore by a molecular motor, the terminase. this promotes expansion of prohead ii leading to the mature capsid. the different assembly steps and the conformational changes accompanying capsid maturation were characterized using proheads i either self-assembled from the overproduced and purified capsid proteins or isolated from a phage mutant. these precursor capsid structures were analysed by small angle x-ray scattering. the d structure of prohead ii and of the expanded capsid were solved from cryo-em. our data show that the assembly process of a large icosahedral capsid can be efficiently reconstituted in vitro. amyloid beta peptide fibril formation modulated by phospholipid membranes e. hellstrand , e. sparr , s. linse lund univ., department of biophysical chemistry, sweden, lund univ., department of physical chemistry, sweden disease-causing amyloid fibril formation can be modulated by many factors including interactions with biological lipid membranes. an increasing amount of evidence suggests that the process of fibril formation in vivo and the mechanism of toxicity both involve membrane interactions. alzheimer's is probably the most well-known amyloid disease and the associated amyloid beta peptide originates from the membrane incorporated amyloid precursor protein (app). we use recombinant abeta m - and abeta m - produced in escherichia coli, which allows us to perform large scale kinetics assays with good statistics where the amyloid formation process is followed in means of thioflavin t fluorescence. the lipid membranes are introduced in the system as large unilamellar vesicles composed of dopc, dppc and sphingomyelin, with and without incorporation of cholesterol. we find that the phase behanviour of the membrane in the vesicles has a large effect on the lag time of the amyloid formation process for both abeta m - and m - . all membranes increase the lagtime to some degree but dppc has the largest effect. by comparing different phases we can conclude that the translational diffusion in the membrane seems to be more important than the acyl chain ordering. furthermore, electrostatics, concentration dependence and membrane addition at different time points in the amyloid formation process have been investigated. equilibrium/non-equilibrium transitions in macromolecule interactions p. dumas , g. gibrat , s. bernacchi , e. ennifar ibmc-cnrs, strasbourg, france, llb (cea/cnrs), saclay, france usually, so called 'relaxation phenomena' occur on a fast time-scale and 'p-jump' or 't-jump' techniques are required to follow such events lasting (much) less than ms. we report that, during stability studies of proteins or nucleic acids, such relaxation events can be observed on astonishing long time-scales. we first performed 'melting studies' with nucleic-acid duplexes by using linear variations of temperature (t) with time (t). we observed that even very low rates dt/dt could lead to a frozen state for temperature values below a sharp temperature range, and relaxation to equilibrium beyond that range. this allows defining a 'relaxation temperature' t r separating the two regimes. numerical simulations very accurately described the related hysteresis phenomenon observed upon a heating-cooling cycle, which is the hallmark of departure from equilibrium. analogous observations were made with protein oligomers submitted to either a variable pressure, or variable concentration in denaturant. importantly, a single theoretical frame predicts that the critical relaxation value x r (x standing indifferently for temperature, pressure or denaturant concentration) depends on ln(dx/dt). one may ask whether some thermosensor rnas known for switching on or off genetic expression by 'feeling' a temperature variation, might also 'feel' dt/dt. if true, the exact switching temperature would depend on dt/dt and faster temperature changes would increase t r . -biomolecular self-assembly - the major component of amyloid plaques in the gerstmann-sträussler-scheinker disease is a prion peptide fragment from - to - residues. here, we present a structural study of prp - in form of oligomers and fibrils by fourier transform infrared spectroscopy (ftir) and atomic force microscopy (afm). after incubation at • c, the unfolded peptide was found to aggregate into oligomers characterized by intermolecular β-sheet infrared bands and by a wide distribution of oligomer volumes. after a lag phase, a conformational rearrangement of oligomers into fibrils, with a parallel orientation of the cross β-sheet structures, was observed. by afm, different morphologies were also detected for fibrils that displayed high heterogeneity in their twisting periodicity and a complex hierarchical assembly. in addition, we also studied thermal and random aggregation. the prp - peptide was found to undergo several aggregation pathways, whose end products display different structural properties and intermolecular interactions. these findings underline the high plasticity of the prion peptide, a peculiar feature of prion proteins to overcome species barriers (natalello et al. j.mol.biol. ; : - ). the role of proline isomerisation in the aggregation process and fibril formation of alpha-synuclein j. meuvis , m. gerard , v. baekelandt , y. engelborghs lab.of biomolecular dynamics, ku leuven, belgium, lab.of biochemistry, campus kortrijk, belgium, lab.for neurobiology & gene therapy, ku leuven, belgium alpha-synuclein (α-syn) plays a central role in parkinson's disease. the aggregation of this protein, which contains five proline residues (p ,p ,p ,p ,p ), is accelerated in vitro by fk binding proteins (fkbps), a family of enzymes with a peptidyl-prolyl cis-trans isomerase activity (ppiase). fkbps catalyze the cis-trans conformational change of proline, often a rate limiting step in protein folding. to elucidate the role of the proline residues in aggregation, we constructed a mutant p( , , , , )a α-syn . the kinetics of the aggregation of the mutant were studied with turbidity and thioflavin t fluorescence (tht). turbidity measurements show the formation of early, tht negative aggregates which is as fast for both wt and mutant. fibril formation however is faster for the proline-deficient mutant. we also studied the effect of fkbp on the aggregation of the mutant. although wt α-syn early aggregate formation is accelerated by the addition of µm fkbp , this effect disappears in the mutant. addition of ( pm- µm) fkbp accelerates the fiber formation of wt α-syn, which is abolished in the mutant. we can conclude that α-syn fiber formation is accelerated for the proline-deficient mutant, which suggests a role for the proline residues in fiber formation. furthermore all accelerating effects of fkbp are abolished in the mutant which suggests that the ppiase activity of fkbp is responsible for the accelerating effect on the aggregation of wt α-syn. materials used as gene delivery vehicles must be able to condense dna into small sizes to facilitate transport and crossing various barriers. one of the polycations investigated for dna compaction is chitosan, which has the advantage of being safe and biodegradable. as a step towards reducing the aggregation behaviour of dna-chitosan complexes, chitosans were modified by grafting peg-chains on the backbone. it is known that the transfection efficacy depends on the chitosan chain length. additionally, the degree of pegylation might influence the condensation process. here a systematic biophysical study of pegylated chitosans and how the interplay between chitosan chain length and degree of pegylation affect the compaction of dna in terms of particle size and structure, stability in pbs and when exposed to serum, and transfection efficacy is presented. three different chain lengths of chitosans are employed, and for each sample three pegylation degrees are investigated and the properties of the dna-pegchitosan complexes compared to complexes formed when employing the original, chitosan for dna compaction. it is found pegylation of chitosans can be used to increase both the stability of the dna-chitosan complexes when exposed to serum as well as increase their transfection efficacy in hek cells. max-planck institute for polymer research, mainz, germany model membranes mimic the essential function of a natural membrane. however, the complexity is reduced in order to allow the study of fundamental processes. tethered membranes consist in principle of a lipid bilayer that is covalently linked to a solid support through a spacer group. this architecture allows the characterization of the membrane itself as well as of incorporated membrane proteins using surface analytical techniques. we have established a versatile system of various anchor lipids, which allow membrane formation on different surfaces. the architectures have been characterized by surface plasmon techniques, neutron reflectivity and electrochemical methods. the membranes are electrically insulating and allow for the functional incorporation of ion channel proteins. polymerizable lipids allow to pattern the membrane and to study lateral diffusion processes. furthermore, the membranes can be used as a sensing platform, where embedded membrane proteins act as actual sensing units. a. perico, s. pietronave, l. arcesi, c. d'arrigo consiglio nazionale delle ricerche (cnr), institute for macromolecular studies (ismac) the electrostatic free energy (fe) of two parallel rigid likecharged polyelectrolytes (pes) is given as a function of the separation distance. for high linear charge density, z , the fe shows a minimum due to the increasing of the counterion condensation and condensation volume as the two pes approach. the interaction fe is governed by a critical linear charge density, z c , inversely proportional to the counterion valence. for highly charged pes (z > z c , like dna), the pes attract the stronger the smaller is the counterion valence, because the fe is dominated by the entropic term due to condensation of counterions in a volume displaying a maximum at short distances. for weakly charged pes (z < z c / ) the pes remain undercritical in the whole separation range and therefore repel. for moderately charged pes (z c / < z < z c ), the infinitely separated pes are undercritical but become supercritical as they approach a critical distance and charge condensation and condensation volume expansion start: in these circumstances the pes may attract if the counterion valence is sufficiently large. in the case of many dna rods, hexagonal clusters may be formed. upon interaction with hydrophobic surfaces, proteins show a tendency to expose regions that are normally buried in the hydrophobic core. unfolding is generally perceived as an undesired process in studies aimed to anchor functional proteins at surfaces. upon an upset of perspective the fine control of the unfolding/re-assembly process could be regarded as a strategy to build up molecular nanostructures for the development of organic-inorganic assemblies. we show that molecular layers patterned at the nanoscale, with longrange order properties extending over the microscopic scale, can be obtained upon adsorption of proteins onto the hydrophobic and ordered surface of pyrolytic graphite. upon adsorption, proteins lose their native folding and polypeptide chains re-assemble on the surface in a layered fashion, forming a molecular bilayer. the first layer, in contact with the substrate, and the second molecular layer show corduroy-like nanopatterns of different periodicity, with a relative orientation between the first and second layer patterns of • . surface-induced protein unfolding and polypeptide chain reassembly according to a layered ordered structure is a rather general phenomenon since it is observed for different proteins irrespectively of their specific structural properties. the possibility of using these ordered molecular structures as templates for the subsequent patterned deposition of supramolecular aggregates will be discussed. understanding protein-protein interactions and assemblies to control the hierarchical building of well-ordered supramolecular structures is highly relevant to new tailormade biomaterials. we previously evidenced that contrary to native calcium-loaded α-lactalbumin (holo α-la), calcium-depleted form (apo α-la) has the ability to selfassemble with lysozyme (lys) to form different supramolecular structures in temperature-dependent manner. in the present work, the events occurring at molecular scale were explored using fluorescence techniques. fluorescence anisotropy and fluorescence lifetime measurements provide a powerful and sensitive mean to measure intermolecular interactions. we showed that lys interacts with both apo α-la and holo α-la to form oligomers, assumed to be heterodimers, at • c and • c. the dissociation constants for dimerization, found to be in the µm range, were sensitive to the ionic strength. correlation time calculations suggest that formed heterodimers holo α-la/lys and apo α−la/lys differed in their shape and/or conformation. such conformation differences could explain why holo α-la/lys complexes are trapped as heterodimers while the apo α-la/lys complexes have the ability to further self-assemble into previously reported various supramolecular structures. polyglutamine aggregation and neurodegeneration g. nicastro, l. masino, a. pastore national institute for medical research, the ridgeway, nw aa london, u.k. polyglutamine (polyq) diseases are rare but dominant misfolding diseases linked to neurodegeneration. they are caused by the expansion of cag codon repeats, which encode polyq tracts in the corresponding gene products. aggregation of polyq proteins is thought to be triggered by polyq expansion but be strongly modulated by the protein context. in the attempt of understanding the molecular bases of polyq diseases, we are studying the structures, interactomes and aggregation properties of selected polyq proteins. here, we present recent work on ataxin- , taken as a representative example of the whole family. ataxin- is a ubiquitin specific cysteine protease, involved in the ubiquitinproteasome pathway and known to bind poly-ubiquitin chains of four or more subunits. the enzymatic site resides in the n-terminal josephin domain of ataxin- . we have characterized, using different biophysical techniques, the structure in solution and the aggregation properties of josephin both in isolation and in a ubiquitin complex. we demonstrate that interaction with ubiquitin strongly modulates the aggregation properties of ataxin- and suggest the importance of protein-protein interactions in preventing aggregation. our study also provides new insights into the molecular mechanisms which determine ataxin- specificity for poly-ubiquitin chains of the correct length and cross-linking. förster resonant energy transfer (fret) from an optically excited to a non-excited molecule has been widely used to probe molecular interactions in living cells. changes in the molecular makeup of a cellular region occurring during the acquisition of fluorescence images place tight constraints on the fret technology and data analysis, which could not be addressed satisfactorily until recently. we will describe a method for imaging protein complex distributions in living cells with sub-cellular spatial resolution, which relies on a spectrally resolved two-photon microscope (raicu et al, , nature photon. : - ) and a simple theory of fret in oligomeric complexes (v. raicu, , j. biol. phys. : - ) . then, we will overview recent results on the determination of the supra-molecular structure and distributions in living cells of oligomeric complexes of some g protein-coupled receptors. observing protein aggregates on surfaces m. rabe, d. verdes, s. seeger institute of physical chemistry, university of zurich, switzerland protein aggregation is an important topic of current protein research as it is associated with several human diseases including alzheimer's disease, parkinson's disease, and type ii diabetes. although protein aggregation mechanisms and conditions have been comprehensively investigated, studies on the formation and the fate of protein aggregates in contact with solid interfaces are scarce. we have comprehensively investigated the structure of protein assemblies that form spontaneously upon protein adsorption on solid interfaces using a surface sensitive fluorescence imaging technique based on super critical angle fluorescence (saf) detection. combining this technique with fret we not only succeeded to detect protein aggregates deposited on surfaces but also to characterize their behavior in real time, i.e., their emergence, growth, or spreading. the model protein bsa, for instance, was found to exhibit a certain tendency for aggregation in the buffer solution. these protein clusters can deposit onto solid surfaces and spread resulting in a large, flat structure after some time. a different possibility how protein aggregates emerge on surfaces consists of a direct deposition of protein monomers to pre existing aggregates. such a growth of protein aggregates on the surface has been observed in a model system using the protein α-synuclein, which is tightly associated with the parkinson's syndrome. we present the results of a spectroscopic ellipsometry (se) study of the adsorption process of yeast cytochrome c (ycc) on gold and graphite substrates, according to methods already applied to study the growth dynamics of organosulphur sams on gold [ ] . se investigation was carried out both in situ, at room temperature during protein deposition, and ex situ. on gold, se data demonstrate the formation of an about - nm thick layer, consistent with the formation of a dense monolayer of ycc molecules, confirmed by afm inspection. both in situ and ex situ measurements were characterized by well defined spectral features related to the soret band. analysis of the fine position of this feature allowed to obtain information on the oxidation state of the iron ion of the heme group. se data suggest that proteins have preserved their native structure. a completely different adsorption mechanism was observed on highly oriented pyrolytic graphite (hopg) [ ] . ex-situ se data on ycc/hopg, supported by afm observations, indicate the formation of an ultrathin molecular layer (∼ . nm) related to complete protein unfolding at the hydrophobic surface. the role of phospholipid anisotropy in the stability of inverted hexagonal phase was considered. the equilibrium configuration of the system was determined by the minimum of the free energy involving the contribution of the isotropic and deviatoric bending and the interstitial energy of phospholipid monolayers. the shape and local interactions of a single lipid molecule were taken into account. the minimization with respect to the configuration of the lipid layers was performed by the monte carlo simulated annealing method. at high enough temperature the lipid molecules attain a shape exhibiting higher intrinsic mean and deviatoric curvatures which fits better into the inverted hexagonal phase than into the lamellar phase. for the mathematical model the advanced geometry with non-spherical cross-section of inverted hexagonal phase was calculated, resulting in lower energy in non-spherical cross-section. theoretical results are in a good agreement with the small angle x-ray scattering experimental data. for a long while the conventional view has been that alzheimer disease is brought about by the beta-amyloid fibrils found in the senile plaques, but more recently it has been suggested that the main neurotoxic species would be the soluble oligomeric species, apparently prone to interact with cell structures and macromolecules potentially inducing neuronal dysfunction. peptide-peptide interactions resulting in self-assembly phenomena of beta-amyloid yielding fibrils can be modulated and influenced by small organic molecules that might also be effective therapeutic tools to ideally target both oligomeric and fibrillar species. in this perspective, polycyclic aromatic molecules are of special interest because they might disrupt the molecular architectures precursors of beta-amyloid fibrils by means of weak, non-covalent aromatic interactions, like stacking interactions. we have performed an in vitro spectroscopic study (light scattering, circular dichroism, ftir and fluorescence) of the effects on beta-amyloid fibrillogenesis of the natural pigment hypericin extracted from hypericum perforatum. our results show that, thanks to its structural characteristics and peculiar spectroscopic features, hypericin can be easily used to in vitro monitor the appearance of initial aggregation states of beta-amyloid peptides and, more importantly, that hypericin can interfere with the early stages of polymerization process, playing the role of an aggregation inhibitor. peptaibols are peculiar peptides produced by fungi associated to plants. they are composed by to amminoacidic residues and exhibit antibiotic and antifungal properties. due to their amphypatic nature, they can form ion channels in biological membranes. by making use of experimental models of biological membranes (biomimetic membranes) currently employed in the laboratory of bioelectrochemistry, and models of plant membranes (corn seed root), that are used in the international laboratory of plant neurobiology (linv), we characterized synthetic peptides such as trichogin gaiv and its shorter homologues ( and residues). we studied these peptaibols in a dioleoylphosphatidylcholine monolayer supported by hg using different electrochemical techniques (ac,vc,eis). the experimental technique employed in the linv (clark microelectrode coupled to mife system) allows to measure oxygen flux in the solution contacting plant cell membranes, after treatment with different peptide concentrations. preliminary results might indicate that short peptides can influence the whole metabolism of the plant and can therefore be used as "elicitors" in order to induce an acquired systemic resistance. supported biomimetic membranes (sbm) were developed for protein-membrane interactions studies. phospholipid vesicles were chemically linked onto amine grafted gold or glass surfaces; after an osmotic choc and liposomes fusion a continuous membrane bilayer was formed. the anchoring phospholipid molecule (dspe-peg-nhs) incorporated into the vesicles allowed the formation of a water-filled compartment between the surface and the bilayer. this first sbm model was used to monitor the membranes binding properties (dependent of calcium) of the adenylate cyclase toxin (cyaa) from bordetella pertussis. the sbm model was improved in order to study the translocation of the catalytic domain of cyaa across the bilayer. naturally, the cyaa catalytic domain, when it reaches the target cell cytosol, associates with intracellular calmodulin (cam) an activator of the adenylate cyclase activity of cyaa. to mimic this biological phenomenon, cam was first immobilized on the surface (gold or glass) and in a second step membrane construction was performed over the cam layer. the formation of the biomimetic membrane onto the cam layer was monitored by spr while membrane fluidity and continuity were analysed by fluorescence. our results demonstrated the potentialities of sbm for the study of protein insertion into and translocation across biological membranes. a multi-resolution approach to the structure and function of integrin αiibβ m. rocco , c. rosano , j. w. weisel , d. horita , r. r. hantgan istituto nazionale per la ricerca sul cancro (ist), genova, italy, university of pennsylvania, philadelphia, pa, usa, wake forest university, winston-salem, nc, usa. integrins are heterodimeric transmembrane receptors involved in mechanical anchoring and two-way signaling. each α and β subunit has a modular structure with a large extracellular portion, a single transmembrane region, and a cytoplasmic domain. integrins activation mechanism is regulated by controversial conformational changes: while crystallography revealed similar bent shapes for resting and primed extracellular region constructs, ligand binding-induced large structural rearrangements in smaller constructs suggested extension, "opening" and tails separation. in a multiresolution approach, we used experimental and computed hydrodynamics to discriminate among αiibβ integrin models built on x-ray, nmr, and em data. in contrast with xray data and d em maps, an extension is needed to match the hydrodynamics of full-length, solubilized αiibβ ; an electron tomography-based model fares better. consistent with that, and with our averaged d em images, a conformational change in the head region (β hybrid domain swingout) coupled to a simple transmembrane helices shift matches priming agents-induced frictional changes in full-length αiibβ . our multi-resolution study thus suggests that in integrins extension and immediate tail separation are uncoupled from head domain rearrangements following activation. -biomolecular self-assembly - stabilizing effects of α s -casein, a natively unfolded protein, on the aggregation of biomolecules a. trapani, r. carrotta, p. l. san biagio, d. bulone ibf-cnr palermo, italy α s -casein is one of the four types of caseins, a group of calcium phosphate-binding proteins that, in the form of micellar aggregates, makes up the largest protein component of bovine milk. the structure of α s -casein is that of a triblock polymer with a hydrophilic tract interposed between two hydrophobic blocks. due to the lack of a compact folded conformation, this protein can be classified as one of the intrinsically disordered (or natively unfolded) proteins. this class of proteins is known to exert a stabilizing activity on biomolecules through specific interaction with hydrophobic surfaces that partially unfolded molecules may expose to the solvent. here we present results on α s -casein effects on the thermally induced aggregation of gluthathione stransferase, a ligand-binding anzyme, and - β-amyloid peptide involved in alzheimer's disease. by means of light scattering and circular dichroism experiments, we attempt to reveal the molecular details of α s -biomolecules interaction. bacterial protein self-assembly on surfaces of well-defined chemistry s-layers are one of the most common cell envelope components of prokaryotic organisms and represent the simplest biological membrane developed during evolution. these (glyco)proteins, which can self-assemble into -d crystalline nanostructures on lipid films, liposomes, and polymers, play already an important role in nanobiotechnology. in this work, we present new findings concerning the recrystallization of bacterial proteins on substrates with defined chemistry. manipulation of the protein-sample interaction was carried out by changing the relative height of oh and ch terminated moieties in self-assembled monolayers (sams). we have found that differences in chain length lead to: i) protein bilayer-protein monolayer transition, ii) preferential protein side adsorption, and iii) increase of the crystal lattice parameters. further manipulation of the protein-sample interaction was achieved by using silane chemistry. we will show that sample hidrophobicity speeds up recrystallization kinetics and reduces the crystalline domain size (and layer compliance). the protein-adsorbed mass per unit area on these substrates is reported for the first time. self-assembly of phenylalanine oligopeptides: insights from experimental and computational studies p. tamamis , l. adler-abramovich , m. reches , k. marshall , p. sikorski , l. serpell , e. gazit , g. archontis dpt. of physics, univ. of cyprus, cyprus, dpt. of molecular microbiology and biotechnology, tel aviv univ., israel, dpt. of biochemistry, univ. of sussex, u.k. the diphenylalanine peptide (ff) forms well ordered nanotubes and its derivatives form nano-assemblies of various morphologies with promising material applications. we demonstrate for the first time by electron and laser microscopy and ftir spectroscopy that the related, triphenylalanine peptide (fff) assembles into rather planar nanostructures, rich in β-sheet. in addition, we conduct . -µs replica exchange m.d. simulations of aqueous ff and fff solutions in implicit solvent. the peptides coalesce into aggregates and participate frequently in open or ring-like linear networks, as well as elementary and network-containing structures with β-sheet characteristics. polar and nonpolar interactions, as well as the surrounding aggregate medium contribute to the network stabilities. within a network, consecutive peptides are linked by head-to-tail interactions; the aromatic sidechains of neighbor peptides assume approximately "t-shaped" orientations. these features are observed in ff crystals and could characterize early formations, or stabilize the mature nanostructures. the fff aggregates acquire higher stability and peptide-network propensity compared to the ff aggregates due to energetic contributions , . chlorophyll biosynthesis is light-dependent in angiosperms because the reduction of protochlorophyllide (pchlide) into chlorophyllide is driven by a photoenzyme, nadph:pchlide oxidoreductase (por). the unique properties of por are due to its ability to assemble into dimers and oligomers within the prolamellar body (plb) membranes of etioplasts studied mainly in leaves of dark-grown seedlings under laboratory conditions. we extended these studies to plant organs developed in the nature: cabbage heads, leaf primordia inside buds, pericarp-covered regions of sunflower cotyledons, potato tubers and seedlings germinating under the soil. in electron microscopic and fluorescence spectroscopic studies we found in many of these organs poorly developed plbs in which por was mainly in monomer state. as a consequence, the chlorophyll accumulation was slow and photo-oxidation processes occurred at illumination. in vitro we artificially induced the aggregation of por monomers into oligomers in glycerol and sucrose containing buffers. this resulted in the increase of the photoreduction rate at the expense of photo-oxidation. these results underline the importance of the self-assembly of por and the plbs in chloroplast development and chlorophyll synthesis in nature. v. vetri , g. ossato , v. militello , m. a. digman , m. leone , e. gratton dsfa, university of palermo, palermo, italy, lfd, university of california, irvine, ca, usa we report an experimental study on concanavalina (cona) aggregation in live cells. in vitro, close to physiological temperature, cona readily forms fibrils involving secondary structure changes leading to β-aggregate structures. the effect of cona on cell cultures and formation of protein aggregates were measured by confocal fluorescence microscopy. in particular, we monitored protein aggregation in live cells by means n&b analysis, cross-n&b and rics. n&b showed the aggregation kinetic and the progressive formation of cona oligomers at cell surface. this suggests that, at cell membrane where local concentration is higher, nucleation sites for aggregation are provided. in parallel, the morphology of the cells changes indicating the progressive cell compaction and death. aggregation and binding of small aggregates to the cell surface were assessed by rics: it is possible to distinguish regions where small aggregates are diffusing and regions where they are bound to the cell. oligomers formation may stimulate non-specific cellular responses due to the exposure of reactive regions of protein structure and of progressive formation of cross−β structures. moreover, aggregates stoichiometry was measured during the kinetic by cross-variance n&b. the two conductive pathways of p x purinergic receptor: different modulation and selectivity r. barbieri , s. alloisio , a. di garbo , m. nobile institute of biophysics, cnr, via de marini , genoa, italy, institute of biophysics, cnr, via g. moruzzi , pisa, italy the p x purinoceptor (p x r) is an atp-gated cation channel that is able to activate a cell permeabilizing pore. p x r cytosolic c-terminal tail is thought to modulate this function. this study was aimed to characterise the biophysical properties of p x r compared to those of the variant lacking the entire c-terminus tail (trp x r) by measuring whole-cell currents and intracellular ca + variations. a mathematical model is used to describe the experimental results. in p x r expressing hek- cells, the potent agonist '-o-( -benzoyl)benzoyl adenosine '-triphosphate (bzatp) -evoked ionic currents depending on concentration and frequency of agonist applications. the currents were strongly inhibited by extracellular mg + in a noncompetitive way. by contrast, in trp x r cells, only high bzatp concentrations elicited small currents not affected by mg + . interestingly, bzatp-induced ca + influx was present both in p x r and in trp x r cells, albeit in the latter the intracellular ca + elevation was smaller. importantly, in trp x r the intracellular ca + rise maintained a competitive mechanism of mg + inhibition similar to that observed in p x r. the experimental data and the modelling findings support the tenet of a functional structure of p x r possessing two distinct conductive pathways. the review of our data on the effects of physical and chemical weak signals on physicochemical properties of water, cell volume, activity and the number of membrane proteins (receptors, ionic channels and enzymes, na + /k + pump and na + /ca + exchanger), intracellular signal systems in norm and pathology (cancer and nerve disorders) would be presented. light microscopic, cell voltage-and patch-clamp, isotope, standard biochemical and genetic engineering methods were used. weak signal-induced effects on cell functional activity (intracellular enzymes activity, the number of functionally active membrane proteins) are realized by changing the physicochemical properties of extra-and intracellular aqua medium. the latter induces the modulation of na + /k + pump-induced cell hydration, which serves as a primary mechanism through which the autoregulation of pump and regulation of membrane excitability and chemosensitivity are realized. by genetic engineering method in oocytes it was shown that the correlation between na + /k + pump and na + /ca + exchanger, which is realized through intracellular messenger systems, plays a crucial role in weak signals transduction in cells and determination of aging-induced increase of cell pathology. listeriolysin pore forming ability in planar lipid membranes at different ph listeriolysin o (llo) is a cholesterol-dependent cytolysin secreted by the intracellular pathogen listeria monocytogenes. its main task is to enable escape of bacteria from the phagosomal vacuole into the cytoplasm. llo exhibits optimal cytolytic activity at low ph but it is still able to bind membranes at physiological or even slightly basic ph values in a cholesterol-dependent fashion. high cholesterol concentrations in the membrane restore the low activity of llo at high ph values. based on this broad ph activity we investigated the electrophysiological properties of pores formed by llo at room temperature and at different phs using planar lipid bilayer technique. llo is able to form pores both at ph . and . with a similar permeabilizing ability and similar heterogeneous conductances in the range of picosiemes to nanosiemens. cholesterol content directly correlates with llo activity but it does not change the pore characteristics. collectively, our results demonstrate that llo activity at physiological ph cannot be neglected and that its action at sites distal to cell entry may have important physiological consequences for listeria pathogenesis. s. aimon, g. toombes, p. bassereau institut curie, paris, france the physics of membrane/channel and channel/channel interactions is difficult to investigate in cells where it is nearly impossible to modify relevant parameters to deduce physics laws. to overcome these difficulties we built a model system in which voltage gated ion channels were reconstituted in giant unilamellar vesicles (guvs) for the first time. as a first step, we successfully expressed kvap (a voltage gated potassium channel) in e-coli. the channel was purified, fluorescently labelled and reconstituted in small liposomes. its functionality was checked with electrophysiology via fusion of these liposomes into black lipid membranes (blm). as a second step, guvs were formed from these small proteoliposomes using electroformation in a buffer containing mm kcl salt. the proper incorporation of proteins into guvs was controlled using confocal microscopy. functional proteins were detected using the patch clamp technique. with our protocol, we are thus able to prepare guvs containing functional voltage-gated ion channels. one goal is now to study the effect of channel activity on its spatial distribution in these guvs. ryr activation in cultured shr cardiomyocytes at the end of the prehypertensive period the rate of [ca + ] i elevation after the ryanodine receptor (ryr) activation by -chloro-m-cresol ( -cmc) and l-type ca + channels (dhpr) activation by bay k was studied in cultured ( days) cardiomyocytes of spontaneously hypertensive (shr) and normotensive rats (wky, wistar) during weeks of postnatal development. the differences in dh-prs and ryrs activities began to be evident after weeks age when cicr formation has finished and became more expressed at the end of prehypertensive period ( weeks). in response to -cmc ( mm), a drastic increase in the rate of [ca + ] i accumulation ( . ± . times) in shr myocytes was registered after days age versus a decrease in the rates of ca + efflux from the sarcoplasmic reticulum of wistar and wky rat cardiomyocytes. bayk ( µm) also induced more sharp [ca + ] i elevation in shr myocytes ( . ± . times) as compared with wistar ( . ± . times) and wky ( . ± . times) ones of the same age. our results argue that in shr and wky cardiomyocytes, as opposed to normotensive wistar rats, gradual growth of dhpr activity is observed, which follows in parallel with cicr formation in the excitation-contraction coupling during early postnatal ontogenesis, and drastic activation of ryr in shr myocytes after the process termination. cytochrome ba from thermus thermophilus belongs to the large family of structurally related heme-copper oxidases. it accepts electrons from cytochrome c at the p-side of the membrane and uses them to reduce oxygen to water. the energy released in this reaction is used for proton pumping across the membrane to form of an electrochemical proton gradient, used by the cell for formation of atp. in this work we followed the kinetics of single-electron injection into the oxidized nonrelaxed state (o h →e h ) of cytochrome ba by time-resolved optical spectroscopy. two main phases of electron transfer were resolved. the first (τ∼ µs) includes oxidation of cu a and simultaneous reduction of both low and high spin hemes. the second (τ∼ µs) reflects reoxidation of both hemes by cu b . this is in significant contrast to the o h →e h transition of aa -type oxidases, where the fastest phase is due to transient reduction of the low-spin heme a only. on the other hand, the single-electron reduction of the relaxed o state in ba oxidase consisted of only rapid electron transfer from cu a to heme b, which is similar to that in aa oxidase. this indicates a functional difference between the relaxed o and the pulsed o h states of cytochrome ba . as opposed to the phospholamban pentamer, sarcolipin forms anion-selective channels in biomembranes l. becucci , c. b. karim , d. d. thomas , g. veglia , r. guidelli chemistry department, florence university, florence,italy, chemistry department, university of minnesota, minneapolis, mn , department of biochemistry, molecular biology, and biophysics, university of minnesota, minneapolis, mn sarcolipin (sln) and the phospholamban pentamer (pln) are two membrane proteins that inhibit ca-atpase of the sarcoplasmic reticulum at low concentrations. in contrast to pln, sln stimulates maximal ca + uptake rates. sln and pln were incorporated in a bilayer lipid membrane (tblm) tethered to a mercury electrode through a hydrophilic spacer. electrochemical impedance spectroscopy measurements show that sln forms channels permeable to chloride ion, weakly permeable to phosphate ion and impermeable to inorganic cations such as na + and k + . a relationship between this property of sln and its regulatory function on ca-atpase of sarcoplasmic reticulum is proposed. atp increases the permeability of a tblm incorporating sln to phosphate ion by associating to sln with an association constant of . µm. an explanation for this behavior is provided. sln can be identified with the " p i transporter" described by a.g. lee et al. conversely, both electrochemical impedance spectroscopy measurements and molecular dynamics simulations provide strong evidence that the pore of the pentameric form of pln does not act as a chloride channel. "social" domain organization and dynamics of nicotinic acetylcholine receptor at the cell membrane f. j. barrantes unesco chair biophys. & mol. neurobiology. univ. nac. sur, b. blanca, argentina a combination of experimental techniques (patch-clamp, confocal frap and fcs, single-particle tracking, highresolution fluorescence microscopy) has been used to analyze the supramolecular organization of the acetylcholine receptor (achr), the dynamics of the receptor at the cell surface, and the kinetics of receptor internalization. changes in cholesterol (chol) content affected muscle and neuronal-type achr organization and dynamics at the cell surface. chol depletion produced gain-of-function of single-channel dwell time. submicron-sized (∼ nm) domains, stable over a period of hours at the cell membrane, could be resolved into achr "nano-clusters" with a peak size distribution of ∼ nm by sted microscopy. chol depletion reduced the number of nanoclusters, increasing their size, and changed their supramolecular "social" organization on larger scales ( . - . microns). frap, fcs and spt experiments provided information on the dynamics of achr nanoclusters, disclosing the dependence of their mobility on chol content and cortical cytoskeleton. chol content at the plasmalemma may thus modulate cell-surface organization and dynamics of receptor domains, and fine-tune receptor channel function to temporarily compensate for acute achr losses from the cell surface. m. czaplinska, k. gwozdzinski, a. koceva-chyla division of research of structure of biopolymers university of lodz, lodz, poland doxorubicin (dox) and paclitaxel (ptx) are anticancer drugs commonly used in chemotherapy of breast cancer therapy, however, the use of these drugs is limited by the risk of developing heart failure. generation of reactive oxygen species contributes to the cardiotoxicity of doxorubicin. nitroxides are low molecular weight, stable free radicals reacting with ros and they present antioxidant properties. the aim of this study was to analyze the effects of pirolid (pd) on the oxidative stress induced by dox and taxane in mcf- breast cancer cell line. results from mtt test revealed that ptx is more cytotoxic towards mcf- cells than dox. the ic was . µm and µm, respectively. pd alone does not influence cell viability. pd in combination with both drugs did not change viability of cells. both drugs increased the level of carbonyl groups in cells. the highest level of peroxide was observed in cells incubated with dox (approx. -fold). nitroxide alone did not influence the level of peroxide in the whole range of concentrations. combination of pd with dox and ptx reduced the level of carbonyls depending on its concentration. pd did not affected on the level of peroxide in cells suspension. dox and ptx increased ( -fold) the level of carbonyls. pd decreased the level of peroxides in cells treated with dox and ptx. the lowest concentration of peroxide was observed at µm of pd. these results show that pd protect mcf- cells against oxidative stress induced by drugs. open channel structure of mscl from fret microscopy and simulation mechanosensitive channels open in response to membrane bilayer deformations occurring in physiological processes such as touch, hearing and osmoregulation. here, we have determined the likely structure of the open state of the mechanosensitive channel of large conductance from e. coli (mscl) in a natural environment using a combination of patch-clamp studies, fret spectroscopy, epr data, molecular and brownian dynamics simulation. structural rearrangements of the protein are measured while controlling the state of the pore by modifying lipid bilayer morphology. fret efficiency changes can be related to distance changes using a monte carlo analysis program in conjunction with detailed orientational analysis. these measurements are used as restraints in all atom molecular simulations in order to determine the likely structure of the open state, whose probable conductance is derived from brownian dynamics simulations. transition to the open state occurs via large rearrangements throughout the protein that create a wide pore nearly a in diameter. both transmembrane helices are found to line part of the pore. the n terminal helix is found to lie along the face of the membrane where it can act to sense membrane tension and directly transfer this to the pore lining helices. the method of coupling spectroscopic data with simulations is likely to be of great value for studying conformational changes in a range of membrane proteins. putative potassium channels in synechocystis sp. pcc v. checchetto, m. zanetti, g. m. giacometti, i. szabò, e. bergantino department of biology, university of padova, italy we are interested in the identification and characterization of potassium channels in the cyanobacterium synechocystis sp. pcc , an organism which is considered the ancestor of plant chloroplasts. a bioinformatic screening of synechocystis proteome identified, among others, two proteins on which we focused our attention. the first one (syncak) displays sequence homology to mthk, a ca + -dependent potassium channel from m . thermoautotrophicum. the second one (synk) is predicted to contain six transmembrane regions and the typical selectivity filter of all potassium channels. our goal is to understand their roles in the physiology of cyanobacteria. we cloned their coding sequences in fusion with gfp and the hybrid proteins were expressed in chinese hamster ovary cells. we evaluated the presence of both proteins in plasma membrane by fluorescence microscopy and then we proceeded to their functional characterization using patch clamp technique.this analysis will allow us to gain information about channel activity, regulation and pharmacology. we also plan to evaluate the importance of syncak and synk channels in photosynthesis. to test the hypothesis that they could be involved in regulating this process, we will produce deletion and site-specific mutants in synechocystis. finally we would also identify the homologous of these channels in the higher plant a. thaliana and obtain some information about their localization and function. j. braunagel max-planck institute for polymer research, ackermannweg , mainz, germany the cyclododecadepsipeptide valinomycin is composed of two amino acids (l-valine and dvaline) and two hydroxyl acids (d-α-hydroxy-isovaleric acid and l-lactic acid). they form a membered ring of alternating amino and hydroxyl acids. in the cyclic structure, the polar groups are oriented towards the central cavity, whereas the rest of the molecule is relatively nonpolar. this enables the complexation of ions and valinomycin acts as a selective ion transporter (k + ) in lipid membranes. when one of the amino acids is exchanged, e.g one l-valine by an l-lysine, selected functionality can be engineered into the depsipeptide while maintaining its ion conducting properties. we induced several modifications into valinomycin, i.e. a biotin binding unit or a ferrocene group to induce an electrochemical active center. the ion conducting properties of the modified ion carriers have been probed in planar lipid bilayers as well as in solid supported membranes. the role of the membrane dipole potential (ϕ d ) is of a particular interest due to a powerful impact of this potential on the membrane permeability and lipid-protein interactions. channel forming activity of gramicidin a, alamethicin, syringomycin e, hpa peptide, and ompf porin are influenced by ϕ d . we have studied the effect of the membrane dipole modifier, phloretin, on the properties of single channels formed by a wild-type alpha-hemolysin in planar lipid bilayers. the single channel of a ∼ ps conductance exhibits transitions into a number of low-conductance states as the transmembrane voltage exceeds ∼ mv (regardless of the voltage polarity). the phloretin addition to the bathing solutions ( µm) (after the hemolysin channel was formed in the membrane) shifts dramatically the channel voltagedependence. transitions to the low-conductance states are observed at ∼ mv. the effect of the phloretin addition was not observed in the case when it was introduced into the bathing solution before alpha-toxin. since phloretin reduces ϕ d , the data may report on the influence of the electric potential profile on the energy of the low-conductance state of the alpha-hemolysin channel. the alternative explanation of this effect consists in a specific interaction between the phloretin and toxin channel. the work is supported in part by rfbr (# - - ), ss (# . . ) , and the program of the ras «molecular and cell biology». atypical mechanism of conduction in potassium channels c. domene , s. furini physical and theoretical chemistry laboratory, department of chemistry, university of oxford, oxford, u.k., department of electronics, computer science and systems, university of bologna, bologna, italy potassium channels can conduct k + ions with rates of up to ∼ ions per second at physiological conditions, and they are selective to these species by a factor of over na + ions. ion conduction has been proposed to involve transitions between two main states, with two or three k + ions occupying the selectivity filter separated by an intervening water molecule. the largest free energy barrier of such a process was reported to be of the order of - kcal mol − . here, we present an alternative mechanism for conduction of k + in k + channels where site vacancies are involved, and we propose that coexistence of several ion permeation mechanisms is energetically possible. conduction can be described as a more anarchic phenomenon than previously characterized by the concerted translocations of k + -water-k + . we exploited the au-deposited self-assembled monolayers of the type: [−s−(ch ) n −ch ] (where n = , and ) with hydrophobically adsorbed redox protein -azurin to verify intrinsic electron transfer mechanisms according to the charge-transfer theory. the enthalpies and volumes of activation were determined through the variation of temperature ( - o c) and pressure ( - mpa) and experimental values were compared with those expected on the theoretical grounds. for the case of n = the activation enthalpy definitely contains a large contribution originated from frictional-like dynamics of protein, and the activation volume has a small positive value. for n = the value of activation enthalpy directly matches / of that for the reorganization energy, and the activation volume attains substantially negative value. for n = we observed the intermediary performance. the whole kinetic pattern is consistent with the smooth changeover between adiabatic and nonadiabatic mechanisms of electron transfer. two gating modalities in the pore of the miniature k + channel kcv kcv is a viral protein that forms functional k + channel in heterologous systems. because of its miniature size ( amino acids) we use kcv as a model system to study and manipulate basic properties of the k + channel pore. by analysing single-channel recordings we highlighted two voltage-dependent modalities of gating in kcv: a slow and a fast gating. the presence of a slow gating is revealed by the very low (in the order of - %) mean open probability. slow gating is not related to the presence of a bundle crossing, as shown by accessibility of the cavity to mts reagents. channel opening might involve the transient formation of salt bridges between residues at the n and c termini of the channel, as suggested by mutational experiments inspired by molecular dynamics simulations of kcv. fast gating, analyzed by beta distributions, is responsible for the negative slope conductance in the single-channel i/v curve at extreme potentials and can be explained by depletion-aggravated instability of the filter region. aca is a type b caatpase with a regulatory n-terminus whose autoinhibitory action can be suppressed by binding of calmodulin (cam). aca n-terminus is able to bind a region of the small cytoplasmic loop connecting transmembrane domains and . to define the role of this interaction in autoinhibition we have analysed a number of single point mutants produced by mutagenesis of aca e -n sequence. mutation to ala of any of acidic residues (e , d , d , d , e , d ) originates an enzyme with normal activity in the presence of cam, but less camstimulated. these results highlight the relevance of a negative charge of the surface area of the small cytoplasmic loop in aca autoinhibition. the most deregulated mutant is d a aca , which is less activated also by controlled proteolysis or by acidic phospholipids; moreover, the phenotype of the d a mutant is stronger than that of d n aca suggesting a more direct involvement of this residue in autoinhibition. of the other mutants (i a, n a, p a, p a, v a, n a), only p a aca has a basal activity higher than that of the wt. these results provide the first evidence that the small cytoplasmic loop of a type b caatpase plays a role in the attainment of the autoinhibited state. complex i, the first member of the respiratory chain, serves as a proton pump catalyzing transfer of two electrons from nadh to ubiquinone coupled with the translocation of four protons across the membrane. so far the mechanism of energy transduction by complex i is unknown. the nadh-binding cavity of complex i has a very prominent feature -the presence of two invariant amino acid residues, glutamate and tyrosine, that are exposed to the solvent and located in the vicinity of the fmn, the primary electron acceptor in the enzyme. it was suggested that they might be involved in the binding of nadh through interaction with its nicotinamide moiety. in this work we assessed the function of corresponding glu from the nuof subunit of e. coli complex i by mutation for glutamine. we showed that the negative charge of glutamate in the catalytic site is needed for the electrostatic repulsion of negatively charged phosphates of nucleotides. this process facilitates release of the product nad + and, as a result, accelerates turnover of complex i. we also found that glutamate, as one of the four negatively charged amino acid residues surrounding the isoalloxazine ring of the fmn at a distance of - Å, has a share of mv of the overall mv depression of the midpoint potential of this redox cofactor. l. erokhova , p. kügler , p. pohl institute of biophysics, johannes kepler university, linz, austria, ricam, austrian academy of sciences, linz, austria the mechanism of water transport through epithelia is still under debate. in the present work we tested the hypothesis of isosmolal water transport using mdck cells stably expressing the human sodium-glucose cotransporter (hsglt ). tagging hsglt with egfp enabled determination of its abundance in the plasma membrane by fluorescence correlation spectroscopy (fcs). by monitoring tiny shifts in the concentration of water-soluble dyes in the vicinity of epithelia, fcs also allowed assessment of the water fluxes through confluent cell monolayers grown on permeable supports. fitting the set of differential equations for the osmotic drift and for the back diffusion to the experimentally determined dye distribution permitted calculation of water flow both in the presence and in the absence of an osmotic gradient. from the calorimetric measurements of glucose transported across the cell monolayer, the water:glucose stoichiometry was derived. dividing the increment in osmotic water flux due to hsglt expression by the number of hsglt copies in the plasma membrane resulted in a single transporter water permeability p f of . x − cm /sec. thus, p f is close to the single channel water permeability of aquaporin- . consequently, even small osmolyte concentration differences between the cytoplasm and the basolateral buffer solution are sufficient to drive a substantial water flux. m. emre, s. kavak cukurova university school of medicine, department of biophysics, balcali-adana, turkey experimental studies have shown that the at -receptor antagonists telmisartan (tel) has a ppar-activating property, but there does not appear to be a class effect. to test telmisartan's importance, we investigated its effect on electrical activities (ea) in diabetic (d) rats. the purpose of this study was to investigate the effects of the tel on ea of diabetic papillary muscle (dpm) with stz-induced. in this study, we used four groups: ( ) nondiabetic control (ndc) group (c), ( ) tel-treated ndc group (c+tel), ( ) diabetic group (d), and ( ) tel-treated d group (d+tel). diabetes was induced by a single i.v injection of stz. in the study, membrane potential (mp) and action potential (ap) recorded after the establishment of diabetes. ) mp was decreased significantly in both tel-treated c and d rats (from - , ± , to - , ± , mv and from - , ± , to - , ± , mv). ) ap unchanged in d group, whereas c+tel and d+tel groups showed increase in ap compared with c and d groups. ) repolarization time was prolonged in diabetic rats. ) in c+tel and d+tel groups depolarization rate values increased significantly. on the other hand, in d group repolarization rate values descreased increased significantly compared to baseline values in tel solution. as a result, our data suggest that the beneficial effects of tel-treatment on the ea of the dpm appear to be due to the diminished k + currents. cytochrome c oxidase is a terminal complex (cco, complex iv) of a respiratory chain that is located in an internal membrane of mitochondria or plasma membrane of bacteria. cco is an electron transfer enzyme that reduces o and uses the redox energy of the o reduction for the proton translocation across the membrane. the electron-and proton-transfer generate a transmembrane electrochemical gradient (∆µh + ) that is used for atp synthesis and all other kinds of work for the cell needs. the proton translocation mechanism of cco requires 'channels' for the h + uptake and expulsion within the enzyme. the proton transfer occurs on a time-scale of micro-to-milliseconds. in order to study the proton transfer in cco, a flow-flash approach based on a time-resolved ftir spectroscopy was developed and applied. our ftir flow-flash approach (the measurement of the reaction of cco with o ) allows to reach a time resolution up to tens milliseconds. with this approach and site-specific mutants of cco where the catalysis is slowed down, separate steps of the proton transfer were studied. the results showed that a unique cross-linked tyr- (in a combination with a time-resolved visible spectroscopy and electrometry) serves as a proton donor for the dioxygen bond cleavage during the o reduction by cco. furthermore, the protolytic transitions of glu- -a key amino acid in the proton transfer mechanism in cco -were shown for the first time. role of calcium ions in nickel potentiation of nmda currents p. gavazzo, i. zanardi, p. guida, c. marchetti biophysics institute ,cnr, genova, italy nmda receptors are glutamate-gated channels distributed throughout the brain in the excitatory synapses and are critical for the nervous system function. they are assembled from two types of subunits, the essential nr and at least one nr (a,b,c,d). nickel (ni) modulates the current flowing through nmda receptors in a different way depending on the nr subunit present. we have recently identified several domains of the channel involved in ni interaction, but many aspects of this modulation remain elusive. in this work we intended to determine the role of calcium (ca) ions in the potentiation induced by ni on the current through nr /nr b recombinant nmda receptors. when ni was applied in the presence of the physiological concentration of ca ( . mm) , a voltage-independent potentiation of the current was observed with a kp of . µm. this effect was progressively reduced by decreasing ca concentrations and it was no more detectable with . mm ca or in the presence of barium (ba, . mm). in this last case the effect of ni on nr /nr b receptors was mainly inhibitory (ki(- mv)= µm). therefore a physiological concentration of ca is necessary to induce ni amplification of the current. many data in the literature indicate a correlation between ca ion entrance through the channel, nmda current facilitation and cytoskeleton; however, in our experiments, the application of the actin perturbing agent cytochalasin-d did not produce major modifications in ni effect. heteromerization properties of voltage dependent potassium channels f. gambale , l. pedemonte , a. naso , i. testa , c. usai , a. diaspro , c. picco istituto di biofisica, cnr, genova, italy, lambs-microscobio, department of physics, genova, italy voltage-gated potassium channels are either homomeric or heteromeric tetramers composed of four α-subunits. in order to bring a contribution to the comprehension of channel heteromerization we have been investigating the properties of two plant voltage-gated k + -channels by using electrophysiological and fluorescence techniques. experiments were focussed on kdc and kdc , coexpressed in xenopus laevis oocytes. kdc , the first potassium channel cloned from daucus carota, belongs to the subfamily of α-modulatory silent channels as it doesn't form functional homomeric channels by itself. on the contrary kdc forms functional heteromeric channels when coexpressed with homologous subunits. kdc , the last k + channel cloned from d. carota, belongs to the kat family and shares an overall identity of % with kat . to correlate kdc functional properties with its localization in oocytes, kdc and/or kdc subunits were labelled with gfp and their properties investigated by confocal microscopy and voltage-clamp. we found that the kdc -egfp fusion protein is not targeted to the plasma membrane unless it is coexpressed with kdc . moreover electrophysiological experiments demonstrated that the heteromeric kdc -kdc channel has altered selectivity and activation properties with respect to homomeric kdc channel. circulating leukocyte sequestration in pulmonary capillaries is arguably the initiating event of lung injury in acute respiratory distress syndrome (ards) [ ] . we present a microfluidic investigation of the roles of actin organization and myosin ii activity during the different stages of leukocyte trafficking through narrow capillaries using specific drugs. the deformation rate during entry reveals that cell stiffness depends strongly on f-actin organization and hardly on myosin ii activity, supporting microfilament role in leukocyte sequestration. in the transit stage, cell friction is influenced by stiffness, demonstrating that the actin network is not completely broken after a forced entry into a capillary. conversely, membrane unfolding was independent of leukocyte stiffness. the surface area of sequestered leukocytes increased by up to % in absence of myosin ii activity, showing the major role of molecular motors on microvilli wrinkling and zipping. finally, cell shape relaxation was largely independent of both actin organization and myosin ii activity, whereas a deformed state was required for normal trafficking through capillary segments [ ] . [ ] g.s. worthen et al., science, , - ( ) excitation-contraction coupling in skeletal and cardiac muscle is tightly regulated by the calcium release channel of the sarcoplasmic reticulum, the ryanodine receptor (ryr). we could previously show that suramin is a potent activator of the ryr via the calmodulin binding site. calmodulin shows dualistic action, i.e. activation or inhibition of the ryanodine receptor, depending on the absence or presence of ca + . screening of suramin analogues identified nf as a use-dependent inhibitor of the skeletal muscle ryr (ryr ). here we show that nf inhibits high affinity [ h]ryanodine binding and single channel recordings of the purified ryr . nf induced a reduction of open probabilities in a concentration dependent manner, with no effect on current amplitude and unitary conductance. importantly, nf triggers flickering episodes of channel openings and closings before the ryr is frozen in a complete non-conducting state, which is fully reactivated by the ryr agonist atp. moreover, zwitterionic behaviour of nf facilitates plasma membrane permeation, which prevented caffeine induced ca + transients in skeletal muscle cells and cardiomyocytes. conversely, ip mediated ca + signals were not altered by nf . this work was supported by herzfelder'sche familienstiftung and fwf . beyond steady-state protein dynamics t. hauß , j. pieper , a. buchsteiner , r. e. lechner , n. a. dencher helmholtz-zentrum berlin für materialien und energie, berlin, germany, technische universität darmstadt, germany, technische universität berlin, germany to study protein dynamics beyond steady-state experiments we have developed a novel laser-pump:neutron-probe experiment which allows us to monitor temporal changes in protein dynamics during a working cycle of a protein. protein dynamics has been extensively studied, but so far, the correlation of internal protein dynamics with the function of proteins was investigated only indirectly in steady-state experiments by variation of external parameters by variation of external parameters like temperature or hydration. the method comprises of an in-situ optical activation of a protein and a time-dependent sampling of the dymamic response using quasi-elastic neutron scattering. with the membrane protein bacteriorhodopsin, a light driven proton pump, we can demonstrate for the first time temporary alterations in the protein dynamics after triggering the working cycle. this observation is a direct proof for the functional significance of protein structural flexibility, in connection with the largescale conformational changes in the protein structure occurring during the operation of a "molecular machine". the slow vacuolar (sv) channels are ubiquitous in all tissues of higher plants. the sv channel is a non-selective cation channel permeable to both monovalent and divalent cations. sv currents recorded in a typical patch-clamp experiment require unphysiologically high cytosolic and low vacuolar calcium concentrations for full activation. we aim at looking for endogenous plant substances which might be able to modify or shift the voltage activation threshold of this channel towards more physiological conditions. flavonoid naringenin [nar] is present in all plant species where it plays a central role in the flavonoid biosynthetic pathway. nar is stored in the vacuoles in glycosylated form called naringin. when nar was added to cytosolic bath solution, we recorded a dose-dependent reversible decrease in sv channel activity. when we investigated the effect of nar on the voltage dependence of the channel, we observed that the activation threshold of the sv channel is shifted towards more positive voltages. our group has evidences that approximately % of the total sv current at high (e.g.> mv) positive voltages is mediated by calcium. therefore, in order to verify whether nar affects both potassium and calcium conductance, we performed experiments by combining the patch clamp technique with fluorescence measurements using the fluorophore fura- : both sv currents and calcium signals were abolished by mm [nar]. determination of calcium currents in cation channels using a novel fluorescence/patch-clamp approach p. v. k. gutla, a. gradogna, a. carpaneto istituto di biofisica, consiglio nazionale delle ricerche, via de marini , genova, italy the patch-clamp technique combined with fura- fluorescence detection is suitable to investigate calcium fluxes. we used the excised patch configuration and focused the photomultiplier to the tip of the recording pipette where the fluorescent dye was present (fluoresence combined with excised patch = flep). this configuration has several advantages, i.e. absence of delay in loading the fluorophore, of interference by endogenous calcium buffers and of photobleaching. here we present an application for the determination of fractional calcium currents (pf) in a plant non-selective cation channel, showing that pf can be modulated by cytosolic calcium and potassium. flep is very efficient for measuring small calcium currents (< pa) of sufficiently long duration; fluorescence signals are amplified by integration in time, as the calcium/fura- complex accumulates at the tip of the recording pipette and diffuses slowly. we propose this technique not only for the study of calcium transport pathways, but also for other transporters of divalent cations as nickel and manganese known to quench fluorescence thus reducing both and nm components. moreover, using the appropriate fluorophore the technique may be extended to further ion species, e.g. bcecf for investigating proton transport pathways. ref: we introduced an original method for the monitoring of the changes in the electrostatic surface potential, using the quenching of the intrinsic tryptophan fluorescence by acrylamide or iodide. this approach opens new way to understanding the dynamic processes within the proteins. our experiments revealed that the conformation of the na + /k + -atpase large cytoplasmic loop (c ) in the presence of the atp (without magnesium) substantially differed from the conformation in the presence of mg + or mgatp or in the absence of any ligand not only in the sense of geometry but also in the sense of the electrostatic surface potential. moreover, our data indicate that the effect of the ligand binding is not restricted only to the close environment of the binding site and that the information is in fact transmitted also to the distal parts of the molecule. this property could be important for the communication between the cytoplasmic headpiece and the cation binding sites located within the transmembrane domain. influx of antibiotics into the periplasm of gram-negative bacteria is facilitated by porins that form channels in the outer membrane. we propose that certain natural antibiotics have been optimized by co-evolution to take advantage of the charge distribution in non-specific porins to achieve binding and thereby facilitating their uptake in bacteria. we investigate the permeation pathways of antibiotics into bacteria by reconstitution of a single porin into an artificial lipid bilayer and measuring the binding of antibiotic molecules through the time-resolved modulation of a small ion current. we have been able to characterize facilitated translocation of several antibiotics through escherichia coli and enterobacter aerogenes porins. noise analysis of ion currents through a porin in the presence of effective antibiotics revealed binding kinetics at a single molecule level. we report for the first time temperature dependent antibiotic translocation that revealed complete energy profile. combining these results with microbiological assays and molecular dynamics simulations, we conclude the molecular mechanism of antibiotic permeation. our approach may contribute to the rational design of new antibiotics against clinical bacterial strains for the most efficient delivery to target sites. mitochondria regulate ca + influx and determine patterns of er ca + refilling in acinar cel o. kopach, i. kruglikov, t. pivneva, n. voitenko, n. fedirko bogomoletz institute of physiology, kiev, ukraine store-operated ca + entry (soce) is mediated by activation of soc-channels of plasma membrane following the emptying of endoplasmic reticulum (er) ca + stores. the soce is required for calcium signaling, secretion of neurotransmitters and proteins, but the mechanisms of natural soce regulation are not well understood. we utilized several imaging methods to measure ca + signals in cytoplasm ( ] mit transients, observed both in egta-and bapta-buffering inside solutions. these data suggest that ca + sequestration by mit is associated with the formation of microdomains and prevents ca + -dependent socc inactivation. we also found that inhibition of mit under prolonged cell stimulation resulted in complete inhibition of soce as well as decrease and deceleration of er refilling. thus, mit regulate calcium recycling and maintain the soce controlling dynamic interplay between soce and sustained er refilling under prolonged stimulation. bioelectrochemical devices composed of au electrodes coated by self-assembled monolayers (sams) of different composition and thickness are ideal systems to probe et patterns and mechanisms for redox proteins. representative proteins, cytochrome c and azurin were studied by using the combinations of four different strategies including the variation of sam thickness ([−s−(ch ) n −ω], with n running over the range to , throughout), solution viscosity (varied by adding of the viscose additive -glucose), temperature ( to o c) and hydrostatic pressure (up to mpa), aiming the identification of different intrinsic et patterns and interplay between them in the framework of generalized charge-transfer theory. we demonstrated the full adiabatic (frictional) control for the case of thinner sams, the intermediate (mixed) regime, and the complete changeover to the nonadiabatic mechanism (long-range tunneling) for the case of thick sams owing to the variation of electronic coupling, in a nice agreement with theoretical predictions. h. nury , f. manon , b. arnou , m. le maire , e. pebay-peyroula , c. ebel institut de biologie structurale (ibs) cea cnrs ujf grenoble france, cea ibitec cnrs ura univ. paris-sud gif-sur-yvette france adp/atp carriers (aacs) are major and essential constituents of the inner mitochondrial membrane. they drive the import of adp and the export of newly synthesized atp. they were described as functional dimers from the s until the structures of the aac shed doubt on this consensus. we aimed to ascertain the published biophysical data claiming that aacs are dimers and to characterize the oligomeric state of the protein before crystallization. analytical ultracentrifugation sedimentation velocity experiments clearly show that the bovine aac is a monomer in -laurylamido-n,n dimethylpropylaminoxide (lapao), whereas in triton x- and reduced triton x- , higher molecular mass species can also be identified. neutron scattering data for monomeric bovine aac in lapao does not give definite conclusions on the association state, because the large amount of detergent and lipids is imperfectly matched by contrast methods. we discuss a possible way to integrate previously published biochemical evidence in favor of assemblies, the lack of well-defined multimers that we observe, and the information from the high-resolution structures, considering supramolecular organizations of aacs within the mitochondrial membrane. local anaesthetic binding to shaker channels: role of aromatic residues j. nilsson, h. ullman, k. sahlholm, p. arhem the nobel institute for neurophysiology, department of neuroscience, karolinska institutet, se- stockholm, sweden local anaesthetic, antiepileptic and antiarrhythmic drugs acting on nav and herg channels have been assumed to bind to aromatic residues in the internal vestibule; to f and y in nav (ragsdale et al., and to y and f in herg (mitcheson et al., ) . despite a lack of such residues in kv channels, local anaesthetics, antiepileptic and antiarrhythmics bind to kv channels with a considerable affinity. to explore the role of aromatic residues for the binding we investigated the effect of bupivacaine, benzocaine, phenytoin and quinidine on shaker channels mutated to residues corresponding to the most c-terminal of the two aromatic residues in the s segment of the nav and the herg channels, (v y and p f respectively). the channels were expressed in xenopus oocytes and the currents measured with the two-electrode voltage-clamp technique. the results suggest that aromatic residues do not increase the binding affinity of the studied compounds to kv channels. rather, the affinity decreases (as reflected in typical k d values for bupivacaine on v f, p f and wildtype channels, being , and µm, respectively). thus, aromatic residues seem not to be necessary for high-affinity binding of the studied compounds to kv channels. how this relates to their suggested roles in the nav and herg channels, remains to be evaluated. (molina et al, ) . the occurrence of a similar behavior in other channels points out to clustering and coupled gating as a potentially important drug target to modulate channel activity. we have identified molecular determinants involved in single and coupled channel gating in kcsa. first, we detected that clustering and coupled gating of kcsa is modulated by anionic lipid. also, a model for the interaction between two kcsa open channels was built. the docking predicts intermolecular sites which includes the non-annular lipid binding site. this explains how an excess of anionic lipid disrupts interactions between channels, destabilizing clustering and coupled gating in kcsa. in addition, the docking model reveals molecular determinants involved in single and coupled channel gating. this interaction involves w , which affects the neighbouring channel through specific interactions in the extracellular mouth stabilizing the selectivity filter in an open conformation. the coupled gating is also explained since this mechanism affects the opposite channel in a mutual manner. finally, mutants kcsa e a and kcsa w a disrupt the coupled gating of kcsa, thus, supporting the model. we think that this coupled gating phenomenon could correspond to the second gate previously detected by fluorescence methods (blunck, et al, ) . supported by grants from the spanish bfu - /bmc and csd - . a. kumar, e. hajjar, p. ruggerone, m. ceccarelli university of cagliari, monserrato, italy the striking presence of outer membrane (om) in gramnegative bacteria of e.coli represents a strong barrier for any molecule to penetrate inside bacteria. in particular for β-lactam antibiotics, which have their target located inside the bacteria; the first step towards reaching inner part of bacterium is the cellular uptake. ubiquitous presence of porins (such as for instance ompf, ompc) in the om, function as a channel facilitating the transport of molecules (such as, for instance antibiotics) across the om. bacteria can exhibit resistant towards antibiotics by: (i) decreasing their uptake by under-expressing the porins or/and (ii) production of inactivating enzymes such as ß-lactamases. to combat the latter mechanism ß-lactamase inhibitors (such as, sulbactam for instance) are prescribed together with the antibiotics. like the antibiotics, the inhibitors must penetrate the om, the main path being through porins. it is thus evident the biological relevance of investigating the mechanisms by which porins can regulate entry/exit across the om. to achieve this goal, molecular dynamics simulations were performed to explore the structure and dynamics of pores formed by ompf and ompc porin. from the analysis of data obtained from our simulations, we identified the key residues buried behind the l loop, which may play be crucial for porins to exert their biological role. as a case study, we report results about the diffusion of sulbactam through the two porins. the pentadecapeptide gramicidin forms a cation-specific ion channel in membrane environment. the two main conformations are the head-to-head helical dimer (hd) known as the channel conformation and the intertwined double helical form (dh) often referred to as non-channel conformation. in this comparative study [ ] , the energetics of single potassium ion permeation by means of the potential of mean force (pmf) for both gramicidin conformations embedded in a dmpc bilayer has been addressed by molecular dynamics simulations. a significantly decreased free energy barrier by ∼ kj/mol for potassium ion passage through dh as compared to hd is reported. favorable electrostatic side chain-cation interactions in hd are overcompensated by phospholipid-cation interactions in dh. the latter are coupled to an increased accessibility of the channel entrance in dh due to distributed tryptophans along the channel axis. this result underscores the importance of the lipid environment of this channel not only for the equilibrium between the different conformations but also for their function as cation channels. y. sawada , m. murase , m. sokabe dept. physiol. nagoya univ. grad. sch. med., nagoya, japan, icorp/sorst cell mechanosensing, jst, nagoya, japan the bacterial mechanosensitive channel of large conductance mscl is constituted of homopentamer of a subunit with two transmembrane inner and outer α-helices, and its d structure of the closed state has been resolved. the major issue of mscl is to understand the gating mechanism driven by tension in the membrane. although several models for the opening process have been proposed with molecular dynamics (md) simulations, as they do not include mscllipid interactions, it remains unclear which amino acids sense membrane tension and how the sensed force induces channel opening. we performed md simulations for the mechano-gating of mscl embedded in the lipid bilayer. upon tension in the bilayer, phe in the outer helix was dragged by lipids, leading to a tilting of the helices. among amino acids in the outer helix facing the bilayer, phe at the water-lipid interface showed the strongest interaction with lipids, thus may work as a major tension sensor. neighboring inner helices cross each other in the inner leaflet, forming the most constricted part of the pore. as tension increases, the crossings move toward the cytoplasm associated with an expansion of the constricted part. during the movement, a hydrophobic water block environment around the constricted part was broken followed by water penetration and permeation. the k + channel kcsa is an integral membrane protein from s.lividans, used as a model system for studies on ion channels and oligomeric membrane proteins. its atomic structure has been solved by x-ray diffraction, which shows an assembly of four identical subunits around a central aqueous pore, including the so-called selectivity filter. this channel is able to permeate k + at high flux rate and it is blocked by na + (physiological blocker). fluorescence, circular dichroism and fourier transform infrared experiments carried out in our laboratory demonstrated that k + and na + are able to bind to kcsa in a competitive manner. this binding is indeed associated with channel conformational changes, which seem to be related to the permeation and blockade processes. to further investigate this phenomenon we carried out a detailed study of chemical and thermal denaturation of wild-type and mutant kcsa channels. these two types of experiments were in agreement and indicate that both cations are able to stabilize the channel through conformational changes, being k + the more efficient one, even more when lipids are present. particularly, mutant channels with a structurally altered selectivity filter show that ion and lipid-induced global conformational changes are intimately associated to the conformation of this selectivity filter (conductive and non-conductive forms). modulation of the voltage-gated sodium channel nav . by rcssii, a toxin from the scorpion centruroides suffusus c. picco , g. corzo , l. d. possani , g. prestipino institute of biophysics, cnr, genova, italy, instituto de biotecnologia, unam, cuernavaca, mexico the main cardiac voltage-gating sodium channel, na v . , generates the fast depolarization of the cardiac action potential and plays a key role in cardiac conduction. its importance for normal cardiac function has been exemplified by the description of numerous naturally occurring genetic variants of the gene scn a, which encodes na v . , that are linked to various cardiac deseases. subsequently, studies of this channel localization have led to its identification in immature and denervated skeletal muscle and in the brain neurons. in our effort to identify high affinity ligands for this channel, we have investigated the effects of the recombinant cssii (rc-ssii), a four disulfide-bridged scorpion toxin isolated from the venom of the scorpion centruroides suffusus. human cardiac sodium channel α subunit scn a was expressed in cho cells and macroscopic na + currents were recorded with patchclamp technique in whole cell configuration. the electrophysiological experiments have highlighted a strong affinity for the channel at low nanomolar concentration. compared with control conditions, rcssii toxin affects in reversible way the kinetics of activation and inactivation and marked decrease the peak na + influx. the extrusion mechanism of substrates in rnd family efflux pumps: a molecular dynamics study a. v the rnd transporters of the acrab-tolc (e.coli) and mexab-oprm (p.aeruginosa) systems are able to export structurally and chemically different substrates outside bacteria through the membrane, being responsible of multidrug resistance. on the basis of crystallographic information, an extrusion process conceived as a three-cyclic peristaltic pumping has been proposed, but further microscopically well-funded investigations are needed to understand the mechanism. using different computational methods like adaptive bias force (abf) and targeted molecular dynamics (tmd), we have investigated the mechanism of substrate uptake and pumping at a molecular level. with the first method we have investigated the passage of antibiotics from the periplasm into the internal pore of the pump, while tmd has been used to assess the effect of conformational changes on the extrusion of drugs (which have been located into one of the proposed binding pockets). comparison between the active pumps acrb and mexb (which show different resistance patterns despite their homology) provide insights into the microscopic details of their functioning. in arabidopsis thaliana there are twenty genes, grouped into three subfamilies, encoding for homologues of animal ionotropic glutamate receptors (iglrs). each protein displays a pore-forming loop, flanked by two conserved helices (plus a third c-terminal helix), a glutamate-binding domain and an n-terminal region. through pharmacological and/or genetic approaches, many physiological functions have been attributed to plant glurs, such as the regulation of cytosolic calcium, photomorphogenesis, water balance and carbon/nitrogen sensing and assimilation. according to the endosymbiotic theory, the cyanobacteria are considered to represent the precursors of the present chloroplasts. the first prokaryotic glutamate receptor (glur ) was identified in the cyanobacterium synechocystis. the putative products of the atglr . and atglr . genes display a possible targeting sequence for chloroplast location and show a high degree of homology with glur . using specific antibodies and confocal microscopy, we have localized the two members of the atglr subgroup , glr . and glr . (splicing variant) , to the chloroplast in arabidopsis and to the inner envelope membrane in spinach. electrophysiological experiments indicate the presence of an activity which is compatible with that of glutamate receptors. furthermore, oxygen evolution measurements suggest that chloroplast-located glutamate receptors may play a role in the regulation of photosynthesis. under extreme conditions many cells control their volume responding to osmotic challenges by unloading or loading solutes to recover their original volume. a faster volume regulatory role triggered by membrane tension has been disclosed for aquaporins in kidney proximal tubule cells, where aqp is the main water channel, using isolated brush border membranes. in conventional osmotic studies in animal cells it is common to disregard internal hydrostatic pressures because they are insignificant compared to osmotic forces. however, by using low osmolarity buffers in small radii vesicles, we detected a rise on the internal pressure that creates surface tension and causes membrane stress, with a negative outcome on aquaporin water permeability. these findings suggested a mechanism for volume regulation in kidney proximal tubule epithelia where massive solute and fluid transport occurs. to further explore aquaporin regulation by membrane tension, yeast cells were used as a model that could bare surface tension some orders of magnitude higher than animal cells due to the existence of a cell wall. the effect of increasing levels of membrane tension on yeast water channel activity was evaluated. an impairment of aquaporin activity correlated with the increase of membrane tension corroborates the volume regulatory role of aquaporin in different cells. deuterium isotope effects on fast gating of the chloride channel clc- g. zifarelli, a. r. murgia, p. soliani, p. michael istituto di biofisica, cnr, via de marini, , i- genova, italy gating of the torpedo cl − channel clc- is modulated by intracellular and extracellular ph, but the mechanism responsible for this regulation has remained so far elusive. using inside-out patch clamp measurements we studied the dependence of the fast gate on ph int and [cl − ] int . only the closing rate, but not the opening rate showed a strong dependence on these intracellular factors. using mutagenesis we excluded several candidate residues as mediators of the ph int dependence. we propose a model in which a proton generated by the dissociation of an intrapore water molecule protonates e leading to channel opening. deuterium isotope effects confirm that proton transfer is rate limiting for gate opening and that channel closure depends mostly on [oh − ]. the model is in natural agreement with the finding that only the closing rate constant, but not the opening rate constant, depends on ph int and [cl − ] int . deletion of the c-terminus destabilizes phosphorylated na/k pump state containing na ions n. vedovato, d. c. gadsby the rockefeller university, new york, u.s.a. the na/k pump's extended c-terminus (compared to the serca ca pump's) links transmembrane helices, and its truncation lowers cytoplasmic na affinity for forming the occluded e p(na ) state. here we test the effects of c-terminal truncations on interactions with external na. we deleted the last (yy) or (kesyy) residues in xenopus α β pumps made ouabain resistant by mutations q r-n d (rd) or c y (c-y), and then used two-microelectrode voltageclamp recording in xenopus oocytes to measure pump currents as mm ouabain-sensitive currents while endogenous na/k pumps were silenced with µm ouabain. inhibition by external na of steady outward pump current ([k] o = mm) at large negative voltages was somewhat weaker in both rd and c-y pumps than in wt pumps, but was severely impaired in all c-terminal truncated pumps. consistent with this, the voltage dependence of transient charge movements under na/na exchange conditions ([k] o = mm) was strongly shifted to more negative potentials in the truncated pumps relative to the parent rd or c-y pumps, shifts comparable to those seen in wt pumps on decreasing [na] o several-fold. together, the results suggest that these c-terminal deletions lower the apparent affinity for external na ions to bind and become occluded in the na/k pump. the c-terminus therefore provides contacts important for stabilizing the occluded e p(na ) conformation, regardless of the route of na ion entry into the binding pocket. muscle contraction is driven by molecular motors that adapt their energy utilization according to the demands made on them. we test the hypothesis that rate constants controlling the biochemical steps involved in atp hydrolysis by myosin atpase are affected by the force of the muscle. here we use fluorescence lifetime imaging microscopy (flim) of a fluorescently labelled atp analogue to investigate changes in the environment of the myosin atpase, caused by different loads applied to skeletal muscle. single muscle fibres were subjected to cycles of stretches and releases in the presence of rigor solution and µm of coumarin-labelled atp. flim acquisition was synchronised with stretch/release cycles and force measurements, which allow us to investigate the effect of strain on the lifetime of the labelled atp bound to the actomyosin complex. characterization of the fluorescence decay by a bi-exponential function resolved the time constant of two populations, namely, free fluorophore (τ = . ± . ns; mean ± s.d.) and fluorescent nucleotide bound to the actomyosin complex (τ = . ± . ns at low strain). these experiments showed that while the time constant of the free fluorophore did not change with force, the time constant of the fluorescent nucleotide bound to actomyosin showed a linear dependence with the force applied to the muscle of . ± . ps/kpa. neck linker docking coordinates the kinetics of kinesin´s heads i. derenyi, a. czovek, g. j. szollosi department of biological physics, eotvos university, pazmany p. stny. a, h- budapest, hungary conventional kinesin is a two-headed motor protein, which is able to walk along microtubules processively by hydrolyzing atp. its neck linkers, which connect the two motor domains and can undergo a docking/undocking transition, are widely believed to play the key role in the coordination of the chemical cycles of the two motor domains and, consequently, in force production and directional stepping. although many experiments, often complemented with partial kinetic modeling of specific pathways, support this idea, the ultimate test of the viability of this hypothesis requires the construction of a complete kinetic model. considering the two neck linkers as entropic springs that are allowed to dock to their head domains and incorporating only the few most relevant kinetic and structural properties of the individual heads, we have developed the first detailed, thermodynamically consistent model of kinesin that can (i) explain the cooperation of the heads during walking and (ii) reproduce much of the available experimental data (speed, dwell time distribution, randomness, processivity, hydrolysis rate, etc.) under a wide range of conditions (nucleotide concentrations, loading force, neck linker length and composition, etc.). besides revealing the mechanism by which kinesin operates, our model also makes it possible to look into the experimentally inaccessible details of the mechanochemical cycle and predict how certain changes in the protein affect its motion. the positive role of noise on the transport efficiency of na, k atpase c.-h. chang , t. y. tsong institute of physics, national chiao tung university & physics division, national center for theoretical sciences, hsinchu, taiwan, institute of physics, academy of sciences, taipei , taiwan na, k atpase is a molecular motor which is able to transport ions through cell membranes, even against the transmembrane ion concentration gradient. while in vivo this nanoscale soft machine consumes atp, it may be driven by external fluctuating electric fields, no matter they are periodic or random. theoretically, the motor conformations can be described by a conformation vector v(t) governed by a multi-dimensional kinetic equation. given an oscillating electric field with a slight fluctuation, the boltzmann distributions of these conformations will change with time. the instantaneous transported ion flux is a functional of the quasi-cyclic trajectory v(t) of this non-autonomous dynamical system. various interesting dynamical properties of this ion pump, including stochastic resonance, can be studied theoretically, some of which have good agreement with recent experimental findings. in situ measurements of the molecular motor of muscle with nanometer-microsecond resolution in a contracting muscle, arrays of the dimeric motor protein myosin ii pull the actin filament towards the centre of the sarcomere during cyclical atp driven interactions. when the external load is smaller than the array force, the sarcomere works as a motor, converting metabolic energy into mechanical work; when the external load is larger than the array force, the sarcomere acts as a brake resisting the load with reduced metabolic cost. to investigate the molecular basis of the work production and the braking action of muscle, we use sarcomere-level mechanics and x-ray interferometry in intact single cells from frog skeletal muscle. during isometric contraction, each motor bears a force of about pn. during shortening against high and moderate loads, the number of myosin motors attached to actin reduces in proportion to the external load while the force per attached motor is maintained similar to the isometric value (piazzesi et al., cell , - , ) . rapid stretches of - nm between each overlapping set of myosin and actin filaments in a muscle sarcomere cause the stiffness of the array of myosin motors to increase up to twice the isometric value within ms (brunello et al., pnas usa , - , ) , indicating that the high resistance of active muscle to stretch is due to recruitment of the second motor domain of the myosin molecules with the first domain already attached to actin. supported by miur and ente crf (italy), nih (usa), mrc (uk), embl, esrf. kinesin- is a molecular motor that moves cellular cargo along microtubules. its functional mechanism is well understood for individual motors. however, the way that many kinesin- motor proteins bound to the same cargo move together is not. we addressed the structural basis for this phenomenon using video microscopy of single microtubulebound full-length motors and various spectroscopy methods were employed to study synthetic peptides derived from hinge- region. these peptides show an unexpected profile of secondary structure forming propensities. video microscopy of single microtubule-bound full-length motors reveal the sporadic occurrence of high compliance states alternating with longer-lived, low compliance states. the deletion of hinge- abolishes transitions to the high compliance state. from the results we hypothesize that strain accumulated during multiple kinesin motility populates the high compliance state by unfolding helical secondary structure in the central hinge domain flanked by unordered regions, thereby preventing the motors from interfering with each other in multiple motor situations. titin is a giant protein of vertebrate skeletal and cardiac muscles. cardiac titin is expressed in two main isoforms: short n b (∼ kda) and long n ba (∼ kda). we have studied changes of titin isoform composition in myocardium of hibernating ground squirrels and spontaneously hypertensive rats (shr). using electrophoresis we have revealed considerable decrease (by - times) in the content of titin relative to myosin heavy chains in shr heart as compared with that for normotensive rats. surprisingly that the data of qrt-pcr showed the increase in mrna content of n ba and n b-isoforms in hypertrophic heart more than times in comparison with norm. we suppose that such a result is an effect of depressed translation of mrnatitin in pathology. we have observed the decrease (by , times) of total titin amount in heart of hibernating animals in comparison with that for summer active animals. however n ba/n b ratio in the heart upon hibernation was increased by times. similar trend was not revealed for the mrna level of corresponding isoforms, although we have showed the decrease of mrna of both titin isoforms in heart of hibernating ground squirrels as compared to their content of summer animals. the decrease in total mrna level may be explained by repressed transcription or mrna degradation in the cell during hibernation. these discrepancies in protein and mrna levels may be considered as the posttranscriptional regulation of titin isoforms expression. actomyosin cross-bridges formed when the globular heads of myosins bind to actin filaments are the molecular engines that drive muscle contraction, fuelled by atp hydrolysis. critical to this process is the change in shape of the cross-bridge and the change in the interactions with actin, in response to force applied to the muscle, and to the status of the nucleotide in the binding pocket. although molecular detail is known from x-ray crystallography and biochemistry, understanding of the interplay between cross-bridge shape and chemical state requires studies in muscle fibres generating force. we use fluorescence life time imaging microscopy (flim) as a probe of the cross-bridge environment.with a fluorescent analogue of atp , fluorescence life-time (flt) changes when the crossbridge binds to actin. now, we show preliminary experiments on the effect of force on flt. the essential light chain of myosin (elc) is a ∼ kda peptide that wraps around a nmlong α-helix of the myosin cross-bridge known as the lever arm which tilts during force generation. using a recombinant elc, labelled with a fluorophore at a strategic cys, we replace the native elc and introduce the fluorescent elc in muscle fibres. preliminary experiments demonstrate that the elc fluorophore also is sensitive to force applied to the muscle fibre. in addition, förster resonance energy transfer occurs between the nucleotide and elc fluorophores, opening the way for studying structural changes in cross-bridges during force generation by fret. muscle contraction: pitfalls in the determination of the contractile response e. grazi dipartimento di biochimica e biologia molecolare, università di ferrara, ferrara, italy the contractile response of an active muscle depends on the load. the load is a force /cross-section. there are three fundamental dimensions: the mass, m; the space, l; and the time, t. from these three dimensions are built up all the physical dimensions. as an example the acceleration, a, is given by, a=l.t − . once the direction and versus are settled the modulus fully defines the physical effect of the acceleration. what about the force? the force, f, is given by, f=m.a. at variance with the acceleration, once the direction and versus are settled, the modulus does not define the physical and the biological effects of the force: the same force is generated by an infinite number of mass-acceleration couples that display different physical and biological effects. the same occurs with the load. thus defining the load that opposes the contractile force does not define the contractile system. in the studies on muscle contraction the acceleration of the load is not considered nor it is provided a way to extract this information. thus these systems are poorly defined from the physical as well as from the biological point of view. models of muscle contraction that consider explicitly both the mass and the acceleration of the load show that, at the same load, the decrease of the acceleration of the load significantly delays the pre-steady state of the contraction and decreases the stiffness of the active fibre. r. shahapure , f. difato , a. laio , d. cojoc , e. ferrari , j. laishram , g. bisson , v. torre int. school for advanced studies, trieste, italy, iit-sissa unit, trieste, italy, lab. nazionale tasc, trieste, italy polymerization of actin filaments is the main source of motility in lamellipodia and is controlled by many regulatory proteins. the underlying molecular mechanisms are only partially understood and now a determination of the dynamical properties of force generation is needed. using optical tweezers we measured with millisecond temporal resolution and pn sensitivity the force-velocity (fv) relationship and the power dissipated by lamellipodia of dorsal root ganglia neurons. when force and velocity are averaged over - s, fv relationships can be flat. on a finer time scale, random occurrence of fast growths and sub-second retractions become predominant. maximal power dissipated by lamellipodia over a silica bead with a diameter of µm is − w. due to the presence of adhesion forces, beads in close contact with a lamellipodium can seal on its membrane reducing the amplitude of brownian fluctuations often by more than times. under these conditions, when lamellipodia grow and push the beads, discrete jumps varying from about to nm are detected. when lamellipodia retract, pulling the beads, no discrete events are observed. our results on the dynamical properties of force generation are: a) force generation is a probabilistic process; b) underlying biological events have a bandwidth up to at least hz; c) fast growths of lamellipodia leading edge alternate with local retractions; d) force generation is produced in discrete steps with varying amplitude up to . pn. pushing on microtubules: dominant spindle centering mechanism in c. elegans embryo? j. pecreaux, s. redemann, a. a. hyman, j. howard mpi-cbg, pfotenhauerstr , dresden, germany asymmetric cell division, where the content of the two daughter cells -as well as their sizes -differ, is found in many organisms. strikingly, the spindle, first centered, starts to be displaced out of the center only in late metaphase. in c elegans embryo, the spindle rocks and is posteriorly displaced during anaphase by force generators asymmetrically localized on cell cortex. prior to anaphase onset, the spindle is usually assumed to be centered by the same pulling force. it thus requires the force generators to be carefully repressed to distribute forces symmetrically. on live embryos, we measured positional fluctuations of centrosomes during metaphase with nm accuracy. fourier analysis shows an extremely accurate centering respect to the number of force generators and microtubules. furthermore, spectrum is close to a lorentzian, modeled by a spring and a dashpot, suggesting a spindle centering more likely by pushing on microtubules than pulling. deviation at high frequencies indicates a subdominant pulling force. rnai of gpr- / , known to control force generation, increases slightly centering accuracy; this result supports the hypothesis of an independent centering mechanism. conversely, zyg- (rnai), a microtubule growing factor, decreases centering accuracy, modeled spring stiffness and damping modulus. conclusion: first, the spindle centering mechanism is independent of cortical pulling force generator. second, microtubules pushing is likely to center the spindle. mechanical forces are important in the regulation of cellular adhesion and migration. the focal adhesion kinase (fak) has been suggested to transduce cellular forces and govern cell migration. to obtain more insight in the functioning of fak, a fret-based optical biosensor for fak was designed to relate integrin-mediated conformational changes in its ferm domain to focal adhesion behavior during cell spreading and migration in living cells. imaging of the kinetics of ferm-based fak conformational changes in spreading cells revealed two consecutive stages of focal adhesion activation. heterogeneous ferm conformational responses were observed in individual focal adhesions of adherent motile cells, with the active ferm conformation being enriched in growing and sliding fas, but not in stable and shrinking focal adhesions. inhibition of the cellular actomyosin system revealed the involvement of rho-rock rather than mlckinduced tension signaling in the modulation of the ferm response. our results place the ferm conformational change of fak at the interface between integrin and force sensing. the time course of inorganic phosphate release in permeabilized cardiac trabeculae of the rat c. mansfield, t. west, m. a. ferenczi imperial college london, u.k. the rate of p i release was determined in permeabilized rat trabeculae. contraction was elicited at • c by laser-flash photolysis of npe-caged atp, and time-resolved p i release was monitored using mdcc-pbp, a coumarin-labelled phosphate binding protein, which increases its fluorescence intensity five-fold upon p i binding. the atpase rate during the first turnover of the total crossbridges (assuming µm myosin heads) was s − . the rate decreased to a steady state of s − after the eighth turnover ( . - . s after activation). this steady state rate is comparable to published values of - s − , made ∼ s after activation using an nadh-linked enzyme assay of adp release. the advantage of using mdcc-pbp is that the control of mechanochemical coupling can be examined from the onset of force production and as it progresses toward the steady state. force production and p i release were simulated using a seven step scheme. force was attributed to the states in the sequence a.m.adp.p i ↔ a.m.adp ↔ a.m.adp, with strain sensitivity incorporated into the isomerisation of a.m.adp. the a.m.adp.p i and a.m.adp states populated rapidly as force was increasing. in contrast, the a.m.adp state accumulated slowly after the force plateau was reached and became the dominant force bearing state at the time of the eighth crossbridge turnover. experiments are on-going to examine how the distribution of a.m states changes in response to rapid length-changes. pulling as a factor in forming the heterophasic structure of immunoglobulin proteins structure of proteins of immunoglobulin superfamily: human igg kuc and muscle protein titin, has been investigated by methods of electron microscopy and diffraction with the use synchrotron radiation. super elasticity of titin, the protein of immunoglobulin superfamily, is a key parameter that determines the mechanical properties of muscle. however, the structural-physical mechanism of titin elasticity under tension remains poorly understood. here both tension transduction and high elasticity of titin are explained in terms of crystalline polymer physics. x-ray data suggest a model of titin as a nanoscale, morphological, aperiodical array of rigid ig-and fn -type domains covalently-connected by conformationally variable short loops. the line group symmetry of the model can be defined as s m with axial translation τ ∞ . homologous domains would have similar stability, but the structure of different domains on stretching is subject to different forces because they have different orientations relative to the axis of the molecule. under the force influence the structure of any domain can become either rigid or flexible depending on its orientation in the titin strand. pulling geometry forms an active axial structure from latent isotropic random coil structure of titin strand. we are suddenly faced with nanophase-separated morphology of igg kuc. study was supported by rfbr grant - - . strain response of myosin essential light chain in permeabilized skeletal muscle fibres d. s. ushakov, d. ibanez-garcia, t. g. west, p. m. w. french, m. a. ferenczi imperial college london, uk we applied fluorescence lifetime imaging microscopy (flim) to investigate the relation between conformation of myosin head and mechanical force in skeletal muscle fibres. recombinant myosin essential light chain (elc) was expressed in e.coli and labelled at cys- with coumarin. the labelled elc was exchanged with native elc in single permeabilized rabbit m.psoas fibres. fluorescence lifetime was measured using leica sp upright confocal microscope equipped with becker & hickl time-correlated single photon counting module and x . na leica planapo dipping objective, with the two-photon fluorescence excitation at nm by sapphire pulsed laser. after acquiring flim images of muscle fibres in relaxed state, the solution was changed to ca-free rigor. further images were acquired in rigor with or without . - % stretch applied by a motor. both single and double exponential fluorescence decay analysis showed that the lifetime in rigor was lower compared to relaxed (about ps difference for single exponential fit) and to rigor fibres under strain (about ps). these data suggest a change in the microenvironment of coumarin induced by nucleotide binding and strain. this change is likely to be due to interaction between c-terminal domain of elc and the n-terminal domain of myosin heavy chain related to the lever arm re-orientation process. supported by bbsrc. alpha-synuclein and its a p mutant affects the actin cytoskeleton structure and dynamics v. sousa , s. bellani , g. ronzitti , f. valtorta , j. meldolesi , e. chieregatti department of neuroscience, hsr, milano, italy, department of neuroscience, iit, genova, italy alpha-synuclein (syn) is a soluble protein abundant in the brain, primarily enriched at pre-synapses. syn overexpression and the expression of its a p mutant participate in the pathogenesis of parkinson's disease. many roles have been proposed for syn, including the regulation of synaptic vesicle pools and of neurotransmitter release. the actin cytoskeleton regulates many aspects of synaptic function and its dysregulation may be a cause of neurodegeneration. working both in cell-free and in vivo conditions we demonstrate that syn and the a p mutant have different effects on the actin cytoskeleton dynamics. our results show that syn binds actin, and decreases actin polymerization rate probably by monomer sequestration. on the contrary, a p accelerates actin polymerization in vitro and disrupts the cytoskeleton of intact cells. in particular, during dynamic cytoskeleton remodeling, a p induces the assembly of discrete actin-rich foci. actin trapping and the impairment of filaments reassembly lead to inhibition of cell movement and of the re-establishment of cell-cell contacts. in a p expressing cells cytoskeleton-based processes, such as cell migration and the exo/endocytic traffic are inhibited. elucidating the dynamics of syn interaction with actin may contribute to the understanding of its role in neuronal physiology as well as in neurodegeneration. on the physics of muscle contraction m. l. shur ural state university, yekaterinburg, russian federation whichever energy source is chosen as an engine, its force will decrease with increasing velocity. this is connected with a limited power of any engine. thus, we state that hill's formula is a mere sequence of the law of energy conservation. to derive a mathematical dependence "force-velocity", all the means of consumption of fuel energy should be determined -in our case, the energy of the atp hydrolyze. moreover, the conformation energy of the crossbridges attached serves as the force source as well. we state that part of the energy release transforms into the energy of oscillations of myosin proteins; the other part goes into thermal energy of the sarcoplasmic solution. interaction of the oscillating myosin system with the sarcoplasmic solution controls the process of force generation by a muscle. it is just this interaction that leads to the temperature dependence of force. the presentation is devoted to constructing a theory based on these simple considerations. a discussion and constructive critic is especially wanted. -biological motility and molecular motors - meiotic nuclear oscillations in the fission yeast schizosaccharomyces pombe are crucial for proper chromosome pairing and recombination. we report a mechanism of these oscillations based on collective behavior of dynein motors linking the cell cortex and dynamic microtubules that extend from the spindle pole body in opposite directions. by combining quantitative live cell imaging and laser ablation with a theoretical description, we show that dynein dynamically redistributes in the cell in response to load forces, resulting in more dynein attached to the leading than to the trailing microtubules. the redistribution of motors introduces an asymmetry of motor forces pulling in opposite directions, leading to the generation of oscillations. our work provides the first direct in vivo observation of self-organized dynamic dynein distributions, which, due to the intrinsic motor properties, generate regular large-scale movements in the cell ( ) m. versaevel, s. gabriele, p. damman university mons-hainaut, mons, belgium the remodeling of blood vessels in response to changes in blood flow is mainly realized by endothelial cells (ecs) that convert mechanical stimuli from flowing blood into changes in cell signaling through a process called mechanotransduction. many of the biological responses to external forces originate at two types of microscale structures: focal adhesions linking cells to their extracellular matrix and adherens junctions that link adjacent cells. this study aims to elucidate the role of the cytoskeleton, cell-matrix and cell-cell junctions in transducing fluid shear stress into intracellular signals in ecs. by using microcontact printing of proteins, we design substrates with defined adhesive islands in order to control shapes of living cells. this confinement of ecs allows to study the organization and the contractile activity of the cytoskeleton in order to redistribute their intracellular forces in response to externally applied forces. we design microfluidic channels with sizes and geometries close to small blood vessels to apply a physiological range of shear stresses on ecs. our results indicate that cells deposited on a precisely defined adhesive area inside microchannels and subjected to shear stress reorganize their cytoskeleton, their focal adhesions and adherens junctions in response to blood flow. drugs interfering with the cytoskeleton are used to underline the role of its different components in the cellular adaptation to the mechanical environment. s. mahdavi , b. ranjbar , s. gharibzadeh , m. toosi , m. javan tarbiat modares university, tehran, iran, amirkabir university of technology, tehran, iran multiple sclerosis (ms) is the main known pathology of myelinating cells. an autoimmune reaction occurs against myelin sheets of neurons, so action potential (ap) propagation along the affected nerve fibers has been destroyed and it causes various disorders. here, we propose a novel strategy for ms symptoms treatment. we modeled neuron by orcad software and simulated the action potential propagation along the axon in normal condition. our model simulated normal neuronal behavior. then we destroyed the myelin sheet as it occurs in ms and observed destroyed ap propagation as it was reported in ms disease. we investigated the effect of changes in the voltage-gated sodium channel (vgsc) threshold on the efficiency of ap propagation. the results demonstrated that reduction of vgsc threshold improves the propagation of ap by increasing the amount of sodium flux during ap propagation. although, some researches have proposed vgsc blocker as ms symptom treatment, our result suggests that the increase of sodium current produced by reduction of vgsc threshold, improves ap propagation and probably cure some ms symptoms. so, we suggest that vgsc gating modifiers can be considered as novel strategy for ms treatment. surely, this results needs to be confirmed by experimental studies. a. gradogna, e. babini, a. picollo, m. pusch istituto di biofisica, consiglio nazionale delle ricerche, via de marini , genova, italy clc-ka and clc-kb are highly homologous cl − channels expressed in the kidney and the inner ear where they mediate transepithelial chloride transport. both channels heteromerize with the beta subunit barttin. mutations in clc-kb and barttin genes lead to bartter's syndrome. we analyzed the modulatory effect of extracellular ca + and h + on clc-k channels using the xenopus oocyte expression system. clc-ka currents increased with increasing [ca + ] ext without full saturation for [ca + ] ext up to mm. however, in the virtual absence of ca + , clc-ka currents are about % of currents measured in mm [ca + ] ext , demonstrating that ca + is not strictly essential for opening. vice versa, clc-ka was blocked by increasing the [h] + ext with an almost complete block at ph . among various reaction models tested, the model that best fitted all state-steady data predicts an allosteric regulation of channel opening by separate binding sites for ca + and h + . moreover, the best fit suggests that one ca + and two h + bind to the channel. kinetic analysis of current responses upon [ca + ] ext and ph jumps confirmed the allosteric character of modulation. in support of the presence of two separate binding sites we identified several mutations that selectively altered ca + or h + sensitivity. our data represent a first step towards a molecular picture of ca + and proton regulation of clc-k channels and suggest that it is of physiological relevance. the extracellular matrix molecule hyaluronic acid modulates l-type voltage-dependent ca + channels e. dvoretskova , g. kochlamazashvili , o. bukalo , c. henneberger , d. rusakov , m. schachner , a. dityatev italian institute of technology, genova, italy, centre for molecular neurobiology, hamburg, germany, institute of neurology, university college london, london, uk we studied the effects of hyaluronic acid (ha), a major extracellular matrix molecule, on activity of l-vdccs in a heterologous expression system and in hippocampal slices. we recorded currents mediated by a major neuronal subtype of l-vdccs (ca v . c, β b, and α δ ) expressed in cho cells. a five-minute application of . mg/ml ha potentiated l-vdcc currents at - , - , and + mv by approximately %. analysis of boltzmann curves showed that ha increased maximal conductance rather than other parameters (v . or k ). treatment with hyaluronidase removed endogenous ha in murine hippocampal slices and specifically impaired long-term potentiation (ltp) induced at ca -ca synapses by repetitive theta-burst stimulation. blockade of l-vdccs reduced ltp in control slices to the levels seen after hyaluronidase treatment. a potentiation of l-vdccs with bay k fully restored ltp after hyaluronidase treatment. removal of ha reduced ca + transients elicited by backpropagating action potentials in individual dendritic shafts and spines of ca pyramidal cells, whereas pretreatment with nifedipine fully occluded this effect. thus, ha potentiates postsynaptic l-vdccs and by this way influences use-dependent synaptic plasticity. whole-cell patch clamp recordings from a variety of human cancer cells showed that functional voltage-gated sodium channel (vgsc) expression occurred specifically with strongly metastatic cells. in addition, where studied, this was accompanied by down-regulation of outward (mainly potassium) currents. this has led to the celex ("cellular excitability") hypothesis of cancer according to which metastatic cell membranes are excitable and this promotes their hyperactivity. importantly, the vgsc genes expressed are embryonic splice variants, which are normally developmentally regulated, hence the phenomenon is 'oncofetal'. in breast cancer, where the predominant vgsc is nav . the neonatal and adult forms are significantly different and this is reflected in channel activity whereby the neonatal vgsc has much slower inactivation kinetics. the double-charge change at position is critical for this difference. the slow kinetics results in much greater influx of na + into cells and one consequence of this is activation of protein kinase a. this is a tonic effect and, under steady-state resting conditions, it results in a positive feedback effect promoting post-translational trafficking of vgsc protein to plasma membrane. the unique amino acid sequence of the spliced region has enabled the production of a polyclonal blocking antibody specific to neonatal nav . . it is concluded that vgscs represent novel biophysical targets for clinical management of metastatic disease. m. pusch cnr, istituto di biofisica, genoa, italy clc proteins form an evolutionary conserved gene-family that comprises members in mammals. four of the human clcs are passive plasma membrane cl − ion channels. the other clcs are expressed in intracellular organelles. clc- and clc- , mutations of which lead to dent's disease, are secondary active cl − /h + antiporters, similar to the bacterial clc-ec , and with identical cl − : h + stoichiometry. cl − and h + transport activity of the exchanger clcs depends on two glut residues. mutating a 'gating glutamate' (e in clc- ) converts the exchanger into anion conductances. neutralizing the 'proton glutamate' (e ), but not its replacement by some other titratable groups, abolishes cl − and h + transport. noise analysis indicated that clc- switches between silent and transporting states with an apparent unitary conductance of . ps, indicating a very large transport turnover. no − uncouples h + transport but mutating the highly conserved s to p, as found in the plant no − / h + antiporter atclca, led to coupled no − : h + exchange. clc proteins are a fascinating example of how a very similar protein architecture can be used to provide either a passive electrodiffusive permeation pathway or a strictly coupled secondary active ion transporter. (supported by telethon italy -grant ggp ). the role of ion dynamics in zebrafish fin regeneration the specific and directional ion transport across cell membranes or tissue layers results in differential accumulation of ions and endogenous electric currents. these phenomena have been shown to be important for vertebrate organs regeneration. however, the specific ion nature of such electric currents remains unknown, as well as the role of cellular ion dynamics during regeneration and the molecular signalling pathway that transduces electric cues into cellular responses. we use zebrafish caudal fin as an adult regeneration model to unveil the specific ion composition of the currents associated with wound healing and regeneration, using a non-invasive ion-specific scanning microprobe setup. our data suggests a role for potassium (k + ), calcium (ca + ) and protons (h + ) at different stages of the regeneration process. k + and ca + extracellular effluxes have both been detected during the wound healing stage. h + efflux is triggered during wound healing and is maintained throughout regeneration. we are validating these data with genetic and pharmacological approaches, as well as advanced ion imaging. overall, our results suggest ion-driven mechanisms underlie adult tissue regeneration and its comprehension may open way for new therapeutic strategies, both in regenerative and developmental medicine and in cancer therapy. cardiac effects of anabolic steroids: an electrophysiological approach anabolic androgenic steroids (aas) have been used by athletes and non athletes for almost five decades in order to improve performance. however, the illicit abuse of high-doses of aas has been attributed as a main cause of several cardiovascular disorders such as arterial hypertension, lipid profile abnormalities, heart failure, hypertrophic cardiomyopathy, arrhythmia and sudden death. the aim of this study was to investigate qt interval and transient outward potassium current (i to ) changes in rats treated with nandrolone decanoate (deca). male wistar rats received weekly mg/kg of deca (n= ) or vehicle (control, n= ). electrocardiogram was recorded weekly, and qt interval was measured. after weeks hearts were excised and single myocytes were isolated from the ventricles of animals. i to was recorded by means of the whole cell patch clamp technique. qt interval was larger in deca group from th to th week (p < . ). analysis of i to showed a decreased current density (p < . ) in ventricular cardiomyocytes of deca group, compared to control group. in conclusion, our results show that alterations on ventricular repolarizaton may constitute an early consequence of the chronic administration of high doses of anabolic steroids in rats, and demonstrated qt prolongation and i to density reduction, which may constitute an important marker of arrhythmia vulnerability and sudden death. -ion channels in channelopathies and cancer - denitrifying bacteria control no and no cytosolic levels by regulating the expression of denitrification gene clusters via redox signalling of specific transcriptional factors that may act as no sensors in vivo. a protein belonging to the subclass dnr (dissimilative nitrate respiration regulator) from pseudomonas aeruginosa has been recently suggested to be a heme containing protein. very recently the three dimensional structure of the apo-form of dnr (in the absence of heme) has been determined by x-ray crystallography, whereas the holo-form (in the presence of heme) has not yet been crystallized. we have investigated the heme local structure in solution of ferric, ferrous, co bound and no bound holo-dnr by x-ray aborption spectroscopy (xas) and we added a kinetic study of the co bound form by means of a flash photolysis setup using uv-visible absorption as a spectroscopic probe. the combination of fe k-edge xanes fingerprints and kinetic study reveal a heme pocket able to bind exogenous ligands like no and co with increased plasticity, thus supporting its role as the cofactor involved in no sensing activity. molecular examination of motifs that lead to the formation of s-nitrosylated proteins i. alicea, e. r. schreiter university of puerto rico rio piedras, san juan, puerto rico physiologically, a wide range of proteins experience structural and functional modifications after the addition of a nitric oxide (no) moiety to cysteine thiol. this posttranslational modification, known as s-nitrosylation, regulates a large number of cellular processes like vasodilatation, cell signaling and others, and the products of s-nitrosylation can be involved during the development of different human diseases. however, little is known about the mechanism by which different proteins specifically bind the no moiety to their cysteine. here we show a bio-statistical analysis of some properties of cysteine that are s-nitrosylated in different proteins, including the pk, electrostatic environment, solvent accessibility of the target cysteine and identity of surrounding amino acids. we also chose model proteins (human thioredoxin and s protein) to make specific targeted amino acid substitutions around selected cysteines to alter the properties described above. the reactivity and stability of these mutant proteins towards s-nitrosylation will be examined. sampling the flexibility of ppar-γ s. aci-sèche , n. garnier , d. genest , s. bourg , c. marot , l. morin-allory , m. genest upr cnrs , orléans, france, fr pcv cnrs , orléans, france, umr cnrs , université d´orléans, france a promising approach to consider the flexibility of proteins in docking studies consists in performing multiple rigid docking on a representative set of the receptor conformations. molecular dynamic (md) simulation is one of the best adapted methods for structural sampling, but exploring the conformational diversity of a protein is computationally expensive. we present a protocol for generating a wide range of conformational states of a receptor using restrained md and a partitioning protocol to select a few representative conformations of the binding site from this md. a way to speed up efficiently md calculation is using an implicit model to represent the solute-solvent interactions. we explore a protocol using a distance-dependant permittivity function to represent solvent effect and an ensemble of controlled restraints applied on a subset of specific atoms in order to prevent artefactual structural distortions, but preserving receptor's flexibility. ten ns simulations have been performed using different sets of parameters and compared to a reference ns simulation with explicit solvent. to select a representative set of conformations, partitioning (k -means algorithm) was applied on the ensemble of simulated conformations. this methodology was applied to the ligand binding domain of peroxysome proliferator-activated receptor-γ. department of biomedical sciences, university of antwerp, antwerp, belgium the complex system of cavities identified in human neuroglobin (ngb) has been postulated to be of functional significance to the putative no dioxygenase activity of the protein. the interconnected hydrophobic cavities may support this catalytic activity by acting as reservoir for reactants and providing preferential pathways assisting product removal from the active site. we thus decided to investigate co rebinding kinetics to ngb embedded in silica gels to expose ligand migration processes in the geminate phase. encapsulation of the co complexes of reduced neuroglobin, leads to a slight increase in geminate recombination after nanosecond laser photolysis. increasing the viscosity of the medium, by soaking the gels in glycerol, completely inhibits escape of the photodissociated ligand to the solvent, and highlights a complex, multiphasic kinetic pattern. this finding can be rationalized by assuming the existence of a discrete set of temporary docking sites, capable of trapping the photodissociated ligand for very long times, up to a few ms after photolysis. g. bartolommei, f. tadini-buoninsegni, m. r. moncelli bioelectrolab -department of chemistry, university of florence, italy ion pumps are integral membrane proteins devoted to ion transport through a lipid membrane phase. the ion pumps ca-atpase and na,k-atpase are prominent members of the p-type atpases family. due to fundamental physiological roles of these proteins, they are very promising drug targets. bioelectrolab has a wide expertise in the study of ion transport by these proteins [ ] . our attention has been recently focused on the interaction of these enzymes with molecules of potential pharmacological interest. frequently, drugs exert an inhibitory action on the transport activity of an ion pump, usually confining it in an inactive conformation. molecules like thapsigargin and cyclopiazonic acid belong to high (nanom) affinity inhibitors of the ca-atpase, whereas clotrimazole and curcumin are medium (microm) affinity inhibitors of both ca-atpase and na,k-atpase. for each of these compounds a mechanism of action is proposed. moreover, recent results concerning ca-atpase inhibition by clotrimazole analogues will be shown: the relevance of this type of molecules is due to their potential employment as an alternative to traditional drugs against malaria parasite. financial support of ente cassa di risparmio di firenze and of miur (prin project) is gratefully acknowledged. [ ] tadini-buoninsegni f., bartolommei g., moncelli m.r., fendler k. . arch. biochem. biophys. : - (review). s. asthana , s. shukla , g. giliberti , f. luliano , m. ceccarelli , r. loddu , p. ruggerone , p. la colla department of biomedical science and technology, università di cagliari, cagliari, italy, department of physics, università di cagliari, cagliari, italy studies of protein-inhibitors interactions are helpful to elucidate the mode of action of ligands and thereby providing clues for rational drug design. bvdv, is an important target of drug discovery activities largely because it is essential for viral replication. in bvdv rdrp no specific nni binding site has been reported till now. experimental results have shown that different class of inhibitors (benzimidazole, imidazoquinolines and pyridoxyquinolines), have resistant mutations located in the finger domain of the rdrp. all the reported mutations are spatially very close to each other. thereby, indicating that binding sites of nni's may lie in the finger domain for these different class of inhibitors.herein, we have utilized docking procedure to investigate binding sites, binding modes as well as binding affinity of different class of inhibitors.we then used all atom molecular dynamics (md) simulations to investigate the stabilizing interaction between inhibitor-receptor pairs. our md results are in good agreement with experimental data and provide deep insights into the dynamical features of the high affinity inhibitorreceptor binding. thus identifying the binding modes of our inhibitors and mechanism leading to inactivity of the enzyme can help us to build a microscopically well-funded picture of the functioning of these enzymes. we present an investigation of the molecular basis of ligand binding and reactivity of heme proteins using computer simulation. a combination of classical molecular dynamics and hybrid quantum-classical (qm-mm) calculations are applied to explore distal and proximal effects on diatomic ligand binding to the heme. trends in binding energies and in the kinetic constants are illustrated through a number of selected examples. an investigation of the interplay between ligand migration and protein dynamics obtained through classical molecular dynamics techniques in combination with advanced sampling tools is also presented to yield information about free energy profiles and possible secondary docking sites. results for truncated n hemoglobin of mycobacterium tuberculosis, presented as an illustrative example, suggest that the truncated hemoglobin n has evolved a dual-path mechanism for selective/distinct migration of o and no to the heme, to achieve efficient no detoxification. finally, we present also an analysis of the molecular basis of hexacoordination in human neuroglobin, which suggest that the flexibility of the cd plays a key role in determining the ligand binding properties. thermodynamic bases of nucleoplasmin-histone complexes recognition by the nuclear transport machinery i. arregi, j. falces, s. bañuelos, m. a. urbaneja, s. g. taneva unidad de biofísica (csic/upv-ehu), departamento de bioquímica y biología molecular, universidad del país vasco, spain the nuclear transport of the chromatin remodeling (nucleoplasmin) and chromatin building (histones) proteins is mediated by importins. nucleoplasmin (np) contains a classical bipartite nuclear localization signal (nls) that is recognized by importin α, while histones present multiple sequence elements (nls-like motifs) for nuclear targeting. besides, ternary importin/np/histone complexes might represent a putative coimport pathway for nuclear import of linker (h ), nucleosomal core (h ah b) histones and their chaperone protein np, enhancing the histone import efficiency. to better understand np and histone recognition by the transport machinery we studied the thermodynamics of complex formation of importin α (a truncated form) and importin β with histones and np, and with np/histone binary complexes by means of isothermal titration calorimetry. data show that importins interact with the two histone types and np, and that importin and histones can simultaneously bind to np. analysis of the binding energetics reveals an enthalpy driven formation of high affinity binary and ternary complexes. we demonstrate that different amount of importin molecules can be loaded on np/binary complexes dependent on the histone type, linker or core, and the amount of the bound histones. g. breuzard, i. di maïo, p. barbier, d. allegro, c. brault, v. peyrot cro inserm u , ufr de pharmacie, marseille, france since a decade, our laboratory has already studied the interaction of tau variant with tubulin (tub) or microtubules (mts) and phosphorylation process on this interaction. indeed, fret assay was a powerful tool to achieve binding parameters between tau and tub in vitro: / with mts stabilized by fluorescein-coupled taxol (flutax- ) as donor and rhodamine-labelled tau (rho-tau) as acceptor, and / in living cells by confocal laser scanning microscopy (clsm) with tau/α-tub fused to egfp/mcherry, respectively. results revealed ± % energy transfer efficiency from flutax- to rho-tau and a donor-to-acceptor distance of ± Å. by titration, the dissociation constant of tau was determined to . ± . µm. a cleavage procedure of αβ-tub was performed to determine the influence of the c-term tails of αβ-tub on the tau-mt interaction. no difference in distances and binding parameters was observed. clsm images displayed a heightened concentration of fluorescent tau in patches along mts. fret experiments revealed in particular higher efficiencies between gfp-tau to mcherry-α tub proteins in these locations. overall, our results suggested no involvement of the hypervariable and highly acidic c-term tails of tub in mt/tau binding. a molecular model is proposed in which flutax- is directly accessible to tau molecules. besides, the modified distribution of fluorescent tau could be implicated in local change of mechanical properties of mts. a. boreham , k. winkler , c. gebhard , k. rueck-braun , p. henklein , e. michalsky , r. preissner , r. misselwitz , a. ziegler , u. alexiev freie universität berlin, berlin, germany, technische universität berlin, berlin, germany, charité-universitätsmedizin berlin, berlin, germany peptide presentation by major histocompatibility complex (mhc) molecules is crucial for immune responses. photocontrol of peptide dynamics by means of photo-switchable peptide analogs will provide insights in peptide dynamics and its dependence on mhc polymorphism ( ) ( ) ( ) . we have designed a hemithioindigo (mhti) photo-switch bearing peptide using the viral epitope rrrwrrltv (plmp ) as a template. incorporation of mhti in the peptide backbone should result only in a minor change of the overall peptide structure given the relaxed conformation of the zisomer. indeed, the human mhc molecule hla-b* can tolerate nonpeptidic elements in plmp , as evidenced by normal peptide binding. using the autofluorescent properties of mhti we determined the stability of the hla-b* /mhti-plmp complex. photoswitching from z →e results in a decrease of hla-complex stability. based on computer modeling this decrease is due to a reduced interaction of the peptide c-terminus with hla-b* . truncated hemoglobins (trhbs) are heme proteins present in bacteria, unicellular eukaryotes, and higher plants. three phylogenetic groups (n, o, and p) have been identified in trhbs. the crystal structure of truncated hemoglobin o of b.subtilis, does not show an evident tunnel/cavity system connecting the protein active site with the solvent, a fact that cannot be easily rationalized considering the very high oxygen association rate. moreover, resonant raman results of the co bound protein, showed that a complex hydrogen bond network exists in the distal cavity, making it difficult to assign unambiguously the residues involved in the stabilization of the bound ligand. for these reasons we performed classical molecular dynamics simulations of the oxy, carboxy and deoxy protein, and computed the free energy profiles associated with ligand migration to the active site. our results suggest that there is a key residue, glne , that may present an alternate conformation in which a wide ligand migration tunnel is formed, consistently with the kinetic data. the results for the co and o bound protein show also that glne is directly involved in the stabilization of the coordinated ligand, playing a similar role as tyrb , and trpg in other trhbs. our results not only reconcile the structural data with the kinetic information, but also provide additional insight about the general behaviour of trhbs. patterned functionalization of surfaces for guided transport on molecular motors tracks m. bhagawati , s. ghosh , t. surrey , j. piehler department of biophysics, university of osnabrueck, osnabrueck, germany, european molecular biology laboratory, cell biology and biophysics unit, heidelberg, germany chelator head groups with multiple nitrilotriacetic acid (nta) moieties have been very well characterized and successfully utilized as high affinity adapters for functional immobilization of oligohistidine tagged proteins to surfaces. we have recently established a generic method for patterning nta functionalized surfaces by selective photodestruction via a light induced fenton reaction. efficiency of different transition metal ions for catalyzing this reaction was tested. functionality of the patterned protein was confirmed using the interaction between interferonα and its receptor. implementation of this technique in a confocal laser scanning microscope allowed us to control surface density of binding sites, providing the possibility to vary the surface concentration of immobilized proteins in a spatially resolved manner. we also applied this approach for exploring guided transport of microtubules by kinesin selectively immobilized onto trisnta patterns. rheumatoid arthritis (ra) is an autoimmune disorder, leading to pathological damage at the level of joints, associated with the hla class ii allele hla-dr . although etiology of ra is unknown, type ii collagen (cii) is a potential antigen candidate and it is believed that t cell responses in collagendependent ra are directed towards the immunodominant pathogenic epitope cii( - ). despite recent advances in characterization of class ii major histocompatibility complex (mhc) and t-cell receptor (tcr) contacts in this epitope, the atomic details of tcr-cii( - )-mhc complex are not known. here, homology modeling and molecular docking studies have been used to derive a three-dimensional model of tcr β chains, obtained from a dr + subject, in complex with cii( - )/hla-dr . the best complex from docking was further refined using molecular dynamics simulations for ns. the proposed model represents a reasonable structural basis for understanding cii( - )-mhcii complex recognition by tcr and for rational design of inhibitors targeting tcr-pmhc interface. probing bio-molecular bonds with magnetic force for biosensor applications a. m. de jong , a. jacob , x. j. janssen , j. m. van noorloos , l. j. van ijzendoorn , m. w. prins eindhoven university of technology, eindhoven, the netherlands, philips research laboratories, eindhoven, the netherlands we investigate new technologies to be applied in next generation biosensors, which not only measure biomarker concentrations but also probe bio-molecular interactions. the concept is based on the response of ligand-receptor pairs to an applied force or torque [ , ] . we use functionalized magnetic beads and magnetic fields to apply translational and rotational forces on the molecular bonds. in a model experiment, polystyrene surfaces were coated with anti-biotin and beads were coated with biotin. after incubation, a constant magnetic force was applied to the beads and the number of bound beads was measured as a function of time. this was repeated for a range of forces. the dissociation rate (k o f f ) is determined for each force and k o f f at zero force is extracted from these data. rotational forces were exerted on protein-g coated beads bound to igg antibodies immobilized on a surface. rotating fields revealed an oscillating behavior, which can be understood from the balance between the applied magnetic torque and the torque due to the deformation of the biological bond. studying interactions between cop regulatory protein and hy transcription factor d. s. dalafave the college of new jersey, ewing, nj , usa this work addresses important questions of protein-ligand interactions and selective protein recognition. proteinligand bindings are crucial for many cellular processes. dependable methods for predicting binding sites would lead to a better understanding of proteins' selective recognition and would, in turn, help research on fighting diseases. presented here is a study of interactions between the wd domain found in a regulatory protein cop and the motif v-p-e/d-Φ-g (Φ=hydrophobic residue) found in a transcription factor hy . cop and hy proteins have opposing roles in developmental regulation. cop can repress hy by directly binding with it. the repression involves specific interactions between the wd domain and the motif v-p-e/d-Φ-g. previous experimental research showed that mutations in the motif's v-p pair resulted in a large decrease in hy repression. to study effects of similar mutations, residues in the motif v-p-e/d-Φ-g were systematically substituted with other residues. interactions between the mutated motif and the wd domain were studied. distributions of binding sites, bond lengths, local shape complementarity, and interaction potentials were modeled for each residue substitution. the study identified binding sites critical for the cop -hy binding. some hy residues in the vicinity of the motif were also found to be important in the binding. the significance of the results for understanding selective protein recognition is discussed. determination of protein-ligand binding thermodynamics by thermal shift assay p. cimmperman, a. zubriene, l. baranauskiene, e. kazlauskas, j. matuliene, d. matulis laboratory of biothermodynamics and drug design, institute of biotechnology, vilnius, lithuania thermal shift assay determines the effect of ligand binding on the protein thermal denaturation equilibrium. several models have been derived to describe the general cases of protein-ligand binding. first, the model describing protein stabilization and destabilization by ligand binding to the native and unfolded states. second, the split of protein melting transitions by tightly binding ligands is presented when the transition of free protein and ligand-bound protein occur separately. mathematical equations describing the protein unfolding and ligand binding thermodynamic parameters at various temperatures are presented. the protein melting temperature shift can be determined by various techniques such as differential scanning calorimetry, circular dichroism, and intrinsic or extrinsic fluorescence. the advantages of fluorescent techniques are presented. the melting temperature shift caused by ligand binding is dependent on the thermodynamic parameters of protein unfolding and ligand binding, including enthalpy, entropy, and heat capacity, thus allowing determination of binding thermodynamics. application of the models in the design of hsp chaperone and carbonic anhydrase inhibitors is discussed. comparison with isothermal titration calorimetry data is presented. recent reports have shown that the bacterial redox protein azurin can enter into cancer cells and induce apoptosis by stabilizing p . the formation of a complex between the two proteins has been demonstrated, but little is known about binding features. for the first time, we show here that azurin binds to the n-terminal region of p with a dissociation constant in the - µm range. trp phosphorescence lifetime measurements revealed conformational changes of azurin induced by the interaction with p ( - ). acrylamide quenching of trp phosphorescence also indicated a significant increase of the overall flexibility of azurin upon binding to p . no change of the fluorescence emission of p ( - ) was detected in the presence of azurin. the latter finding suggests that w of p is not directly involved in domain binding to azurin, indicating that the binding site is distinct from that of mdm . the present results may assist the design of novel cancer treatments based on p stabilization by azurin. a. eleta , r. georgieva , h. bäumler , j. l. toca-herrera cic biomagune, donostia-san sebastián, spain, charité-universitätsmedizin berlin, berlin, germany human serum albumin (hsa) is the most abundant nonglycosilated plasma protein in the human body. this multifunctional protein has ligand-binding and transport properties, antioxidant functions and enzymatic activity [ ] . bilirubin interacts with albumin in order to be transported from the blood to the liver where it is secreted. the interaction occurs specifically in hsa i-domain of its three domains [ ] . in our work, we investigate the interaction between hsa and bilirubin by quartz crystal microbalance with dissipation (qcm-d) and atomic force microscopy (afm) [ , ] . albumin was adsorbed on negatively charged silicon oxide. however, hsa was removed after rinsing with pbs. hsa adsorbed on positively charged polyelectrolyte multilayers leads to a stable layer of surface mass density of ng/cm . cross linked albumin with glutaraldehyde after its adsorption on nh -terminated thiols is also stable reaching surface mass density of ng/cm . a preliminary bilirubin adsorption results on cross linked hsa substrate show that ng/cm is immobilized. taking into account that hsa-bilirubin stoichiometry is : , the outcome demonstrates that the % of hsa i-domains remain active. antimicrobial peptides (amps) are short positively charged polypeptides. they are important due to their potential to provide an alternative to conventional therapy against bacterial infections. rbpi is a kda peptide based on the nterminal region of the neutrophil bactericidal/permeabilityincreasing protein (bpi). it was shown that this amp possesses bactericidal effects on gram-negative bacteria and higher affinity for lipopolysaccharide (lps), neutralizing its effect. the peptide use against meningitis, is in phase iii clinical trials. here, we demonstrate that rbpi promotes aggregation of negatively charged large unilamellar vesicles (luv) and lps aggregates, by dynamic light scattering, while for zwitterionic phosphatidylcholine (popc) luv the size remains unchanged. the aggregation increases with peptide concentration until peptide promotes massive aggregation followed by sample flocculation/precipitation. with the rbpi -lipid interaction there is a progressive change in the zeta-potential of the luv systems and lps aggregates. luv systems composed of phosphatidylglycerol (popg) and popc:popg mixtures have higher zeta-potential variations than popc luv. for lps aggregates, rbpi neutralizes the surface charge and at higher peptide concentrations overcompensates it. results demonstrate that the interaction of the peptide rbpi with lps aggregates and luv systems has electrostatic and hydrophobic contributions. serratia marcescens heme acquisition system: heme transport and protein:protein interactions m. delepierre unité de rmn des biomolécules cnrs ura , institut pasteur, paris, france heme transport systems in bacteria are required and might be potential target for antibacterial drugs. the heme acquisition system, has, exists in pathogenic as well as in opportunistic bacteria but only for the latter one extensive studies have been conducted, constituting as such a model system. the outer membrane receptor hasr, the central component of this system, functions in synergy with a secreted high affinity heme binding protein, the hemophore hasa. hasa extracts heme from host hemoproteins and returns it to hasr. then, the energy given by a protein complex of the inner membrane is used to allow heme entrance across the bacterial membrane and to eject the empty hemophore from the receptor. reconstitution of this heme acquisition system in e coli, overexpression and purification of its various components have allowed us to obtain sufficient amount of protein to perform nmr and biophysical studies to analyse at the molecular level the different steps of heme acquisition by hasr. protein-protein interactions (ppi) are the central pillar supporting most of biological functional activity on the molecular level. a binding event between two proteins typically consists of two stages: ) diffusional search of the binding partners for each other, and ) specific recognition of the compatible binding surfaces followed by the formation of the complex. we focus here on the non-specific component of ppi, which refers to all physico-chemical properties of the binding partners (such as size, charge, isoelectric point, hydrophilicity etc.) that are independent of the exact details of their binding sites, but which could in turn affect their localization or diffusional search for one another. it is known that proteins co-localize due to segregation into different cellular compartments, sequestration via anchor and scaffold proteins or even chemical modifications. we suggest that the non-specific component of ppi determines in part the co-localization and clustering of the binding partners, which then directly in a non-specific fashion influences their interactions. we examine the possibility that such signature might be encoded within the experimental d structures of a large set of known mutually interacting proteins. we provide preliminary evidence that this indeed may be the case, and corroborate our findings by using different statistical tests to compare those features of the known interacting partners, and ascertain correlations and commonalities between them. a link between hinge-bending domain motions and the temperature dependence of catalysis in ipmdh i. hajdú, a. szilágyi, j. kardos, p. závodszky institute of enzymology, hung acad sci, budapest, hungary enzyme function depends on specific conformational motions. since conformational flexibility strongly depends on temperature, temperature dependent enzyme kinetic studies with measurements related to dynamics can give us some insight at atomic level into these functionally relevant motions. the catalytic efficiency (k cat /k m ) of -isopropylmalate dehydrogenase for its substrate (ipm) has unusual temperature dependence, showing a local minimum at • c. in search of an explanation, we measured the individual constants k cat and k m,ipm as a function of temperature, and found that the van't hoff plot of k m,ipm shows a sigmoid-like transition in the - • c temperature range. by means of various measurements including h-d exchange and fret, we showed that the conformational fluctuations, including hinge-bending domain motions increase more steeply with temperature above • c. the thermodynamic parameters of ligand binding determined by itc as a function of temperature were found to be strongly correlated to the conformational fluctuations of the enzyme. because the binding of ipm is associated with a hinge-bending domain closure, the more intense hinge-bending fluctuations at higher temperatures increasingly interfere with ipm binding, thereby abruptly increasing its dissociation constant and leading to the observed unusual temperature dependence of the catalytic efficiency. a simulation approach to multiple sclerosis: study of a peptide with a pharmaceutical potential c. guardiani , s. marsili , p. procacci , r. livi centro dinamiche complesse, università di firenze, italy, dipartimento di chimica, università di firenze, italy, dipartimento di fisica, università di firenze, italy multiple sclerosis (ms) is an autoimmune disease of the central nervous system, leading to premature death. one of the potential targets of the autoimmune reaction is the myelin protein mog that has been crystallized in complex with the - c ms-autoantibody. the analysis of contacts and buried surface area combined with an alanine scanning computation reveals the key role of mog fragment - for the interaction with - c . a docking simulation shows that the - fragment, excised from mog, and kept in crystal-like conformation, is still capable of fitting into the binding pocket of the antibody. we then studied, through replica exchange molecular dynamics simulations, the structural equilibrium distribution of the free peptide and of a number of analogs stabilized by a disulfide bond. we found that the free peptide yields a significant fraction of crystal-like conformations and the proportion of native-like structures is further increased by the disulfide bridge. when we tried to dock the centroids of the most populated clusters to - c , we discovered the existence of a docking funnel whose bottom is populated by stable complexes where the peptide occupies the same spatial region as in the crystal. we therefore conclude that the mog - fragment may be used to develop a diagnostic assay or a drug for ms. the escherichia coli membrane insertase yidc reversibly binds its substrate pf coat protein u. gerken, s. winterfeld, a. kuhn institute of microbiology and molecular biology, university of hohenheim, germany the membrane insertase yidc of e. coli belongs to the oxa family of mitochondria and plays an essential role in facilitating the insertion and assembly of membrane proteins. we have previously shown with detergent-solubilized (c pc) yidc, labelled with ans, and pf coat that the initial step of the membrane insertion process, the binding of the substrate pf coat to yidc, is reversible [biochemistry , - ( ) ]. the dissociation constant k d for that particular system is about µm. in order to obtain data for the native system we used in this study membrane-reconstituted (dopc and dope/dopg) yidc. the effect of the initial binding was examined in vitro by fluorescence quenching of the tryptophan (trp) residues of yidc which are highly sensitive fluorescent probes for changes of the tertiary structure. quenching of the trp fluorescence after titration with a trp-free pf mutant indicates a change in the yidcs tertiary structure upon binding to its substrate. the binding data show a k d value in the range of . - . µm. the influence of different environments (lipid membranes, ddm micelles) on the secondary structure of yidc as well as on the yidc large periplasmic domain p was investigated by circular dichroism (cd). the cd data show that the secondary structure of yidc changes upon reconstitution into a membranes when compared to the detergent solubilized state. particularly, the p domain is considerably affected by the detergent c pc. b. karasulu, b. erman, o. keskin koc university, istanbul, turkey histone proteins are fundamental to the cells since they are involved in cell regulatory processes, such as chromatin regulation, gene silencing and transcription, cell cycle control, and epigenetics, which are controlled via post-transcriptional modifications of the histone protein tails. these modifications are categorized under four main groups: methylation, acetylation, ubiquitination, and phosphorylation. among them methylation has been very recently proven to be reversible with the discovery of histone demethylase proteins and this modification type has been extensively studied, because the abnormal methylation rates cause the excess proliferation of the cell, which, in turn, triggers the cancer. therefore, understanding of the details of reaction mechanisms of histone methylation/demethylation dynamics provide the required knowledge basis for preventing the abnormal methylation rates by designing proper inhibitor (drug) molecules. in this study, we display possible reaction mechanisms (such as amine oxidation via lsd ) for the demethylation of specific histone tail proteins. we try to explain the role/importance of residues that take place in or near to the reaction pocket for the demethylation reaction. we also carry out md simulations and reaction path (free energy profile) analysis using free energy perturbation (fep) method for qm/mm hybrid systems in order to compare different possible reaction pathways. the sonic hedgehog (shh) signalling pathway plays an important role both in embryonic development and in adult stem cell function. inappropriate regulation of this pathway is often due to dysfunction between two membrane receptors patched (ptc) and smoothened (smo) , which lead to birth defects, cancer or neurodegenerative diseases. however, little is known about ptc, the receptor of the shh protein, and the way ptc regulates smo, the receptor responsible for the transduction of the signal. to develop structure-function studies of these receptors, we expressed human ptc (hptc) in the yeast saccharomyces cerevisiae. we demonstrated that hptc expressed in a yeast membrane fraction is able to interact with its purified ligand shh, indicating that hptc is produced in yeast in its native conformational state. using surface plasmon resonance technology, we showed that fluorinated surfactants preserve the ability of hptc to interact with its ligand after purification. this is the first report on the heterologous expression and the purification of a native and stable conformation of the human receptor ptc. this work will allow the scale-up of hptc production enabling its biochemical characterization, allowing the development of new therapeutic approaches against diseases induced by shh signalling dysfunction. dissecting the colicin translocon c. l. johnson , a. solovyova , p. callow , s. a. holt , l. a. clifton , k. weiss , j. h. lakey inst. for cell and molecular biosciences, newcastle univ., uk, inst. laue langevin, grenoble, france, isis, rutherford appleton lab., didcot, uk, oak ridge national lab., centre for structural molecular biology, oak ridge, usa pore-forming colicin n hijacks e. coli outer membrane protein ompf and exploits it as both a receptor and translocator to cross the outer membrane [ ] . it is currently a matter of debate if the translocation route is through the ompf lumen or the interface between ompf and the lipid bilayer. recent electron microscopy data from our laboratory suggests the latter route for translocation [ ] . the colicin n/ompf complex in detergent has been studied by sans to examine the translocation pathway undertaken by colicin n. by using a combination of deuterated ompf and hydrogenated colicin we have been able to derive a low resolution structure of individual proteins in the binary complex. low resolution structural studies supplemented by targeted mutagenesis and screening techniques including itc, potassium efflux assays and auc have allowed us further our understanding of colicin n translocation. dual-color fluorescence cross correlation spectroscopy (fccs) has been used to explore the molecular dynamics at immune cell surfaces, with a particular focus towards the regulation mechanisms of natural killer (nk) lymphocytes. nk cells are critical mediators of anti-viral immunity and protectors against cancer spread. their activity is governed by a fine-tuned balance between inhibitory and activating receptors, where ly a and kir receptors represents the inhibitory ones. their ligands are mhc class i receptors. fcs is a technique based on the analysis of intensity fluctuations of fluorescent molecules excited by a focused laser beam. the technique offers information about molecular dynamics at the single molecular level, in the nanosecond to millisecond range. dual color fccs expands fcs by correlating the intensity from two different colors. by labeling two potential interaction partners with dyes emitting at different wavelengths, the amount of interaction can be determined. here, we will report on recent fccs data exploring the interaction between the inhibitory receptors and their ligands, as well as different labeling strategies used to enable these measurements. effect of osmolytes on the dhfr activity, structure and dynamics b. legrand , s. renaud , m. collen , c. tascon , s. bonnassie , e. gautier , j. mellet , c. blanco , e. le rumeur , j.-f. hubert , a. bondon rmn-ilp, duals, cbp, umr cnrs , univ. de rennes , france osmolytes are small molecules accumulated by a wide variety of organisms in response to hyperosmotic stress. they contribute to save the cellular integrity and to stabilize the macromolecules from environmental stress. dihydrofolate reductase activity is inhibited by several osmolytes. we studied the impact of osmolytes on the dhfr structure and dynamics by various techniques. we observe that substrate (dhf) and cofactor (nadph) diffusions are quite different in glycerol and betaine despite similar viscosities. we demonstrate that the overall structure is maintained at high osmolyte concentrations while no direct interactions can be detected with the enzyme. the k o f f of substrate analogues decreases with increasing the osmolyte concentrations. the enzyme dynamics, in various media, has been compared with the dhfr behaviour in water described in the literature. the osmolyte impact appears only partly conditioned by its viscogenic properties which reduce the molecules diffusion and the k o f f of the product controlled by the m loop of the dhfr. we suggest that the osmolytes decrease m loop mobility. comparing the results obtained with different osmolytes, we offer a better understanding of the osmolyte nature dependence of the dhfr inhibition. estrogen receptor (er) is a well characterized member of the nuclear receptor superfamily that modulates the expression of estrogen-responsive target genes in response to estradiol and other natural and synthetic chemicals mimicking the estradiol structure. in human, two ers, erα and erβ, lied on two distinct chromosomes, are known. er exhibits several functional domains: two conserved domains, a short domain c involved in dna-binding and a large domain e/f responsible for ligand-binding and hormone-dependent transcription activation, are linked by a hinge domain d; a poorly conserved a/b domain, at the n terminus, mediating interactions with the general transcription machinery, is involved in hormone-independent transcription activation. upon estrogen binding, ers can specifically bind to a dna fragment, called estrogen response element ere, and activate the transcription. the optimal ere sequence consists of two six base-pair half-sites, aggtca, organized as inverted repeats with a three base-pair spacing. in this study, we have investigated, by fluorescence methods, the effect of kcl concentrations, on the protein conformational flexibility and the thermodynamic stability of hers -eres complexes. we show, here, that electrostatic interactions, inside hers, contribute to its conformational flexibility and its thermal stability. moreover, the specific interaction between hers and eres is poorly sensitive to changes in ionic strength, in opposite to unspecific complexes. kinetic and structural explanation for the low enantioselectivity of human -phosphoglycerate kinase p. lallemand , j. rouhana , l. chaloin , b. roy , s. arold , t. barman , c. lionne cpbs umr , bd henri iv cs , montpellier cedex , france, ibmm umr , cbs umr l-nucleosides comprise a new class of antiviral and anticancer agents that are converted to pharmacologically active nucleoside triphosphates in vivo. the last step of the cascade may be catalyzed by -phosphoglycerate kinase (pgk), an enzyme that has low specificity for nucleoside diphosphate: ndp + , -bisphosphoglycerate ↔ ntp + -phosphoglycerate. here we compare the kinetics of formation of the complexes of human pgk with different d-and their mirror images, l-nucleoside diphosphates, and the effect of -phosphoglycerate thereon. two types of experiment were carried out: equilibrium experiments allow the estimation of dissociation constants, and stopped-flow experiments the transient kinetics of the interactions. in addition, by the rapid-quench-flow technique, we compare the kinetics of the phospho-transfer and product release steps with each d-or l-nucleotide. crystallographic and molecular modelling studies allow defining the structural reasons for the low enantioselectivity of pgk. the aim of this basic work on the mechanism of action of human pgk with non-natural nucleotides is to obtain information for the optimization of therapeutic nucleoside analogues that are poorly phosphorylated by pgk. anrs is gratefully acknowledged for financial support. a new itc assay for measuring high-and lowaffinity protein-ligand interactions g. krainer , j. fanghänel , s. keller leibniz institute of molecular pharmacology (fmp), berlin, germany, bayer schering pharma ag, berlin, germany isothermal titration calorimetry (itc) is the gold standard for the quantitative characterisation of protein-ligand and protein-protein interactions [ ] . however, reliable determination of the dissociation constant (kd) is typically limited to the range µm > kd > nm. nevertheless, interactions characterised by a higher or lower kd can be assessed indirectly, provided that a suitable competitive ligand is available whose kd falls within the directly accessible window [ ] . unfortunately, the established competitive itc assay requires that the high-affinity ligand be soluble at high concentrations in aqueous buffer containing only minimal amounts of organic solvent. this poses serious problems when studying protein binding of small-molecule ligands taken from compound libraries dissolved in organic solvents, as is usually the case during screening or drug development. here we introduce a new itc competition assay that overcomes this limitation, thus allowing for a precise thermodynamic description of high-and low-affinity protein-ligand interactions involving poorly water-soluble compounds. we discuss the theoretical background of the approach and demonstrate some practical applications using examples of both high-affinity (kd < nm) and low-affinity (kd > µm) protein-ligand interactions. a molecular dynamics study of the cftr nucleotide binding domains interaction v. martorana , r. noto , o. moran cnr-istituto di biofisica, palermo, italy, cnr-istituto di biofisica, genova, italy the cystic fibrosis transmembrane conductance regulator (cftr), the dysfunctional protein in cystic fibrosis, contains two transmembrane domains, two nucleotide-binding domains (nbds), and a regulatory domain. opening of the pore have been linked to the atp-driven tight dimerisation of nbd. we have studied the nbd -nbd interactions on wild type and cystic fibrosis-related mutations by steered molecular dynamics simulations (smd). a fully solvated dimer, including the two bound atps, was separated by pulling one monomer with an external, increasing force. interestingly, the force needed to break the mutated (g d) dimer is significantly smaller than in the wild type case. the effect of a cftr potentiator, the genistein, has also been tested by repeating the smd simulations with the small molecule docked at the interface between the two nbd domains. to test the validity of our results we have repeated the separation process for different simulation lengths and force strengths. the amount of distortion on the pulled nbd domain has also been studied. this work is partially supported by the italian cystic fibrosis foundation (prog ffc # / ), with the contribution of "mille bambini a via margutta"onlus"blunotte", "lega italiana fc toscana" stability and aggregation studies of human septin (sept ) j. n. a. macêdo, r. c. garratt, a. p. u. araújo instituto de física de são carlos, usp, são carlos, brazil the septins are a conserved family of nucleotide binding proteins firstly identified in saccharomyces cerevisae as proteins required for the completion of the cell cycle. septins are implicated in several cellular functions such as cytokinesis, vesicle trafficking, exocytosis, cytoeskeletal dynamics, cell polarity and sperm motility. furthermore, they are associated with alzheimer's and parkinson's disease. sept was identified in rat brain being highly enriched in presynaptic nerve terminals. it colocalizes with synaptophysin and dynamin i and is associated with synaptic vesicle. in this work, human sept without its n-terminal domain (sept gc) was expressed in e. coli and purified by affinity and size exclusion cromatographies. structural stability studies were performed with recombinant sept gc using circular dichroism spectroscopy (cd), right-angle light scattering, and fluorescence spectroscopy. the sept gc cd spectrum showed a conformational transition from a predominantly α-helical starting structure to one dominated by β-sheet, just above physiological temperatures. the formation of irreversible aggregates, detected by light scattering, and their ability to bind thioflavin-t suggested that sept forms amyloidlike structures, as has been previously observed for human septins and . our data suggest that amyloid formation by isolated septins in vitro may be a general phenomenon whose physiological relevance needs to be further investigated. pln is an antimicrobial peptide produced from lactobacillus plantarum nric . analog pln (pln a) and a ser-derived (pln s, tyr to ser replacement) were synthesized on solid phase and their interactions with biomembrane model systems and inhibitory property on s.aureus and p.aeruginosa were investigated by cd, leakage assays, fluorescence spectroscopy, and differential scanning calorimetry. both peptides share the same unordered structure-like cd spectrum in aqueous solution, but a helicoidal induction in the presence of negative vesicles were observed, however pln s showed lower helical content than pln a. the ser-derived peptide presented % decrease of its leakage activity in different liposome compositions, a threefold increase in the dissociation constant from the liposomes than pln a, and close to % reduction for the inhibitory activity against p. aeruginosa growth. we can conclude that besides the electrostatic contacts between the amphipathic &alpha-helix, formed by the cationic residues from pln and negatively charged phospholipids, the pln -membrane binding is a process driven for the hydrophobic interactions from non-polar residues. in this case, there was a significant contribution of the tyr residue, which must be allocated in a lipid interface, described as the preferential position to this residue in membrane proteins. supported: fapesp label free detection using deep-uv laser-based fluorescence lifetime imaging microscopy q. li, s. seeger physikalisch-chemisches institut, universität zürich, winterthurerstrasse , ch- zürich, switzerland label free detection based on native fluorescence excited at uv region shows great potential for the life sciences. it offers simple, low-cost and fast method for sensitive detection of important biological analytes without modification. in this contribution we present a deep uv fluorescence lifetime imaging microscopy system (duv-flim) based on a picosecond deep uv laser using time-correlated single-photon counting method. the described setup is well-suited for biological applications for ultrasensitive detection. we have showed single uv dye (bmq) and single protein (ecβ gal ) molecules detection using duv-flim. further, the label free detection of protein interaction between ecβ gal and monoclonal anti-ecβ gal has been demonstrated by means of steady-state and time-resolved fluorescence spectroscopy. we achieved detection sensitivity for the ecβ gal/ anti-ecβ gal pair down to the nanomolar concentration range. we also extended this method to study the interaction of therapeutic drug porphyrin with bsa protein. fluorescence resonance energy transfer between protein and alexa fluor has been investigated using duv-flim. the intrinsic fluorescence and fluorescence lifetime changes of donor biotin β-galactosidase have been measured. energy transfer efficiency and donor acceptor distance have been obtained. fluorescence images of acceptor af due to fret have been observed when excited at nm. the monomeric heme-containing indoleamine , dioxygenase (ido) catalyses the oxidative cleavage of the indole moiety of l-tryptophan (l-trp). enhanced l-trp degradation contributes to various physiological disorders including depression or failure of the immuno-regulating system. the regulation of ido by inhibitors is extensively studied. however, the catalytic mechanism of ido on the molecular level is still unknown. in addition to l-trp, a wide range of substrates are degraded including d-tryptophan, melatonine or tryptamine. in contrast, indole or histidine do not function as substrates for ido. to understand the determinants for substrate specificity, we investigate the interaction of the heme iron, the heme-bound ligand and the substrate. we use (time-resolved) uv/visible and fourier transform infrared (ftir) spectroscopy over a wide temperature range ( - k) to monitor the binding of diatomic ligands to the heme iron and the binding of different substrates to coligated ido. changes in the spectra upon addition of l-trp are analyzed and compared to those induced by other substrates or inhibitors. archaeal protoglobin d-structure: novel ligand diffusion paths and heme-reactivity modulation protoglobin (pgb) from methanosarcina acetivorans c a, a strictly anaerobic methanogenic archaea, is the latest entry in the hemoglobin superfamily. our previous crystallographic studies on pgb have shown that protoglobinspecific loops and a n-terminal extension completely bury the heme within the protein matrix ( ) . access of o , co, and no to the heme is granted by protoglobin-specific apolar tunnels reaching the heme distal site from locations at the b/g and b/e helix interfaces. here we report structural and kinetic data on pgb mutants engineered to probe the protein structural and kinetic properties. six crystal structures (pgb mutants: ∆ (missing nter residues), y(b ) →a, y(b ) →w, f(b ) →w, f(g ) →w, i(g ) →f) show that the mutations engineered essentially restrict access to ligand tunnel . an accurate molecular level description of the signaling mechanism in ca + transducers necessitates the knowledge of the kinetics and energetics of conformational changes associated with ca + binding to various calcium binding proteins. with this in mind, we have developed an approach that combines the laser-induced photolysis of photolabile "caged" ca + compound, dm-nitrophen, with the photothermal beam deflection (pbd) technique to determine thermodynamic profiles associated with the ligand binding to calcium chelator, edta, and ca + sensor, calmodulin. this approach allows us to monitor time profiles of volume and enthalpy changes on the microsecond to millisecond timescale. the initial pbd study of ca + photo-relase from ca + loaded dm-nitrophen reveals the presence of two phases. the first step takes place within first µs upon and is associated with a volume decrease of - ml mol − and enthalpy change of kcal mol − . on the longer timescale (τ = µs), the second event with a positive volume change of ml mol − and enthalpy change of kcal mol − was detected. on the other hand, ca + photorelease in the presence of calmodulin is accompanied with an additional phase with a distinct lifetime and volume and enthalpy changes that reflects the metal binding to calmodulin and concomitant structural changes. antimicrobial peptides: linking partition, activity and high membrane-bound concentrations antimicrobial peptides (amps) have been intensively studied at micro and macroscopic levels for over twenty years. knowledge from these two domains has, however, contributed little to a comprehensive understanding of amp action; rather, in vivo amp performance has been only remotely correlated to biophysical properties. we focus on the assessment of peptide accumulation on bacterial membranes as an example of this separation: amp-bilayer interactions have been subject to extensive biophysical characterization, but conversion of that information into educated estimates of in vivo membrane-bound amp concentrations is lacking. this has led to the overlooking of important factors for activity. using simple partition models we were able to analyze available information on amp activity and interaction with membranes to show that unexpectedly high membranebound peptide concentrations are likely in vivo and may, in some cases, be required for triggering bacterial death. e. e. schäfer , s. schuy , a. janshoff georg august-university göttingen, germany, johannes-gutenberg-university mainz, germany retrovirus entry into cells occurs through fusion of the lipid bilayers that surround the virus and the lipid bilayer of the host cell. fusion proteins, present on the surface of the virus membrane play an essential role in the early stage of virus entry. understanding of the molecular mechanism is important for the design and function of modern fusion inhibitors. in this project we analyze the fusion of active conformation of the envelope gp from the human and simian immunodeficiency viruses (hiv and siv). during the infection process gp undergoes a sequence of conformational changes where the n -terminal nhr develop a trimer pre-hairpin intermediate. afterwards the three chr fold back towards the central nhr and a six helix bundle is formed. this rearrangement forces the viral and the host membrane into close contact and fusion pores may induce membrane fusion. this decisive molecular step in retroviral fusion has been modeled by rational design of lipopeptide assemblies that mimic a coiled-coil structure serving as a receptor for potential antagonists. for this purpose, sslbs were functionalized in an in situ coupling reaction with peptides originating from the nhr (n-peptides) of siv and hiv to monitor the interactions with the specific chr peptides (c and t ). binding of antagonists to surface confined coiled-coil structures has been quantified by ellipsometry, quartz crystal microbalance and was visualized by atomic force microscopy. a. rupprecht , e. sokolenko , e. e. pohl institute of cell biology and neurobiology, charité -universitätsmedizin, berlin, germany, frumkin institute of physical chemistry and electrochemistry russian academy of sciences, moscow, russia the production of reactive oxygen species (ros) in mitochondria is very sensitive to the proton motive force and can be decreased by mild uncoupling, mediated e.g. by uncoupling proteins (ucp) . in contrast, the activation of uncoupling proteins by ros as a negative feedback loop is a highly controversial hypothesis. ucp activation in mitochondria by -hydroxy- -nonenal (hne, aldehydic product of lipid peroxidation) was first demonstrated by echtay et al. . here we investigate the ability of hne to activate and/or to regulate the expression of ucp in two different systems: (i) in lipid membranes reconstituted with recombinant ucp and (ii) in primary neuronal cells. total bilayer conductance was enhanced in the presence of hne, but this effect was independent on ucp and ucp . the results concerning the hne-mediated ucp expression after induction of ros-production and/or after exogenous addition of hne are discussed for three brain-associated proteins (ucp , ucp , ucp ) in view of their possible functions. . beck, v et al. . faseb j. : - . . echtay, k. s. et al. . t. rudack, j. schlitter, k. gerwert ruhr university, department of biophysics, nd north, bochum, germany the gtpase ras p which is linked to the membrane via a lipid anchor, is a crucial switch in the cellular signal transduction processes that control cell growth and proliferation. docking simulations can be seriously hampered by the great difficulty to accurately estimate the ligand-protein binding affinity constant k, which is usually derived via the computation of the binding free energy ∆g. unfortunately, due to the involved logarithmic relationship, errors of less than . kcal/mol in the computation of ∆g result in about one order of magnitude inaccuracy on k. this can hinder computational methods from discriminating micromolar from nanomolar compounds. to improve the reliability of docking prediction, we make use of enhanced sampling methods, ranging from steered molecular dynamics to metadynamics . we also test several descriptors, such as the recently developed path collective variables , to identify the most suitable reaction coordinates accounting for binding and unbinding processes. using these approaches we investigate ligand docking and undocking and we attempt to describe at an atomistic level the kinetics of binding, which we intend to exploit for drug design purposes. here, an example of application in drug design is reported. . isralewitz, b. et al.; j. mol. graph. model. , , - . . laio, a. and parrinello, m.; pnas , , - . . branduardi, d.et al.; j. chem. phys. , , . prediction of protein-protein complex structures using wang-landau simulations a. solernou, barcelona supercomputing center, jordi girona , barcelona, spain protein-protein interactions are essential in the majority of life processes, so they have keen interest in several knowledge areas. however, experimental data on protein complexes is being produced at a quite low pace in comparison to that of the individual components. thus, in recent years attention has focused into computational approaches to the proteinprotein docking problem. a variety of docking protocols have been recently reported, sharing usually the following strategy: fast rigid-body search of the interacting subunits, followed by scoring and refinement of the interfaces. although this kind of strategy has proven some good results in the capri blind test it has two main limitations. on one hand one should include full flexibility on the protein structures. on the other, the evaluation should be made with free energy calculations instead of using a scoring function. in this work we propose to search the native complex structure in the minimum of the free energy landscape. we use the coarse grained potential unres to get the potential energy of the possible conformations. they are generated changing the dihedral angles, the side chain rotamers, and lastly the mutual orientation using a new sampling protocol we have developed (rotation-based uniform sampling; rotbus). finally, the free energy calculations are performed using an omp parallelization of the wang-landau algorithm. s. shukla , s. asthana , g. giliberti , f. luliano , m. ceccarelli , r. loddu , p. ruggerone , p. la colla department of biomedical science and technology, università di cagliari, cagliari, italy, department of physics, università di cagliari, cagliari, italy the virus encoded rdrp has emerged as a prime target in the search for specific hcv and other flaviviridae antivirals. recently, the determination of the hcv rdrp structure in complex with certain benzimidazoles has been reported, these nni's bind to the surface of the thumb domain, thereby disrupting its interaction with the finger domain, which is necessary for catalytic activity. on the other hand, we have found that, in bvdv, the mutations conferring resistance to same class of inhibitors lie in the finger domain of rdrp, indicating that the mode of inhibition of benzimidazole class of compounds is different in both hcv and bvdv rdrp. herein, we have applied docking approaches (binding orientation), molecular dynamics (md) simulations combined with metadynamics to elucidate the microscopic mechanism of the interactions between the ligand and the receptor in order to identify features barely seen in experiment. the recently designed algorithm overcomes the time scale problem by accelerating properly defined reaction coordinates. it mimics the real dynamics of a ligand staying or leaving the receptor and in doing so reconstructs the free energy surface, which in turn gives an idea of the residence time of the inhibitor in the cavity.finally we identified the binding modes and different mechanism of inhibition of benzimidazole class of compounds in rdrp of two closely related rna viruses. . c. seifert , w. stacklies , f. graeter protein mechanics and evolution, bioquant, inf bq , heidelberg, germany, ag graeter, picb, yueyang lu, shanghai , china we use a new method that detects force distribution in proteins. based on molecular dynamic simulations, changes in inter-atomic forces are calculated, here caused by different ligands. these changes will then be analyzed to detect a signal transfer through a protein initiated by the binding of a ligand to the protein. chaperones are ubiquitous proteins, which help other proteins to fold into a native conformation. they are able to refold misfolded proteins with a great variety of mechanisms. in this project, our group focuses on molecular dynamic simulations of htpg, an e. coli homolog of the human hsp (heat shock protein kda). the full structure of htpg was published by shiau et al. in , but the mechanism of how htpg performs its function is still not understood. the folding mechanism is an atp driven reaction cycle, in which the functional entity is a homodimer of htpg. we separate the cycle in three main states: apo, adp-and atpbound state. conventional molecular dynamics simulations are used to build a stable simulation system and provide structure trajectories and primary information about the behavior of htpg in its different states of the folding process. experiments indicate that the molecular movement is atp driven. we use force distribution analysis to elucidate how atp effects htpg conformation and dynamics. the small size of myoglobin makes it the preferred candidate to investigate the structure-function paradigm. in its interior five docking sites have been identified and for long time these xenon cavities have been classified as packing defects. recently, it was shown that they might be involved in ligands migration path. however, some questions regarding its role as oxygen carrier no scavenger remain yet open as well as the microscopic mechanism regulating these biological functions. in this work we made use of standard md simulations of solvated myoglobin to characterize internal cavities. our principal results is that we have found several secondary cavities showing volume and occurrence at least comparable to that of xenon cavities. in order to analyze and rationalize internal cavities we applied special cluster-analysis: we classified all cavities with respect to the position, size and occurrence ascribing them to different clusters. this analysis highlights possible intrinsic migration paths for small ligands within the protein matrix controlled by spontaneous fluctuations of the protein itself. moreover, we identified some key residues playing a fundamental role in controlling internal pathways. our suggestion that the secondary cavities constitute the preferred path for ligand escape is also supported by explicit metadynamics simulations of ligand escape. a. varga , p. lallemand , j. szabó , p. závodszky , m. vas , t. barman , l. chaloin , c. lionne institute of enzymology, brc, hungarian academy of sciences, budapest, hungary, cnrs-université montpellier -université montpellier , institut de biologie, umr , montpellier, france -phosphoglycerate kinase (pgk) is a promising candidate for the activation of nucleotide analogues used in antiviral and anticancer therapies. pgk is a key enzyme in glycolysis; it catalyses the reversible reaction , -bisphosphoglycerate + adp ↔ -phosphoglycerate + atp. here we explored the catalytic role in human pgk of the highly conserved lys that has been proposed to be essential for pgk function, by a transient and equilibrium kinetic study with the active site mutant k a. by the stopped-flow method we show that the kinetics of substrate binding and the associated protein isomerization steps are fast and identical for the wild-type pgk and mutant k a. by the use of a chemical sampling method (rapid-quench-flow) under multi and single turnover conditions and in both directions of the reaction, we show that the rate-limiting step with wild type pgk follows product formation, whereas with the mutant it is the phospho-transfer step itself that is rate limiting. these data are supported by saxs measurements which showed no direct role of lys in the domain closure of the enzyme i.e. the isomerisation step of the reaction. the results are explained by the structural data of the enzyme. characterization of different recombinant nrp proteins and interactions with heparin k. a. uniewicz, y. ahmed, d. g. fernig school of biological sciences and centre for glycobiology, biosciences building university of liverpool, l zb liverpool, u.k. neuropilin- (nrp ) is a mammalian membrane glycoprotein involved in tip sprouting processes like angiogenesis and neurogenesis. it has been shown that the interaction of nrp with heparin/heparan sulfate is implicated in its enhancement of growth factor signalling, however the mechanism is not yet known. commercially available extracellular domain of nrp is available either as a truncated his-tagged human protein (hnrp ) or as the rat protein fused to histagged human fc and expressed as a dimer (fcrnrp ). biochemical properties such as kinetics of heparin binding and structural requirements for sugar binding together with biochemical tests of protein properties were performed in order to characterise these commercial proteins. fc rnrp shown high affinity to heparin (kd= . nm) and required a minimum of dp to effectively compete with fc rnrp binding to immobilised heparin. competition experiments with various modified sugars show that interaction of fc rnrp with heparin is highly ionic and dependent on the position of sulfate groups along the heparin chain. the hnrp did not bind to heparin immobilised via nhs-biotin, though it did bind to some heparin affinity chromatography matrices. organic compounds of tin are among wide spread environmental pollutants. due to physical and/or chemical actions, poly-substituted alkyltins speciate into less substituted, more toxic species. tetra-or tri-alkyltins show a marked delay in their toxic action with respect to the corresponding di-and mono-alkylated analogs. it has been hypothesized that the delayed toxicity may results from the progressive dealkylation of alkyltins in more toxic di-and mono-derivatives, which bind and inhibit essential enzymes. it has been proposed that alkyltins preferentially target enzyme sulphydryl groups. previously, we showed that a nine amino acid linear peptide (i lgcwcylr ) containing a cxc motif is able to bind and dealkylate tri-substituted alkyltin compounds into the corresponding dialkyl derivatives. here, we investigated the time dependence of the degradation of the most common alkyltin derivatives by this peptide. we monitored the reaction kinetics using the intrinsic fluorescence of the tryptophan residue in position of the peptide. we found that all of the alkyltins analyzed are progressively degraded to dialkyl derivatives, following a pseudo-enzymatic reaction mechanism. the end-point of the reactions was the formation of a covalent complex between the disubstituted alkyltin and the peptide. these data agree with the speciation profiles proposed for polysubstituted alkyltins in the environment and reveal a possible biotic degradation pathway for these toxic compounds. parkinson's disease (pd) is a multifactorial neurodegenerative condition characterized by the progressive loss of dopaminergic neurons in the substantia nigra and by the presence of intracellular inclusions, composed predominantly of fibrillar alpha-synuclein (as). post-mortem studies indicate the presence of oxidative damage in the nigral neurons. dopamine oxidation, which leads to the formation of highly reactive quinones (daq), may account for the specificity neurodegeneration observed in pd. daq have many potential protein targets for chemical modifications. among them, we focused our attention on dj- , of which mutated forms have been found in familial cases of pd. a possible function of dj- is its redox-dependent chaperone activity that could prevent as aggregation and fibril formation. in the present work, we analyzed the structural and functional modification induced on dj- by daq. n-hsqc spectra of dj- were recorded in the presence of different amounts of daq and chemical shift perturbations were used to identify the dj- residues target of daq and their relative reactivity toward daq. aggregation assays were also performed to evaluate the functional effects of the daq modifications on the chaperone activity of dj- . rabies virus (rabv) infects neurons exclusively and causes lethal encephalitis. pathogenic rabv strains favor neuronal survival, whereas non-pathogen strains lead to neuronal apoptosis. the use of recombinant rabv showed: / the g protein determined the induction of the survival or death phenotypes; / the last cooh amino acids of the g protein cytoplasmic domain (cytog) are critical. these residues form a binding site for pdz domain (pdz-bs). one of the amino acid differences between survival and death gproteins are located in this pdz-bs. results of two-hybrid experiments showed that cytog survival interacted pdz domain of ser/thr kinases (mast), while cytog death interacted also with additional pdz containing host proteins. to understand the fine structural basis for the specificity of the pdz-cytog complexes, we determined the d structure and the dynamics of the mast-cytog complexes by nmr. the structures, as well as the affinities and constant kinetics, of the mast pdz complexes with both cytog are similar. we conclude that difference by one aa in the pdz-bs of the two strains cannot drastically modify the interaction with mast -pdz, in agreement with the two-hybrid data. preliminary results suggest that the interaction of cytog death with one additional cellular partner blurs the pro-survival signals engaging the infected cells through apoptotic trails. functional protein immobilization on glass-type surfaces s. waichman, m. bhagawati, y. podoplelova, j. piehler institute of biology, department of biophysics,university of osnabrück , barbara st. osnabrück, germany the immobilization of membrane proteins onto solid supports enables protein interactions and conformational changes to be probed by spectroscopic techniques under highly defined conditions. here, we present a novel method for covalent protein immobilization on glass-type surfaces using a bottom-up approach. in this approach the 'phosphopantetheinyl group of coenzyme a (coa) was transferred to the acyl carrier protein-derived ybbr tag of the target protein by means of the phosphopantetheinyl transferase sfp. the glass-support was rendered biocompatible by coating it with an ultrathin layer of peg (polyethylene glycol), followed by functionalization with coa through maleimide chemistry. immobilization of ybbr-tagged proteins in presence of sfp was followed in real time by label-free detection using reflectance interference spectroscopy. the immobilization procedure was thus systematically optimized by means of binding specificity, enzyme activity and functionality of the immobilized protein. this approach was employed for immobilizing the type i interferon ifnα in order to probe ligand recognition by ifnar and ifnar and ligand-induced ternary complex formation. the versatility of this technique was further enhanced by its combination with photopatterning methods. this immobilization technique can provide a beneficial tool for bioanalytical and biophysical applications at the single molecule level. r. vijayan, p. c. biggin department of biochemistry, university of oxford, south parks road, oxford, ox qu, uk ionotropic glutamate receptors mediate excitatory synaptic transmission in the brain and are heavily implicated in memory and learning as well as in numerous neuropathological conditions. one family of iglurs, the kainate receptors, show unusual sensitivity to changes in external ion species resulting in an apparent requirement for both sodium and chloride ions for activation. our recent work revealed the location and selectivity of the cation binding sites. despite this progress, it is still unclear how the cation binding sites confer sodium selectivity and how apparent affinity for chloride is influenced by the presence of cations. we have attempted to address these questions by performing extensive free energy calculations using all-atom molecular dynamics simulations. the rank order of cation binding obtained from relative binding free energy calculations is in agreement with experimental measurements of apparent affinity. these calculations also reveal that the pair of cation binding sites in the dimer interface act independently. binding free energy calculations performed using a reduced model of the binding site show that cation selectivity can been attributed to both the rigidity and high charge density of the binding sites. finally, a potential of mean force derived from umbrella sampling simulations indicate that the presence of cations stabilize the anion binding site considerably. the effect of toxins on the inorganic phosphate release during the actin filament formation a. vig, t. kupi, g. hild, m. nyitrai university of pécs, faculty of medicine, department of biophysics, szigeti str. , pécs, hungary actin can be found in monomeric (g-actin) and filamentous (f-actin) form in eukaryote cells under physiological circumstances. the first step of actin polymerisation is the formation of actin nuclei by atp-binding actin monomers. the next step in is the elongation when monomers are associated to the previously formed nuclei or to the ends of the growing filaments. after the association of monomers the bound atp is hydrolysed to adp.p i , and with first order kinetics the release of inorganic phosphate occures. the rate constant of the release is , s − , which is a slower process than the hydrolysis itself ( , s − ).phalloidin, a cyclic peptide from amanita phalloides can bind to the filaments and stabilizes their structure. jasplakinolide is another cyclic peptide from marine sponge (jaspis johnstoni) which binds actin filaments. the aim of this study was to investigate whether the binding of toxins to the newly created filaments has an effect on the kinetics of the inorganic phosphate release or not. we used absorption photometry measurements to measure the rate of phosphate release. phalloidin decreased the rate of the release substantially. although the effect of jasplakinolide was weaker, the results showed that the binding of these toxins to the actin can modify the rate of the release of inorganic phosphate from the filaments. these observations are in agreement with the molecular mechanisms by which these toxins stabilise the actin filamens. fluorescent proteins (fps) are invaluable fluorescent markers in cell biology. however, their use is often limited by photobleaching of the chromophore, notably in single-molecule, time-resolved or super-resolution imaging studies. we will present the crystallographic studies at near atomic resolution of a photo-activatable fluorescent protein irisfp that has been observed in a transient radical state en route to photobleaching. we took advantage of x-rays to populate the radical, which, under illumination with visible light, presumably forms with low probability from the triplet state. the combined x-ray diffraction and in crystallo spectroscopic data (from uv-vis and raman spectroscopies) reveal that radical formation in irisfp involves strong but reversible distortion of the chromophore, suggesting a transient loss of pi-conjugation. these results will help unravel the mechanisms of blinking and photobleaching in fps, which is of importance to rationally design variants of higher photostability. optimizing photoactivatable fluorescent proteins for live-cell imaging recently, novel fluorescent proteins (fps) have been reported which perform spectral changes in response to irradiation with light of a particular wavelength. reversibly photoactivatable proteins switch between a bright and a dim fluorescent state. this process is accompanied by photoisomerization of the protein chromophore. irreversible photoactivation results from photochemical processes within the protein, e.g., photolysis of an amino acid side chain, or an extension of the alternating π-electron system of the chromophore by a β-elimination reaction. fps have became valuable tools in live-cell imaging because they allow intracellular protein labeling by using them as fusion tag, and photoactivatable fps are powerful tools for application in novel subdiffraction imaging techniques. however, the available fps still offer potential for improvement in various ways. they frequently show a tendency to aggregate or oligomerize, incomplete chromophore maturation, fluctuating emission and low photostability. here, we will present our recent progress towards engineering the 'perfect' fp. simultaneous intracellular chloride and ph measurements using gfp-based sensor in ldcv d. arosio , f. ricci , l. marchetti , l. albertazzi , f. beltram italian institute of technology, udr pisa, italy, nest, scuola normale superiore, pisa, italy, nest, infm cnr, pisa, italy chloride ion participates in many physiological functions including control of neuronal resting potential, charge balance during endosome acidification, and regulation of cell volume. as a consequence dysfunctions in regulating membrane chloride permeability lead to severe diseases including motor disorders, cystic fibrosis and epilepsy. at present processes regulating intracellular chloride ion concentrations are still widely unexplored mainly as a consequence of limiting methods to quantify chloride fluxes in living cells. in the present work a highly specific, genetically encoded sensor is developed for detecting simultaneously intracellular ph and chloride concentration. the sensor is obtained by fusion of a red fluorescent protein (dsred-monomer), insensitive to chloride and ph, to a gfp variant containing a specific chloride-binding site (gfp-chl). dsred-gfp-chl binds the chloride ion following a fluorescence static quenching mechanism, which allows measurements of intracellular ph in a chlorideindependent manner. the sensor has been successfully tested in different living cells, in a ph range - and chloride concentration up to mm. for the first time, to the best of our knowledge, it allowed to measure the chloride concentration of dense core vesicles in the secretory pathway. applicability to high-throughput screening, range of validity and accuracy of time-lapse maps will be discussed. we aim to understand the basis of the photophysical changes in fluorescent proteins (fp) induced by reactive oxygen species (ros). indeed, ros might be involved in photochemistry of fp, leading to their photobleaching or photoconversion. in addition, fp may be used to investigate cellular events like phagocytosis or mitochondrial activity, where ros are naturally produced. in the latter cases, an accurate analysis of fp's fluorescence signals requires the full knowledge of reactions between ros and fp. in the future, this work may help in developing either photoresistant or photoswitchable fp and improving their use for imaging under oxidative stress conditions. using γ-radiolysis as a quantitative source of ros, we investigated the reactions of oh and o − radicals on the cyan fluorescent protein (cfp) and the modifications of the cfp's photophysical properties by oh radicals were explored in detail (submitted). in order to address the corresponding chemical changes in the protein, we devised a mild proteolysis protocol that for the first time offers a peptide mass fingerprint almost covering the cfp sequence (alvarez et al. biochemistry ). then, we achieved the meticulous characterization of the cfp oxidation products by mass spectrometry and proposed a mechanism to account for their formation by pulse radiolysis. since the cloning of the green fluorescent protein from aequoria victoria, numerous screens have been performed to improve the brightness of this protein, its spectral variants and fluorescent proteins from other species. the improvement is evaluated by comparing fluorescence intensity of individual bacteria or colonies. in this way also expression level, folding and maturation efficiency, and thickness of the bacterial colony contribute. here we report a screening method that, in addition to fluorescence intensity, quantifies the excited state lifetime of a fluorescent protein, which is independent of intensity or expression level, and provides a direct measure for the quantum efficiency of the fluorescent protein. the novel approach was used to screen a library of cyan fluorescent protein (cfp) variants randomly mutated at position , and , yielding an improved bright cyan fluorescent protein named, mposeidon, with a markedly increased fluorescence lifetime and quantum yield, increased photostability and improved fluorescence intensity in vivo. it is shown that mposeidon is the brightest cyan fluorescent protein in mammalian cells. in addition, several lifetime variants were identified that can be used for lifetime unmixing. it is demonstrated that three cfp variants can be separated and their distribution quantified in a single detection channel. a. r. faro , p. carpentier , d. bourgeois , e. rosny irtsv, cea, ufj, grenoble, france, ibs, cea, cnrs, ufj, grenoble, france, ibs, cea, cnrs, ufj, grenoble, france, ibs, cnrs, ufj, grenoble, france enhanced yellow fluorescent protein (eyfp) is extensively used as a fluorescent marker. like other photo activatable fluorescence proteins (pafp), it exhibits photo-switching properties. however, the mechanism by which fluorescence can be swiched on or off upon light irradiation is not fully understood at the molecular level. the bright to dark conversion involves a protonation step and structural rearrangements of the chromophore, but it is not clear which of these two steps is the triggering event. to answer this question, we carried out photo-switching experiments at cryotemperatures. our data suggest that a photo-induced protonation step (probably in the triplet state) is the primary event in the bright to dark conversion. our results may bear relevance to other pafps, such as iris, eos, dronpa, padron or kaede. how misfolding and aggregation of proteins constitutes a toxic insult to neurons remains largely unknown. in order to obtain insight into the molecular biology of neurodegenerative disease, we have developed a number of gfp-based biosensors for the detection and quantification of cellular clearing mechanisms for aggregated proteins. the high load on these protein quality control mechanisms, and their failure to meet normal physiological demand ultimately results in a "de-compensation" condition from which the nerve cell cannot recover. our fret/flim-based bioassays visualize protein ubiquitination and degradation; proteasomal activity; foldase activity using a folding-impaired gfp mutant which gains fluorescence conditional on the upregulation of chaperone activity; chaperone binding to unfolded proteins; and autophagosome formation/lysosomal integrity via the targeted and sensitive fret-based measurement of ph changes. these sensors are employed in cellular model systems for parkinson's and alzheimer's disease, and amyotrophic lateral sclerosis (als) to delineate the molecular pathway of cellular demise, and to gain a mechanistic understanding of the toxicity of protein aggregates and the basis for the vulnerability of neurons. millisecond photo-switching dynamics of e q gfp mutants for sensor and imaging applications m. collini , v. quercioli , l. d'alfonso , g. baldini , b. campanini , s. bettati , g. chirico dipartimento di fisica, università di milano bicocca, italy, dipartimento di biochimica e biologia molecolare, università di parma, parma, italy e q mutants of the green fluorescent protein are known to possess photo-chromic properties: the anionic emission, primed by a pump nm laser beam, can be switched between two levels of different brightness by irradiation with a blue, ∼ = nm, probe laser light. we have studied here the amplitude and the dynamics of the brightness enhancement of the e q mutant of gfpmut . the fluorescence emission increases almost threefold, under saturating probe laser excitation, for pump excitation intensity in the linear regime. two characteristic activation times, estimated by means of modulated two colour fluorescence correlation spectroscopy, are detected in the - ms range, independent of solution temperature and viscosity. the brightness enhancement factor and the characteristic activation times depend markedly on the solution ph. these results indicate that this mutant can be used as a high sensitive intracellular marker for local proton concentration and for modulated excitation imaging. the advent of fluorescent proteins (fp) gave researchers the opportunity to study proteins in situ. fluorescence resonance energy transfer (fret) benefited from this. cell fixation is a commonly used approach when working with microscopy. however, we have found that fret efficiency (e) in cells transfected with cerulean and venus chimeras could not be reproducibly measured after fixation. to evaluate this problem in detail, we measured e of cerulean-venus standard constructs by acceptor photobleaching fret, intensity-based ratiometric fret and flim-fret. the constructs were produced as standards (biophys j, , , ) with , and % e values, comprising donor-acceptor separations of , and amino acids, respectively. transient transfection of the fusion plasmids was performed into hela cells and e was measured in live and pfa or methanol fixed cells. literature e values were reproduced when measuring live cells. conversely, cell fixation caused a deviation of e values. methanol fixed cells showed e between - % for all the constructs. the effect of pfa fixation on both fluorescence intensity and fret varied vastly among independent experiments regardless of the measurement modality. thus, fixation should be avoided due to the effects it has on fp's fluorescence and consequently on fret efficiency. to explain the improvement in the fluorescence properties of cerulean when compared to ecfp, we have determined the x-ray crystallographic structures of these two proteins at physiological ph, and performed molecular dynamics simulations. both proteins exhibit a structural heterogeneity in the nterminal half of their seventh strand, which forms a specific set of van der waals interactions with the chromophore. the critical h d mutation present in cerulean induces a modification of these interactions, and allows the chromophore to be more planar and better packed, albeit only intermittently. as a consequence, the probability of non-radiative decay is significantly decreased. our results highlight the considerable dynamical flexibility that exists in the vicinity of the tryptophan-based chromophore of these engineered fluorescent proteins, and provide insights which should allow the design of mutants with enhanced optical properties. the fluorescence lifetime of green fluorescent protein g. jung biophysical chemistry, saarland university, saarbruecken, germany biotechnological design of the green fluorescent protein (gfp) and the discovery of other proteins boosted the development of the life sciences in the past decade. tracking protein movements and high resolution microscopy are only a few recent applications which were realized by fluorescent protein technology. among these examples, the switching between two chromophore state is exploited. our aim is to establish fluorescent proteins for bioanalytical fluorescence lifetime measurements. despite the progress in other fields, quantification with gfp still imposes practical problems [ ] . in the past, we observed that the uv-light driven decarboxylation of the nearby aminoacids glu distorts the fluorescence lifetime of gfp [ ] . we found out that this chemical reaction also occurs under excitation of the anionic chromophore state with a high quantum yield [ ] . recently, we could show by time-resolved spectroscopy that, indeed, the more susceptible state for this kind of photoconversion is the anionic chromophore state [ ] . suppression of this reaction therefore enables the design of autofluorescent proteins which can be used e.g. for the quantification of ions and which are beneficial as donors in fret applications. the fluorescent properties of tryptophan residues (w) in proteins are highly dependent on their immediate protein environment. however, the multi exponential decay of single w proteins is not completely understood. the most cited hypothesis contributes a multi exponential decay to the existence of several micro conformations (rotamers) of the w residue within the protein matrix. to determine rotamers we apply a method based on dead-end elimination (dee) and molecular dynamics simulations (md). low energy rotamers are calculated by dee while dynamics and further refinement is accomplished using md. the method was applied on several test cases including the protein mutant bc-csp l e from bacillus caldolyticus, which contains a solvent exposed w residue. as resolved by x-ray crystallography, this w residue occupies two conformations. using dee and md we were able to retrieve the w conformations found in the x-ray structure. the results demonstrate the ability of the method to obtain valuable w rotamers, both for solvent shielded as exposed residues. the determined conformations were compatible with the findings based on a method using replica exchange simulations. k. nienhaus , h. nar , r. heilker , j. wiedenmann , g. u. nienhaus inst. of biophysics, universität ulm, ulm, germany, dept. of lead discovery, boehringer ingelheim, biberach/riss, germany, national oceanography center, university of southampton, southampton, uk, inst. of applied physics, universität karlsruhe (th), karlsruhe, germany eqfp is a red fluorescent protein (rfp) with the chromophore in a co-planar trans orientation, whereas the cis isomer is preferred by most other rfps. by using x-ray crystallography, we determined the structures of the dimeric variants d eqfp and d eqfp at high resolution (up to . Å). for d eqfp , we had previously seen a redshifted species upon irradiation with -nm light. concomitant changes in the raman spectrum were interpreted as evidence of a trans-cis isomerization of the chromophore. here we have combined x-ray crystallography and site-directed mutagenesis to assess whether we can create a stable fluorescent, red-shifted eqfp variant with a cis chromophore. in a first step, we introduced the n s substitution. this variant, d rfp , is highly fluorescent, with the absorption (emission) maximum red-shifted by ( ) nm. with an additional s c mutation, the chromophore is found completely in the cis form. the variant, rfp , is highly fluorescent, with excitation and emission maxima at and nm. still further red shifts appear to be in reach. k. nienhaus , v. adam , d. bourgeois , g. u. nienhaus of biophysics, universität ulm, ulm, germany, esrf, grenoble, france, institute of applied physics, universität karlsruhe (th), karlsruhe, germany dendra is an engineered, monomeric gfp-like protein that belongs to a sub-class of fluorescent proteins undergoing irreversible photoconversion from a green-to a red-emitting state upon exposure to purple-blue light. this process occurs in the neutral state of the chromophore and is known to result from backbone cleavage accompanied by an extension of the delocalized π-electron system. we have measured the x-ray structure of green dendra and performed a comprehensive characterization of the optical absorption and fluorescence properties of the protein in both its green and red forms. the structure, which is very similar to those reported for the closely related proteins eosfp and kaede, revealed a local structural change next to the chromophore, involving mainly arg and a water molecule. we propose that this structural change explains the blue shift of the absorption and emission bands, as well as the markedly higher pks of the hydroxyphenyl moiety of the chromophore. the -fold enhancement of the neutral species in dendra at physiological ph accounts for the observed higher photoconversion yield of this protein in comparison to eosfp. photochromic green fluorescent protein mutants: chromophore states unveiled by raman spectroscopy s. luin, v. voliani, g. lanza, r. bizzarri, r. nifosì, p. amat, v. tozzini, m. serresi, f. beltram nest, scuola normale superiore, cnr-infm and italian institute of technology, pisa, italy the most widespread genetically-encodable fluorescent markers used for studies in living cells and tissues belong to the green fluorescent protein (gfp) family. reversibly switchable fluorescent proteins (rsfps) were developed that can undergo repeated transitions between different states, e.g. bright and dark forms. this property makes rsfps particularly attractive as active labels in biological systems for selective photolabeling applications or subdiffraction imaging. we shall present pre-resonant raman results unveiling the photophysical mechanism underlying the observed photochromic behavior. the variation of spectral properties before and after photoconversion of chemically-synthesized isolated chromophores under different protonation and/or isomerization have been analyzed, and compared to results obtained for the case of complete folded proteins comprising the same chromophores. experimental results have been analyzed within a time-dependent density functional theory, allowing us to assign all relevant vibrational modes. these results make it possible to discriminate between the effect of cis-trans isomerization and of diverse protonation states of the chromophore in the photoproducts of these proteins. s. luin et al, j. am. chem. soc. , - ( ). r. bizzarri et al., anal. bioanal. chem. , - . a combined study of the interaction of outer membrane proteins with cephalosporin antibiotics m. lovelle, i. barroso, m. j. feio, p. gameiro requimte , fac. ciências, universidade do porto, portugal gram-negative bacteria characteristically are protected by an outer membrane that serves as a selective permeation barrier. most of the β-lactam antibiotics appear to penetrate the outer membrane through these non-specific channels, and it becomes important to understand the possible interactions between β-lactams and the porin. fluorescence techniques have been largely used to characterize both the conformation and the dynamic behavior of large biological structures such as membranes and proteins. the fluorescence of the tryptophan residues is quenched in the presence of the different cephalosporin antibiotics. this reaction between the excited state of the fluorophores and the drug can be described as a formation of a non-fluorescent complex. the dependence of the fluorescence intensity upon quencher concentration for static quenching is proportional to the binding constant for complex formation. since β-lactam susceptibility is closely related to the presence of these non-specific porins, minimum inhibitory concentration (mic) by micro-broth dilution in microplate were used to assess the bactericidal activity of cephalosporin antibiotics upon on escherichia coli strain bl (de ) and a series of bl (de ) mutated in different outer membrane proteins. this combined study of the interactions at single molecular level and at in vivo level provides new insights for a better understanding of the antibiotic translocation. when used in combination with e.g. a cyan fluorescent protein, keima offers the unique opportunity to perform dual color fluorescence cross-correlation spectroscopy using a single laser line to excite both fluorophores. the molecular determinants of the large stokes shift of mkeima have been characterized structurally by combining x-ray crystallography with in crystallo uv-visible absorption, fluorescence and raman spectroscopy. our results reveal a ph-dependant "reverse chromophore protonation" of mkeima, driven by the key residue asp . moreover, the chromophore protonation state is shown to be coupled with different chromophore conformations, cis conformation at ph . , and mostly trans conformation at ph . . these results will help unravel the mechanisms of fluorescence in fps, which is of importance to rationally design and develop new fluorescent markers. recently reversibly switchable fluorescent proteins (rsfps) have become a new branch of the green fluorescent protein (gfp) like family. the rsfps may be reversibly switched between a fluorescent and a non-fluorescent state by irradiation with light of distinct wavelengths. the key process of this switching behaviour is a light induced change of the chromophore between a cis-and a trans-isomeric state. the proteins are becoming increasingly important for diverse applications like protein tracking, sub-diffraction resolution microscopy and data storage. based on the switching mechanism, we created novel rsfps with unique and improved characteristics. padron and bs-dronpa are two of these new switchable proteins. padron features a reversed switching mechanism as compared to all other green rsfps known to date while bsdronpa exhibits a very broad absorption spectrum extending into the uv. utilizing the characteristics of both proteins, we performed multi label single detection colour microscopy as well as dual colour sub-diffraction microscopy, the latter with a resolution of nm. further, we recently introduced the first monomeric rsfp exhibiting red fluorescence: using a semi rational mutagenesis approach on the non-switchable mcherry, we generated the switchable monomeric protein rscherryrev. the use of this protein in single molecule switching microscopy allowed us to record time-lapse live-cell images of the endoplasmic reticulum with sub-diffraction resolution. a spectroscopic approach to the study of chromophoric dissolved organic matter (cdom) in the sea c. santinelli, r. lavezza, l. nannicini, a. seritti cnr-ibf, via moruzzi , pisa, italy dissolved organic matter (dom) in the ocean represents the largest reservoir of reactive carbon on the earth. it is produce at each level of the marine food web and it represents the food for heterotrophic bacteria, which can use the different pools of dom (labile, semi-labile, refractory) with a large range of turn-over times (from minutes to millennia). dom plays a central role in the global carbon cycle and it has a high ecological significance. chromophoric dom (cdom) is the fraction of dom capable of adsorbing light at uv and visible spectral regions. it determines the underwater light availability in open and coastal regions with important implication on primary production and biological activity. it is photodegraded to co , co, with a significant impact on the role of the ocean as source and/or sink of atmospheric co and to highly reactive compounds, dangerous for marine organisms. in its pool "humic-like" and "protein-like" fluorophores have been individuated. cdom optical properties (absorption and fluorescence) together with doc data collected in some key regions of the mediterranean sea will be presented and discussed, in order to (i) investigate the powerful of cdom optical properties to get information on cdom "quality" (i) asses the role of dom in carbon export at depth, (iii) underline the main unresolved question on dom and cdom dynamics in the ocean, with particular emphasis to their biological lability. mechanism and applications of photo-and redox-switchable fluorescent proteins s. j. remington, j. n. henderson, x. shu, j. lohman institute of molecular biology, university of oregon, u.s.a. photoswitchable fluorescent proteins have significant advantages over conventional fluorescent labels, and in a revolutionary application, now allow cell biologists to exceed the diffraction limit in light microscopy by a factor of ten. atomic resolution crystal structures and time resolved spectroscopy of both reversible (mtfp . ) and irreversible (pa-gfp) photoswitchable fluorescent proteins in the light and dark states explain the long term stability of either state, as well as how illumination at the appropriate wavelength causes the molecules to switch between states. the photoswitching mechanisms will be discussed in terms of photochemistry, light induced chromophore isomerization and excited state proton transfer (espt). mutagenesis of espt pathways provide insight into the nature of the ratedetermining steps in proton transfer and lead to practical applications, such as redox-sensitive gfp biosensors. k. i. willig, b. hein, s. w. hell max-planck-institute for biophysical chemistry, goettingen, germany stimulated emission depletion (sted) nanoscopy is a light microscopic technique offering a resolution far beyond the diffraction limit: the excitation beam is overlapped with a doughnut-shaped, red-shifted sted beam, which switches off the ability of the molecules to fluoresce in the outer region of the excitation spot. scanning the nanosized focal spot through the sample renders sub-diffraction images with a sub-second frame rate. we used the yellow fluorescent protein citrine to image individual structural elements of living mammalian cells: vimentin and the tubular network, structures of the cytoskeleton, were recorded with a lateral (x,y) resolution < nm inside the living cell, corresponding to a -fold improvement over that of a confocal microscope. also, time lapse sted imaging of dendritic spines of yfppositive hippocampal neurons in organotypic slices outperforms confocal microscopy in revealing important structural details. as an alternative to fluorescent proteins we used a genetically encoded protein tag which can be labelled with modified organic dyes within living samples. thus nanoscale imaging of structures in the interior of living cells greatly expands the scope of light microscopy in cell biology. pulling membrane tubes from solid-supported lipid bilayers with atomic force microscopy j. w. armond , j. v. macpherson , m. s. turner department of physics, university of warwick, u.k., department of chemistry, university of warwick, u.k. information on the elastic and dynamic properties of membranes is essential for a thorough understanding of biological processes such as exocytosis, endocytosis, cell division, and pore formation. we use an atomic force microscope to pull on solid-supported lipid bilayers and observe that an energy barrier must be overcome prior to the formation of membrane tubes. we have modified elastic models of lipid bilayer vesicles to describe the free energy of a planar lipid bilayer in adhesive contact with a surface. from this model we are able to extract force-distance curves for the formation of a membrane tether, including the associated energy barrier. the experimental data can thus be understood in a quantitative fashion. this work enables the atomic force microscope as a quantitative instrument for measuring membrane pulling mechanics, and in future work will allow two-dimensional mapping of elastic properties under tension. from pores to micelles -a peptide-membrane interaction study t. l. andresen, j. r. henriksen technical university of denmark, dtu nanotech, frederiksborgvej , b , roskilde, denmark. email: thomas.andresen@nanotech.dtu.dk. research in the partitioning of peptides into lipid membranes has been intense for several years. along with scattering techniques and nmr, isothermal titration calorimetry (itc) has proven to be a powerful tool for thermodynamic characterization of peptide-membrane interactions. we have studied two antimicrobial peptides, mastoparan-x and melittin, and found that these peptides induce a range of structural transitions of popc and popc/popg membrane systems at different peptide-lipid ratios. we have found that itc can be used to elucidate the threshold where transitions occur, including the threshold for pore formation and micellation. this has been achieved using a thermodynamic model based on gouy-chapman theory, which provides the partitioning constant of the peptide-membrane interaction and thereby the concentration of peptide on the membrane surface. the structural changes have furthermore been visualized by cryo-tem. we have further investigated the pore formation in detail and found that the thermodynamic parameters of pore formation can be fully characterized using a system specific temperature where the enthalpy of peptide partitioning becomes zero. this allows for an exclusive study of the pore formation process. lactoferrin is a glycoprotein with two globular lobes, with of two domains each. since its discovery, the research on antimicrobial properties has been extended to peptides derived from this protein. the largest family studied so far is known as lactoferricin b, obtained from the protein by digestion with pepsin. more recently, a new family of antimicrobial peptides derived from lactoferrin was discovered by bolcher et al and named lactoferrampin (lfampin). the original sequence of lfampin contained residues - from the n domain of lactoferrin. we studied peptides derived from lfampin obtained by extension and/or truncation at the c-or n-terminal sides, keeping the essential characteristics, in order to unravel the main structural features responsible for antimicrobial action. the peptides were tested against model membranes. the ability to adopt helical conformation was followed by cd, the perturbation of the membrane phase transition by dsc, the energetics of interaction by isothermal titration calorimetry (itc), the partition of the peptide to the membrane by trfs and the importance of charge effects assessed by zeta potential measurements. the results are discussed and compared to the antimicrobial and hemolytic activities obtained by microbiology techniques. defensins are small cysteine-rich cationic proteins or peptides found in both vertebrates and invertebrates. they can be active against bacteria, fungi and many enveloped and non-enveloped viruses; thus, being also classified as antimicrobial peptides (amps). in the present work a comparative study between psd (a plant defensin with antifungic properties obtained from the garden pea pisum sativum) and hnp (a human neutrophils defensin) was conducted, in order to shed some light on the mechanism of action at the molecular level of these defensins. large unillamelar vesicles with different lipid compositions were used for this purpose as biomembranes model systems; namely, popc/cholesterol (characteristic of the outer leaflet of vertebrates cell membranes) and popc/ergosterol (fungal) mixtures. changes on the intrinsic fluorescence of the tryptophan residues present in these peptides upon membrane binding/insertion were followed by fluorescence spectroscopy. experimental results show the affinity of both defensins for mammalian and fungal sterols. the partitioning behavior of psd showed a high selectivity for ergosterol rich membranes, while hnp has preference for cholesterol rich membranes. preliminary characterization of atomistic computer models of galactolipid bilayers k. baczynski, m. pasenkiewicz-gierula faculty of biochemistry, biophysics and biotechnology, jagiellonian university, krakow, poland the main lipid component of thylakoid membranes are galactolipids, which constitute more than % of its lipid composition. the most common types of galactolipids found in thylakoid are monogalactosyldiacylglycerol(mgdg), whose headgroup consists of a single molecule of beta-d-galactose and digalactosyldiacylglycerol(dgdg), whose headgroup consists of two galactose molecules: alpha-d-galactose and beta-d-galactose linked by o-glycosidic bond majorty of thylakoid galactolipid have gamma-linolenic acid both in the sn- and sn- position. atomistic computer models of mgdg and dgdg molecules have been constructed and parametrized in the opls-aa force field. the molecules were used to construct three bilayer systems for molecular dynamics (md) simulations:( ) composed entirely of dgdg molecules,( ) composed entirely of mgdg molecules,( ) composed of a mixture of dgdg and mgdg molecules the ratio : . the systems have been md simulated using the gromacs . . package. the generated trajectories were analysed to determine a number of structural parameters among them: membrane thickness, average area per lipid, electron density profile accross the bilayer, order parameters, organisation of the bilayer/water interface as well as several dynamic parametrers like diffusion coefficients, lifetimes of conformational states, lifetimes od lipid lipid interactions. single mixed-lipid guv method reveals interaction of viper venom with lipid membranes n. m. ayvazyan, n. a. zaqaryan, n. a. ghazaryan dpt. biophysics, yerevan state university, armenia studies on the interaction of snake venom and organized lipid interfaces have been conducted using a variety of systems, including bilayer lipid membranes (blms), small and large unilamellar vesicles. giant unilamellar vesicles (guvs) with a mean diameter of µm have a minimum curvature and mimic cell membranes in this respect. guvs are ideal for studying lipid/lipid and lipid/protein interactions using microscopy techniques with membrane fluorescence probes. guvs were formed from the total lipid fraction from bovine brain by the electroformation method, developed by angelova and dimitrov ( ) . vipera lebetina obtusa venom was added to the sample chamber before the vesicles were formed. the membrane fluorescence probes, ans and pyrene, were used to assess the state of the membrane and specifically mark the phospholipid domains. ans and pyrene allows us to quantify the fluidity changes in the membrane by measuring of the fluorescence intensity. the presence of viper venom in guvs media reveals a noticable decreasing of membrane fluidity compare the control, while the binding of fluorophores with guvs modified by venom lead to appearance of channel activity. it was recognized early that the vipers' venom components preferred an organized lipid substrate near the lipid's phase transition and were particularly active against micellar lipids. these studies also emphasize the importance of a membrane surface curvature for its interaction with enzymatic components of venom. the attenuated total reflection fourier transform infrared (atr-ftir) spectroscopy is ideal for highly absorbing samples such as water suspensions or even bulk water due to the small light penetration depth. it is also suited for experiments on lipid membranes in excess water. for example, we have studied the hydrogen bonding (h-bonding) between cholesterol (ch) and phosphatidylcholine (pc) or sphingomyelin (sm), which could be important for the stabilization of the cholesterol-rich lipid domains. the atr-ftir method enabled comparison of the carbonyl band shape for pc/ch samples in excess h o or d o, and has confirmed similar behavior for both [ ] . secondly, we were able to analyze the amide ii band for sm/ch samples in excess h o. the observations confirm the presence of h-bonds between ch and n-h group in sm [ ] . another example is the study of the interlamellar water structure, which could influence the water-mediated phenomena in membranes. h-bonds in interlamellar water in partially hydrated lipid multibilayers are stronger with respect to bulk water. in contrast, we show by atr-ftir spectroscopy that the h-bonds are weaker in multibilayers in excess water [ ] . the bactericidal activity of antimicrobial peptides is linked to their ability to perturb the permeability of bacterial cells. they often show α-helical conformation, and many peptides have a kink in the middle of this structure, caused by pro or gly. in order to understand the role of the kink we designed various analogues of p , in which the central pro residue was moved from its central position, or removed altogether (in analogue p f). displacement of the pro residue caused a decrease of the antimicrobial activity, and an increase in the toxicity against erythrocytes, with the less active and more toxic peptide being p f. fluorescence studies suggest that both p and p f bind on the membrane surface. fluorescence experiments show that the activities of the two analogues correlate with their affinity for different kinds of lipid bilayers: the kinked p peptide has a dramatically higher affinity for negatively charged vesicles (mimicking the composition of bacterial membranes) than for neutral liposomes (similar to mammalian cells), while analogue p f exhibits comparable affinities for both membranes. therefore, our data suggest that the central pro-induced kink is involved in selectivity, inhibiting peptide binding to the membranes of eukaryotic cells. d. behn , h. hoffmeister , r. witzgall , c. steinem institute for organic and biomolecular chemistry, university of göttingen, germany, institute for molecular and cellular anatomy, university of regensburg, germany polycystin- , encoded by pkd , is an integral membrane protein with a size of kda and amino acids. the protein, which is known to be a ca + permeable, non selective cation channel, interacts with several proteins such as polycystin- , pigea or pacs- /- etc. the interaction takes place through a proposed coiled-coil domain of polycystin- located at the c-terminus of the protein.if mutation of either pkd or pkd (gene product of polycystin- ) occurs, the interaction between the proteins is disturbed leading to increased formation of renal cysts. this autosomal polycystic kidney disease (adpkd) is one of the most common genetic diseases causing renal failure due to the enormous cyst formation. in this study, the interaction of the c-terminus of polycystin- with its postulated specific interaction partners has been investigated in terms of its biological relevance. binding affinities as well as kinetic parameters of the interaction were determined. in particular, the interaction of c-polycystin- and pigea immobilized on solid supported membranes with c-polycystin- has been quantified by means of the quartz crystal microbalance (qcm) method. a dissociation constant of about nm was obtained. the results are compared with those obtained by surface plasmon resonance (spr) technique using a different immobilization strategy. correlation of the lateral structure of lipid bilayers and monolayers using two photon excitation fluorescence microscopy l. a. bagatolli membrane biophysics and biophotonics group/memphys -center for biomembrane physics, bmb, university of southern denmark most of the reported fluorescence microscopy applications on langmuir lipid films are focused in obtaining fluorescence intensity images using particular fluorescence probes. in this type of experiments the probes are generally utilized to obtain "contrast" between membrane regions (lipid domains) displaying dissimilar physical properties. the obtained information largely depends on the partition of the fluorescent probes for the lipid domains and the obtained fluorescence intensity pictures are not providing any information about the local physical properties of the lipid film. local physical properties of these distinct regions can be determined by exploring fluorescent probe related parameters such lifetime, emission shift, polarization. however these fluorescent probe's properties are almost unexplored in this type of experiments. this presentation will focus in describing a new experimental setup that includes a specially designed film balance on top of a custom built multiphoton excitation fluorescence microscope. this setup allows obtaining laur-dan gp images ( ) many studies on phase separations of lipids in bilayers and their leaflet asymmetry make use of fluorescence techniques. we used a dithionite assay, steady-state fluorescence anisotropy and fluorescence resonance energy transfer (fret) for characterization. dithionite assay was performed to measure the fluorophore distribution in the inner and outer leaflets of symmetric membranes. for low concentrations, the fluorophore is distributed almost homogeneously, whereas for concentrations > . mol % it accumulates in the outer leaflet. we discuss these effects taking into account the presence of multilamellar liposomes and dynamic effects produced by higher local bending elasticities. the results point out possible artifacts in the use of fluorophores to characterize bilayers under the assumption of their homogeneous distribution. dithionite relative bilayer permeability is discussed. the results won by fret and steady-state fluorescence anisotropy regarding lipid phase transitions are in good coincidence to each other and to results reported in the literature. the photosensitizing properties of three chlorins are compared in solution and when incorporated in dioleoylphosphatidylcholine vesicules. in solution, they possess a similar efficacy to generate singlet oxygen and a similar ability to induce the peroxidation. however, the role of the photosensitizer localization within the lipidic bilayer is strongly highlighted, when chlorins are incorporated in liposomes, both by the changes in order of peroxidation efficacy but by the measurements of the photoinduced permeation of the liposomes. the results are discussed in relation with the technology of photochemical internalization, pci. then, using giant unilamellar vesicles, we asymmetrically oxidize the membranes. we observed different shape transitions, such as budding, typical of membrane curvature modifications. the asymmetry of the shape transitions are in accordance to a lowered effective spontaneous curvature of the leaflet targeted. this effect is interpreted as a decreased preferred area of the targeted leaflet compared to the other, due to the secondary products of oxidation. permeabilization of guv by photooxidation is interpret as the opening of a pore above a critical membrane tension due to the budding of vesicles. the effective spontaneous curvature of photosensitized vesicle at lysis was estimated. additionally photooxidation was shown to be fusogenic. influence of polyphenol extracts from fruits on biological and model membranes d. bonarska-kujawa, h. pruchnik, j. sarapuk, j. oszmiański, h. kleszczyńska univ. of environmental and life sciences, wroc law, poland biological activity of polyphenol extracts from apple, strawberry and chokeberry was studied. polyphenols were shown to be good antioxidants and to act as antyhemolytic agents. both the activities are the result of polyphenols incorporation into biological membranes. to check potential changes they induce in membranes some experiments were performed with the use of erythrocytes, and lipid membranes. it was found that the extracts studied induced shape transitions of erythrocytes. they were classified according to the bessis-brecher scale. strawberry extract induced mainly discocytes and discoechinocytes. populations of discocytes, echinocytes and some discoechinocytes were found on applying apple extract, while chokeberry induced mainly the formation of echinocytes and spheroechinocytes. the results evidence that the polyphenols incorporate into the external monolayer of lipid bilayer of the erythrocyte membrane. the results of the fluorimetric experiments showed that all the extracts changed fluidity in the hydrophilic part of rbc membrane. the changes observed were biggest for chokeberry extract and smallest for strawberry one. incorporation was also followed by changes in electrical resistance of black lipid membranes and in shifting the temperatures of main transition (t m ) and pretransition (t p ) in liposomes. this work was sponsored by ministry of science and education, scientific project no. n n . a. boll , n. czudnochowski , m. geyer , c. steinem institue of organic and biomolecular chemistry, university of göttingen, germany, mpi for molecular physiology, dortmund, germany hiv- tat belongs to the accessory proteins of hiv and has regulatory functions. tat is concentrated in the nucleus and nucleolus of infected cells. the protein is composed of amino acids with a molecular weight of kda. tat is a transcriptional activator protein, which stimulates rna polymerase ii-mediated transcription elongation. therefore, tat interacts with cyclin t and binds to the tar rna element. tat has different domains. with respect to the interaction with lipid membranes, the most important structural motif is its basic region, including arginine and lysine residues. the peptide derived from this basic region belongs to the cell-penetrating peptides and is able to translocate through lipid membranes. the major aim of this study is to investigate the influence of full length hiv- tat on artificial lipid membranes. as a starting point solid-supported membranes composed of -palmitoyl- -oleoyl-sn-glycero- phosphocholine (popc) immobilized on silicon dioxide were used. the influence of the lipid head groups on the interaction with tat was analysed by using membranes composed of the negatively charged lipid -palmitoyl- -oleoyl-sn-glycero- -[phospho-l-serine] (pops) in a mixture with popc. fluorescently labelled tat was used to localise the protein in the membrane. the impact of tat on lipid membranes was investigated by fluorescence and atomic force microscopy, showing that it is capable of perturbing lipid membranes. g. bocchinfuso , a. palleschi , b. orioni , g. grande , f. formaggio , c. toniolo , y. park , k. s. hahm , l. stella univ. of rome tor vergata, dept. of chemistry, italy, dept. of chemistry, univ. of padova and cnr, italy, chosun univ., rcpm, south korea. most antimicrobial peptides exert their activity by perturbing the permeability of bacterial membranes, but the molecular details of this process are still debated. here, we compare fluorescence experiments and molecular dynamics simulations regarding the interaction of two antimicrobial peptides (pmap- and trichogin ga iv) with lipid membranes, showing that their mechanisms of bilayer perturbation are significantly different. pmap- , a cationic peptide member of the cathelicidin family, associates to the membrane close to its surface and parallel to it, and in this arrangement it causes a severe perturbation to the bilayer, both regarding its surface tension and lipid order. on the other hand, trichogin ga iv, a neutral peptide member of the peptaibol family, undergoes a transition from a surface-bound state to an inserted orientation. in the first arrangement it does not cause any strong membrane perturbation, while in the second orientation it may span the bilayer from one side to the other, despite its relatively short length, by causing a significant thinning of the membrane. lipopolysaccharide (lps) is the main component of the outer membrane of gram negative bacteria. lps is also known as endotoxin because of its potency to induce sepsis, a serious source of mortality in many clinical cases. among lps-neutralizing agents, polymyxin b (pxb) is considered as the "gold standard" due to its high efficiency of binding and detoxification of endotoxin. in this work, we have further explored the interaction of pxb to lps from the rough mutant of salmonella minnesota (re-lps) both in the gel and in the liquid crystalline phase, using isothermal titration calorimetry (itc) and fluorescence based techniques. the effect of pxb binding on lps-membrane integrity was determined with a fluorescence quenching assay treating vesicles of lps labeled with nbd-pe with dithionite. thermodynamic parameters associated with the binding process as well as binding stoichiometry were obtained from itc experiments. finally, itc was conducted with enterococcus faecalis and salmonella typhimurium, as representative examples of a gram negative and a gram positive bacterium respectively. pressure jumps -an accessible trigger for biomolecular transformations high pressure can be used to induce many structural changes in biological systems: from triggering phase changes in model membranes to causing protein unfolding, in fact any change involving a volume reduction is promoted by pressure. as well as being broadly applicable, pressure changes can be applied very quickly both up and down in contrast to other structure change triggers such as temperature jumps. fast pressure jumps allow the trigger to be decoupled from structural changes, so with fast structure probe techniques such as time resolved x-ray diffraction, the out-of-equilibrium evolution of these systems can be monitored. despite great advantages, high pressure remains under-utilised primarily due to its technical difficulty. in response to this technology vacuum a high pressure user facility based around a pressure jump cell for small and wide angle x-ray diffraction has been commissioned at beamline i , diamond light source, uk and will be freely available to the user community. the cell is highly robust requiring virtually no user intervention during an experiment and the pressure system is computer controlled with a graphical user interface and is integrated with the beamline. pressures between and . gpa are accessible and jumps can be carried out in approximately ms at temperatures from - to • c. sample changing has been made simple and fast with a dedicated sample loading port and modular sample holders allow optimisation for a broad range of samples. the transformation of vesicle and lateral distribution of mobile membrane inclusions b. bozic institute of biophysics, faculty of medicine, university of ljubljana, lipičeva , si- ljubljana, slovenia membrane inclusions such as membrane embedded peptides or proteins exhibit curvature dependent interaction with the surrounding lipid matrix due to the mismatch between their intrinsic curvature and the local membrane curvature. this interaction causes an inhomogeneous lateral distribution of the inclusions and a corresponding adjustment of the vesicle shape. by taking into consideration that the membrane free energy includes elastic energy of the lipid bilayer and a contribution due to an inclusion-membrane interaction, the configurations of lipid vesicles with mobile inclusions have been studied theoretically. the variational problem to calculate equilibrium vesicle shapes is solved by applying a ritz method based on an expansion in spherical harmonics. in general, vesicle shapes adjust to the presence of inclusions by increasing regions with favorable curvature and decreasing regions of unfavorable curvature in such a way that the lateral distribution of inclusions becomes inhomogeneous. if the number of inclusions or the inclusion-membrane interaction exceeds a certain value, the prolate shapes become globally stable. investigating the structure of pores formed by antimicrobial peptides using epr spectroscopy m. bortolus , k.-s. hahm , a. l. maniero universita' di padova, padova, italy, chosun university, kwangju, south korea spin label electron paramagnetic resonance (epr) is a spectroscopical technique effective to study the molecular mobility of membrane components and the membranepeptide interactions, as the timescale of epr is optimally matched to the rotational motions of lipids in membranes. we applied epr to study the pore-forming mechanism of two antimicrobial peptides (amp) that create pores of different dimensions when interacting with liposomes. hp ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) is derived from the n-terminus of helicobacter pylori ribosomal protein l , and hpa is an hp ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) analogue where gln and asp at positions and were substituted by trp. we studied the interaction of the two amp with zwitterionic and negatively charged vesicles, doped with phospholipids spin labelled in the lipid head or at the c or c positions; the different phospholipids allow us to study the peptide-membrane interaction at different depths relative to the membrane surface. we studied the interaction of the amp with vesicles following the influence of amp on label mobility as a function of temperature and membrane depth. we also prepared spin-labelled dmpc/dhpc bicelles, doped with lanthanide ions (dy + /tm + ) that allow us to macroscopically orient the system using the magnetic field of the epr spectrometer. we studied the interaction of amp with the oriented bicellar system monitoring the effect of amp on the order parameter of the phase. a. chattopadhyay centre for cellular and molecular biology, hyderabad, india we addressed the organization and dynamics of the human serotonin a receptor fused to enhanced yellow fluorescent protein (serotonin a r-eyfp) expressed in cho cells. serotonin a receptors are prototypical members of the gprotein coupled receptor superfamily and represent a prime target for therapeutic actions of several anxiolytic and antidepressant drugs. interestingly, we observed significant retention in fluorescence of serotonin a receptors upon triton x- treatment of intact cells at low temperature demonstrating their detergent insolubility. we analyzed the role of cholesterol in the plasma membrane organization of the serotonin a receptor by fluorescence recovery after photobleaching (frap) measurements with varying bleach spot sizes. our results show that lateral diffusion parameters of serotonin a receptors are altered in cholesterol-depleted cells in a manner that is consistent with dynamic confinement of serotonin a receptors in the plasma membrane. interestingly, results from frap measurements performed under conditions of mild cytoskeletal destabilization suggest that receptor signaling is correlated with receptor mobility, in agreement with the 'mobile receptor hypothesis'. our current work is focused on exploring the oligomerization of the receptor using photobleaching anisotropy measurements and indicates the presence of constitutive oligomers of the serotonin a receptor in live cells. expression and reconstitution of connexin in pore-suspending membranes c. carnarius , s. kaufmann , m. tanaka , c. steinem institute of organic and biomolecular chemistry, university of göttingen, tammannstrasse , göttingen, germany, institute of physical chemistry, university of heidelberg, im neuenheimer feld , heidelberg, germany the intercellular communication and electronic coupling between adjacent cells of vertebrates are mediated by gap junctions. these proteins are composed of two connexon hemichannels, whereas each connexon consists of six connexin subunits. each subunit is characterized by two conserved motives: two extracellular loops and four transmembrane α-helices. in this study, we focused on cx . to obtain large amounts of this protein, we expressed cx and cx +gfp in a rather new expression system: dictyostelium discoideum. in contrast to human tissue cultures, the system allows for high cell densities up to million cells per ml and the cells can be cultivated by fermentation. cx +gfp was successfully visualized in d. discoideum by confocal laser scanning microscopy, where it was preferentially found in the plasma membrane.after the cells were harvested, plasma membranes were prepared and both proteins (cx and cx +gfp) were verified by western blot analysis. the proteins were solubilized by addition of % n-octyl-β-d-glucopyranoside and purified by ion metal chelate affinity chromatography. the activities of both proteins were confirmed by a cytochrome c assay. after the purification of both proteins, they were reconstituted in µm-sized pore-suspending membranes. in the near future, we plan to determine the mobility of cx and cx +gfp in these membranes by fluorescence recovery after photobleaching. o. cañadas, c. casals dpt. biochemistry and molecular biology i, and ciber enfermedades respiratorias, complutense university of madrid, madrid, spain inhaled bacterial lipopolysaccharide (lps) may incorporate into the lung surfactant monolayer. in this study, the effect of smooth lps (s-lps) on the surface activity of lung surfactant was evaluated. to that end we investigated the behavior of dppc films containing s-lps, with and without surfactant protein a (sp-a) in the subphase, using epifluorescence microscopy combined with a surface balance. our data show that s-lps injected into the subphase incorporated into dppc films forming mixed dppc/s-lps monolayers. cospread s-lps fluidized the dppc monolayer as demonstrated by epifluorescence images and changes in the compressibility modulus of the monolayer as a function of s-lps molar fraction (x s−lps ). the presence of low amounts of s-lps in the monolayer promoted early collapse, preventing high surface pressures to be reached. moreover, s-lps hampered the re-spreading of dppc molecules during dynamic compression at s-lps concentrations as small as x s−lps = . . such inhibitory effects could not be relieved by repeated compression-expansion cycles or by adding surfactant protein a. however, sp-a facilitated the squeeze-out of s-lps from dppc/s-lps mixed monolayers, suggesting that sp-a is an s-lps scavenger. cholesterol displaces ceramide from its tight packing with sphingomyelin in the absence of ld phase j. v. busto, j. sot, j. requejo-isidro, f. m. goñi, a. alonso unidad de biofísica (csic-upv/ehu) and departamento de bioquímica, u. país vasco (upv/ehu), spain a set of biophysical approaches have been applied to study the phase behaviour of palmitoylsphingomyelin (psm)/cholesterol (chol) model membranes upon palmitoylceramide (pcer) addition. fluorescence spectroscopy of di- -aneppdhq-stained psm/chol vesicles reveals no segregation of large liquid-ordered (l o ) microdomains. in contrast, the formation of disperse, compositionally homogeneous l o psm/chol ( : ) nanodomains over a psm gel (l β ) phase is proposed. dsc measurements show that pcer addition to vesicles with coexisting l o and l β phases (low [chol] ) induces the formation of large gel-like psm/pcer microdomains, coexisting with a l o psm/chol phase. the ∆h for the psm/pcer phase at high psm/(chol+pcer) ratio is close to that of the binary mixture in the absence of chol, supporting immiscibility, but no displacement, between chol and pcer-rich phases. on the contrary, both confocal microscopy of guvs and the dsc data coincide in showing that a rise in pcer increases the gel-like phase to a lower extent than in the pure psm/pcer mixture, revealing some cholinduced restriction. in the presence of a pure l o psm/chol phase (high [chol] ), pcer addition is unable to induce the formation of large psm/pcer microdomains. the present data support the role of chol as the key determinant in controlling its own displacement from l o domains by ceramide. hydroquinones modifying lipid membrane morphology c. di vitta , l. marzorati , v. rebbin , s. s. funari iqusp, university of são paulo, são paulo, brasil, hasylab, hamburg, germany quinones are important molecules in cells. flavina, ubiquinone and coenzyme q, coq, are associated with electron transfer. coq, having a hydrophobic tail, is soluble in the internal lipid membrane of mitochondria. their reduction leads to the equivalent hydroquinones. we synthesized novel alkylthioquinones aths, to investigate their interaction with phospholipids. one or more long hydrophobic chains attached to the quinone ring alter their hydrophobicity and electron availability. we aimed at their influence on the structure of membranes. different phs highlights the charge/polarity effect on the interaction and on the morphology of the bilayer. for that, pope and popc, lipids with different charges, but identical chains were selected. x-rays diffraction on pope/ , bath, / at different temperatures and ph, showed cubic phases coexisting with the usual phases formed by simply hydrated pope, at different phs. for investigation of the charge/polarity, we turned to the popc/ , bath system (smaller charge/polarity). despite decrease in temperature of phase transition and dimensions of the lattice, the structures were the same as in hydrated lipid, illustrating a less significant effect than on the similar lipid pope. another additive, , ath, mixed with pope showed the effect of multiple thioalky chains. no morphology change was seen, compared to pure lipid. interestingly, despite both additives differ by one thioalkyl chain only, their influence on the pope matrix is so different. cross-linking of phospholipid membranes by calcium-sensitive synaptotagmins synaptotagmins are vesicular proteins implicated in many membrane trafficking events. they are highly conserved in evolution and the mammalian family contains isoforms. we now show that the tandem c domains of several calcium-sensitive synaptotagmin isoforms tested, including drosophila synaptotagmin, rapidly cross-link phospholipid membranes. in contrast to the tandem structure, individual c domains failed to trigger membrane cross-linking in several novel assays. large-scale liposomal aggregation driven by tandem c domains in response to calcium was confirmed by the following techniques: turbidity assay, dynamic lightscattering and both confocal and negative stain electron microscopy. high-resolution cryo-electron microscopy revealed that membrane cross-linking by tandem c domains results in a constant distance of approximately nm between the apposed membranes. our findings show the conserved nature of this important property of synaptotagmin, demonstrate the significance of the tandem c domain structure and provide a plausible explanation for the accelerating effect of synaptotagmins on membrane fusion. membrane proteins can be challenging samples to work with and as such often require the use of multiple techniques. here we present an insight into the structure of the antimicrobial peptide melittin in lipid membranes using various techniques. we explore the conformational changes of membrane-bound melittin and its interaction with model membrane lipid systems with a series of spectroscopic methods that can be used in parallel. the techniques used include linear dichroism and ft-ir for orientation information and circular dichroism for conformation information. we use dynamic light scattering for molecular sizing, fluorescence emission for information on peptide environment, thin-layer chromatography for lipid identification, analytical ultracentrifugation to identify oligomerisation state and calorimetry to investigate the thermodynamics. we observe how the physical properties of both the peptide and the membranes affect the insertion kinetics of the peptide in the membrane. phospholipid membranes dynamics: molecular dynamics vs neutron scattering v. conti nibali , m. tarek , u. wanderlingh , g. d'angelo department of physics, faculty of science, university of messina, italy, unité mixte de recherche cnrs/uhp , université henri poincaré, nancy, france collective dynamics and single-particle dynamics of hydrated multilamellar phospholipid bilayers ( , -dimyristoylsn-glycero- -phosphatidylcholine, dmpc) have been studied by means of all-atom molecular dynamics simulations. here we report results of a gel phase bilayer at k and of a liquid crystal phase bilayer at k. coherent and incoherent dynamic structure factors and meansquare displacements have been calculated from the trajectories for both the inplane and out-of-plane lipid dynamics. moreover, the results have been compared to recent quasi-elastic and inelastic neutron scattering data. optical tweezers allow trapping of particles of different types in a wide range of sizes [ ] . among these, unilamellar vesicles are of interest as they are known to be effective vectors in drug delivery and they are also studied as models for cell membranes. this work focuses on the interactions between optically confined unilamellar vesicles and their cross-linking by proteins [ ] . synaptotagmins are vesicular proteins implicated in many membrane trafficking events having an accelerating effect on the membrane fusion. calcium-sensitive synaptotagmins are thought to confer calcium sensitivity to the fusion of secretory vesicles with target membranes [ ] . the novel optical assay reported here allowed us to visualize the cross-linking of nm liposomes mediated by synaptotagmin and calcium. the use of the optical tweezers approach to investigate the function of other fusogenic proteins related to exocytosis (i.e., snare proteins) is discussed as well. antimicrobial peptides (amps) have received increasing interest as the search for new potential antibiotics has become imperative due to increasing bacterial multiresistance. acylating the natural occurring polymyxin b (pmb) significantly enhances antibacterial activity towards two representative gram-negative bacteria e. coli and k. pneumonia compared to the nonacylated pmb. the aim of the presented work was to study various biophysical parameters, such as partitioning coefficients, zeta potentials and effective charges of a selected amp, mastoparan-x (mpx) and a propanoylated (pampx) and octanoylated (oampx) analogue, respectively. fluorescence spectroscopy, isothermal titration calorimetry and ζ-potential measurements were the main techniques used for the measurements. we employed different luv model systems; a partially charged (popg/popc : ) and a neutral system (popc). for the neutral luvs there was an increase in partitioning with increasing length of the acyl chain, whereas partioning into the partially charged luvs was governed by a balance of hydrophobic and electrostatic contributions. the selectivity for the partially charged luvs over the neutral luvs was in the order pampx, mpx and oampx. the modeled effective charge for the peptide followed the same trend as the partitioning coefficient for the three peptides for the respective luv systems. does the lysosomal membrane need triglycerides? a spectroscopic study of a simple model membrane l. duelund , k. pakkanen , m. vuento , j. h. ipsen memphys -center for biomembrane physics, university of southern denmark, odense, denmark, nanoscience center, university of jyväskylä, jyväskylä, finland lysosomes are intracellular organelles in which proteins and other macromolecules are degraded. morphological and functional changes in different compartments of the endocytic pathway are connected to several diseases. a crucial step in understanding biogenesis of lysosomes and their role in disease conditions, is to characterise the properties of the lysosomal membrane. by the use of tlc we have found that lysosomes contain non-neglible amounts of triglycerides (tg). to investigate how the presence of tgs could influence the lysosomal membrane, we have investigated the properties of a mixture of popc and triolein, as a simple model for the lysosomal membrane. we found the system to form two types of popc-rich membranes. these were determined as co-existing phases based on their spontaneous and stable separation and named heavy and light phase according to their sedimentation behaviour. by using epr and fluorescence spectroscopy the physical properties, including order, fluidity and water penetration, of these phases were found to differ markedly despite of their almost identical composition. the results suggest that presence of tgs on lysosomal membranes could have a crucial effect in the barrier functions and thus, the integrity of the organelle. model membranes with the capacity to align in magnetic fields such as bicelles and magnetically sensitive vesicles are of high interest, since their macroscopically alignment allows for the determination of structure, dynamics and topology of molecules within the membrane [ ] . we performed p solid state nmr on a magnetically sensitive lipid possessing a large positive magnetic anisotropy introduced in form of a biphenyl unit during lipid synthesis in one of its acyl chains ( -tetradecanoyl- -( -( biphenyl) butanoyl)-snglycero- -phosphocholine (tbbpc)). the phosphorus lineshapes of tbbpc mlvs studied at various magnetic fields ( . t, . t, . t, . t), revealed a drastic change in shape upon exposure to fields > . tesla: resonances resulting from phospholipid molecules oriented perpendicular to the magnetic field decrease whereas resonances resulting from parallel oriented molecules increase. this is a sign for magnetically induced vesicle deformation from vesicle to oblate ellipsoid shape. factors influencing this drastic deformation, such as magnetic anisotropy, membrane elasticity, lipid chain length and field dependency are discussed based on existing theories [ ] . hepatitis g virus (gbv-c/hgv) is an enveloped viruses belonging to the flaviviridae family. clinical findings have suggested that in people co-infected with gbv-c/hgv and human immunodeficiency virus (hiv), delayed progression of aids has been observed ( ). the mechanism by which this virus may inhibit the progression of aids remain to be elucidated. enveloped viruses acquire their lipid membranes by budding through host cellular membranes ( ) , in this process; fusion peptides play an important role. study of the interaction of hgv-c peptides with lipid membranes could lead us useful information about the mechanism that takes place. in this work we present a study on the effects of e peptides on the fluidity of and polarizability of model membranes (dmpc and dppc liposomes) by dsc and fluorescence polarization. both techniques showed that the presence of the studied sequences in phospholipid mixtures already affected the thermotropic properties of the gel to liquid-crystalline phase transition. systems were thermodynamically charac- small angle neutron scattering (sans) studies have been performed to study the structural changes induced in membranes of vesicles prepared from phospholipid and mixed phospholipid-sterol mixtures, in the presence of different concentrations of the anti-fungal drug, amphotericin b (amb).the vesicles, sonicated to a mean size∼ nm, were prepared using dimyristoylphosphatidylcholine(dmpc) or dmpc-cholesterol or dmpc-ergosterol mixtures -with both of the mixed systems involving mol% sterol. analysis of the sans data show that when the concentration of amb added is just above the drug's cmc (∼ µm) there is an increase in the membrane thickness of both the dmpc-chol and dmpc-erg vesicles (both cases + Å), but the thickness of the pure dmpc vesicle membranes remains the same as in the absence of amb. when amb is added at a concentration in excess of its cmc (∼ µm), the mixed-sterol vesicles show the same changes in membrane thickness as observed with the lower amb concentration, and the pure dmpc vesicles again remain unaffected. on the basis of these studies, therefore, there appears to be no difference in the structural changes induced by the insertion of amb into the model fungal cell membranes (mimicked by dmpc-erg vesicles and those resulting from its insertion into the model mammalian cell membranes (mimicked by dmpc-chol vesicles). the third transmembrane helix of bacteriorhodopsin, also known as phlip, is a unique model system for studying the interactions of a natural transmembrane domain with lipid membranes: depending on ph, the water-soluble peptide either adsorbs superficially or inserts as a transmembrane helix on addition of lipid vesicles [ ] . published values for the free energies of these processes were based on a stoichiometric model invoking two distinct sets of binding sites [ ] . however, discrepancies between data obtained from different experimental techniques and inconsistencies between experimental and expected temperature dependencies suggest that these values should be taken with caution. we therefore reassessed membrane interactions of phlip using titration calorimetry and fluorescence spectroscopy. if electrostatic effects at the membrane surface are taken into account, the data can be described quantitatively by a partition equilibrium, but not by a stoichiometric binding model. the thermodynamics of membrane partitioning differ substantially from those determined previously [ ] and draw a different picture of peptide-lipid interactions. beyond deepening our insights into the first step of the two-stage model of membrane protein folding, this also sheds light on the ability of phlip to drag cargo molecules across lipid membranes. adsorption of monolysine and polylysines at the surface of lipid membranes of varied composition was studied by two methods. both methods -the electrokinetic one, measured the surface potential of liposomes, and the intramembranouse field compensation sensitive to boundary potential of planar bilayer lipid membranes -detected the positive changes of the potentials for membranes with negatively charged component (cardiolipin, cl). neither monolysine, nor polylysines adsorbed at neutral membranes (phosphatidylcholine, pc). pentalysine show the difference between these potentials for the membranes composed from cl/pc mixtures. this difference is attributed to dipole part of boundary potential and indicates the changes in lipid packing. polylysines show high affinity to the membrane and saturation with plateau. these saturation levels correspond to surface charge densities . and . c/m for oligomers with and units, and . c/m for polymers with and units. these values do not depend on the ionic strength of background electrolyte but proportional to the content of negatively charged components in the lipid bilayer. the polylysine layer at the mica surface was studied by atomic-force microscope (afm) technique. it was shown that pentalysine molecules cover the surface by the layer of . nm thickness and polylysines of high molecular weight by the layer up to nm. effects of lysolipids on the mechanical stability of lipid membranes j. r. henriksen , l. feldborg , j. h. ipsen , t. l. andresen technical university of denmark, dtu nanotech, frederiksborgvej , b , roskilde, denmark., university of southern denmark, department of physics and chemistry, memphys center, campusvej , odense m, denmark lysolipids (lpcs) and fatty acids (fas) are the natural products formed by the hydrolysis of phospholipids. lpcs and fas form micelles in solution and thus act as detergents in the presence of lipid membranes. in the present study we investigate the detergent strength of a homologous series of lysolipids (lpcs) on popc lipid membranes by use of isothermal titration calorimetry (itc) and vesicle fluctuation analysis (vfa). the membrane partition coefficient (k) and critical micelle concentration (cmc) are determined and found to obey an inverse proportionality relation (cmc x k ∼ constant). the partition coefficient and critical micelle concentration are used for the analysis of lpc's effect on the membrane bending rigidity. the dependence of the bending rigidity on the lpc membrane coverage has been analyzed in terms a phenomenological model based on continuum elastic theory which yield information about the curvature inducing properties of the lpc molecule. the results reveal: (i) an increase in the partition coefficient with lpc acyl-chain length and (ii) the degree in acyl chain mismatch between lpc and popc determines the magnitude of the membrane mechanical perturbation per lpc molecule on the membrane. patch clamp electrophysiology remains the gold-standard for ion channel research because of the information richness of the data produced. automated planar patch clamp devices, with their higher throughput and high data information content, have made the technique accessible to a wider audience. nanion technologies offers two planar patch clamp workstations combining higher throughput with high data quality. the port-a-patch records from a single cell at a time and the patchliner from up to cells simultaneously with high success rates (typically - %). both, the port-a-patch and the patchliner are bench top patch clamp rigs which uses a planar borosilicate glass chip for obtaining a giga-seal for electrophysiological recordings. suction applied from the underside of the chip is used to attract a single cell to the recording site without the need for optical visualization. both workstations have been successfully used for whole cell, perforated patch, and cell attached recordings as well as for guv-bilayer recordings. due to the versatility of nanion`s products it is possible to study a wide variety of ion channels including nav, kv, cav, ligandgated, herg or reconstituted proteins such as ksca, cx , ompc. special features unique to nanion products, including but not limited to internal solution exchange and temperature control, expand the experimental possibilities. waves on lipid monolayers j. griesbauer, s. boessinger, a. wixforth, s. f. matthias univiversity of augsburg, experimental physics i, biological physics group, augsburg, germany " for the sake of illustration we shall try to provide a physical basis for the equations, but must emphasize that the interpretation given is unlikely to provide a correct picture of the membrane." [ hodgkin&huxley, ] to explain the occurrence of reversible heat production during nerve pulse propagation it has been suggested by multiple authors [wilke,kaufmann,heimburg] that travelling sound waves and not ion channels may offer a better explanation than the hodgkin&huxley theory. therefore, if sound waves are an essential feature of the nerve membrane they should also appear on lipid monolayers where ion conductivity is evidently absent. here we demonstrate that sound waves can be excited on a lipid monolayer by using a set of planar electrodes incorporated into the monolayer and driven by an alternating voltage. not only do our results indicate propagating waves on lipid monolayers in accordance with their thermodynamic predictions, but importantly, no significant attenuation is detected proposing an adiabatic phenomenon. in order to provide evidence that our physical explanation provides a correct picture of the membrane, direct detection of the waves was done, whereas a clear transduction of signals was shown. finally, the impact of toxins, neuropharmaca and anaesthetics needs to be integrated in our physical picture of the nerve membranes, what can easily be done now and could deliver a new approach for understanding their physical mechanism. our earlier studies showed that organometallic compounds (otc) in the presence of uvc radiation enhanced the degree of phosphatidylcholine (pc) liposome oxidation, whereas quercetin effectively protected the membrane. the present investigation is concerned with the effect of uvb and otc (chlorides of diphenyl-and dibutyltin, triphenyl-and tributyltin), both in combined action and separately, on oxidation of erythrocyte proteins, pc liposome and albumin. the degree of oxidation of proteins and liposomes induced by otc and radiation, also in the presence of selected antioxidants (trolox, quercetin) was determined on the basis of changes in the number of sulfhydryl groups, carbonyl groups and malone dialdehyde, respectively. the studies indicate that uvb induces pc liposome and erythrocyte oxidation, whereas in the case of albumin it causes both an increase in the number of c=o groups and free sh groups (most probably, braking sulphonic bridges). otc compounds interact with membrane biomolecules both as weak pro-and antioxidants, also in combined action (uvb plus otc). the prooxidative effects are markedly diminished by application of antioxidants. the action of quercetin results from its ability to incorporate into membranes and formation of complexes with otc (liposome>erythrocyte>albumine). this work was supported by grant n n . a phospholipase c/ sphingomyelinase from pseudomonas aeruginosa has been assayed on giant unilamellar vesicles (guv) consisting of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and cholesterol, at equimolar ratios. the enzyme activity modifies the chemical composition, thus the physical properties of the bilayer, and conversely the latter influence the enzyme activity. biochemical assays of enzyme activity, together with confocal fluorescence microscopy examination of guv provide novel information about the system. the original lipid composition in the absence of enzyme gives rise to lateral phase separation of liquid-ordered and liquid-disordered domains in the guv. the two enzyme end-products, diacylglycerol and ceramide, have opposite effects on the bilayer physical properties, the former abolishes lateral phase separation, while the latter generates a new gel phase. morphological examination of individual guv shows that the enzyme binds preferentially the more fluid (or more disordered) domains, and that, in most cases, it causes the fluidification (disordering) of the other domains.. cholesterol incorporation into lipid bilayers, in the form of multilamellar vesicles or extruded large unilamellar vesicles, has been quantitated. to this aim, the cholesterol contents of bilayers prepared from phospholipid:cholesterol mixtures ( - mol% cholesterol) have been measured and compared with the original mixture before lipid hydration. there is a great diversity of cases, but under most conditions the actual cholesterol proportion present in the bilayers is much lower than expected. the maximum solubility of chol in bilayers containing saturated pc or pc with less than four unsaturations is - mol%, while in polyunsaturated pc, e. g. dic : , and in sphingomyelin the maximum chol contents is mol%. a quantitative analysis of the vesicles is thus required before any experimental study is undertaken. membrane fusion assay based on poresuspending lipid bilayers i. höfer, c. steinem institute of organic and biomolecular chemistry, university of göttingen, germany membrane fusion has attracted significant interest due to its biological relevance. thus in recent years a great variety of fusion assays has been developed. since the process of membrane fusion is not yet fully understood, our aim is to develop a new fusion assay based on pore-spanning membranes, which have been proven mechanically stable, highly insulating and rather tension free. the application of these so-called micro-blms should allow simultaneous monitoring of lipid mixing, content release as well as electrical readouts. furthermore both membrane sides can be addressed individually to apply transmembrane potential or fusion modulating compounds. first results show that the micro-blms provide the opportunity to investigate lipid bilayer fusion by means of fluorescence microscopy. the formation of porespanning membranes is achieved by the painting technique of dphpc doped with oregon green dhpe dissolved in ndecane. the addition of large unilamellar vesicles doped with texas red dhpe allows direct observation of single fusion events. lipid mixing during fusion leads to a decrease of the donor fluorescence (oregon green) and an increase in acceptor fluorescence intensity (texas red) in the plane of the planar bilayer due to fret. in future work simultaneous monitoring of lipid-mixing and content release combined with electrical measurements are planned to gain further insight into different intermediate steps of membrane fusion. the kinetics of membrane-peptide folding and orientation m. r. hicks department of chemistry, university of warwick, uk. understanding interactions of peptides with lipid membranes is essential if we are to be able to design new and better antibiotic peptides. there are different steps in these interactions and following the kinetics of these processes requires that we combine the information from different biophysical techniques. here we present data on the kinetics of peptidemembrane interactions using circular dichroism to report on conformation and linear dichroism to report on orientation of the peptides in/on membranes. these are combined with other techniques such as dynamic light scattering and fluorescence spectroscopy to elucidate the mechanisms of action of the peptides. one of the most important aspects of membrane-active peptide design is that of specificity. we investigate specificity for different types of membranes by using libraries of lipids with different properties of charge, chain saturation and curvature stress. in this way one can test for effects of e.g. negatively charged head groups found in bacterial membranes or cholesterol found in animal and human membranes. using these approaches it is proposed that we will be able to modify peptide sequences, test what part of the kinetic processes are affected and subsequently use this information to design new and better antibiotics. k. kubica, h. misiak wroclaw university of technology, wroclaw, poland reversible membrane electroporation, that can be observed as a effect of electric field influence on biological membranes, finds application in cell delivery of biologically active compounds. the membrane susceptibility for creation of stable pores, depends on electric field parameters applied to the membrane as well as on the membrane environment (ionic strength, ph, temperature) and membrane molecular composition. we have already shown that the changes of van der waals lipid chain interaction effect the process of pore formation. there are known factors which increase or decrease the membrane respond to electric field. because pores appear in lipid membrane matrix it make sense to study this phenomenon using model planar lipid membranes. as lipids are very receptive on oxidizing factors we decided to study the relation between lipid oxidation and membrane reaction on electric field. with the use of four electrode galvanostat we are examining the influence of factors initiating lipid oxidation (uv, fe + ) and the efficiency of some antioxidant compounds of lipid protection on electric properties of lipid membrane. the product of lipid oxidation is determined by characteristic reaction with tiobarbituric acid. we want to determine the role of polyunsaturated fatty acids on membrane properties also. results of our work will be useful in optimization of medicine distribution, aided by electric field, into cells. budding of giant phospholipid vesicles induced by β -glycoprotein i j. kovacic, b. bozic, s. svetina institute of biophysics, faculty of medicine, university of ljubljana, ljubljana, slovenia β -glycoprotein i (β gpi) is classified among amphitropic proteins: in its inactive form it is dissolved in plasma and in its active form it is bound to membrane. in comparison to other amphitropic proteins it exhibits a distinct membrane interaction behavior. a patch of positively charged amino acid residues contacts anionic phospholipids via electrostatic interactions, and a hydrophobic loop anchores the protein into the outer leaflet of the membrane via hydrophobic interactions. the binding constant depends on physical properties of the membrane such as membrane lipid composition, surface potential, lipid packing density and curvature. the binding of an amphitropic protein alters the membrane's spontaneous curvature as well as the difference between the equilibrium areas of membrane leaflets. theoretical model has been developed to predict vesicles shape transformations realized by the formation of buds. it was shown that the effects are stronger for flaccid vesicles which were therefore chosen for our experimental investigations. vesicles were composed of % pops and % popc, and flaccidity was achieved by an osmotic adjustment. the solution containing β gpi was subsequently injected to a chamber with flaccid vesicles by a syringe pump. observation took place under a phase-contrast microscope. experiments showed a concentration dependent occurrence of buds. their number and size were related to the degree of vesicle flaccidness. the interplay between detergent cohesion and peptide adsorption on the structure and dynamics of a glycophorin a tm-detergent complex j. khao, j.-p. duneau, j. n. sturgis lism, cnrs -aix marseille university, marseille, france in spite of the major interest of membrane proteins at functional, genomic and therapeutic scales, their biochemical and structural study remain challenging as they require, solubilization in detergent micelles. the complexity of this task arises from the structural dependence of membrane proteins on their anisotropic environment and in particular by a delicate balance between different physico-chemical properties. in order to study these properties in a small protein detergent complex, we have used molecular dynamics simulations on the transmembrane part of glycophorin a (gpatm) solubilized in micelles of the detergent di-hexanoyl-phosphatidylcholine(d pc). we show that the molecular aggregates organizes to give distinct populations of detergent molecules. those molecules which loosely interact with the peptide are preferentially involved in highly cohesive inter-detergent interactions that impose a global bilayer structure to the micelle. interaction profiles of other detergents with gpatm depend upon the nature of residues along the surface of the peptide. this topology dependence leads to different modes and strength of interactions that ultimately constrain the orientation of the micelles around the peptide. this simple model illustrates how differential detergent selectivity for faces and strong constraints coming from purely environmental features could influence transmembrane helix packing, membrane protein structure and assembly. in this paper we present a study of a new family of bolaamphiphiles. these amphiphiles are unsymmetrical bolalipids having one sugar polar head and the second glycine betaine polar head. they are potentially useful for pharmaceutical or cosmetics applications as vectors for drugs. therefore it is important to investigate their self-assembled properties. the chemical variations that we introduced in this new family concern the length of the main chain that connects two polar heads as well as the length of the side chain placed on the position of the sugar moiety. another variation concerns the introduction of a diacetylenic unit into the main chain in order to rigidify it. we have performed the saxs (small angle x-ray scattering) measurements on the dehydrated compounds as a function of temperature and observed the lamellar liquid crystalline structures. we also measured the saxs spectra of aqueous solutions of these compounds that have shown lamellar l α structure in all cases. these measurements are compared with polarised optical microscopy measurements that confirmed our interpretation. formation of membrane tubular structures induced by phase separation in giant vesicles y. li, r. lipowsky, r. dimova max planck institute of colloids and interfaces, science park golm, potsdam, germany tubular membrane structures (also known as tethers) exist widely in eukaryotes. abundant work has been done on tube extrusion from cells and model membranes under the application of external forces. we present a novel system allowing tube formation in the absence of external forces. aqueous solutions of two chemically dissimilar polymers, polyethylene glycol (peg) and dextran are encapsulated in giant vesicles, a cell-size model system. the exposure of vesicles to hypertonic solutions induces phase separation of the internal aqueous polymer solution. the excess membrane area created by this vesicle deflation, engages in the formation of tubular structures. membrane tube formation and phase separation are coupled processes. hydrodynamic flows and changes in the membrane spontaneous curvature during phase separation might be the driving forces for tube formation. the tubes are rather stable: without external perturbation, they can exist for several days. they prefer to be located in the peg-rich phase at low polymer concentration. at high concentration, they are absorbed at the interface of the liquid phases to lower the surface energy of the system by decreasing the contact area between the liquid phases. the membrane tubes can be retracted back to the vesicle surface by increasing the membrane tension via vesicle aspiration. membrane tubes, which can form and be retracted easily, might be relevant to lipid storage in cells. insight into the antimicrobial mechanism of a de novo auto-assembling peptide b. legrand , m. laurencin , e. duval , c. zatylny , j. henry , m. baudy-floc´h , a. bondon rmn-ilp, umr cnrs -univ. de rennes , france, icmv, umr cnrs -univ. de rennes , france, pemm, umr ifremer -univ. de caen, france a short ( residues) de novo antimicrobial peptide (k ) composed of a cationic polar head and a hydrophobic tail was studied. it exhibits a broad spectrum of antimicrobial activity on bacteria. no haemolytic activity or cytotoxicity on eukaryotic cells are observed at mic. when bacteria are lysed by the k , spherical objects are observed on the sem micrograph. k was structurally studied using various membrane mimetic media such as different micelles and small unilamellar vesicles (suv) of different composition. cd revealed that k adopt various structures (random, β-turn, α-helix) in the presence or the absence of detergents or phospholipids. nmr structures confirmed the α-helical structure of k hydrophobic tail in presence of sds. k self-assembles at high concentration as observed by sem and dls. we suggest that k may act as a surfactant building mixed microsomes composed of peptide and lipids. this destabilization mechanism of the bacterial membrane support the "detergent like model" previously described in the literature. oxidative stress and the membrane dipole potential; modulation with tocopherol s. le-nen-davey , b. m. davis , j. l. richens , k. a. vere , m. w. tilley , p. g. petrov , c. p. winlove , p. o'shea biomedical physics group, university of exeter, uk, cell biophysics group, university of nottingham, uk tocopherol, a component of vitamin e well know for its antioxidant properties, has recently been thought also to influence the structure of cellular membranes. tocopherol treatment reduces hyperglycaemia induced oxidative stress and the associated endothelial dysfunction which is a precursor to the vascular complications of diabetes. tocopherol and insulin interactions are also modified by hyperglycaemia. it is unclear whether these clinically important effects arise from the anti-oxidant and/or structural properties of tocopherol. we have therefore measured the dipole and surface potentials of phosphatidylcholine vesicles containing different amounts of cholesterol, ketocholesterol and tocopherol. we have also investigated the effects of hyperglycaemia, ketocholesterol and tocopherol, alone and in combination, on microdomain formation and interactions of insulin within the membranes of cultured endothelial cells. both sets of experiments indicate that tocopherol causes significant modifications of the membrane dipole and surface potentials. the physiological significance of these changes will be discussed. t. d. lazzara , a. janshoff , c. steinem georg-august university göttingen, institute for organic and biomolecular chemistry, tammannstr. , , germany, georg-august university göttingen, institute for physical chemistry, tammannstr. , , germany cell penetrating peptides (cpp) have been shown to penetrate cellular membranes. they have been of interest for their ability to translocate not only themselves through cellular membranes, but also carry along with them, cargo as large as iron nanoparticles. the exact entry mechanism remains unclear, but has been shown to vary with peptide sequence. their translocation properties have been demonstrated through different experiments involving vesicles, cells and living animals. we plan on using nano-black lipid membranes (nano-blm), which span the pores of nanoporous materials as a model system to study the interaction between cpp and lipid membranes. the nanopores can be used as cellular containers whose interior surface can be functionalized with receptors for biotin-streptavidin recognition. we hope that this system will provide greater control over experimental variables, such as the type of cpp and lipid used, as well as provide kinetic data that can be used to evaluate cpp activity and the kinetics of cargo transport. ethanol induces phospholipid acyl chain interdigitation. while much is known, important issues remain unclear, such as the role of lipid domains. the main purpose of this study was to follow, in real time, the changes induced by ethanol on supported lipid bilayers with nano to microdomains by in situ atomic force microscopy. to this goal, a pure lipid in the fluid phase (dopc), a pure lipid in the gel phase (dppc), binary phospholipid mixtures with gel/fluid phase coexistence, and cholesterol-containing, raft -forming mixtures (dopc/dppc/cholesterol and dopc/sphingomyelin/cholesterol) were investigated. from the height differences observed upon ethanol addition to pure lipids (dppc and dopc), and to dopc/dppc mixtures, it is shown that in the binary system the interdigitation of the fluid phase occurs prior to the gel phase. however, for the lipid rafts mixtures the simultaneous interdigitation of both raft and non raft portions of the membrane is observed both in mica and silicon substrates. for all compositions studied, domain formation or rearrangement accompanied by lipid bilayer expansion occurs as a consequence of interdigitation. these results show the ability of ethanol to influence the bilayer properties in different ways according to membrane composition. ethanol may exert its biological effects by reducing bilayer thickness, and also by changing membrane proteins conformation and lateral distribution, as a consequence of the altered properties of the lipid bilayer. interactions between non-steroidal anti-inflammatory drugs and a pc/cholesterol bilayer m. markiewicz , t. librowski , p. serda , m. pasenkiewicz-gierula faculty of biochemistry, biophysics and biotechnology, jagiellonian university " department of pharmacodynamics, medical college, jagiellonian university " regional laboratory, jagiellonian university, krakow, poland. the non-steroidal anti-inflammatory drugs (nsaids) are among the most frequently prescribed and used drugs [ ] . there are several side effects connected with frequent use of nsaid, mainly gastrointestinal ulcers and bleedings. a plausible molecular mechanism of these effects are direct interactions of nsaids with gastric phospholipids [ , ] . the influence of three well-known non-steroidal antiinflammatory drugs with a diverse gastric toxicity (aspirin, ketoprofen and piroxicam) and three newly-synthesized xanthone derivatives (belonging to the nsaids) on the structure and dynamics of lipid bilayers was studied using small angle x-ray scattering and molecular dynamics simulations. the results showed some correlation between nsaid toxicity and its binding to the lipid polar groups. this binding increases membrane fluidity by reducing its density due to an increased membrane surface area. a reduced lipid packing in the membrane most likely increases gastric mucosa permeability, which can result in a decreased resistance of the gastric mucosa to luminal acid. we studied the partition of the anionic amphiphile pyrenesulfonate (psa) into mlv and luv (produced by extrusion), composed by zwitterionic popc and zwitterionic/anionic mixtures with pops (until mol %). psa is an anionic amphiphile that mimics several xenobiotics (e.g. pharmaceuticals, pesticides) and endogenous substrates that interact with biological membranes. we found increasing b. lorenz, s. schuy, a. janshoff georg august university, göttingen, germany cell-cell and cell-virus interactions are ubiquitous in living organisms. the analysis of the forces acting between two cells or a cell and a virus gives insight into details of these interaction processes such as their stochastics, cooperativity, reversibility, and energy landscape. using colloidal probe microscopy in conjunction with solid-supported lipid bilayer techniques, we mimic the contact between two membranous compounds displaying sponge glycolipids or viral peptides on their surfaces. after spreading functionalized lipid bilayers on both -a colloidal probe and a silicon wafer surface -the molecular interactions are quantified by means of force-distance curves. by probing the dynamic interaction strength between the viral peptides n and c we aim for a deeper understanding of the role of these peptides in the complex process of the formation of the prehairpin intermediate as the key step in retroviral fusion. as far as the cellular interaction of marine sponge cells is concerned, we intend to investigate the strength, specificity, and the ca + dependency of the self-recognition between sponge glycans. the systems advantages are the flexibility of the membrane composition and the control over the distribution of receptor molecules in the membranes. since adhesion in biological systems relies heavily on cluster formation within the biomembrane, we plan to mimic the clustering by "printing" interaction domains and comparing the results to homogeneous samples. development of video-rate imaging microscope using laurdan and its applications to lipid raft t. ohba, k.-i. muto, t. kiuchi, k. ohki department of physics, graduate school of science, aobaku, sendai, japan - , ohki@bio.phys.tohoku.ac.jp various biological functions are located on cell membranes, and the biomembranes maintain heterogeneity as microdomain even in its dynamic structure. existence of such domain was reported as phase-separated 'rafts' in a cell. and the bio-functions of membrane protein are affected by physical property of its surrounding lipid bilayers. laurdan is a useful fluorescence dye to monitor membrane fluidity, and we have developed an instrument to image spatial and temporal change in membrane fluidity by use of laurdan. in order to examine role of 'micro-domain' in biomembrane, we applied this imaging instrument to a giant liposome, cho cells and pc d cells. fluorescence microscope was equipped with a home-build dual-view optical unit. microscopic image of membranes stained with laurdan is separated into an image at nm and an image at nm by the combination of monochromatic filters and dichroic mirrors. the each image is focused on an image plane of a ccd camera side by side. and generalized polarization (g.p.) image is calculated from those images by personal computer according to the definition of g.p. the g.p. imaging at video rate was applied to a giant liposome of composed of dmpc and dmpe in order to observe phase separation. it was also examined that specific interaction of sphingomyelin and cholesterol in living cho cells and neuritis protrusion from raft region of pc d cells stimulated by neuron growth factor. current fluctuations in biological lipid membranes from human cell lines s. nuschele, a. wixforth, m. f. schneider university of augsburg, experimental physics , univer-sitätsstrasse , d - augsburg, germany lipid membranes can undergo phase transitions at physiological temperatures. not only do single lipid component membranes show melting behaviour but also biological lipid membranes from eukaryotes as well as from prokaryotes. performing calorimetric and monolayer studies we are able to detect phase transitions in extracted membranes from different human cell lines (e.g. keratinocytes and melanoma cells). close to and in the melting transition regime we measured distinct channel like current fluctuations. the opening times of these current fluctuations can be predicted based on the lateral compressibility measured from the monolayer isotherm and agree well with the experimental data [*]. the applied method of extraction excludes the presence of functional proteins in the membrane rendering the lipid bilayer as the source of the observed current fluctuations. the molecules are composed of single hydrophobic tail and two hydrophilic aldonamide-type groupings (gluconyl c -dga or lactobionyl c -dla) linked by the propylene chain at the nitrogen atom. the micellization processes of c -dga and c -dla were studied by means of itc. the critical micelle concentrations, the enthalpies (∆h m ) and the entropies (∆s m ) of micellization as well as the contributions of the headgroups to the gibbs free energies (∆g m (hy)) were calculated. qspr analysis was also used to predict cmc of studied compounds. the interactions of c -dga and c -dla with model membranes (dppc and dppc/chol. bilayers) were studied by means of dsc. using quantum computations some basic molecular properties were calculated. the conformational space was explored using molecular mechanics. obtained results were compared with those for analogical compounds with single head groups, e.g. with also synthesised by us n-alkanoyl-nmethyllactitolamines (c n mela) and common sugar-based surfactants c gluc and mega- . enfuvirtide (t- ) was the first hiv- fusion inhibitor peptide approved for clinical use. t- is an inhibitor still under evaluation. previous studies, based on tryptophan intrinsic fluorescence, showed that the peptides interact differentially with membrane model systems (luv) with different lipid compositions. studies with human blood cells were necessary to further establish the role of membranes on these peptides mode of action. an experimental strategy was applied based on the membrane dipole potential, as measured by the fluorescent probe di- -anepps. human erythrocytes and peripheral blood mononuclear cells (pbmc) were successfully labeled. for both systems, a fusion inhibitor concentration-dependent decrease on di- -anepps fluorescence excitation ratio (a measure of the spectral shift and dipole potential) was observed. the quantitative analysis of these variations indicated that t- has an approximately ten-fold higher affinity towards erythrocyte and pbmc, when compared with enfuvirtide. this is in agreement with the previously known adsorption of t- on cholesterol-rich membrane domains and with its higher partition constants. hiv associates with erythrocytes in vivo, which can constitute a route to deliver peptide to the viral membranes (also rich in cholesterol). lymphocyte membranes can concentrate and accelerate the drug interactions with its molecular target, gp in its exposed conformation. oxidation of low density lipoprotein (ldl) is known to be a key step in atherogenesis, leading to inflammation, proliferation and apoptosis of cells of the arterial wall. these effects are largely exerted by oxidatively fragmented phospholipids, which are highly exchangeable between cells, tissues and lipoproteins. in particular, pgpc has been identified in minimally modified ldl and has been reported to elicit a wide range of pathophysiological responses in vascular cells, e.g. the activation of apoptotic signaling pathways. we investigated here the behavior of the fluorescent oxidized pgpe-alexa compared to dhpe-bodipy in artificial supported lipid bilayers with different cholesterol contents. the two labeled lipids differ in the type of membrane insertion: while dhpe-bodipy is anchored to the membrane via two fatty acids, pgpe is incorporated with only one fatty acid. the second chain is an acyl fragment in sn- position, which represents the oxidation product of an unsaturated acyl chain. with increasing cholesterol content we observed a decrease in the diffusion coefficient for both lipids. interestingly, the diffusion of the oxidized lipid was reduced in a higher degree compared to that of the non-oxidized lipid. the calculated ratios of the diffusion constants of pgpe-alexa and dhpe-bodipy suggest a different type of interaction with cholesterol. membrane proteins account for a third of all proteins encoded for by the human genome, and play a vital role in a number of cellular processes. few membrane protein structures have been determined to date in comparison to soluble proteins. this discrepancy is due to experimental difficulties in preparing membrane protein samples for structural analysis. traditional techniques for studying membrane proteins by x-ray crystallography or solution nmr use detergent solubilised proteins which can differ from their native confirmations. solid-state nmr allows the study of transmembrane proteins in lipid bilayers, representing a more native like environment in which to obtain biologically relevant structural information. we have been working on the development of reliable methods for reconstitution of transmembrane peptide into liposomes using glycophorin a as a model tm protein. using reconstitution methods based on the removal of detergent by bio-beads, we have used electron microscopy to screen the ideal conditions for insertion of gpa into liposomes in preparation for mas nmr experiments. em has allowed us to identify conditions favourable for insertion of peptide into lipid vesicles and those that result in aggregation. in order to confirm the secondary structure and insertion of gpa into lipid vesicles, techniques such as cd, ocd, ftir and dls have been used to provide quantitative information in addition to the visual results from em. nonesterified fatty acids regulate a broad spectrum of metabolic activities and are involved in many physiological, pathological and/or pharmacological processes in living cells. they spontaneously transfer between donors and acceptors such as fatty acid binding proteins and lipid membranes. we focus on protein-lipid dispersions of human serum albumin (hsa) and sterically stabilized liposomes (ssl) composed of dppc and appropriate amount of peg: -dppe in which stearic acids (sa) are inserted either in the protein or in the ssl. exploiting the fact that hsa has a single tryptophan residue and that the intrinsic trp-fluorescence emission signal is quenced by the presence of sa, the kinetics of sa transport between hsa and peg: -grafted dppc membranes is studied by means of fluorescence. it is found that the transfer of sa between hsa and ssl is a first-order process and the kinetics of transfer depends on the type of donor and acceptor matrix, on the temperature (i.e., on the physical state of the lipid bilayers), and on the grafting density of the peg-lipids at the protein/lipid interface. indeed, in the absence of polymer-lipids, the rate of transfer increases with temperature in both directions of transfer and it is faster for the passage from dppc bilayers to hsa. the presence of polymer-lipids reduces the rate of transfer both in the mushroom and in the brush regime of the polymer-chains, especially for lipid membranes in the fluid phase. a. orth , w. römer , l. johannes , c. steinem institute for organic and biomolecular chemistry, university of goettingen, germany, laboratoire trafic et signalisation, institut curie, paris, france shiga toxin (stx) from shigella dysenteriae is an ab -class bacterial toxin. infections with stx lead to the haemolyticuraemic syndrome, which is known to be a major cause for renal failure at an early age. the interaction of the homopentameric b-subunit (stxb), which is responsible for binding and intracellular transport of the holotoxin, with its cellular receptor, the glycosphingolipid gb , is the first step for endocytosis of the toxin. one b-subunit can bind up to gb -molecules to form stxb-gb -clusters causing negative curvature of a membrane. the binding of stxb to giant unilamellar vesicles, composed of dopc, cholesterol and gb induces tubular membrane invaginations, which were also found in experiments with energy-depleted hela-cells. in recent studies, protein and lipid reorganization processes after attaching stxb to solid supported membranes, composed of dopc/sphingomyelin/cholesterol/gb have been investigated. the compaction of gb -molecules led to an additional stxb-gb -enriched phase, which was also observed in lipid monolayers at the air/water interface. in this study, the influence of stxb on model membranes will be investigated combining the advantages of free-standing lipid membranes with those of ssms. the impact of stxb on pore-suspending membranes will be followed by confocal laser scanning mi croscopy and atomic force microscopy. a. robaszkiewicz , c. spickett , p. sicinska , g. bartosz , m. soszynski department of molecular biophysics, university of lodz, lodz, poland, strathclyde institute of pharmacy and biomedical sciences, glasgow, u.k. hypochlorite generated in vivo under pathological conditions is a known oxidant, able to initiate lipid peroxidation process, which affects the stability of biological membranes. the aim of this study was the analysis of the products formed during the reaction of hypochlorite with phosphatidylcholines containing unsuturated fatty acid residues ( -stearoyl- -oleoyl-, -stearoyl- -linoleoyl-, stearoyl- -arachidonylphosphatidylcholine) and their effects on the human erythrocytes. using electrospray mass spectrometry we observed complete conversion of the lipids into chlorohydrins, which resulted in the decrease of the rotational correlation time and rotational motion freedom of liposomes estimated by epr using spin probes ( -and doxylstearic acid). unilamellar chlorohydrin liposomes had lower diffusion coefficient for calcein than liposomes made of parent lipids. flow cytometry demonstrated fast incorporation of uni-and multilamellar chlorohydrin liposomes labeled with nbd-pe into erythrocytes. this effect was accompanied by the formation of the erythrocyte subpopulations of higher volume, decrease of the rate of fluorescein diacetate hydrolysis, estimated by flow cytometry, and increase of affinity and maximal velocity of the membrane enzyme acetylcholinesterase. extensive bilayer perforation coupled with the phase transition region of an anionic phospholipid k. a. riske , l. q. amaral , m. t. lamy depto. biofisica, universidade federal de sao paulo, sao paulo, brazil, instituto de fisica, universidade de sao paulo, sao paulo, brazil at low ionic strength dimyristoylphosphatidylglycerol (dmpg) exhibits a broad phase transition region characterized by several superimposed calorimetric peaks. peculiar properties, such as sample transparency, are observed only in the transition region. we use differential scanning calorimetry, turbidity and optical microscopy to study the narrowing of the transition region with the increase of ionic strength. upon addition of salt, the temperature extension of the transition region is reduced and the number of calorimetric peaks decreases until a single cooperative event is observed in the presence of mm nacl. the transition region is always coupled with a decrease in turbidity, but a transparent region is detected within the melting process only in the presence of up to mm nacl. optical microscopy of giant vesicles shows that bilayers first rupture when the transition region is reached and subsequently lose optical contrast. fluorescence microscopy reveals a blurry image in the transparent region, suggesting a different lipid self-assembly. overall sample turbidity can be related to the bilayer optical contrast. our observations are discussed in terms of the bilayer being perforated along the transition region. in the transparent region the perforation is extensive and the bilayer completely loses the optical contrast. financial support: fapesp. label-free imaging of biological membranes using surface imaging techniques j. l. richens, k.-a. vere, p. o'shea cell biophysics group, university of nottingham, nottingham, uk surface plasmon resonance (spr) is a detection technique traditionally used for specific protein detection, which is now exploited routinely as a generic label-free sensor. spr responds to changes on a metal surface conditioned to sense the binding of analytes. thus, the composition of the external medium and metal surface will be fundamental to the signal output. here we use a model phospholipid membrane system to investigate the effect of altered buffer and surface compositions on spr signals. comparisons are undertaken between continuous gold surfaces and gold nanoparticles. we demonstrate that surface electrostatics and the salt composition and molarity of a buffer all have significant impacts on spr output. the association of respiratory syncytial virus matrix protein with membrane microdomains the association of the matrix protein from respiratory syncytical virus with membranes has been characterised by tensiometry, brewster angle microscopy, and atomic force microscopy following deposition of langmuir monolayers onto modified silica substrates. association of the protein with monolayers containing phosphocholines and cholesterol leads to the formation of materials with new properties that differ from those of either of the pure components. the behaviour of the protein in monolayers rich in cholesterol and sphingomyelin exhibits a significant concentration dependence. at low concentrations, the protein exhibits a simple partioning behaviour. at higher concentrations, lipids are extruded from the monolayer below a critical surface area. these findings are discussed in relation to the recently published structure of the protein (pnas, , , - ) , the documented formation of viral filaments during key stages of the infection cycle and the isolation of the protein from detergent-resistant membrane fractions. structure and dynamics of closed melting membranes v. m. rondelli , s. santorsola , p. brocca , e. del favero , l. cantù , m. zimbone dep. of chemistry, biochemistry and biotechnologies for medicine, university of milan, segrate, italy., dep. of physics, university of catania, catania, italy. we studied the dynamical and structural properties of large unilamellar vesicles (≈ nm luvs) of phospholipids (dmpc, dc pc and dmpc : dc pc = : molar mixture) in the temperature range around the chain-melting transition, ≈ • c wide, with . • c resolution and . • c accuracy. small-angle (saxs) and wide-angle x-ray scattering (waxs) measurements show that across the transition the vesicle behaves as an 'evolving membrane', passing through several different states, each of them being characterized by different proportions of coexisting fluid-and gelchains molecules. noteworthy, no kinetics has been detected. on the same samples, a unique and very sensitive laser light scattering technique allows to determine the characteristic times of thermally induced shape fluctuations, connected to the elastic properties of the membranes. results indicate a clear softening of the membranes in correspondence to the chain-melting transition, as indicated by a manifold increase of the corresponding fluctuation characteristic time. meanwhile the overall size of the vesicle is not sensibly changed. this softening is likely to be due to the presence of structural defects, eventually driving to local morphological modifications. thermodynamics characterization of isolated lung surfactant assembled as lamellar bodies e. x. rodriguez , r. alvarez , r. barrio , c. irles , a. ortega biochemistry depto., school of medicine, inst. of physics unam, nat. inst. of perinatology, mexico city, mexico lungs are a large extension of ∼ m of one layer cells, which is structured as compacted sacs called alveoli, where lung function takes place: the gas exchange. alveolar endotelium is formed by two kinds of cells: neumocyte type i (nti), which covers ∼ % of the alveolar surface where gas exchange occurs and, ntii where lung surfactant (ls) is produced. ls are a mixture that lay over a water film in the luminal surface of the alveoli and it is thought to decrease the surface tension to avoid alveolar collapse during expiration. ls are made of phospholipids and proteins, which are determinant for the structure of ls under particular conditions of pressure and temperature. ls inside the cell is packed in organelles called lamellar bodies (lb), and is released to the water/air interphase in other conformation. in the present study we isolate lb from pig's lung and study lb thermodynamic characteristics by microcalorimetry in the presence and in the absence of structural proteins. phase transition profile of lb with and without proteins is basically the same; while ls in the alveolar lumen have a higher transition temperature (tm). mayor changes in tm are observed between lb and ls from alveolar lumen. although ls lipid composition in and outside the cell is assumed to be the same, the ground for the differences in tm under these two conditions is unknown. m. rodrigues , g. rádis-baptista , b. g. de la torre , m. castanho , d. andreu , n. c. santos instituto de medicina molecular, portugal, pompeu fabra university, spain, federal university of cearà, brasil nucleolar-targeting peptides (nrtps) have been recently designed by structural dissection of crotamine, a toxin from the venom of a south america rattlesnake (radis-baptista et al. . j med chem : ) . at µm concentration, nrtps penetrate different cell types and exhibit exquisite nucleolar localization. the aim of this work is to decipher the molecular mechanism for the translocation of nrtps into cells. quantification of partition into membranes was carried out, based on intrinsic tyrosine fluorescence. the role of the bilayer phase, anionic lipids, reducing agents and peptide concentration on the extent and kinetics of partition were studied. both nrtp and nrtp exhibited high partition to popc (neutral) lipid vesicles (k p ≈ × ), which was enhanced by the anionic lipid popg for nrtp , but not nrtp . the peptides showed a decrease in partition for popc:cholesterol (liquid ordered state) or dppc (gel) membranes. depending on the lipid composition, the peptides either increased or decreased their quantum yield upon membrane insertion. quenching experiments with acrylamide showed no peptide aggregation in solution. once the translocation mechanism is fully understood we will test nrtps as carriers of relevant cellular cargos, evaluating their potential clinical application in drug delivery or gene therapy among other applications. the ingestion of trans fatty acids (tfa) formed during the partial hydrogenation of vegetable oils has been linked to a detrimental impact on health by an, as yet, unknown mechanism. we synthesized deuterated analogs of -elaidoyl- -stearoylphosphatidylcholine (t : - : pc) that contains a single "unnatural" trans double bond and -oleoyl- -stearoylphosphatidylcholine (c : - : pc) that contains a single "natural" cis double bond. solid state h nmr, complemented by molecular dynamics (md) simulations, was then employed to compare molecular organization in model membranes prepared from these isomeric molecules. analysis of spectra recorded as a function of temperature reveals a higher chain melting temperature for the trans isomer, indicating tighter molecular packing in the gel state. in the liquid crystalline, however, the difference between the trans and cis isomers is subtle. order as probed by the perdeuterated [ h ] : sn- chain, and corroborated by computer simulation, coincides within < %. only in the conformation of the double bond is an appreciable difference implied. thus, our results contradict the conventional view that tfa resemble saturated fatty acids, which is > % more ordered. (supported by acs, prf -ac .) photooxidation and lateral membrane diffusion of dipole molecules v. s. sokolov , e. a. sokolenko , a. a. lents , p. pohl a.n frumkin institute of physical chemistry and electrochemistry, russian academy of science, moscow, russia, institut fuer biophysik, johannes kepler universitaet linz, austria the photodynamic oxidation of phloridzin, of the styryl dye di- -anepps and of unsaturated lipids was monitored "online" by measuring the collapse of the dipole potential which has been introduced to the membrane by these molecules. their photodamage occurred at different rates when the target molecules and the singlet oxygen generating photosensitizer (phthalocyanin) were adsorbed to the same or to opposite sides of the planar lipid bilayer. the difference in the oxidation rates were attribute to singlet oxygen transport through lipid bilayer and therefore we were able to estimate the permeability of lipid bilayers to singlet oxygen. however, the apparent permeabilities derived from experiments with different targets were differ from each other. therefore, we tested the hypothesis that the lateral membrane diffusion of target molecules and oxidation product may have biased our analysis. in line with this anticipation we found that the apparent permeability is dependent on the size of the planar bilayer. the development of a new mathematical model, which takes the mobility of all reacting species in the aqueous and lipid environments into account, allowed estimation of the real membrane permeability to singlet oxygen. it appeared to be very close to that of oxygen in the ground state. a number of complex three-dimensional lyotropic liquid crystal phases are already known, such as the bicontinuous cubic phases, but so far only a single example has been found -a cubic phase of spacegroup fd m -of a structure based upon a complex close packing of inverse micelles ( ) . we now report the discovery ( ) of a novel lyotropic liquid crystal phase, of space group, p /mmc, whose structure is based upon a hexagonal close packing of identical quasi-spherical inverse micelles. the model membrane system consists of a hydrated mixture of dioleoylphosphatidylcholine, dioleoylglycerol, and cholesterol. this novel phase has a number of unique features which may render it useful for a range of applications. firstly, it is the only known self-assembled lyotropic phase whose structure consists of a periodic close packing of identical inverse micelles. secondly, it is stable in excess aqueous solution, which is very important for potential biological or biomedical applications. references ( ) v. luzzati, v., r. vargas, a. gulik, p. mariani, j.m. seddon, and e. rivas, biochemistry , - ( ) . dystrophin is a rod-shaped muscular subsarcolemal protein. its deficiency is one of the root causes of duchenne muscular dystrophy. dystrophin rod domain contains homologous repeats, where the sub-domain constituted by the repeats to (r - ) was reported to bind actin and membrane lipids. we analyzed the interaction of r - with lipid monolayers. to better understand the assembly mechanism of this protein with lipids, we studied its adsorption behavior at the air-liquid and lipid/liquid interface using ellipsometry, surface pressure, and atomic force microscopy (afm). using two different mixtures of phospholipids, ellipsometry and pressure surface data show that r - interacts with the lipid monolayers, but is inserted into the monolayer formed by dopc-dops while it lies below the monolayer of dopc-dope. this indicates that r - interacts more strongly with dopc-dops than with dopc-dope. afm images show that the pressure of lipid monolayer influences the organization of r - . when the lipid surface pressure is mn/m, r - forms a striking network, indicating a protein-protein interaction in addition to the protein-lipid interaction. this unique behavior of one part of the central domain of dystrophin may explain its key role in muscle cell. studies on polyphenol extracts from helichrysum l., fagopyrum mill., crataegus l. and hypericum l. were performed in order to find if they may be applied as a natural free radical scavengers protecting biological membranes against peroxidation. ghosts erythrocytes were used in the experiments. they were suspended in tris-edta solution, ph , , then.irradiated with a bactericidal lamp without (control) or with a proper amount of the extracts studied. the product of lipid peroxidation was malonic dialdehyde (ma). the colour reaction of ma with thiobarbituric acid (tba) enables to determine the concentration of ma spectrophotometrically. it was found that peroxidation increased with the irradiation time. however, it significantly decreased when concentrations of poliphenols increased. the best antioxidtive property was found for hypericum l. the antioxidative efficiency sequence of the plant extracts studied was the following: hypericum l. > crataegus l. > fagopyrum mill. > helichrysum l. the results obtained indicate that polyphenol extracts exhibit excelent antioxidative properties that make them good free radical scavengers for efficient protection of biological membranes. this work was sponsored by ministry of science and education, scientific project no. n n . interaction of cationic porphyrins with neutral and negatively charged liposomes i. voszka , g. corradi , p. maillard , h.-j. steinhoff , g. csík institute of biophysics and radiation biology, semmelweis university budapest, hungary, research institute for solid state physics and optics, hungarian academy of sciences, budapest, hungary, institute curie, section de biologie, orsay, france, fachbereich physik, universitat osnabrück, germany porphyrin derivatives are used in photodynamic therapy of tumors. the knowledge of the photosensitizer location within the cell is important. the effect of porphyrin derivatives containing cationic side groups was examined on neutral and negatively charged liposomes by esr. the esr signal was first examined as a function of temperature and porphyrin concentration in the dark. a significant change related to the appearance of quasi free spin labels was obtained for spin probes at the th carbon atom and was more expressed for the asymmetrical derivative. illuminating the samples the esr amplitude decreased for all positions of the spin probe but to different extent. the effect was more expressed in case of the symmetrical derivative, especially for label positions and . for the asymmetrical derivative the effect changed from moderate to weak from the th to the th position. this indicates that the asymmetrical derivative is incorporated nearer to the lipid head groups while the symmetrical one may be located deeper in the membrane. under oxygen-free conditions both derivatives showed weaker but still pronounced effects.. single channel recording of α-hemolysin in nanopore-spanning tethered bilayer lipid membranes n. t. thet , i. pfeiffer , w. knoll , i. köper max planck institute for polymer research (mpi-p), ackermannweg , mainz, austrian research centers gmbh -arc, tech gate vienna, vienna, austria artificial lipid bilayer membranes mimic biological cell membranes in many aspects and can be used to study functional processes such as ion channeling, signal transducing, transport of nutrients etc. as most of the functions of a cell are accomplished by membrane proteins, research has been ongoing in studying and characterization of membrane proteins embedded in model lipid bilayer membranes. black lipid membranes (blms) were studied for decades but their potential for practical applications is hindered, mainly due to their lack of stability and limited lifespan. recently, tethered bilayer lipid membranes (tblms) proved to have long life span of up to several months without significant changes in membrane resistance and capacitance. in order to combine advantages of blms and tblms, we have designed a membrane system which is freely spanned across a single nanopore in a silicon nitride membrane. this system not only mimics cell membranes, but also it allows control over the chemical composition of buffer with unlimited ionic reservoirs on both sides of the membrane. this freestanding tblm maintains structural stability and lifetime of up to hours without significant decrease in its structural integrity and electrical sealing. for the validation of the tblm formation, we have inserted well known α-hl pores. we are able to control the amount of α-hl pores insertion and measure single channel ion transport across the tblm by α-hl pores using a capacitor feedback amplifier. the language of shape: biological reactions are dramatically affected by the shape of lipid membrane d. stamou university of copenhagen, copenhagen, denmark a plethora of biological process are taking place on the surface of lipid membranes. as a rule membranes in vivo are curved in a variety of complex geometries. here i will present a quantitave study on the influence of membrane curvature on protein-membrane and membrane-membrane interactions. to gain systematic access to a continuum of membrane curvatures we immobilized liposomes on a surface at dilute densities. using confocal fluorescence microscopy we imaged single liposomes of different size, and therefore different curvature, and monitored their interaction with a binding partner (proteins or other liposomes). i will discuss unpublished data on two important classes of biomolecular interactions that exhibited dramatic curvature dependence: a) snare-mediated docking and fusion b) anchoring of peripheral proteins. the following references provide partial information on the single-liposome assay: a. zettergren, c. gudmundsson, t. nylander, e. sparr physical chemistry , lund university, lund, sweden there is accumulating evidence of substantial amounts of phospholipids in the cell nuclei , although the function of these lipids is still not fully understood. it has been shown that the chromatin complex composed of dna, rna and proteins also includes phospholipids, and that rna colocalize with these . although the rna-phospholipid interactions may have important implications to biological function, in gene therapy and in medicine, very little work has been dedicated to the characterization of rna interaction with phospholipids. the objective of this work is to investigate the adsorption behavior of short single stranded bases long rna (ssrna ) molecules (similar to mirna) to lipid monolayers at the air-water interface as well as to study how the presence of rna affect the domain formation in the monolayers using fluorescence microscopy. monolayer studies have shown adsorption of ssrna to monolayers consisting of zwitterionic dppc as well as to monolayers consisting of cationic dodab. the adsorption behavior of these very short nucleic acids differ significantly from the adsorption process for longer nucleic acids as for example a base pairs long ds dna (dsdna ) which has been used as a reference system . the presence of ssrna significantly changes the compression isotherm of both dppc and dodab monolayers. plant defensins are cysteine-rich antimicrobial peptides found in various plant species. they share a common threedimensional structure, stabilized by eight disulphide-linked cysteines consisting of three antiparallel β-strands and one α-helix. most plant defensins show antifungal activity with no effect on mammalian and plant cells. expression of these peptides in plant tissue is induced by pathogen infection. the mechanism of defensin action is based on membrane permeabilization. this occurs through an interaction with high affinity binding sites on fungal membranes, resulting in alteration of membrane potential. the genes encoding for a peach ppdfn and a grape vvamp defensins were expressed in e. coli and purified to homogeneity. they were tested for antimicrobial activity against some fungi and showed to have an inhibitory effect on the spore germination. biophysical analysis showed that defensins were able to interact with artificial membranes. binding of defensins to membranes was dependent on lipid composition, increasing with the sphingolipids content. interaction between peptides and sphingolipids could lead to insertion of the defensins into the membrane resulting in its destabilization. extracts of the plant perilla frutescens have many uses in the asian kitchen, e.g. as a popular garnish used in sushi. the plant is also employed in eastern traditional medicine to treat a variety of ailments including colds, food allergy and depression. two of the main constituents of the plant are limonene and perilla aldehyde. by bio-oxidation, these molecules can be converted to perillic alcohol and perillic acid. these cyclic terpenes possess antibacterial and anticarcinogenic properties. the modes of action for these compounds are at present not understood, but their remarkably diverse pharmacological properties suggest that they might target the phospholipid matrix of the cellular lipid membrane. indeed, the cyclic terpenes can bind to, and alter the physiochemical properties of the lipid bilayer of the membrane, the effects of which can cascade down to several essential cellular processes. here, we use molecular dynamics (md) simulations to investigate the effect of limonene and its derivatives on the properties of lipid bilayers including the changes in acyl chain order parameters, bilayer thickness and the area per lipid. md can afford molecular-scale dynamic information, often not easily accessible from experimental measurements. this information can be used to interpret existing experimental data obtained by e.g. isothermal titration calorimetry, electron paramagnetic spectroscopy and differential scanning calorimetry. catalysis in the membrane: interfacial mechanism of phospholipase a h. p. wacklin , r. k. thomas institut laue langevin, grenoble, france, physical and theoretical chemistry laboratory, oxford university, u.k. phospholipase a cleaves the sn- acyl chains of membrane phospholipids and performs a range of physiological functions. one of the least well understood aspects of the its mechanism is how its activity is regulated by the interaction with the substrate membrane. we have used neutron reflection to monitor changes in membrane structure during lipid hydrolysis [ , ] . the penetration depth of pla depends on lipid packing and increases during the lag phase of porcine pancreatic pla . by using a selectively deuterated lipid substrate, d -popc, we determined the relative membrane-water partitioning of the lipid products in-situ. the lyso-lipid product partitions into the solution phase, while fatty acid accumulates in the membrane and increases the affinity of pla [ ] . pla is inhibited at ph , which is consistent with protonation of the catalytic histidine. however, irrespective of ph, pla is fully activated by me-betacyclodextrin, which facilitates the release of the lyso-lipid from the enzyme-substrate complex. me-beta-cyclodextrin does not interact directly with the membrane surface or the substrate lipids, indicating that product release occurs outside the immediate membrane-water interface. diffraction limits resolution in optical microscopy, but many interesting biological problems occur on shorter (molecular) length scales. recently, methods to circumvent the diffraction limit have been presented. fluorescence photoactivation localization microscopy (fpalm) uses activation of many small subsets of photoactivatable or photoswitchable fluorescent probes (pafps) to generate images with effective resolution in the tens of nanometers. pafp molecules are photoactivated, imaged, localized, and photobleached in small numbers. the process is repeated for many subsets to build up data on thousands to many hundreds of thousands of molecules. the positions of all localized molecules are used to construct an image of the sample with resolution limited not by diffraction, but by the localization precision and molecular density. results will be shown from a variety of biological systems, including live and fixed cells expressing a variety of pafp-tagged proteins. bi-plane fpalm can image in three dimensions with demonstrated resolution of nm x nm x nm. polarization fpalm can image both molecular positions and anisotropies simultaneously with lateral resolution of ∼ - nm. using these powerful capabilities, many potentially interesting biological problems can be addressed. binding of the hiv- ncp on oligonucleotides at the single-molecule level j. godet, p. didier, a. jouonang, y. arntz, y. mély laboratory of biophotonics and pharmacology, umr cnrs , university of strasbourg, france the nucleocapsid protein (ncp ) of the hiv- is a small basic protein which plays key functions in the viral life cycle. the activity of ncp mainly rely on its potent rna-and dna-chaperone activities that direct the rearrangement of numerous nucleic acid molecules into their most stable conformation. two main features of nc's chaperone activity are its abilities to aggregate and destabilize nucleic acids. in addition, the rapid kinetics of ncp interaction with nucleic acids was recently proposed as another major component of nc's chaperone function based on the apparent correlation between an indirect measurement of the nucleic acid dissociation kinetics of ncp and its overall chaperone activity. but so far, no direct measurement of the on/off rates of ncp binding to oligonucleotides was performed. in the present work, we realized single molecule fluorescence resonance energy transfer (smfret) measurements to probe the transient interactions of a tmr-labelled ncp with a short cy -labelled dna oligonucleotide confined into nanovesicles. after confirming the efficiency of the ncp /odn complex encapsulation into the nanovesicles by fcs, the vesicles were tethered to the surface for immobilization. integrity of the entrapping vesicles attached on the surface was confirmed by afm. finally, the association and dissociation constants retrieved from these smfret measurements were discussed in the context of the dna-chaperoning activity of the protein. high-resolution spatiotemporal organization of the integrin lfa- m. f. garcia-parajo , t. s. van zanten , g.-j. bakker , r. diez-ahedo , a. cambi , c. g. figdor bionanophotonics group; ibec, barcelona, spain, tumour immunology dept; ncmls, nijmegen, the netherlands lfa- is a leukocyte-specific integrin involved in different steps of the immune response. on monocytes, lfa- plays a key role in the regulation of monocyte-endothelial interaction during rolling, arrest and extravasation into the underlying tissue. i n-vivo experiments showed that blood borne lymphocytes can 'switch' within seconds from rolling to arrest. furthermore, tem observations of pro-active, ligandindependent nanoclusters confirmed that affinity and clustering are complementary processes required in adhesion. yet, the mechanisms leading to fast-switching remain obscure. in our group, we used a combination of single molecule fluorescence techniques to study the spatiotemporal organization of lfa- on monocytes. we performed optical nanoimaging of lfa- nanoclusters in relation to membrane rafts with a resolution of nm and accuracy of ∼ nm. in quiescent cells, lfa- nanoclusters do not associate with membrane rafts and diffuse freely on the membrane. binding of the integrin to its ligand icam- induces the formation of microclusters that further associate with rafts and exhibit reduced mobility, consistent with cytoskeleton interactions. our work highlights the markedly different spatiotemporal organization of lfa- that might explain its concerted action to form larger and stable platforms on the cell surface required for rapid and effective cell adhesion. c. ciobanasu, u. kubitscheck rheinische friedrich-wilhelms-universität bonn, germany cell penetrating peptides like the hiv tat peptide have the property to rapidly translocate the cell membranes and the capability to deliver a wide range of cargoes. the mechanism of the membrane translocation is still under investigation and object of considerable controversy. we applied and single-molecule and confocal laser scanning microscopy (lsm) to study peptide-membrane interactions. electron-multiplying ccd cameras yield images of single fluorescent molecules with a time resolution in the range of a few milliseconds only, which allows the tracking of fluorescently labelled peptides and lipids at bio-interfaces in realtime with a localization precision of a few nanometers. we formed giant unilamellar vesicles (guvs) from different lipid mixtures and examined their interaction with fluorescently labeled tat peptides. we found that the passive peptide internalization process depends on lipid composition, charge of the lipid bilayer, and the ionic properties of the medium. a translocation of cationic tat peptides was observed in membranes containing at least mol% of lipids with a phosphatidyl ethanolamine or a high mol fraction of the phosphatidyl serine head group. in salt-free solution tat efficiently bound to guvs, however, in a physiological nacl solution tat binding was completely abrogated, but the peptides efficiently equilibrated across the guv membrane. new approaches to measure interactions in the live cell plasma membrane g. j. schütz biophysics institute, johannes kepler university linz, austria in my lecture, i will show examples how to obtain insights into the organization of the cellular nanocosm by single molecule experiments. our primary goal is an understanding of the role of such structures for immune recognition. brightness and single molecule colocalization analysis allows us to study stable or transient molecular associations in vivo. in particular, i will present results on the interaction between antigen-loaded mhc and the t cell receptor directly in the interface region of a t cell with a surrogate antigenpresenting cell. in addition, we developed a method for in vivo micropatterning of plasma membrane proteins to measure molecular interactions. the method allows identifying and characterizing interactions between an arbitrary fluorescence labeled protein ("prey") and a membrane protein ("bait") directly in living cells. cells transfected with a fluorescent fusion protein of the prey are plated on micropatterned surfaces functionalized with specific antibodies to the extracellular domain of the bait; the fluorescence copatterning is used as readout for the interaction. we applied this tool for the study of the interaction between cd -the major coreceptor for t cell activation -and lck, an important tyrosine kinase in early t cell signaling. in addition to the well-known zinc-clasp structure, we found strong contributions of lck membrane anchorage for the binding of the two proteins. developing a fluorescent redox sensor for monitoring metal-ion mediated catalysis in biosystems a. rybina , a. kiel , b. thaler , a. sprödefeld , r. krämer , d. p. herten bioquant and, department of inorganic chemistry, heidelberg university, germany a fluorescent redox sensor is an electron photo-switching device that can be used for the characterization of the redox state of a given environment. it combines a fluorescent fragment with a redox-active unit that senses the media by a redox reaction and controls the light emitting properties of the fluorophore. such reversible sensor can help to examine the electrochemical state and changes in biological systems during biochemical processes in real time. recently a new fluorescent molecular sensor with a redox-active hydroquinoneunit covalently linked to fluorophore rhodamine b was developed. (kierat r.m. et al., bioorg. med. chem. lett., -accepted) . the reduced hydroquinone-form of the sensor is fluorescent while its oxidation to benzoquinone-derivative leads to a significant decrease of the fluorescence. although the above method shows great promise for applications in biological systems, the exact mechanism of this process is not fully understood yet. we use fluorescence spectroscopy to investigate oxidation reactions on the ensemble and single-molecule level and study kinetic rates. the proposed strategy is to use cu (ii) complex as oxidation mediator immobilized on surface via dna linker to examine the oxidation mechanism. fluorescently labeled atp as a probe of the outer mitochondrial membrane barrier: role of vdac fluorescence correlation spectroscopy (fcs) was applied for studying the distribution of fluorescently labeled atp (bodipy-atp) in isolated mitochondria. the setup and peak intensity analysis (pia) was described in our recent paper (perevoshchikova et al. biochim.biophys.acta : . the binding of bodipy-atp to mitochondria was maximal in the non-energized state, whereas the addition of succinate (respiratoty substrate) or atractyloside (adenine nucleotide translocase inhibitor) led to a decrease in the binding. nadh reduced the fcs signal from bodipy-atp added to non-energized mitochondria more than nad+ did under the same conditions suggesting the control of nucleotide transport through voltage-dependent anion channel (vdac) residing in the outer membrane. konig's polyanion also decreased the bodipy-atp binding to mitochondria with the effect being reduced by alamethicin or digitonin. control experiments showed that bodipy-atp did not bind to liposomes showing minor role of unspecific binding. it was suggested that bodipy-atp in combination with fcs can be used to monitor the functional state of mitochondrial vdac which is considered to be a principal regulator of mitochondrial function. fluorescence correlation spectroscopy studies of lysozyme partition to phospholipid vesicles a. m. melo , a. coutinho , m. prieto cqfm and in, ist, - lisboa, portugal, dqb, fcul, - , lisboa, portugal binding to membrane lipids has been increasingly recognized as an important step in the aggregation and cytotoxicity of several amyloidogenic proteins [ ] . in addition, it has been recently reported that membranes containing negatively-charged phospholipids can also trigger rapid amyloid-like fiber formation by a variety of several nonamyloidogenic proteins, such as cytochrome c and lysozyme [ ] . our study aims to elucidate the factors that govern the formation of these lipid-protein complexes. given the importance of electrostatic interactions between the proteins and the acidic phospholipids in the putative membrane-induced protein misfolding step, it is essential to first characterize quantitatively the protein partition behavior towards liposomes prepared with variable anionic lipid content. in this study, lysozyme was chosen as a model protein and fluorescence correlation spectroscopy (fcs) was used to monitor its binding to liposomes after its conjugation to alexa fluor . most organic dyes labelling techniques produce a mixture of populations of molecules labelled with a different number of fluorophores. the influence of this poli-dispersity of labelled molecules on the protein partition behaviour will be explored, namely the ability of the fcs technique to detect the production of non-competent membrane-binding species. t. wohland , p. liu , x. shi , y. h. foo , t. sudhaharan , s.-w. chong , v. korzh , s. ahmed chemistry dept., singapore nat. univ., medical biology inst., singapore, molecular & cell biology inst., singapore biomolecular interactions have been measured mostly under in vitro conditions because of higher accuracy and ease of measurement. however, it has become clear that the cellular environment has an important influence on these interactions. it was therefore necessary to develop new tools to allow the measurement of interactions in cells and organisms. recently, we have developed a modality of fluorescence cross-correlation spectroscopy (fccs) called single wavelength fccs (sw-fccs), which uses one-photon excitation to excite two fluorophores with overlapping absorption but separable emission spectra. sw-fccs has been used to determine e.g. dimer fractions of proteins in live cells. here we aim to extend the use of sw-fccs to cells and organisms. in the first part we determine the dissociation constants of a small rho-gtpase (cdc ) with an effector domain (crib) and two effectors (n-wasp or irsp ). in the second part we measure the binding between cdc and a scaffolding protein (iqgap ) in dependence of their expression levels in cho cells and in live zebrafish embryos. by using gfp/mrfp fusion proteins we can excite both fluorophores at nm and detect them separately in two different wavelength channels. we quantitatively determine the dissociation constants and compare their differences in vitro, in cells, and in embryos. these experiments demonstrate that sw-fccs is a powerful biophysical tool for the quantitation of biomolecular interactions in cells and organisms. addressing plasma membrane structure at the nanoscopic length-scale s. wieser, m. axmann, g. j. schütz biophysics institute, johannes kepler university linz, altenbergerstr. , a- linz, austria there is increasing interest in a detailed understanding of the structure and dynamics of the cellular plasma membrane, primarily based on recognizing its essential role for controlling cellular signaling processes. we employed single molecule fluorescence microscopy to study diffusion of cd , a gpi-anchored protein, in the plasma membrane of living t cells at sub-wavelength resolution, both on the cell body and on tunneling nanotubules connecting cells. the lateral motion of this single fluorescence labeled molecule was imaged on a millisecond time scale. within the experimental errors, no indications for confined diffusion for cd on the cell body in t cells have been found. furthermore by separating longitudinal and transversal mobility, we found isotropic diffusion behavior on the surface of tunneling nanotubules. in both studies we analyzed the mean square displacement as a function of the time-lag and the distribution of displacement steps. however, a closed analytical theory for these analysis is only available for the simplest models. to address a suspected diffusion process we reasoned that a full analytical description may not be required; it may well be sufficient to compare the experimental data with monte carlo simulations of the process. we demonstrated the working principle for this simulation based analysis for free diffusion, hop diffusion and transient binding of the tracer molecule to slowly moving receptors. n. chakroun , f. fraternali , m. malfois , h. rezaei , c. a. dreiss king's college london, u.k., diamond light source, didcot, oxfordshire, u.k., inra, jouy-en-josas, france prion(prp) diseases are fatal neurodegenerative diseases affecting mammals including human and sheep.they are characterized by the accumulation of extracellular βrich fibrillar deposits of a structurally modified form (prp sc ) of the cellular prp c .despite the increasing interest for prp diseases,the mechanism of prp c /prp sc conversion is still unknown.studies on prp diseases suggest that neurotoxicity arises from small pre-fibrillar oligomers.we have used a range of biophysical techniques combined with molecular dynamics simulations (md) to resolve the oligomerization pathways of sheep prp (sprp).we have shown that under well established conditions, sprp oligomerizes into three oligomers, which form in parallel.in addition, we have now identified the minimal region of sprp leading to the same oligomerization profile of the entire sprp,namely h h .low resolution shapes of sprp, h h and resulting oligomers have been determined by small-angle x-ray scattering.time-resolved studies have been used to follow the oligomerization of sprp and h h monomers into the oligomers.the conversion of sprp sc at the molecular scale was studied by md.simulations of the h h region recreating experimental conditions revealed a complete unfolding of h helix followed by h helix.these crucial steps are followed by the formation of β-sheet structures leading to a stable βrich double hairpin structure. single-molecule force spectroscopy investigation of the conformational equilibria of alphasynuclein m. brucale , a. rampioni , m. sandal , i. tessari , l. tosatto , l. bubacco , b. samorì istituto di biochimica g.moruzzi, università di bologna (italy), dipartimento di biologia, università di padova (italy) alpha-synuclein (asyn) is an abundant intrinsically disordered protein (idp) primarily located at presynaptic terminals. mutations in the gene encoding asyn have been linked to early-onset parkinson's disease (pd). by means of single molecule force spectroscopy (smfs) experiment, we show how the conformational equilibrium of monomeric wild type (wt) asyn shifts toward more compact structures in several unrelated conditions linked to pd pathogenicity [ ] . the conformational heterogeneity of pathological alpha-syn mutants a p, a t and e k has also been characterized, revealing marked differences in the conformational behaviors of the mutants with respect to wt asyn [ ] . all the mutants show a distinctively higher propensity, with respect to wt, to acquire a monomeric compact conformation that is compatible with the acquiring of beta structure. the same smfs experimental methodology is then used to characterize the conformational behavior of wt and mutant asyn in a variety of conditions, in an attempt to gain insight about the multiple and contrasting parameters controlling the equilibrium. in vitro protein folding studies using chemical denaturants have contributed tremendously to our understanding of the folding thermodynamics and kinetics of water-soluble proteins. this is not the case for integral membrane proteins, which constitute about one third of all eukaryotic proteins and more than half of all validated and potential drug targets. fully reversible denaturant-induced unfolding remains limited to a few β-barrel porins, whereas the much larger and more relevant class of α-helical membrane proteins has thus far evaded this approach. we report here the first example of an α-helical membrane protein that can be unfolded completely and reversibly by a chemical denaturant: mistic, a -residue protein from bacillus subtilis [ ] , dissociates from detergent micelles or lipid vesicles and assumes an unfolded monomeric state on titration with urea. using spectroscopic and microcalorimetric techniques, we exploited this unique property to provide (i) a quantitative comparison of membrane protein stability in different membrane-mimetic systems; (ii) an experimental test of controversial predictions [ ] regarding the folding core of mistic; and (iii) a convenient setup to study the spontaneous, translocon-independent membrane insertion of this unusual membrane protein. the mechanical functioning of biological tissues is important from many viewpoints such as diseases, clothing and even food. the protein collagen makes up the greater part of these tissues, and is remarkable for its many uses in the body, however there are at least two other major components. one of the most interesting properties of these tissues is their non-linear behavior under stress. this behavior is essential to prevent a catastrophic failure such as an aneurysm. at least three major theories have been proposed within the past few years to explain this behavior, but have been impossible to verify. in order to determine a correct description of the mechanical structure of the tissue we have been using cutting edge technological solutions to address the single molecules within the extra cellular matrix. this technique combines optical methods with single molecule force spectroscopy, allowing stiffness measurements over the nanoscale as well as determining the individual protein tensions within the extra cellular matrix. the results show that this method can be used to determine the network properties even in the complicated aortic wall enabling better understanding of disease states, which in this case include marfan's syndrome and ascending aortic aneurysms. beta amyloid peptide abetapy - shows a faster aggregation kinetics than abeta - c. d'arrigo , m. tabaton , a. perico institute for macromolecular studies, national research council, genova, italy, department of neurosciences, university of genova, genova, italy we test directly the differences in the aggregation kinetics of three important beta amyloid peptides, the full-length abeta - and the two n-terminal truncated and pyroglutamil modified abetapy - and abetapy - , found in different relative concentrations in the brains in normal aging and in alzheimer disease. we find by following the cd signal and the tht fluorescence of the solution in phosphate buffer, a substantial faster aggregation kinetics for abetapy - . this behavior is due to the particular sequence of this peptide which is also responsible of the specific oligomeric aggregation states, found by tem, during the fibrillization process which are very different from those of abeta - , more prone to fibril formation. in addition abetapy - is found here to have an inhibitory effect on abeta - fibrillogenesis, coherently with its known greater infective power. this is an indication of the important role of this peptide in the aggregation process of beta-peptides in alzheimer disease. the puzzle of the anomalously long denaturation kinetics of green fluorescent protein (gfp) mutants still is largely unveiled. in this study we have followed the effect of mutation h g on the stability of gfpmut (mut g) in the presence of guanidinium hydrochloride (gdnhcl). different techniques of fluorescence spectroscopy have been employed in order to obtain information concerning the unfolding event: time resolved fluorescence, fluorescence correlation spectroscopy (fcs), and fluorescence anisotropy. the substitution of the histidine with glycine affects protein stability versus ph: in particular mut g kinetics is not ph dependent and at basic ph values the protein is less stable. the fluorescence properties (quantum yield, lifetime) and the rotational correlation time are unchanged during the unfolding dynamics, while the number of fluorescent proteins decreases exponentially. an extrinsic probe, bound to cysteine , has been employed in order to gain more insights on the unfolding process, monitoring the stability of a different region of the protein. in particular, it has been found that a softer region is present around cysteine in both gfp variants, showing that the unfolding process does not follow a simple two step mechanism. recently, negatively-charged membranes were reported to catalyze amyloid fiber formation by amyloidogenic peptides/proteins and also to induce formation of "amyloidlike" fibrils by nonamyloidogenic proteins. here, we used an approach which combines steady-state and time-resolved fluorescence measurements to obtain structural information about these supramolecular assemblies and to gain insights about the factors that control their formation. by exploring a wide range of lipid concentrations, the interaction of alexa -lysozyme with phosphatidylserine-containing membranes was found to be a complex multi-step process, critically dependent upon the protein-to-lipid molar ratio (p/l) used. upon increasing the total lipid concentration in solution, there was a balance between an increased overall protein binding to the lipid vesicles and a progressive protein dilution on the membrane surface. as the surface potential of the vesicles decreased upon increasing the protein interfacial coverage of the liposomes, the protein binding mode was found to switch from a peripheral binding of lysozyme to the anionic headgroups (at low to intermediate p/l) to a partial insertion of the basic protein into the hydrophobic core of the membrane (at a high p/l). it is hypothesized that disruption of the protein tertiary structure might be a stepwise process beginning with loosening of the structure caused by its deeper insertion in the membrane bilayer. unexpected scaling laws in the mechanical unfolding of single protein molecules m. clusel, e. i. corwin, h. lannon, j. brujic center for soft matter research, physics department, new york university, new york, ny, usa it is a question of fundamental importance to understand the response of proteins to a stretching force, particularly in the case of mechanically active proteins, such as those in muscle fibers. we aim to understand how the structure and topology of a protein affect its resilience to external forces and presumably its function. owing to recent advances in single molecule force-clamp spectroscopy using the atomic force microscope (afm), we are now able to probe the structure and dynamics of single proteins under a constant stretching force by measuring their end-to-end length over time. the probability distribution of unfolding times at a given force allows us to estimate the strength of the protein in terms of a characteristic energy barrier, while the shape of the distribution provides a window into the microscopic mechanism by which the protein breaks apart. here we show a novel scaling of the unfolding kinetics with the stretching force, which deviates significantly from the currently accepted bell model. instead, we propose a physical picture for forced unfolding that is analogous to the mechanics of fracture. v. garcía-gonzález, j. mas-oliva instituto de fisiología celular. universidad nacional autónoma de méxico. méxico, d.f. méxico. studies focused on the thermodynamic and kinetic analysis have demonstrated that transfer of neutral lipids such as cholesterol esters through an aqueous phase is a highly costly biophysical event. therefore, nature has developed a series of lipid transfer proteins such as the cholesterylester transfer protein (cetp) designed to efficiently lower the energy barrier for transfer of cholesterol-esters between lipoproteins through an aqueous environment. employing circular dichroism we evaluated the secondary structure stability of a small peptide derived from the carboxy-end of cetp (helix y ) in a wide range of ph's. the percentage of α-helix is diminished only at extreme temperatures and acidic ph's in a reversible way. we report that while a mixture of phosphatidylcholine/cholesteryl-ester forms large aggregated particles independently of ph, inclusion of helix y to the mixtures close to neutral ph's allows the formation of small micellar-like structures confirmed by dynamic light scattering and electron microscopy. these results suggest that helix y when close to physiological ph values presents the ability to organize a micellar structure around itself. this type of organization allows the process to dramatically lower the energetic barrier for lipid transfer through aqueous media, phenomenon directly related with the facilitation of lipid transfer between lipoproteins. mimicking metastasis by a novel microfluidic approach there is increasing evidence that cancer metastasis shares commonalities with thrombosis. the von-willebrand-factor (vwf), a mechanical active blood clotting protein appears to be a particular potent candidate to bridge the gap between clotting and cancer extravasation. modeling the crucial physiological conditions of the blood circulatory system, for an in situ study of blood clotting-metastasis connections is not only, absolutely necessary, but also a challenging task. here, we present acoustically driven flow as a novel microfluidics method for mimicking the blood flow. this method enjoys very beneficial advantages of possibility of handling very little volumes of fluids, together with freedom to model most of the geometries present in our microcirculatory system. one technologically challenging, yet physiologically important factor, is the hydrodynamic condition in a bifurcated vessel, where complex shear profiles arise. we present a model to mimic these conditions and discuss the impact of hydrodynamics on vwf mediated cancer cell adhesion in bifurcated vessels of our microcirculatory system. protein structural changes occurring in flows stresses inherent to viscous fluid flow have previously been associated with protein unfolding, although structural changes have not been well documented as a function of relevant hydrodynamic parameters. we have used raman spectroscopy to monitor the structure of various protein solutions in situ for multiple flow scenarios within a concentric cylinder fluidic device ( ) . the flows, which ranged from circular couette to wavy taylor-couette flow, were characterised experimentally using particle image velocimetry. several proteins were observed to change conformation when exposed to these flows, although the nature of these changes was protein specific. shearing hen egg white lysozyme in water altered the protein backbone structure, while similar shear rates in a % glycerol, % water solution affected the solvent exposure of the side chain residues near the exterior of the α-domain. the solventdependent response may be due to the flow topology, viscous stress, or the surface hydration properties. comparison of spectra acquired at different time points, including before and after flow, confirmed that the observed changes are reversible and independent of fluid stress exposure time. the scripps research institute, la jolla, ca, usa. intrinsically disordered proteins are increasingly found to play major roles in cell biology and disease. we are utilizing single-molecule fluorescence methods to probe these complex and highly dynamic molecules, allowing more direct measurements of structural distributions and dynamics, while avoiding loss of information due to ensemble averaging. in one example, we investigated the structural dynamics of sup -nm, whose regulatable amyloid formation is believed to have functional significance in yeast. using a combination of single-molecule fret as a molecular ruler, coincidence to interrogate intermolecular interactions, and correlation analysis to probe conformational dynamics, we showed that the monomeric protein populates an ensemble of compact and rapidly interconverting conformations. a particularly interesting feature of intrinsically disordered proteins is that they are relatively unstructured in isolation, but can gain stable structure by interaction with binding partners. in this context, we used single-molecule fluorescence to characterize the complex folding pathway for the parkinson's-related idp alpha-synuclein induced by binding to a lipid-mimic. this combined single-molecule fluorescence methodology provides a powerful approach for detailed studies of the coupling of folding and dynamics with interactions and biology of this important class of proteins. m. ito , j. johansson , r. stromberg , l. nilsson department of biosciences and nutrirtion, karolinska institutet, huddinge, sweden, department of anatomy, physiology and biochemistry, swedish university of agricultural sciences, the biomedical centre, uppsala, sweden amyloid β-peptide (aβ) is a - amino acid polypeptide and known to aggregate and form insoluble amyloid fibril, which is regarded as a primary cause of alzheimer's disease (ad). the discovery of practical and effective treatments and drugs for ad has been waited eagerly. in a recent experimental study, it was suggested that stabilization of the helical conformation of the aβ middle region, which strongly favors collapsed coil formation in the extracellular environment, would reduce the aβ fibril formation. based on the experimental evidence, inhibition of the unfolding of the aβ α-helix can be a forceful strategy to repress the aβ fibril formation resulting in prevention of ad. however, the detailed mechanism of the unfolding of the aβ α-helix has remained unclear, because the x-ray or the nmr structure of the aβ α-helix in aqueous solution has not been reported due to its instability. the aim of this study is to find effective ways to inhibit the unfolding of the aβ middle region, which is a prerequisite for the aβ fibril formation. in this study, we attempted to elucidate the molecular mechanism of the aβ unfolding by molecular modeling and molecular dynamics (md) simulations. the md simulations were performed for α-helical structures of a wild-type aβ( - ) model and a mutant aβ( - ) model. linker average hydrophilicity as a tool to discriminate between extended and non-extended calcium binding proteins a. isvoran , e. quiniou , c. craescu , l. mouawad west university of timisoara, department of chemistry, pestalozzi , timisoara, romania, inserm u , centre universitaire paris-sud, bâtiment , orsay, france the ef-hand calcium binding proteins (cabps) may exist either in an extended or a compact conformation, sometimes correlated with their functions. for the cabps with know structure and function, calcium sensors are usually extended and calcium buffers compact, hence the interest to predict the form of the protein starting from its sequence. in this study we used two different procedures, the sosuidumbbell algorithm and a novel procedure that is based on the linker average hydrophilicity (lah). the two procedures were tested on known-structure cabps and then applied to unknown-structure centrins. the so-suidumbbell algorithm yielded the right conformations for of the known-structure proteins and predicted that all centrins should are compact. the lah procedure discriminated well between the extended and non-extended forms of all the known-structure cabps and it reflected well the phylogenetic classification of centrins being a simple and powerful means to discriminate between extended and nonextended forms of cabps. only few residues that constitute the linker are responsible for the form of the cabp, showing that this form is mainly governed by short-range interactions. (http://u .curie.u-psud.fr/modelisation/lah) lipid and protein organization of hepatitis b antigen characterized by fluorescence spectroscopy v. greiner , c. egelé , s. oncul , f. ronzon , c. manin , a. klymchenko , y. mély laboratoire de biophotonique et pharmacologie, umr cnrs, faculté de pharmacie, université de strasbourg, france, sanofi pasteur, av. marcel mérieux, marcy l'étoile, france. hepatitis b surface antigen (hbsag) particles are nm lipoprotein particles, mainly composed of the major s surface viral protein containing trp residues and yeast-derived lipids. since the structure of these particles is still missing, we further characterized them by fluorescence techniques. fcs indicated that the particles diffuse mainly as monomers and contain about proteins per particle. fluorescence quenching and time-resolved fluorescence experiments showed that the fluorescence signal is largely dominated by the trp residues at the protein surface. moreover, time-resolved anisotropy measurements indicate that the protein motion is restricted and that the surface trp residues exhibit both local and segmental motions. the lipid part of the particles has been studied by environment sensitive -hydroxyflavone probes and viscosity-sensitive dphbased probes, and compared to lipid bilayers and low density lipoproteins (ldls), taken as models. the results suggest that the lipid part of hbsag is closer to ldls than to model lipid bilayers. we present an extensive calorimetric study of bovine alphalactalbumin for various ca++ content. equilibrium dsc raw data are analyzed and the melting temperature tm, the specific heat jump deltacp, the heat of unfolding deltahm are directly extracted. the binding of calcium on the native (n) state greatly stabilizes the protein, essentially by the enthalpic difference between the unfolded (u) and n states. we show that subsequent addition of calcium in the mm range stabilizes further the n state. the equilibrium calorimetric measurements are completed with out of equilibrium stopped flow refolding kinetics by cd spectroscopy performed at different temperature and ca++ concentrations. we discuss the possible stabilization mechanisms compatible with our measurements. protein unfolding/refolding in cellular compartments: application of luciferase constructs our studies show that a reporter enzyme, firefly luciferase, can be used for evaluation of the stress-induced proteotoxicity within different cellular compartments such as the nucleus, cytoplasm or mitochondria. in transfected mammalian cells, firefly luciferase is localized in microsomes. we engineered plasmid constructs encoding luciferase with inserted specific sequences that ensure its cytoplasmic or intranuclear, or intramitochondrial localization. in addition, we fused luciferase to the green fluorescent protein (gfp) that enables to visualize patterns of the compartment-targeted product. using such gfp-labeled constructs we had a possibility to monitor protein unfolding, aggregation and refolding in the cytoplasm, nucleus and mitochondria of transfectants exposed to either stressful conditions. gfp-luciferase expressed in mammalian cells behaves as a relatively labile protein which can undergo reversible unfolding and aggregation in response to heat shock, atp depletion or action of toxic agents. in the case of cell recovery, refolding of denatured luciferase is carried out at the chaperone machine. we explored unfolding/refolding of gfp-luciferase in the cytoplasm, nucleus and mitochondria of ischemia-stressed rat cardiac cells and in several cancer cell lines treated with hyperthermia or some chemotherapeutic drugs. the results obtained have revealed intriguing correlations between the proteotoxic impact within either compartment and the viability of treated cells. amyloidogenic and conformational properties of proiapp and iapp in the presence of lipid bilayers s. jha , d. sellin , r. seidel , r. winter biophysical chemistry, department of chemistry, tu dortmund university, otto-hahn str. , d- , dortmund, max-planck-institute for molecular physiology, otto-hahn str. , d- , dortmund, germany the islet amyloid polypeptide (iapp), which is considered as the primary culprit for β-cell loss in type diabetes mellitus patients, is synthesized in the β-cells of the pancreas from its precursor, the pro-islet amyloid polypeptide (proiapp). proiapp is co-processed in the secretory granules and co-secreted to the extracellular matrix together with insulin as iapp. here, we compare the amyloidogenic and conformational properties of proiapp and iapp in the presence of lipid membranes, which have been discussed as loci of initiation of the fibrillation reaction. the two peptides show an enhanced amyloidogenic propensity in the presence of negatively charged membranes and similar secondary structural properties. however, proiapp shows a much less amyloidogenic propensity, probably due to the increased net charge on proiapp, compared to iapp. unlike iapp, proiapp forms small oligomeric structures at the lipid interface, having heights of ∼ . nm. this morphological distinction can be attributed to the presence of the pro-region, flanking the amyloidogenic iapp. the addition of proiapp to iapp marginally delays iapp fibrillation, probably by interfering with the interaction between amyloidogenic iapp cores of distinct iapp molecules. thus, it appears reasonable to speculate that the pro-region of the proiapp could serve to delay the fibrillogenesis of iapp at negatively charged lipid bilayers. the role of transmembrane domain interactions in the kinetics and folding of cpt z. a. jenei , k. borthwick , v. a. zammit , a. m. dixon chemistry dept., univ. of warwick, coventry, uk, clinicalȃsciencesȃres.ȃinst., warwick univ., coventry, uk carnitine palmitoyltransferase i (cpt ) enzymes are polytopic integral membrane proteins in the outer membrane of mitochondria (omm). cpt controls the rate of entry of long-chain fatty acids into the mitochondrial matrix for βoxidation. the two catalytically active isoforms, cpt a and cpt b, are different in their inhibitor binding kinetics and structure (interaction between n-and c-segments, interactions of transmembrane domains (tmd)). it has been suggested that inter-and intramolecular tmds interactions are important for cpt a, but not for cpt b function. cpt a has also been implicated in formation of oligomeric complexes through its tm segments. the study of tm helix-helix interactions in cpt isoforms could lead to a better understanding of their function and inhibitor binding kinetics, and will contribute towards the design of pharmacological strategies aimed at modulating the activities of cpt enzymes in conditions such as diabetes. to investigate the ability of the tmd in cpt to self-associate and the order of any oligomers formed, several biochemical and biophysical techniques have been used. we found the self-association of rcpt a tmds (tm , tm ) to be different as measured using the in vivo tox-cat assay. chemical cross-linking and analytical ultracentrifugation studies demonstrated formation of both trimers and hexamers by the rcpt a tm peptide. these results provide further evidence that tm plays role in formation of a channel in the omm. self-assembly of transmembrane domains in cpt : role of gxxxg(a) motif in possible channel formation z. a. jenei , k. borthwick , v. a. zammit , a. m. dixon department of chemistry and, warwick medical school, university of warwick, coventry, uk carnitine palmitoyltransferase a (cpt a), a membrane protein that controls the rate of oxidation of long-chain fatty acids, is of key importance in diabetes and has recently been reported to exist as an oligomer in vivo. we have investigated full-length cpt a and find that the protein exists as a hexamer in liver mitochondria. using mutants of cpt a expressed in yeast mitochondria, we have localised key protein interactions in the hexamer to the transmembrane (tm) domains of the protein. detailed study of the tm domains in isolation, in both e.coli membranes and detergent micelles, demonstrated that while tm shows little self-assembly, tm displayed significant self-association. biophysical analyses of a tm -derived synthetic peptide revealed oligomerization behaviour identical to native cpt a in mitochondria, providing a strong link between tm helixhelix interactions and cpt a hexamer formation. this is significant in light of a recent suggestion that cpt a oligomerization may lead to formation of a channel in the mitochondrial outer membrane through which acylcarnitine gains access to the inter-membrane space. our data supports this new theory, and we go on to demonstrate experimentally the structural determinants of hexamer (channel) formation, specifically gxxxg(a) motifs in the tm domain which pack favourably in the hexamer and stabilize the oligomer. investigation of flexible loop role in structure and thermodynamic stability of firefly luciferase p. maghami, b. ranjbar, s. hosseinkhani department of biochemistry and biophysics, faculty of basic sciences, tarbiat modares university, tehran, iran protein folding, like any chemical process, consists of two fundamental components, thermodynamics and kinetics, which determine the stability of the folded state and the pathway of folding, respectively. these processes are currently too difficult to be solved de novo by purely computational methods. experimental evidence is required to simplify the problem via protein engineering .in this research, the wild type firefly luciferase (photinus pyralis) and some of its mutants were over expressed and purified. then their unfolding thermodynamics were examined, using circular dichroism and conventional fluorescence measurements. the unfolding equilibrium constant were measured over a complete rang of denaturant conditions. the measurements were shown structural and physico-chemical changes between wild-type and mutant proteins. exploring intrinsic disorder of unstructured membrane proteins by surface polymer physics intrinsically unstructured proteins (iups) are considered as a separate class within the protein world because they lack a well-defined folded structure. because iup's function is indeed directly linked to structural disorder, they are assumed to be natively unfolded. several physicochemical techniques are available to discriminate the degree of disorder. however a clear structural classification is still lacking. in this context, polymer physics emerges as a powerful tool for getting inside on the conformational abilities directly related to structural disordered of iups. in the present contribution, surface pressure and ellipsometry experiments in conjunction with polymer physics have been used to infer structural data in terms of molecular conformation and flexibility of a membrane protein essential for bacterial division, zipa. this protein has been pointed to posses a high molecular flexibility and to adopt a random coil conformation. folding dynamics of peptides studied by timeresolved infrared spectroscopy c. krejtschi , o. ridderbusch , r. huang , l. wu , t. a. keiderling , k. hauser institute of biophysics, university of frankfurt, germany, department of chemistry, university of illinois at chicago, usa peptides are ideal model systems to study protein folding mechanisms. ir techniques provide both the necessary time resolution as well as the structural sensitivity. we initiate rapid heating by laser-excited ns temperature jumps (∼ • c) and study fast ns-to-µs relaxation dynamics [ ] . the dynamics of the α-helix to random coil transition of polyglutamic acid was analyzed under reversible folding/refolding ph-conditions. the observed relaxation kinetics allowed separation of the folding and unfolding process with additional use of ftir measurements in thermal equilibrium. sitespecific dynamics were monitored by use of isotopic labeling for a β-hairpin peptide whose conformation is stabilized by a hydrophobic core. various single and cross-strand isotopically labeled variants were analyzed. the isotope-edited kinetics show variations in local structural stability of the hairpin backbone. our data support a multistate dynamic behavior, and the site-specific kinetics are consistent with a hydrophobic collapse hypothesis for hairpin folding [ ] . small heat shock protein hsp was predicted to belong to the family of intrinsically disordered proteins. one of the features of these proteins is that they do not demonstrate cooperative thermal transitions on heating. we applied different methods (dsc, ftir and intrinsic tryptophan fluorescence) to investigate the thermal unfolding of hsp . dsc results have shown that thermal denaturation of hsp begins from o c and occurs, with very low cooperativity, within a broad temperature region (up to o c and above). the thermal unfolding of hsp is fully reversible. the ftir data show that heating of hsp from to o c results in complete disappearance of α-helices (from - % to ) and the decrease in β-sheets content from to %. studies on the temperature dependences of tryptophan fluorescence have shown a significant red shift of the spectrum. these changes occurred within temperature region from to o c with midpoint at ∼ o c. probably, this transition can be explained by destruction of β-sheets around trp , the only trp residue of the α-crystallin domain of hsp (other trp residues of hsp are localized in the n-terminal domain). the data obtained confirm the suggestion that hsp is a protein, whose significant part is intrinsically disordered. we propose that, on heating, the α-crystallin domain containing β-sheets melts at higher temperature than the n-terminal domain containing the most of α-helices. t. rosenkranz , a. katranidis , d. atta , j. enderlein , i. gregor , m. grzelakowski , p. rigler , w. meier , j. fitter isb- : molecular biophysics, research centre jülich, germany, institute of physics, biophysics/complex systems, georg august university, göttingen, germany, institut für physikalische chemie; universität basel, basel, swizerland the protein folding mechanism is the missing link in the biological flow of information from the dna to its specific function. since most of proteins within a cell consist of more than one domain studies on this protein class are of major importance. it is a common feature of multidomain proteins to aggregate under refolding conditions, which hinders a refolding. molecular encapsulation of single molecules prevents aggregation. by immobilizing the nanocapsule the observation period in a wide field microscope will be extended, so that slow or rare folding events can be detected. a major goal of this study is to investigate polymeric vesicles with respect to their suitability for protein folding studies [ ] . polymer vesicles maintain their structural integrity even under harsh unfolding conditions. furthermore the nanocontainer proved to be permeable to guanidinium hydrochloride. using encapsulated phoshoglycerate kinase, labeled with atto- , a dye which experiences fluorescence quenching by photo-induced electron transfer (pet) with tryptophans, we demonstrate the remarkable properties of polymeric nanocontainers. applying pet as a folding probe we detected multiple unfolding/refolding transitions of single proteins. proteins frequently become irreversibly modified by carbonylation, a process of introducing the carbonyl group (carbon monoxide) in a reaction with reactive oxygen species (ros) such as superoxide, peroxide or ozone. the main targets for carbonylation in proteins are amino-acid side chains of lysine, arginine and proline. products of carbonylation are aminoadipic semialdehyde from lysine (asa) and glutamic semialdehyde (gsa) from arginine and proline. importantly, carbonylated proteins are marked for proteolysis by the proteasome, but can escape degradation and form aggregates that can be cytotoxic. carbonylation increases with the age of cells and it is associated with ageing and age related disorders such as alzheimer's disease, parkinson's disease and cancer. we have used the molecular dynamics method to study the stability of carbonylated proteins villin headpiece and ubiquitin. simulations were run after mutations of arginine, proline and lysine into gsa and asa had been performed. in addition, we have used thermodynamic integration on lysine, arginine, proline, asa and gsa residues in order to estimate their solvation free energy (related to relative hydrophobicity and hydrophilicity). our results suggest that carbonylation markedly decreases the overall stability of proteins, and that one potential reason for that may be a disruption of the balance between hydrophilic and hydrophobic regions in the protein. a. martino, d. crane, i. m. feavers, b. bolgiano division of bacteriology, national institute for biological standards and control, potters bar, uk the sensitivity to protein's secondary structure and progress in computational calculations have made circular dichroism (cd) an attractive technique to explore the optical properties of three promising vaccine candidates to neisseria meningitidis. clinical batches of a c-term deleted form of nada (genome-derived neisseria antigen -gna ) and the fusion proteins gna - (fp- ) and gna - (fp- ) were therefore studied. increases in temperature and denaturant concentration on secondary structures and folding/unfolding profile were monitored by cd in the far and near uv regions and complemented by trp fluorescence spectroscopy data. furthermore, epitope conformational changes on binding activities to immune-sera were investigated. the calculated secondary structure content was in broad agreement with the available predicted or solved protein structures. upon temperature incubation, a structural transition from a highly α-helical nada to a more unordered conformation, with a mid point at ∼ • c, was observed. fp- and fp- maintained their conformation up to • c or m guhcl in the case of fp- . unfolding was not always reversible. reductions in binding to monoclonal ab titrated along with increasing unfolding. folding/unfolding studies have proven useful in better understanding the solution behaviour and extent of folding of proteins. cold denaturation of yfh offers the clue to understand the effect of alcohols on protein stability s. r. martin , v. esposito , p. de los rios , a. pastore , p. a. temussi national institute for medical research, the ridgeway, nw aa london, u.k., laboratoire de biophysique statistique, sb/itp, ecole polytechnique fédérale de lausanne (epfl), ch- , lausanne, switzerland, dipartimento di chimica, università di napoli federico ii, via cinthia, i- napoli, italy although alcohols are well known to be protein denaturants when present at high concentrations, their effect on proteins at low concentrations is much less well characterized. here we present a study of the effects of alcohols on protein stability using yfh . exploiting the unusual property of this protein of undergoing cold denaturation around • c without any ad hoc destabilization, we determined the stability curve on the basis of both high and low temperature unfolding in the presence of three commonly used alcohols: trifluoroethanol,ethanol methanol. in all cases, we observed an extended temperature range of protein stability as determined by a modest increase of the high temperature of unfolding but an appreciable decrease in the low temperature of unfolding. we suggest that alcohols, at low concentration and physiological ph, stabilize proteins by greatly widening the range of temperatures over which the protein is stable. our results also clarify the molecular mechanism of the interaction and validate the current theoretical interpretation of the mechanism of cold denaturation. biomolecular sciences and biotechnology tor vergata moro , rome, italy cholesterol plays an important role in regulating the structural properties of phospholipid and non-phospholipid membranes. in this study we have applied in situ energy dispersive x-ray diffraction (edxd) to investigate the effect of cholesterol on the structure of different phospholipid and non-phospholipid oriented membranes. in detail, phosphatidylcholine (pc) bilayers and niosomal membranes, made of a non-ionic surfactant centre for bioactive chemistry, department of chemistry department of chemistry this process is initiated at nuclear envelope remnants (ners) in the presence of atp and gtp. the mvs can be divided in two main populations: mv and mv . mv has a classical lipid composition while mv is enriched in phosphoinositides (pips: pi, pip, pip and pip ). ners have an unusual lipid composition, enriched both in cholesterol and pips. physicochemical properties of the pips were investigated as a function of ph and temperature (t) using nmr, saxs and dls to map out their phase state. pips-water dispersions are observed in lamellar, hexagonal or isotropic phases depending on t and ph. in parallel, model membranes mimicking mv and ners lipid composition were studied by h and p nmr. mv modelling shows a complex behaviour of pips on pc membranes: they order or disorder membranes, whereas the order of pc/pi/pip/pip membrane is lower than that of pc or pc/pi membranes c. manzo, t. s. van zanten, m. f. garcia-parajo bionanophotonics group, ibec-institut de bioenginyeria de catalunya, barcelona, spain membrane proteins play a fundamental role in intra-and inter-cellular functions. in particular, the proteins lateral mobility in the fluid membrane environment is crucial for the regulation of several mechanisms, as receptor-mediated signal transduction and establishment of immunological synapses. these mechanisms are controlled through protein crowding and reduced lateral diffusion, which induce macromolecular associations and limit the application of conventional single molecule fluorescence techniques. to measure proteins mobility on living cells membrane, we developed a fluorescent correlation spectroscopy (fcs) setup in which the sample illumination is obtained through near-field scanning optical microscopy (nsom) probes. the use of nsom probes is particularly suited for the observation of dynamics on the cell membrane and overcomes the drawbacks of other techniques. in fact, through a shear-force-based position control, the probe is kept at a fixed distance from the membrane and its sub-wavelength aperture (∼ nm) reduces the illumination area, allowing the observation of highly crowded regions of the membrane. on the basis of preliminary results, the nsom-fcs is expected to provide an additional insight on the proteins trafficking at the membrane level. the technique also presents several potential developments, as the further reduction of the illumination area and two-colors correlation. single molecule fluorescence microscopy of the store-operated calcium channel subunit orai j. madl, j. weghuber, d. bergmair, m. fahrner, m. muik, c. romanin, g. j. schütz johannes kepler university, institute for biophysics, linz, austria store-operated calcium entry (soce) is essential for many cellular signalling processes. the essential pore forming subunit of soce channels in the plasma membrane is orai . here we present single molecule fluorescence microscopy of orai which was performed in order to directly visualize the stoichiometry of mobile orai pores. the protein was fluorescently labeled with monomeric gfp. a novel single molecule fluorescence approach, toccsl (thinning out clusters while conserving the stoichiometry of labeling), was used for the determination of the stoichiometry. this technique allows reducing the density of fluorescently labeled molecules without affecting the stoichiometry of labeling. density reduction is achieved by completely photobleaching a defined area within the plasma membrane; nonbleached gfp-orai aggregates enter the bleached region subsequently by diffusion. our data indicate that there are different populations of orai present in the cell: most of orai is located in the plasma membrane. a second population of orai -mgfp was found to be localized in intracellular vesicles. a significant fraction of the plasma membrane orai exhibits a diffusive movement. we found by analyzing the bleaching characteristics of single orai -mgfp aggregates that in resting cells mobile orai is predominantly dimeric. a. kobitski , a. nierth , m. helm , a. jäschke , g. u. nienhaus university of ulm, germany, university of heidelberg, germany, university of karlsruhe, germany rna molecules have attracted enormous attention in recent years, and various novel roles were revealed for rna in biological processes. ribozymes are a class of rna molecules capable of catalyzing chemical reactions. we have studied a diels-alderase (dase) ribozyme, a small artificial -mer ribozyme, which is capable of catalyzing carbon-carbon bond formation between an anthracene diene and a maleimide dienophile in multiple turnovers. single-molecule fluorescence resonance energy transfer was employed to investigate the intramolecular dynamics of this rna molecule as a function of mg + ion concentration. folding into a functional state occurs via an intermediate state, and continuous fluctuations between these two states were observed on the ms time-scale at the midpoint concentration of mg + ions. an effect of substrates binding on the folding and catalytic reaction of the dase ribozyme is in the focus of our recent research with the ultimate goal to obtain a detailed structural view of the single-molecule conformational changes that accompany the catalytic reaction. a. katranidis , r. schlesinger , k. nierhaus , i. gregor , m. gerrits , g. bueldt , j. numerous studies showed that protein folding and maturation can differ substantially between de novo synthesized proteins and in vitro refolded proteins. here we present an approach employing a two color single molecule sensitive fluorescence wide-field microscope in order to visualize surface tethered fluorescently labeled ribosomes and de novo synthesized gfp molecules in real time [ ] . fluorescence of co-translational folded proteins was observed from mature fluorescent gfp molecules which carry additional amino acids at the c terminus remaining linked to the ribosome. thus it was possible to co-localize fluorescence from labeled ribosomes and from gfp molecules. we demonstrate that the green fluorescence protein mutant gfp emerald is produced with a characteristic time of five minutes. the fastest gfp molecules appeared already within one minute. processes precedent to chromophore formation, such as polypeptide synthesis and protein folding, are fast and last not longer than one minute. in fluorescence spectroscopy, photobleaching is a process which leads to irreversible loss of fluorescent properties of a dye molecule, usually due to photochemical reactions. it is especially important for fcs experiments on slow-diffusion systems since for high excitation intensities it can have a strong impact on fluorescence intensity correlation function. usually it is observed as apparent shortening of the mean diffusion time of the dye molecules. the behavior of tmr-labeled fd-virus rods in water solution under various excitation conditions was investigated. the experiments were conducted for low ( : ) and high ( : ) tmr:virus ratios and for increasing laser intensities. the correlation function was measured in the experiments. the results were fitted using origin software to estimate the influence of photobleaching, and compared with computer simulations. a strong effect of photobleaching was visible for rods labeled with a single dye molecule, while rods labeled with tmr molecules showed little to no bleaching. a prolongation of characteristic diffusion times for highly labeled virus rods in comparison to low-labeled ones was also observed. k. toth , a. gansen , a. valeri , v. böhm , c. a. seidel , j. langowski abt. biophysik der makromoleküle, deutsches krebsforschungszentrum, heidelberg, germany, lehrstuhl für molekulare physikalische chemie, heinrich heine universität, düsseldorf, germanythe nucleosome has a central role in the compaction of genomic dna and the control of dna accessibility for transcription and replication. we studied the effect of dna sequence and selective histone acetylation on the structure, stability and disassembly of the mononucleosomes. quantitative single molecule fret measurements between dyes attached to different parts of the nucleosome permitted us to detect the equilibrium between several subpopulations of reconstituted nucleosomes in solution. we obtained that the heterogeneity and stability of the samples are correlated with each other and influenced both by the dna sequence and the histone acetylation. the path of the linker dna is more sensitive to all studied effects than the dna on the core. intermediates of the disassembly pathway were identified and characterized. j. strömqvist , s. johansson , y. ohsugi , k. andersson , l. xu , m. kinjo , p. höglund , j. widengren experimental biomolecular physics, kth, stockholm, sweden, department of microbiology and cell biology, karolinska institutet, stockholm, sweden, laboratory of molecular cell dynamics, hokkaido university, sapporo, japan dual-color fluorescence cross correlation spectroscopy (fccs) has been used to explore the molecular dynamics at immune cell surfaces, with a particular focus towards the regulation mechanisms of natural killer (nk) lymphocytes. nk cells are critical mediators of anti-viral immunity and protectors against cancer spread. their activity is governed by a fine-tuned balance between inhibitory and activating receptors, where ly a and kir receptors represents the inhibitory ones. their ligands are mhc class i receptors. fcs is a technique based on the analysis of intensity fluctuations of fluorescent molecules excited by a focused laser beam. the technique offers information about molecular dynamics at the single molecular level, in the nanosecond to millisecond range. dual color fccs expands fcs by correlating the intensity from two different colors. by labeling two potential interaction partners with dyes emitting at different wavelengths, the amount of interaction can be determined.here, we will report on recent fccs data exploring the interaction between the inhibitory receptors and their ligands, as well as different labeling strategies used to enable these measurements. dynamic multiple-target tracing probes spatiotemporal cartography of cell membranes in order to decipher the non random and non homogeneity of the plasma membrane organization, we had performed fluorescence correlation spectroscopy measurements on live cells. this allowed us to establish the presence of nanoscale confining structures and to demonstrate their implication in signaling process . complementing these studies, we present here a new analytical method, namely multiple-target tracing (mtt) which takes advantage of the high resolution provided by singlemolecule sensitivity to generate dynamic maps at high densities of tracked particles. introducing deflation by subtracting detected peaks allows detecting peaks of lower intensity. we achieved an exhaustive detection of particles with performances reaching theoretical limits, and a reconnection of trajectories integrating the statistical information from past trajectories. we demonstrate the potential of this new method of analysis by applying it to the epidermal growth factor receptor labeled with quantum dots, in the plasma membrane of live cells. this has allowed us to build up a global representation of molecular dynamics in cell membranes. dual polarisation interferometry (dpi) is a surface analytical technique capable of dynamically measuring biophysical parameters of conformational change in biomolecular interactions. the technique measures three key parameters, namely layer thickness, layer density (ri) and mass, thereby enabling the resolution of conformational changes involved during binding. a number of different applications are presented. protein-protein interactions: understanding the biophysical nature of protein interactions can deliver insights into the mechanisms by which proteins interact, thereby elucidating protein function. dpi enables correlation between binding affinity and conformational change, greatly enhancing the study of structure-function relationships. lipid layers: the birefringence mode of dpi can be used to study the formation of lipid bilayers and biomolecular self-assembly. it is possible to use a combination of bilayer refringence and mass to study phase transitions associated with protein or peptide binding to the lipid bilayer. the individual stages of adsorption, absorption and micellisation can be distinguished. carbohydrate interactions: dpi uses a planar glass surface and a wide range of coupling chemistries. a carbohydratespecific surface can be used to study a wide range of biomolecular interactions, such as lectins, acidic proteins, extracellular matrix signaling and interactions with complex membranes. the experimental characterization of the elementary conformational steps constituting the protein folding pathway remains a big challenge. quenching of the triplet state of tryptophan by close contact with cysteine has been shown to provide a new tool for measuring the rate of intramolecular contact formation -one of the most elementary events in the folding process -in peptides and proteins using only natural probes. here we show an extensive study on a stabilized mutant of the second beta-hairpin of gb domain. steady state fluorescence and kinetics of contact formation between a natural tryptophan and a cysteine added to the c-terminal are measured for different temperatures and solvent conditions. we separately address the contributions of different structural elements to the overall stability of the hairpin. we extract kinetics parameter for contact formations in the unfolded state, formation of the hydrophobic core and tails pairing in the folded state. by means of a fragment peptide terminated with a tryptophan and a cysteine, we also estimate the structural propensity of the turn region. the data coherently combine with a simple model previously developed to describe the dynamics of unstructured chain [biophys. j. , ( )], here modified with the addition of attractive interactions between specific residues. catalytic power of partially denatured enzymes: implementation of molten-globule-like states m. shushanyan , d. e. khoshtariya , m. makharadze , t. tretyakova , r. van eldik institute of molecular biology and biophysics, gotua , tbilisi, georgia, department of physics, i. javakhishvili tbilisi state university, tbilisi, georgia, department of chemistry and pharmacy, university of erlangen-nürnberg, germany impact of nonspecific moderate denaturants, urea and dmso, on the kinetic (functional) and thermodynamic (stability) patterns of a hydrolytic enzyme, α-chymotrypsin (α-ct) has been investigated. furthermore, an impact of urea and guhcl in combination with of temperature on the kinetic pattern of carboxypeptidase a has been examined. for the case of α-ct, in particular, we have observed about tenfold increase of the apparent mickaelis constant when going from to m urea ( o c), whereas the value of catalytic constant remained almost unchanged, indicating that the protein is not unfolded even under those severe conditions. the matching microcalorimetric experiments revealed that both the temperature-induced melting temperature and transition enthalpy decrease gradually with the increase of the additive concentration. with m urea the peak-shaped calorimetric feature disappears totally. however, catalytic power of α-ct was preserved owing to its catalytic constant. for other studied enzyme/substrate/denaturant arrays diverse kinetic peculiarities due to mgls have been observed. rubredoxins are a class of iron-containing proteins whose biological role on electron transfer processes and metal binding is still unclear. the unfolding dynamics of the rubredoxin mutant rda c from the mesophile desulfovibrio vulgaris (dv) was studied on the temperature range from • c to • c and along time at • c. resonance energy transfer (ret) was used to determine the donor (d; trp ) to acceptor (a; , -iaedans) distance. from • c to • c the d-a distance increased Å. however the random coil expected d-a distance was only achieved after heating the protein solution during . hours at • c. from uv-vis absorption data it's clear that this protein is capable of maintaining its iron-sulfur center at • c. by melting the protein at the same temperature all iron-centers disintegrate and the protein unfold after . hour. the trp fluorescence also shifts nm to the red reflecting the partial exposition of the indole ring to the solvent. from fluorescence and anisotropy decay curves a breathing type movement of the protein structure was observed between • c to • c without lost of significant secondary structure. this structure flexibility should play an important role on the thermal stability of the dvrd the antimicrobial peptide novicidin (nc) -modified from the sheep self defense peptide smap- , for reduced mammalian cytotoxicity -is a cationic peptide (net charge +∼ . ) that adopts random coil structure in solution, but an α-helix in the presence of lipid vesicles. the conformation of nc in presence of the lipids dlpc, dlpg, dmpc, dmpg, dopc, dopg, dope, and dops in different combinations reveal the lipid selectivity, affinity, and phase dependent changes with varying l/p ratios and temperatures, observed from cd spectroscopy. it is understood that the conformational change is dependent on chain length and head group of the lipid. apart from the in vivo results on the nc activity, studies using qcm-d, dual polarisation interferometry, and calcein release assay reveal the kinetics and concentration dependent activity of nc in lipid bilayers and vesicles. preliminary studies on orientation of nc in various lipid environments using ssnmr, lsnmr, and molecular dynamics simulations apparently suggest that nc may form toroidal pores/detergent effects depending on the chain length of the lipids. further experiments on nc in presence of lipids using dsc, itc, ssnmr, oriented cd, ld, and confocal microscopy to determine the structure, thermal stability, orientation in lipid bilayers, and thereof the action of nc will help in proposing a comprehensive model for its mechanism of action in model membranes. dielectric method for measuring glass transition and denaturation temperatures of hydrated proteins g. e. thomas , s. bone , g. drago institute for bioelectronic and molecular microsystems, bangor university, gwynedd, uk., applied enzyme technology, pontypool, uk.the flexibility of protein structures is important in allowing the variety of motions, covering a wide range of magnitudes and frequencies, essential to biological activity. high frequency dielectric measurements can be used to study the flexibility of proteins by probing the relaxation of dipolar constituents of their structures. hydrated proteins exhibit a broad dielectric loss extending over the frequency range from mhz to ghz which can be decomposed into a number of constituent dispersions. one of these dispersions, with a relaxation time of ∼ ns, has been attributed to the relaxation of protein backbone peptide groups in the protein interior. in the work reported here, this dielectric dispersion was investigated as a function of temperature for the enzyme glucose oxidase. two critical temperatures were identified as the glass transition and denaturation temperatures, both of which were found to decrease with increasing protein water content. the results were consistent with a scheme in which the hydrated glassy protein undergoes a change in structural mobility at the glass transition temperature and experiences an irreversible change in conformation at a higher denaturation temperature. both glass transition and denaturation temperatures are key indicators of protein stability and are important in the production and storage of protein based pharmaceuticals. a. szymańska, k. kacprzyk, g.Ślósarek department of molecular biophysics, a. mickiewicz university, umultowska , - poznań, poland aggregation dynamics of proteins plays an important role in molecular biology and medicine as it permits explanation of several disease states. an interesting problem is to find out in which conditions the interactions of the protein molecules lead to formation of ordered structures and in which to disordered ones. in this study, dynamic light scattering, circular dichroism and also congo red dye experiments were performed to analyze various structural states of lysozyme induced under different concentration of ethanol solvent. at low ethanol concentration the attractive interaction between the protein macromolecules dominate. after addition of more ethanol solvent, the translational diffusion coefficient was much smaller than that for lysozyme solution at zero ethanol concentration. it can be explained by the structural transformation of the polypeptide chain leading to a partially folded conformation needed for oligomerization and fibrillation process. on the basis of the cd experiments we concluded that the ethanol solvent induces changes in secondary structure of lysozyme solution. on addition of % v/v ethanol solution the intramolecular hydrogen bonds were destabilized. above this ethanol concentration, β -sheet were the dominant secondary structure of lysozyme in solution. the phase diagram illustrating the formation of: monomers, oligomers at various structural states, protofilament formation state, protofilament and amyloid fibrils was constructed. key: cord- - s ok uw authors: nan title: abstracts of the th annual symposium of the protein society date: - - journal: protein science doi: . /pro. sha: doc_id: cord_uid: s ok uw nan c-terminus glutamine-rich sequence deleted) to elucidate the role of metalloprotein hpn-like by fluorescence resonance energy transfer (fret) (figure ) [ ] . we found the selective coordination of ni(ii) and zn(ii) to the purified sensors and in e. coli cells. surprisingly, specific interaction between the fret sensors and bi(iii) was observed. our fret analysis confirmed the role of hpnl for ni(ii) storage and revealed the potential association of hpnl with bi-based antiulcer drugs in cells. pb- rna fate is controlled by highly-regulated rna binding proteins molecular mechanism that links the mrna-degradation pathway with extracellular signaling networks through the reversible unfolding of a rna binding domain (rbd). rna binding is also controlled by ph conditions. this finding becomes relevant for rbps such as t-cell intracellular antigen (tia- ), which shuttles between two cellular compartments (nucleus and cytoplasm) with slightly different ph values. in fact, rna binding by tia- is modulated by slight environmental ph changes due to the protonation/deprotonation of tia- histidine residues [ , ] . the ph dependence of the tia- /rna interaction provides a new insight into the function of tia- in recognizing new rna targets [ ] , like the ' terminal oligopyrimidine tracts ( tops) of translationally-repressed mrnas. along with tia- , the rbp hu antigen r (hur) is involved in the assembly/disassembly of cytoplasmic stress granules (sg), which arise as a protective mechanism by preventing mrna decay under stress situations. despite wide acceptance that rbps harboring aggregation-promoting prion related domains (prds), such as tia- , stimulate rapid self-association and formation of sgs, we propose that scaffolding sgs may be driven by rbds, since prd-lacking rbps, like hur, often form oligomers [ , , ] and are included in sgs. under continuous stress, the transition from the physiological to pathological aggregation of rbps in sgs may depend on post-translational modifications of rbds. rna-binding proteinopathies, characterized by the nucleation of irreversible sgs, are often found in neurodegenerative diseases. altogether, resulting insights into rna biology suggest that highly-regulated rbps determine mrna fate from synthesis to decay. a threat to million people in underdeveloped nations around the world, african trypanosomiasis (sleeping sickness) is a neglected tropical disease (ntd) caused by the protozoan parasite trypanosoma brucei (t. brucei). t. brucei is transmitted to humans via the tsetse fly, and replicates in the blood before crossing into the brain, causing death for the infected individual. current treatments that are available for african sleeping sickness are highly toxic and usually difficult to administer past the blood-brain barrier. it is our belief that coupling less toxic compounds with efficient drug delivery systems will contribute to the development of the most effective drug against african sleeping sickness. our goal was to determine a novel and effective chemical inhibitor with the potential to prevent the replication of t. brucei in the human body. the enzyme target for inhibition studied in this research was -phosphogluconate dehydrogenase ( pgdh), a cytosolic enzyme in the pentose phosphate pathway (ppp) of t. brucei. pgdh is essential in the ppp due to its ability to oxidize -phosphogluconate into ribulose- -phosphate, which is essential for the formation of nucleotides. primer overlap extension polymerase chain reaction (pcr) was used to synthesize the coding dna sequence of the pgdh gene, which was then cloned into a pnic-bsa inducible expression plasmid with an n-terminal histidine tag, by way of ligation independent cloning. the protein was then expressed in bl (de ) escherichia coli (e. coli) cells and purified via nickel column affinity and size exclusion fast protein liquid chromatography (fplc) to perform inhibition assays. through virtual screening, various ligands obtained from the chembridge library and nih clinical collection) were docked into the active site of the crystal structure of tb pgdh (pubchem identification pgj) using gold molecular docking software. the top scoring compounds were selected by utilizing parameters such as hydrophobic interactions, hydrogen bonds, and van der waals forces. the compounds with the best scores that also satisfied lipinski's rule of criteria for druggability were then tested in spectrophotometric enzyme inhibition assays monitoring the absorbance of nadph at nm. compounds that show inhibitory activity in the assays will be taken to higher levels of testing to determine their effect on t. brucei in other organisms. nmr studies of the structural influence of phosphopantetheinylation in nonribosomal peptide synthetase carrier proteins and impact on binding affinities andrew goodrich , dominique frueh nonribosomal peptide synthetases (nrpss) are modular enzymatic systems responsible for the production of complex secondary metabolites in bacteria and fungi. each module is comprised of (at least) three core domains whose combined action leads to the selection, activation, and incorporation of a single small molecule into a growing peptide. central to each module is the carrier protein (cp), which is first primed via attachment of a '-phosphopantetheine moiety (ppant arm) to a conserved serine to generate the active holo form. an adenylation (a) domain then covalently attaches an amino or aryl abstract acid onto the ppant arm via formation of a thioester. the cp then shuttles activated monomers and growing peptides between the active sites of catalytic domains in both the same and adjacent modules. during cp priming and peptide elongation, a cp thus exists in multiple different post-translational states and interacts with numerous catalytic domains. understanding how nrpss are able to efficiently orchestrate this series of sequential protein-protein interactions between a cp and its partner catalytic domains is key to unraveling the molecular mechanism of nrp synthesis. using a combination of isothermal titration calorimetry and nuclear magnetic resonance (nmr) titrations, we found that converting a cp from the apo to holo form alters its affinity for its partner a domain. this change in binding suggests a means by which directionality in protein-protein interactions is achieved in nrpss. however, we also found that a domain binding affects the same subset of residues in both the apo and holo forms. in order to identify the molecular features underpinning this difference in affinity, we solved the nmr solution structures of the apo and holo forms of the cp. here, we present the solution structures of an apo and holo cp and discuss them in light of their differential binding to an a domain. functional analysis of of conditional analog-sensitive alleles of essential protein kinases in the fission yeast schizosaccharomyces pombe. juraj gregan , mfpl/imp, the genome of the fission yeast schizosaccharomyces pombe encodes for protein kinases that are essential for viability. studies of the essential kinases often require the use of mutant strains carrying conditional alleles. to inactivate these kinases conditionally, we applied a recently developed chemical genetic strategy. the mutation of a single residue in the atp-binding pocket confers sensitivity to small-molecule inhibitors, allowing for specific inactivation of the modified kinase. using this approach, we constructed conditional analog-sensitive alleles of essential protein kinases in the fission yeast s. pombe. i will present the functional analysis of these mutants during meiosis. peptide conjugates: from self-assembly towards applications in biomedicine ian hamley university of reading, dept of chemistry self-assembling peptides and their conjugates offer exceptional potential in nanomedicine. i will present some of our recent work on nanoscale assembled peptides and their conjugates, focussing on lipopeptides [ , ] and peg-peptide conjugates [ ] . pegylation is an important technique in the development of conjugates for applications in therapeutics. it is found to greatly influence self-assembly of peptides and proteins -one example from our own work is a peptide which itself forms twisted fibrils but when peg is attached, self-assembly of the conjugate leads to spherical micelles [ ] . the conjugate can be enzymatically degraded using alpha-chymotrypsin, releasing the peptide. this nanocontainer delivery and release system could be useful in therapeutic applications. thermoresponsive telechelic peg/peptides with hydrophobic dipeptide end groups (di-tyrosine or di-phenylalanine) were developed, one of which shows a de-gelation transition near body temperature and which may be useful in bioresponsive delivery systems [ ] . examples from our recent work on self-assembling lipopeptides will also be outlined. our focus is to investigate potential relationships between self-assembly and bioactivity, in particular in the fields of regenerative medicine [ ] [ ] [ ] [ ] [ ] , antimicrobial systems [ , ] and immune therapies [ ] . been shown to become derivatized with argpyrimidine, a prominent nem that occurs on arginine residues [ ] , in certain human cancer tissues and cell lines [ , ] . this nem was linked to the elevated antiapoptotic activity of the protein [ , ] , whereby modification of arg- appeared to be of particular significance [ ] . in this work, hsp homogeneously modified with argpyrimidine at position is generated for the first time. using expressed protein ligation [ ] , the first semisynthesis of the unmodified protein is achieved as well. our approach, which combines organic chemistry, peptide synthesis and protein synthesis, enables complete control over protein composition and thus can provide previously unattainable insight into the properties of this vital chaperone following nonenzymatic modification. the synthesis of argpyrimidine-modified hsp and the progress towards structural and functional characterization of the protein will be presented herein. kunitz-type protease inhibitors belong to a widespread protein family present in many plant species and play an important role in plant defense against insect pests and pathogens. members of this family are typically inhibitors of proteases of serine class. interestingly, a few members were identified as inhibitors of proteases of cysteine class, however, they have not been functionally and structurally characterized. our study is focused on kunitz-type inhibitors of cysteine proteases (pcpis) from potato (solanum tuberosum). a series of kda pcpis was purified using a multi-step chromatographical protocol, and two most abundant and effective isoinhibitors named pci - and pci were characterized in detail. they were screened against a broad panel of model cysteine proteases and digestive cysteine proteases from herbivorous insects. pci - and pci exhibit different inhibitory specificity pattern and potency up to the nanomolar range. both isoinhibitors were crystallized and their spatial structures were solved and refined at . Å (pci - ) and . Å (pci ) resolutions. a position of reactive sites against cysteine proteases on the conserved b-trefoil fold scaffold was proposed. the work provides the first analysis of pcpis with respect to the structure-function relationships and evolution within the kunitz-type inhibitor family. role of the abcc transporter in the mode of action of the bacillus thuringiensis cry ac toxin in the diamond back moth plutella xylostella protonation pattern influence actively properties of molecules and play an essential role in biochemical mechanisms. for an accurate determination of the protonation equilibria, the absolute proton solvation free energy needs to be known. the determination of this energy represents one of the most challenging problems in physical chemistry. this is particularly difficult for protons solvated in water, where the solvation is dynamically performed by different water clusters and the proton is not attached to a single solvent molecule. the proton solvation is notably important in order to quantify mechanisms of proton transfer and such processes have been investigated for a long time based on different approaches, often leading to contradictory conclusions. a rigorous and accurate protocol for computing proton solvation in solvents of different nature is of prime importance for applied (pharmaceutical and material science) and fundamental sciences. in this study, proton affinities, electrostatic energies of solvation and pka values of a reference set of organic molecules are computed in protic and aprotic solvents. proportional to the free energy of proton dissociation, the pka value calculation is therefore strongly dependent on the free energy of proton solvation. such energy is then determined in acetonitrile (acn), methanol (met), water and dimethyl sulfoxide (dmso) in order to obtain the best possible match between measured and computed pka values. the computation of these values is based on a combination of quantum chemical (qc) and electrostatic approaches by using a thermodynamic cycle connecting gas-phase and solvent-phase of proton dissociation. the computed proton solvation energies in acn, met, water and dmso of the present study are very precise (rmsd much lower than ph value). they will be a basis for better understanding of proton solvation and help to predict pka values of organic compounds in different solvents more precise. biochemical characterization of two evolutionary distant ten-eleven translocation enzymes and their utility in -methylcytosine sequencing in the genomes at single-base resolution subtypes leading to an inability to perceive pain and painful neuropathies, respectively. however, as nav ion channels are intimately involved in almost all aspects of physiology, only the most selective inhibitors would be suitable as drug leads. disulfide-rich venom derived mini-proteins from cone snails and spiders are being actively pursued as novel therapeutics for pain, because of their high selectivity and potency at human ion channels, including sodium channels (nav). two main strategies of inhibition have been identified; blocking the pore and interacting with the voltage-sensor domains (vsd) surrounding the pore. the ion-conducting pore is highly conserved between all sodium channel subtypes whereas the voltage-sensor domain binding sites are less conserved. therefore, inhibition of a specific nav isoform is more achievable using inhibitors that modulate vsds than with pore blockers. gating modifier toxins from spider and cone snail venom inhibit nav . and nav . by interacting with the vsd. they appear to reach their target by partitioning into the lipid membrane surrounding the ion channel, thus enabling access to the vsd. toxin pharmacology may therefore not only be driven by the peptide-ion channel interactions, but also including the lipids surrounding the channel protein, a feature that is very much under explored. it is therefore apparent that peptide-lipid interactions in combination with peptide-channel interactions need to be considered when designing potent inhibitors. using a range of biophysical techniques, including surface plasmon resonance and nuclear magnetic resonance, we are studying the interactions underpinning the mechanism of action between toxins and membranes and toxins and ion channels. initial results show that the lipid composition surrounding ion channels play a major role in terms of toxin:lipid interaction and that these interactions can be used in combination with traditional structure-activity relationship studies to design selective and potent nav inhibitors, which will be discussed. we believe that our studies will ultimately delineate what drives toxin pharmacology and nav subtype selectivity and will lead to improve rationally engineering of novel therapeutics for the treatment of pain. micelles promote aß assembly into pore-forming oligomers montserrat serra-batiste , mariam bayoumi , margarida gair ı , mart ı ninot-pedrosa , giovanni maglia , nat alia carulla institute for research in biomedicine (irb barcelona), biochemistry, molecular and structural biology section, university of leuven, the formation of amyloid-b peptide (ab) oligomers at the cellular membrane is considered to be a crucial process underlying neurotoxicity in alzheime rs disease (ad). - therefore, it is important to understand how oligomers form within a membrane environment. using solution nuclear magnetic resonance (nmr) spectroscopy, combined with size exclusion chromatography (sec), we have studied the two major ab variants-ab and ab , the latter having a more prominent role in ad than the former-under carefully selected micelle conditions intended to mimic a membrane environment. our results indicate that after an incubation period, ab , but not ab , assembles into oligomers with specific structural properties, which we have named stabilized micelle oligomers (smos). smo complexes incorporate into lipid bilayers as well-defined pores, a feature linked to neurotoxicity. these results have important implications in the ad field as they provide a new perspective on how ab oligomers cause neurotoxicity. indeed, our findings constitute a first step towards the establishment of a new therapeutic target for ad. dimer formation. it should be noted that this nb peptide contains the autophosphorylatable ser- associated with phk activation, and phosphorylated nb; peptide was considerably less effective in promoting b-dimer formation than non-phosphorylated peptide. these results suggest a role for ser- autophosphorylation in mediating homodimeric b subunit interactions within the phk complex, and augment previous studies on the activation of phk by phosphorylation in which changes at the nterminus of b are critical in the activation of the catalytic g subunit. summing these results leads to a new model of activation. in this model, in the inactive state, the nonphosphorylated n-terminus of b interacts directly or indirectly with the regulatory c-terminal domain of the g subunit, inhibiting catalytic activity. upon phosphorylation of the n-terminus of b, three important events occur: ) the interaction between b and g is disrupted, ) the b subunits of the holoenzyme self-associate, and ) the catalytic domain is activated. thus, we envision that the n-terminus of b acts as an allosteric switch, with activation triggered by phosphorylation of this region, causing disruption of its previously inhibiting interactions with g and promotion of b b dimerization to stabilize the activated conformation of g . the research was supported financially by the university of kansas medical center biomedical research training program and nih grant dk . pb- hssb is involved in the cellular response to oxidative dna damage christine touma , nicolas paquet , derek j. richard , roland gamsjaeger , , liza cubeddu , school of science and health, university of western sydney, queensland university of te chnology, school of molecular bioscience, university of sydney cellular dna is subject to oxidative damage in the presence of reactive oxygen species. the , -dihydro- -oxoguanine ( -oxog) adduct is the most common form of oxidative damage and results in g:c to t:a transversions; these lesions are normally processed by the base excision repair (ber) pathway. singlestranded binding (ssb) proteins of the oligonucleotide binding domain family are heavily involved in dna repair processes, which involve the detection of dna damage and recruitment of repair proteins to the site of damage. using immunofluorescence we demonstrate that hssb (a novel human ssb) levels increase in response to oxidative damage (h ). cells depleted of hssb are hypersensitive to oxidative damage and are also unable to efficiently remove -oxog adducts. we show that hssb forms dimers and tetramers under oxidative conditions and that this oligomerisation is likely mediated by inter-domain disulfide bond formation. furthermore, using surface plasmon resonance, we also show that oxidised hssb binds to -oxo-g damaged ssdna with higher affinity than non-damaged ssdna, indicating a direct role for oxidised hssb in the recognition of -oxo-g lesions. as oxidative stress is associated with aging, cancer and alzheimer's disease, understanding the molecular mechanisms of how cells repair oxidative dna damage will be crucial in the development of potential therapeutic treatments. epidemic typhus, which is caused by the bacterial pathogen rickettsia prowazekii, is a menacing disease world wide that the nih lists as one of america's greatest biological weapons threats. this research seeks to find novel inhibitors of b-ketoacyl-acp-reductase (fabg), an enzyme that catalyzes one of the reactions in the fatty acid synthesis type ii system in bacteria. this pathway is essential for survival in bacteria. the fabg enzyme uses nadph as a substrate, which facilitates the binding of the second substrate, acetoacetyl-acp into the active site. the acetoacetyl-acp is subsequently reduced into b-hydroxyacyl-acp. the coding dna sequence for the rpfabg protein was cloned into a pnic vector and transformed into e.coli bl (de ), then the protein was expressed and purified using metal affinity and size exclusion chromatography methods. high throughput molecular docking software (gold) was used to screen a commercial library of ligands against the acetoacetyl-acp region of the active site. the ligands with the best gold scores were selected to be tested in vitro. spectrophotometric enzyme inhibition assays were performed to determine whether the drugs could inhibit rpfabg activity. chlorogenic acid, a previously known inhibitor of homologous fabgs, was tested along with the other potential drugs, and was determined to have moderate inhibitory effects on rpfabg. loop modeling using icm software was performed in order to create a prediction of the complete rpfabg structure, including the disordered loops that are not a part of the f i pdb structure. co-crystallization of rpfabg with both substrates was carried out in order to obtain a structure, but only nondiffracting crystals resulted. further inhibition assays and crystallography trials are being performed in order to continue the search for a novel inhibitor of rpfabg and ultimately a treatment for epidemic typhus. the university of hong kong bioconjugation of proteins has emerged as a useful tool in the study of biological systems. there is an increasing need to develop new synthetic technologies for the bioconjugation reaction of proteins, and metal-catalyzed site-selective modification of proteins has attracted considerable interest in recent years. we have developed a ruthenium glycosylated porphyrin-catalyzed carbenoid transfer reaction for the site-selective modification of proteins. we firstly applied the catalysis to the selective modification of the n-terminus of peptides. by using ruthenium glycosylated porphyrin as catalyst, the n-terminus of a number of peptides can be modified through carbenoid n-h bond insertion in aqueous media with moderate to excellent conversion. the reaction is highly selective, for example, the reaction with ytsssknvvr, which contains various types of oxygenhydrogen and nitrogen-hydrogen bonds possibly available for carbenoid insertion, catalyzed by the ruthenium glycosylated porphyrin gave the n-terminal-modified product with > % conversion and without the formation of other modified peptides including doubly modified and oxygenhydrogen bond insertion products. we next extended the n-terminal modification method to proteins. eventually success was attained in the modification of rnase a and insulin. the reaction of rnase a with a diazoacetate mediated by ruthenium glycosylated porphyrin gave corresponding n-terminal-modified protein with % conversion. we also achieved a bioconjugation to ubiquitin via ruthenium glycosylated porphyrin-catalyzed alkene cyclopropanation in aqueous solution in two steps: ( ) incorporation of an alkenic group by the reaction of n-hydroxysuccinimide ester with ubiquitin and ( ) cyclopropanation of the alkene-tethered lys ubiquitin with the fluorescent labeled diazoacetate in the presence of a catalytic amount of ruthenium glycosylated porphyrin. the corresponding cyclopropanation product was obtained with % conversion based on maldi-tof mass spectrometry. in conclusion, we developed a ruthenium porphyrin-catalyzed siteselective modification of peptides and proteins in aqueous media. the method provides an entry to new bioconjugation reactions for protein modifications using metalloporphyrins as catalysts. uridine monophosphate synthase: architecture versatility in the service of late blight control francisco tenjo castaño , , manuel garavito , , leonor garc ıa , , silvia restrepo , barbara zimmermann biochemistry and molecular biology research group, universidad de los andes., mycology and plan pathology laboratory, universidad de los andes uridine monophosphate synthase (umpase), a bifunctional enzyme in the de novo pyrimidine biosynthetic pathway, is a protein comprised of orotate phosphoribosyl transferase (oprtase) and orotidine monophosphate decarboxylase (odcase). different fusion orders of the two domains have been documented to exist in nature. in some organisms oprtase and odcase are monofunctional proteins, and act as a complex. here, umpase from solanum tuberosum (potato) and from phytophthora infestans (an oomycete) were examined. p. infestans causes late blight disease in s. tuberosum, destroying crops and increasing production costs. since pyrimidines are fundamental cellular components, we have proposed that umpase could serve as a target to control p. infestans infection. the enzymes from p. infestans and s. tuberosum differ in their fusion order of oprt and odc. the study of these two umpase could facilitate the design of species-specific inhibitors, and might shed light on the effect of fusing umpase domains in one order or the other. to this end we carried out bioinformatic and biochemical characterization of the enzymes. sequence analyses showed residue differences among the p. infestans umpase sequences from three strains: , and t - . strain t - was found to have a duplicated umpase, but neither sequence corresponded to the ones predicted previously from the genome. a recombinant umpase from strain was expressed in bacteria and purified but it showed low solubility and was inactive in vitro. the recombinant umpase from the strain complemented both oprtase and odcase deficient e. coli strains. a soluble, active, recombinant protein was expressed and purified in the presence of high salt and the product ump (specific activity . lmol min- mg- ). the sequence skq was found at the c-terminus of the p. infestans umpase sequences and resembles a peroxisome signal peptide (skl). the predicted hydrophobicity of this umpase and its architecture (oprt at the c-terminus and odc at the n-terminus) resembles that of the umpase from leishmania donovani, which has been localized to the peroxisome. we suggest that p. infestans umps could also be located in this organelle. in contrast to the oomycete enzyme, s. tuberosum umpase is highly soluble, and has a higher specific activity (vmax . lmol min- mg- ). we measured the kinetic parameters km(orotate) . lm, km(prpp) . lm, and found that it exhibited product inhibition by pyrophosphate. in conclusion, the different architectures of the two umps might be related to distinct biochemical characteristics, further supporting this protein as a good candidate for p. infestans control. we present computer simulation studies of three different antimicrobial peptides we have been studying by md computer simulation in collaboration with experimentalists. the first is daptomycin, a potent lipopeptide currently licensed to treat infections caused by multi-drug-resistent bacteria. the mechanism of action of daptomycin is currently not completely understood. we have solved the nmr structure of this molecule, and attempted to determine the size of its oligomer by small angle neutron scattering (sans) supported by computer simulation. feglymycin is a -amino-acid peptide with a high percentage of unusual amino acids such as -hydroxyphenylglycine and , -dihydroxyphenylglycine. feglymicin inhibits mura and murc enzymes which are involved in bacterial peptidoglycan synthesis, while also displaying anti-hiv activity by interaction with the viral envelope protein gp . a previous x-ray structure shows the molecule forming a dimer. here, the molecule was studied by nmr in water and dmso. in water, the molecule is clearly at least a dimer, while in dmso it is a monomer. we have performed noe refinement simulations in order to elucidate a structure, however, due to a lack of long-range noe contacts, a unique structure cannot be determined. labyrinthopeptin a is a lantibiotic that contains labionin, a unique carbacyclic posttranslationally modified amino acid that links the protein backbone in three different locations. labyrinthopeptin a has shown promising activity as a pain killer. starting from the x-ray structure, we present results from the first md simulation studies of this unique peptide. because of the extensive cross-linking, this peptide is observed to be highly rigid in its native form. simulation results of mutants are also presented. antibiotics with new mechanism of action are urgently required to combat the growing health threat posed by resistant pathogenic microorganisms. here we report the discovery of a new peptidomimetic antibiotic (l - ), which is active with a minimum inhibitory concentration (mic) in the low nanomolar range, only against pseudomonas sp., and with a non-membrane-lytic mechanism of action. a drug target identified both in a forward genetic screen for resistance determinants and by photoaffinity labeling is the ß-barrel protein lptd, which plays an important role in lps transport and the outer membrane biogenesis. the x-ray structure of lptd in complex with lpte from shigella flexneri shows a stranded b-barrel linked to a periplasmatic n-terminal jelly-roll domain. interestingly the homology model structure for lptd from pseudomonas shows a significant difference: an insertion of around amino acids in the n-terminal domain. the results of our attempts to purify and characterize this large outer membrane protein and to determine the binding site of the peptidomimetic antibiotic will be shown. the theory of how life on earth begun still remains unclear. nevertheless, according to some theories, at the beginning level proteins did not emerge as a complex globular forms as know today. at the times, when solely rna molecules stored both genetic information and catalyzed the chemical reactions in primitive cells, peptides acted as a proteins nowadays [ , ] . literature postulate that the possible role of primordial short peptides was to catalyze reactions in rna-world, as they possess an excellent ability to self-assemble into well-ordered nanostructures [ , ] . elementary functional loops (efls) can be considered as a small structures (blocks) having specific signatures and providing functional residues important for binding/activation as well as principal chemical transformation steps of the enzymatic reaction [ ] . p-loop efl is a widespread structure across vast majority of protein families such as motor domains, aaa , reca, pepck and many others. sequential alignment of these protein families reveals existence of a conserved p-loop motif, that is able to bind atp molecule. we investigated the structure and atpase activity of peptides, which sequences possessed strongly conserved gxgk[t/s] motif from ploop. the goal of our work was to check if peptides corresponding to the most conserved p-loop motif fragment are able to bind and hydrolyze atp molecule. all peptides under study were chemically synthesized and their structures was investigated by nmr spectroscopy. the ability to bind atp molecules was analyzed by using hplc chromatography. results of our study show, that peptides with conserved p-loop motif have a suitable structures to promote binding of the molecules with phosphate group, but cannot accelerate pyrophosphate hydrolysis process. conference participation for w. _ z. supported by the fp project mobi health (grant agreement no ). computational resources were provided by the informatics center of the metropolitan academic network (ic man task) in gdansk, poland. ck is a ubiquitous serine/threonine protein kinase, being one of the most pleiotropic of all protein kinases . ck plays a key role in cell growth, differentiation, cell death and survival, and become the therapeutic target in cancer treatment, since its level is significantly increased in cancer cells . halogenated ligands have been widely developed as potent inhibitors of protein kinases. among them , , , -tetrabromobenzoteriazole (tbbt) is one of the first potent and selective inhibitor of ck a, directed towards the conserved atp binding site . to assess contribution of electrostatic interactions to the specificity and strength of binding of multi halogenated inhibitors by a protein kinase, we have studied interaction between ck a and nine benzotriazole derivatives, representing all possible patterns of halogenation on the benzene ring. herein, we present results that support existence of two alternative regions that are involved in ligand binding. aspartic acid is known for its function in coordination of a mg ion, which is required for atp binding . asp has been identified in crystal structure of ck :tbbt complex (pdb j , fig. ) as the charged residue closest to tbbt. there is also lys proximal to tbbt, interaction with which may favor anionic form of ligands (pk for tbbt < ), however it is involved in the intramolecular salt bridge, and thus its mutation may significantly change stability of the protein. crystal structure of tbbt complexed with ck (pdb: j ). residues with a distance to tbbt (magenta) shorter than a are shown. red residue is negatively charged, blue ones are protonated. abstract comparison of kdiss values determined for ligands at ph and at ph shows that strength of the complex significantly varies upon deprotonation of the triazole ring. this confirms former hypothesis that a negatively charged ligands cluster at the atp binding site region proximal to lys , which is beneficial both to the specificity and to strength of the binding. we have also observed for the tested ligands variations in their binding to either wild type protein and its d n mutant (with less negative charge distributed over atp binding site). all ligands displaying higher pka for dissociation of the triazole proton bind to the mutant visibly weaker than to the wild-type protein. altogether reveals the predominance electrostatic intermolecular interactions. although, negatively charged ligands most probably cluster at the atpbinding site proximal to lys , beneficial for the strength of binding, the less dissociated forms are favored due to unfavorable interactions of the anionic form of ligands with asp . there are many virulence factors produced by these strains, many of which are encoded on mobile genetic elements. psms are of specific interest because these virulence factors are encoded on the core genome of the bacteria and therefore all strains of staphylococci bacteria produce some variation of psms with a variety of biological functions. the specific mechanism by which psms act as virulence factors has been poorly understood until recently. biological functions of psms include cell lysis, biofilm formation and the ability to kill neutrophils after phagocystosis. these toxins are of special interest to our research group due to their genetic similarities to certain bacteriocins, namely leaderless bacteriocins. both groups of peptides are ribosomally synthesized with a n-terminal formyl methionine and secreted from the bacteria by atp-binding cassette (abc) transporters without any leader sequence or signal peptide. abc transporters may also play a role in immunity towards psms and leaderless bacteriocins. these similarities led our group to investigate the solution structure of these peptides through nuclear magnetic resonance (nmr). isolating psms from the producer organisim, s. aureus, typically involves lengthy extractions and low yields. for these reasons, we opted to chemically synthesize the desired peptides using solid phase peptide synthesis (spps). utilizing a variety of spps techniques, psm a and psm a were successfully synthesized, however, due to the hydrophobic nature of psm b , an alternate genetic approach was devised to isolate psm b . formation of a fusion protein between psm b and the small ubiquitin like modifier (sumo) protein allowed for heterologous expression. upon cleavage of the fusion protein with sumo protease, and subsequent purification and isolation of the cut peptide, psm b was obtained. as previously reported, the psms were found to be alpha-helical in structure inducing solvents. a series of dimensional ( d) nmr experiments were ran to determine chemical shift assignments and to obtain noe data. importing the chemical shift assignments and noe data into the structure calculating software, cyana, we were able to elucidate the solution structure of psm a and psm a and we are currently working towards the elucidation of psm b . the synthesis, isolation, characterization and solution structures of the aforementioned psms will be discussed here. transition metals are critical for enzyme function and protein folding, but their excess can mediate neurotoxic oxidative processes [ ] . as, energy production involves oxidative phosphorylation, a process requiring a continuous flow of electrons, mitochondria are particularly vulnerable to oxidative damage [ ] . as such, mitochondria are the major sites of reactive oxygen species (ros) generation, which are produced as byproducts of the electron transport chain. since free iron and certain ros can engage into potentially deleterious processes such as fenton reaction, mitochondrial iron homeostasis must be tightly controlled, and dysregulation of iron metabolism in this organelle has been associated with various diseases, including friedichs ataxia (fa), alzheimer's, and other neurodegenerative disorders [ ] . engineering an efficient mitochondriatargeting, cell-permeable vector is a challenge due to the fact that mitochondrion is impermeable to a wide range of molecules. the development of delivery vectors has been made possible by a greater understanding of mitochondrial structure and chemical features of molecules that selectively localize within this organelle. from these findings, two generalized requirements for mitochondrial localization are delocalized positive charge and lipophilicity [ , ] . targeting iron in this organelle is proposed as a means to ameliorate fa symptoms. desferrioxamine (dfo) is a bacterial siderophore with high affinity for iron, but low cell penetration. we prepared conjugates of dfo with mitochondria penetrating peptides and studied their iron-binding characteristics in vitro. the lipophilic and charged peptides tat - (h-arg-lys-lys-arg-arg-gln-arg-arg-arg-oh) [ ] , a (h-cha-arg-cha-lys-cha-arg-cha-lys-nh ) [ ] , ss- (h-dmt-arg-phe-lys-nh ) [ ] and ss- (h-phe-arg-phe-lys-nh ) [ ] , are known to permeate cytosolic and mitochondrial membranes. they were prepared and conjugated to dfo in solid-phase [ ] , an alternative synthetic route. once detached from the resin, fully deprotected, purified and characterized by means of lc/ms and aminoacid analysis, it was observed that the dfo-conjugated peptides displayed iron-binding abilities identical to the free chelator dfo. dfo-conjugated peptides were also able to quench the iron-catalysed oxidation of ascorbate (a model of oxidative stress in plasma of iron-overloaded patients), as probed by a high throughput fluorimetric method [ , ] . these results indicate that our synthesis and conjugation strategy were successful in preserving the iron-binding moiety and the antioxidant ability of the free chelator dfo. the proteolytic activity and oligomerization status of the human htra protease functioning as a tumor suppressor of an n-terminal domain not required for proteolytic activity, a central serine protease domain and a cterminal pdz domain. the latter serves as a substrate or regulator binding domain and may participate in oligomerization. htra s, its short natural isoform, lacks the pdz domain which is substituted by a stretch of c-terminal amino acid residues, unique for this isoform. down-regulation of htra in tumors, shown by other groups and us, suggests htra s involvement in oncogenesis [ ] . htra acts as a proapoptotic protein and is suggested to function as a tumor suppressor. it promotes cytotoxicity of etoposide and cisplatin in lung cancer cell lines [ , ] . to date, htra has been poorly characterized from the biochemical point of view, mainly due to the fact that it is difficult to purify recombinant htra . we were able to express in bacterial system and purify htra in quantities sufficient to perform structural studies. the aim of this study was to characterize and compare the proteolytic properties and quaternary structure of the htra isoforms. both studied isoforms lacked the n-terminal domain. htra with the pdz domain removed (htra -dpdz) and htra s (htra s) were fully active at a wide range of temperatures and their substrate affinity was not impaired. this indicates that the pdz domain is dispensable for htra activity. as determined by size exclusion chromatography, htra formed stable trimers while both htra -dpdz and htra s were monomeric. this suggests that the presence of the pdz domain, unlike in other human htras (htra and htra ), influences htra trimer formation. the unique c-terminal sequence of dn-htra s appeared to have little effect on activity and oligomerization [ ] . cyclodextrins (cds) are cyclic oligosaccharides that have been recognized as useful pharmaceutical excipients. in aqueous solution cds are capable to form complexes with various ligands, hosting inside their cavity either a whole molecule, or part of a ligand. inclusion complexes with cds offers a variety of physicochemical advantages over the biologically active ligands, including the improved aqueous solubility, solution stability or an increase of bioavailability. ck is an ubiquitous, highly pleiotropic and constitutively active ser/thr protein kinase. halogenated benzotriazoles have been developed as potent and selective inhibitors of this enzyme. the interaction of the catalytic domain of human protein kinase ck with a series of brominated ligands, which represent all possible patterns of halogen substitutions to the benzene ring of benzotriazole, was previously studied by microscale thermophoresis (mst) [ ] . this method alloweddetermination of binding affinities for seven ligands, all of which were found consistent with the values determined independently by isothermal titration calorimetry (itc). however, a very limited aqueous solubility of some brominated benzotriazoles may decrease their bioavability, thus affectingtheir apparent activity [ ] . to overcome this limitation, the aqueous solubility of halogenated benzotriazoles in the presence of cyclodextrins has been tested. the formation of inclusion complexes with b-cyclodextrin (b-cd), hydroxypropylb-cyclodextrin (hp-b-cd) and g-cyclodextrin (g-cd) in aqueous solutions, followed by uv-vis spectroscopy, substantially improved the solubility of tbbt and its derivatives. the interaction between protein kinase ck and cyclodextrins, and also with their inclusion complexes with halogenated benzotriazoles, was followed with the aid of the microscale thermophoresis. the results obtained clearly demonstrate that the binding of halogenated benzotriazoles by ck is only moderately affected by cyclodextrins. oligonucleotide-based molecular circuits offer the exciting possibility to introduce autonomous signal processing in biomedicine, synthetic biology, and molecular diagnostics. here we introduce bivalent peptide-dna conjugates as generic, noncovalent, and easily applicable molecular locks that allow the control of antibody activity using toeholdmediated strand displacement reactions. employing yeast as a cellular model system, reversible control of antibody targeting is demonstrated with low nm concentrations of peptide-dna locks and oligonucleotide displacer strands. introduction of two different toehold strands on the peptide-dna lock allowed signal integration of two different inputs, yielding logic orand and-gates. the range of molecular inputs could be further extended to protein-based triggers by using proteinbinding aptamers. insights of a novel kind of cell wall binding domain that cleaves the peptidoglycan muropeptide: the cw_ motif noem ı bustamante , , manuel iglesias, noella silva-mart ın, isabel uson, pedro garc ıa, juan hermoso, marta bruix, margarita men endez institute of physical-chemistry 'rocasolano', csic, institute of physical-chemistry 'rocasolano', csic, ciber of respiratory diseases (ciberes), center of biological research (cib), csic, enzybiotics constitute a hopeful alternative to current treatments to fight against bacterial infections. phage endolysins are consider as enzybiotics due to their capacity to cleave the peptidoglycan (pg) of gram-positive bacteria in a generally species-specific manner and kill bacteria when exogenously added ( , ) . the cpl- endolysin, a lysozyme encoded by the pneumococcal cp- bacteriophage, is a remarkable exception among all the pg hydrolases produced by streptococcus pneumoniae and its bacteriophages due to its capacity of degrading pneumococcal cell walls containing either choline or ethanolamine ( , ) . this fact confers to cpl- the advantage of displaying a broader microbicide spectrum comparing to choline binding proteins ( ) . this behavior results from the acquisition of a cell wall binding module (cwbm) made of three identical repeats of amino acids each (cw_ motifs), with unknown specificity and totally unrelated with the choline-binding motives present in pneumococcal hydrolases. interestingly, cw_ repeats have been identified in many putative proteins potentially involved in cell wall metabolism (pfam entry: pf ) from different species of gram positive and gram negative bacteria, and some bacteriophages ( ) . preliminary studies of thermal stability in presence of a small cell wall structural-analogue (glcnac-murnac-l-ala-d-isogln) point to the muropeptide as the cell wall target recognized by cw_ motifs ( ) . in this communication we have gone in depth in the characterization of cw_ repeats. we present the first crystal structure of the cw_ motif, which reveals a three-helical bundle folding. using std_nmr spectroscopy the epitope of binding of the disacharide dipeptide to this repeats has been identified. interestingly, the b anomer of the murnac moiety, the form present in the peptidoglycan, seems to be preferentially recognized with respect to the a anomer. finally, a docking model of the complex cw_ /gmdp compatible with std results was built allowing to identify the major contacts between the protein and the muropeptide and to propose the relevant role of a conserved arginine residue in this interaction. energy-dependent aaa proteases carry out regulated proteolysis to ensure protein quality control and post-translational regulation of many cellular processes. control of proteolysis occurs primarily at the level of substrate recognition, which can be modulated by adaptor proteins. the clps adaptor protein enhances and inhibits degradation of different classes of substrates, and thus triggers a specificity switch in clpa. whereas the mechanism for substrate delivery by clps has been described in detail, the inhibition mechanism is poorly understood. we show that clps inhibits ssra substrate recognition and processing, instead of simply preventing substrate binding. we demonstrate that clpa engagement of the clps n-terminal extension (nte) is necessary, and may even be sufficient, for inhibition. in addition, we find that inhibition of substrate processing requires a longer nte, as compared to inhibition of substrate recognition. interestingly, the nte length required for inhibiting substrate processing is also necessary for suppression of the clpa atpase rate. furthermore, preliminary data suggests that clps slows down substrate translocation. these results support a model where there is an ssra•clpa•clps inhibitory complex in which the clpa pore engages the clps nte. this engagement of the nte causes suppression of atpase activity, and therefore slower substrate translocation and processing. this model illustrates how an adaptor protein can inhibit recognition of one type of substrate while efficiently promoting degradation of a different substrate. single-molecule assay development for studying human rna polymerase ii promoter-proximal pausing rna polymerase ii (polii) pausing has been shown to play a significant role in transcription regulation of elongating polii complexes in a large number of metazoan and mammalian genes ( ) . the traditional understanding of transcription regulation in mammals involved controlling polii recruitment to promoters and controlling initial steps at the promoter, including pre-initiation complex formation and promoter escape. most works investigating promoter-proximal polii pausing have employed chromatin immunoprecipitation followed by sequencing to determine polii localization or in vitro transcriptional assays using nuclear extracts analyzed with radio-active gel electrophoresis. in order to gain greater mechanistic insight into the regulation of promoter-proximal polii pausing, we have been developing a diffusion-based single-molecule method using alternating laser excitation on the micro-second timescale (msalex). the method detects rna transcripts generated by a reconstituted human polii system in vitro using complementary doubly dye-labeled single-stranded dna (ssdna) probes. the human gene hspa b for heat shock protein (hsp ) is used as a model system due to its extensive characterization in drosophila. the method would provide a rapid, sensitive and robust avenue to screen for protein factors regulating promoter-proximal polii pausing. controlling of the pic composition using the reconstituted system allows for dissection of the functional roles of different pic components in facilitating regulation of polii pausing. we have demonstrated the hybridization of double dye-labeled ssdna probe to complementary ssdna mimicking rna transcripts and to transcripts generated with bacterial rna polymerase. also, a functional reconstituted human polii system has been verified using radioactive polyacrylamide gel electrophoresis of transcripts from in vitro transcription assays. malaria is a major global health problem. in , there were an estimated million case of malaria and deaths, most of them children under years old [ ] . among the malaria species that affect humans, plasmodium falciparum is the most deadly form. since no efficient vaccine is available yet, the fight against malaria includes vector control, protection from mosquito bites and artemisinin combined therapy. however, resistances to all known treatments have been observed. therefore, new antimalarial strategies involving novel targets and new mechanisms of action are needed. during its life cycle, in erythrocytic stage, which causes all the malaria symptoms, plasmodium falciparum relies on phospholipids to build the membranes necessary for daughter cell development. approximately % of parasite phospholipids consist of phosphatidylcholine (pc) and phosphatidylethanolamine (pe) synthesized by the parasite through the de novo kennedy pathways. in the pathway of phosphatidylcholine biosynthesis, the second step catalyzed by ctp:phosphocholine cytidylyltransferase [ec . . . ] is rate limiting and appears essential for the parasite survival at its blood stage [ ] [ ] . we are focused on the structural characterization of this enzyme, the identification of effectors by fragment-based drug design approach (fbdd) and then their optimization to eventually design a lead. the first reported crystal structure of the catalytic domain of the enzyme target (pfcct) has been solved at resolution . Å, enzyme-substrates complexes (cmp-, phosphocholine-and choline-bound forms) at resolutions . - Å and an enzyme-product (cdp-choline) complex structure at resolution . Å that give detailed images of binding pocket, demonstrate conformational changes between apo-and holo-protein forms and provide the information about the mechanism of the catalytic reaction at atomic level. the fbdd method uses a library of small molecules (fragments) with molecular weight that does not exceed da to explore target binding sites. although fragments often have too low affinities to evoke a biological response, their probability of binding is high because they are small enough to prevent unfavorable interactions with target protein-binding sites. moreover, they represent more attractive and synthetically tractable starting points for medicinal chemistry compared to more complex compounds. as the affinity is low, fragment screening usually depends on detecting binding rather than inhibition. screenings of a fragment library ( molecules) has been performed by fluorescence-based thermal shift assay and nuclear magnetic resonance saturation transfer difference (nmr std) [ ] . this combination of techniques identified so far fragment hits that are currently evaluated for their binding modes and affinities. co-crystallization of the protein-fragments complexes is carrying out to provide accurate information on the molecular interactions. topology of interactions will be used to rationally monitor every iterative round of the optimization process allowing subsequent rational design. [ ] world health organization, world malaria report (who press, geneva, switzerland), http://www. who.int/malaria/publications/world_malaria_report_ /wmr- -no-profiles.pdf?ua protein scaffolds play a crucial role in signaling pathways by generating signal specificity and increasing signal efficiency and amplitude. engineered protein scaffolds can be used as key regulators for signal transduction in artificial signal transduction cascades where they can regulate in-and output of the network. in this research a - - protein scaffold is developed which induces dimerization of proteins mediated by the small molecule stabilizer fusicoccin. as proof of principle caspase is used to constitute proximity induced dimerization. dimerization of caspase leads to its activation and consecutively initiates the caspase cascade involved in the programmed cell death pathway. caspase does not naturally bind to - - proteins, therefore the caspase monomer is conjugated to a - - binding motif which is known to bind into the binding grooves of a - - dimer. this interaction can be stabilized by the small molecule fusicoccin. we showed that upon addition of small molecule fusiccocin caspase dimerization is induced, resulting in caspase activity which is measured using a synthetic caspase substrate. moreover the biphasic effect of the - - scaffold could be proven. additionally, the activated caspase is also able to cleave its natural substrate caspase , downstream in the caspase cascade. these results indicate that the - - platform is a versatile small molecule induced dimerization platform which can be used as tool for engineering of a synthetic signaling network. the g e variant of the apoptosis inducing factor, responsible of a rare encephalopathy, is hampered in nad /h binding luca sorrentino , laura rigamonti , mirvan krasniqi , alessandra calogero , vittorio pandini , maria antonietta vanoni , alessandro aliverti the apoptosis inducing factor (aif) is a highly conserved mitochondrial flavoprotein known to play two opposite roles in eukaryotic cells: in mitochondria it is required for efficient oxidative phosphorylation (oxphos), while, when released into the cytoplasm, it triggers caspase-independent apoptosis ( ) . the mechanism of aif-induced apoptosis was extensively investigated, whereas its mitochondrial role is poorly understood. there are many evidences of aif importance for mitochondrial correct morphology and functions and recently the discovery of its direct interaction with chchd , a key regulator of respiratory complexes subunits import and folding in mitochondria, was reported ( ) . a unique feature of aif, probably pivotal for its vital function, is the ability to form a tight, air-stable charge-transfer (ct) complex with nad and undergo dimerization. although some aspects of aif interaction with nad / h have been analyzed, its precise mechanism is not fully understood. we investigated the effect of the pathogenic g e replacement, associated with oxphos defect and neurodegeneration ( ) , to understand how it could alter aif properties at the molecular level. to do so, we analysed how the wild type and the g e forms of murine aif interact with nad /h and nicotinamide mononucleotide (nmn / h), finding that the pathogenic replacement resulted in a dramatic and specific decrease of the rate for ct complex formation and consequent protein dimerization only in the case of the physiological ligand. our results demonstrate that the adenylate moiety of nad /h is crucial for the ligand binding process and that the g e replacement causes an alteration of the adenylate-binding site of aif that drastically decreases the affinity for and the association rate of the ligand. in addition, we shed new light on the mechanism of the dimerization process, demonstrating that fad reduction rather than nad /h binding initiates the conformational rearrangement of aif that leads to quaternary structure transitions. taken together, our results contribute to define how aif works at the molecular level in binding nad /h and undergoing dimerization and also point out that the g e replacement, responsible of a rare neurodegenerative disease, has the selective effect of slowing down the formation of aif dimeric ct complex. dipartimento di bioscienze, universit a degli studi di milano, dipartimento di scienze veterinarie e sanit a pubblica, universit a degli studi di mical, from the molecule interacting with casl, indicates a family of conserved cytoplasmic multidomain proteins that catalyze a nadph-dependent f-actin depolymerization activity through their essential n-terminal fad-containing monooxygenase-like domain (mo) in response to semaphorin signaling [ ] . this domain is followed by calponin homology (ch) and lim domains, proline-and glutamate-rich regions and a c-terminal coiled-coil motif that mediate the interaction with various proteins (e.g: crmp, casl, plexin, g proteins, ndr) [ ] . to contribute to establish the catalytic properties of mical mo and their modulation by the additional domains and by the interacting proteins, we have produced and are characterizing the human mical (mical-fl) and forms containing the mo [ ] , mo-ch and mo-ch-lim domains. all mical forms contain stoichiometric amounts of fad in the mo domain and zn ions in the lim domain. mical-mo catalyzes a nadph oxidase (h o -producing) activity. the ch, lim and c-terminal domains lower its catalytic efficiency (kcat/km, nadph) mainly due to an increase of km for nadph. the kcat is similar for all forms excepted for mical-fl where a -fold drop is observed, in agreement with the proposed autoinhibitory function of the c-terminal domain [ ] . the ph dependence of the kinetic parameters of mo, moch and mochlim is complex suggesting that it does not reflect the ionization state of individual groups, but rather the overall protein charge. mical-mo, -moch and -mochlim catalyze a nadph-dependent f-actin depolymerization with a similar apparent km for actin. f-actin (but not g-actin) stimulates the rate of nadph oxidation by increasing kcat and lowering knadph. the extent of nadph oxidation exceeds total f-actin which is in contrast with the proposal of specific modification of actin met or met reported for drosophila and mouse moch [ ] [ ] , but it suggests that f-actin stimulates the nadph oxidase activity or a case of substrate recycling. accordingly, with hmical mo and moch several actin residues are oxidized beside met and met . thus, the ch and lim domains do not seem to be important for the mical-actin interaction and actin modification may be mediated by in situ h o production. in hek t and cos- cells mouse collapsin response mediator protein- (mcrmp ) interacts with mical inhibiting h o production [ ] , suggesting that crmp could be a hydroxylatable substrate of mical-mo. we have produced the same mcrmp form ( - aa) and we have shown that under conditions that limit non specific interactions a mild stimulation (up to %) of nadph oxidation is observed. f-actin reversed the effect of mcrmp suggesting their competition for mical. these results suggest that crmp , a major microtubules regulator, is not the substrate of the mo domain, but actin and microtubules cytoskeleton components may be linked through the formation of crmp-mical complex in response to semaphorin-plexin signaling. experiments are in progress to complete the characterization of mochlim and full length mical forms. green fluorescent protein (gfp), owing to its genetically encoded strong fluorescence, has become one of the most important tools in modern biology [ ] . enhanced gfp (egfp, f l/s t-gfp), frequently used variants of this protein, is thermodynamically more stable and -times brighter than gfp [ ] . due to the improved fluorescent properties, egfp is commonly used as a fluorescent intracellular marker in bio-imaging in vitro and in vivo. despite sustained interest of the scientific community and numerous practical applications, the actual biological role of gfp remains elusive. recent reports put forward a hypothesis of antioxidant and photo-protective functions of gfp [ ] . in this study, we focused on the photo-protective role of egfp against reactive oxygen species (ros) photo-generated by visible light in water suspensions of nano-particular nitrogen-doped titanium oxide (n-doped nano-tio ), that is in the system: 'n-doped nano-tio )/visible light'. n-doped nano-tio (sumitomo tp-s ) was chosen as a photo-catalyst, since it is widely accepted that nitrogen doping enhances visible light photoactivity of tio . -hydroxy- , , , -tetramethylpiperidine-n-oxyl (tempol), a paramagnetic water-soluble compound, belonging to the nitroxide class o superoxide dismutase (sod) mimetics, was used as a target for photo-generated ros. a solar simulator, with the flux output intensity of kw/m , was used as a visible light source. electron spin resonance (esr) was employed to monitor the changes in the paramagnetic signal of tempol exposed to the action of ros in the absence and presence of egfp. in the absence of egfp and after min of illumination, due to a combined action of superoxide (o •-) and hydroxyl (oh•) radicals generated by the system 'n-doped nano-tio )/visible light, the esr signal of um tempol decayed by %. moreover, the growth of a new signal, interpreted as -oxo- , , , -tetramethyl- -piperidinyloxy (tempone), resulting from the attack of oh•radicals on tempol, was also observed. in contrast, in the presence of egfp ( . um) , the ros-induced decay of the esr signal of tempol was markedly smaller, not exceeding %. concomitantly, the growth of the esr signal of tempone was also partially inhibited ( % smaller amplitude), as compared to the process performed in the absence of egfp. in summary, our results point to a significant inhibition of the photodecomposition of tempol in the presence of egfp and support the hypothesis of the protective role of this fluorescent protein against ros generated by the system 'n-doped nano-tio )/visible light'. school of chemistry, national university of ireland galway, school of biochemistry and immunology, trinity college dublin by studying a variety of anionic ligands and their interactions with cationic cytochrome c, we are building knowledge of protein recognition geared towards regulating activity. in previous work it was shown that psulfonatocalix [ ] arene selectively binds to, and encapsulates, three lysine side chains on cytochrome c . here, the binding of two small molecule ligands to cytochrome c was investigated. nmr spectroscopy was used and in one case, a crystal structure of the complex was obtained (fig ) . the calixarene bound to cytochrome c, reveals a crystal packing assembly that suggests it is a key mediator of crystal formation. nmr data analysis indicates the calixarene's binding site on cytochrome c. the pillararene, a relatively new class of compound, is a symmetrical arrangement with a p-rich cavity , related structurally to calixarenes. this suggests good host-guest complexation properties. previously, the carboxylatopillararene showed selective binding to arginine, lysine and histidine . with this ligand, an interaction with cytochrome c was observed and a complex formed. additionally, biphasic binding behaviour was observed through analysis of the chemical shift perturbations. this may indicate more than one binding event taking place. the data from these studies indicate that recognition is occurring and again that lysine side chains play an essential role. the enzyme dihydrofolate reductase (dhfr) is necessary for the growth and development of all organisms. the structure and function of escherichia coli dhfr have been characterized in buffer. however, dhfr exists in living cells, where the protein concentration can exceed g/l. we know that weak, non-specific chemical interactions with cytosolic proteins alter protein conformation and dynamics, , both of which are expected to influence dhfr catalysis. investigators have examined steady-state enzyme kinetics under crowded conditions, but conclusions can be conflicting. , here, the effects of crowding on e. coli dhfr catalysis are assessed through specific activity measurements in solutions of synthetic polymers. these kinetics studies are complemented by in-cell and in vitro f nmr data from fluorinated tryptophan residues. preliminary results suggest that the effects of polymeric crowders on dhfr activity are non-monotonic, which may arise from the polymer's transition from the dilute to semi-dilute regime. the data suggest that synthetic polymers are not a valid representation of the cellular interior. biotechnology department, university of verona calcium (ca ) is one of the most important second messengers in eukaryotes. ca binding proteins can be subdivided into two categories: "ca buffers" that modulate ca ion concentrations in cells, and "ca sensors" that decode ca signals in a wide array of physiological processes in response to external stimuli. calmodulin (cam) is the prototypical example of ca sensor proteins in both animals and plants. in addition to conserved cam, plants possess a unique family of cam-like proteins (cmls). many of these cmls still remain uncharacterized and the investigation of their biochemical and biophysical properties will provide insight into ca signalling in plants. herein, a detailed characterization of arabidopsis thaliana cml is reported. cml is a protein of amino acids with a theoretical molecular weight of , da and % amino acid sequence identity with atcam . cml is predicted to have one functional ca binding site despite the presence of three ef-hand motifs (prosite). we overexpressed cml in e. coli and analyzed its biochemical and biophysical characteristics, i.e. calcium affinity and stoichiometry and eventual changes in conformation, thermal stability and proteolytic susceptibility upon ca binding. isothermal titration calorimetry (itc) and nuclear magnetic resonance (nmr) spectroscopy identified one ca binding site in cml and showed that ca and mg compete for the same binding site. the kd values determined by itc established that cml has higher affinity for ca than for mg . our data were consistent with the sequence based prediction of one functional calcium binding site. differential scanning calorimetry (dsc) showed that ca and mg have the same stabilizing effects on protein folding. apo-cml undergoes two thermal unfolding transitions, but in the presence of ca or mg only one unfolding event at an intermediate temperature occurs. limited proteolysis experiments showed that ca binding affords protection against cml digestion by trypsin. surprisingly, cml exhibits very few conformational changes upon calcium binding, which were evaluated by ans fluorescence and stokes radius measurements in the apo-and ca bound-forms. these results suggest that cml does not show the characteristics of a classical ca sensor protein. to better understand the physiological role of cml in plants, in vivo analysis will be performed. pb- fbp controls the hepatocyte morphology through rho signaling jun zhang , mingming ling , qianying zhang , yunhong wang , deqiang wang the department of cell biology and genetics, the formin-binding protein (fbp ) widely expressed in eukaryotic cells was previously identified to play a role in morphological maintenance in hepatocyte, but the molecular mechanism keeps still unclear so far. in the present investigation, it was found that rho family proteins cdc /rac signaling was involved in the morphological regulation controlled by fbp . knockdown of endogenous fbp expression with rnai technique or dominant negative mutant of fbp could trigger the cell morphological remodeling from the epithelioid to fibroid following the significant down-regulation of cdc / rac activities and dephosphorylation of paxillin. while the rho protein specific activator could restore the cdc /rac activities, and in turn abrogated the silence effect. overexpression of wild type fbp could not result in any of the morphological transition. furthermore, withdrawal of the silence could induce morphological recovery when the fbp expression, cdc /rac activities and paxillin phosphorylation were restored to the normal level. the experimental evidences strongly indicated that fbp was implicated in morphological control probably via rho signaling pathway in hepatocyte. key words: fbp ; rho signaling; paxillin; morphological control; hepatocyte this work was supported by a grant from national natural science foundation of china (nsfc, no. cytochrome c oxidase (cco) is the final enzyme in the respiratory chain of mitochondria but also an integral part of the metabolism of many types of bacteria. in a complex, stepwise redox-reaction, cco catalyzes the reduction of molecular oxygen to water and utilizes the resulting free energy to pump protons across the membrane thereby creating an electrochemical gradient [ , ] . to investigate proton pumping spectroscopically it is possible to label the entrance of the proton entrance channel with fluorescein, a ph sensitive dye, which allows determining time resolved local changes in proton concentration at the cytoplasmic cco surface and related properties. it has already been shown that the redox state of copper and heme centers affects such properties at the cytoplasmic surface. [ ] this study is a theoretical approach to investigate changes of pka values of the fluorescein label at the entrance of the k-channel for different protonation pattern in both oxidized and reduced cco by performing molecular dynamics (md) simulations. further work is based on calculations of pka values of the fluorescein using software karlsberg [ , ] . methods for genetically and synthetically manipulating protein structure enable a greater flexibility in the study of protein function. we have shown that using inteins as traceless, cleavable purification tags enables the separation of full length unnatural amino acid (uaa) containing proteins from their corresponding truncation products. this method has been used to incorporate uaas in previously unattainable positions in a variety of proteins using a myriad of uaas, inteins, and purification tags. in other applications, we have used e. coli aminoacyl transferase (aat) to selectively modify the n-termini of proteins with uaas in denaturing conditions and conditions that maintain folding. applications of particular interest include overcoming the need for an n-terminal cys residue in expressed protein ligation, transfer of reactive handles for "click" chemistry labeling of proteins, and transfer of fluorogenic molecules for photophysical experiments. we have found that aat can transfer protected cysteine, homocysteine, and selenocysteine to expressed proteins. after ligation, these residues can be converted to met or ala, making the ligation traceless. we continue to develop variants of aat to broaden the substrate scope of both its transferred substrate and n-terminal recognition element. in addition, expressed protein ligation is being used to incorporate backbone modifications, such as the thioamide, into various positions in the protein calmodulin to determine how these modifications can impact the structure and function of an ordered protein. in general, by working at the interface of several protein modification technologies, we have made beneficial discoveries that might be missed by more focused approaches. function and modularity of cw_ motives in the c-terminal region of the endolysin cpl- encoded by the cp pneumococcal bacteriophage manuel iglesias-bexiga , , noelia bernardo-garc ıa , rub en mart ınez-buey , noem ı bustamante , , guadalupe garc ıa , , marta bruix , juan hermoso , margarita men endez , dept. of biological physical-chemistry, iqfr-csic, ciber of respiratory diseases (ciberes), department of crystallography and structural biology, iqfr-csic, bacteriophage lytic murein-hydrolases have been proposed as enzybiotics, an efficient way to fight bacterial infections. however, the use of these enzymes is normally restricted to gram-positive bacteria since the outer membrane of the gram-negative bacteria hampers the access of the hydrolases to the peptidoglycan substrates. all the murein hydrolases reported in the pneumococcal system, both from host or phage origin, depend on the aminoalcohol choline to be fully active. there is only a unique exception to this rule, the cpl- lysozyme. this hydrolase is encoded by the lytic pneumococcal phage cp- and, instead of the common cell wall binding module (cwbm) that recognizes choline, cpl- harbors a completely different cell wall binding structure. recent studies have revealed that reducing the net charge of the cwbm, from . to . , leads to an improvement in the antibacterial activity of cpl- ( ) . the cwbm of cpl- is composed by three identical repeats of amino acids, the cw_ motives, and it folds both in the presence and in the absence of the n-terminal catalytic module ( ) . this module shows the capacity of recognize the glcnac-murnac-l-ala-d-isogln muropeptide (gmdp), structurally related with the peptidoglycan basic unit ( ) . here, we report the high resolution structure of the cell wall binding module of the cpl- endolysin. each cw_ repeat is composed of a bundle of three a-helices with a highly negative electrostatic charge at the surface. the strong inter-repeat interactions and the high ionic strength used in the crystallization conditions allow them overcoming the electrostatic repulsions inducing a closed-packed structure with a three-fold symmetry. the module dimensions ( x x Å) and the repeat arrangement in the crystal structure are inconsistent with the gmdp binding characterization, the activity displayed by cpl- truncated variants with one or two cw_ repeats, or the experimental determined hydrodynamic properties. using the small angle x-ray scattering (saxs) technique and the atsas computational platform ( ), a different arrangement of the cw_ repeats is envisaged in solution (fig. ) , whose rather opened structure ( x x Å) is consistent with the experimental data. additionally, employing the saxs-based structure and the honeycomb structure proposed for the peptidoglycan, a model, where each cw_ repeat of the cell wall binding module fit in adjacent glycan chains, has been derived. in , the protein structure initiative (psi) was started as to determine three-dimensional structures of proteins within every family. once solved, structures are deposited into the protein data bank (pdb) and termed structural genomics (sg) proteins. as of june , there are over , sg proteins deposited in the pdb and most of them are of unknown or uncertain biochemical function. in addition, many of these sg proteins have a putative functional assignment based on their sequence and structural similarities with proteins of known function; such comparisons can be made against large databases using programs such as blast or dali. however, these putative functional assignments are often incorrect. this project analyzes members of the crotonase superfamily (cs). the cs consists of five diverse functional subgroups that are well characterized structurally and functionally, representing different types of reactivity, including hydrolase, isomerase, hydratase, and dehalogenase activities. this superfamily also contains at least sg proteins, so it is ideal to test predictions of protein function. our approach is based on local structure matching at the computationally predicted active site. first, partial order optimum likelihood (pool) is used to predict the functionally important residues of each sg protein and of the proteins of known function in the superfamily. next, structurally aligned local sites of activity (salsa) is used to align the predicted catalytic residues of the well-characterized members in the superfamily. from this analysis we generate chemical signatures for each functional subgroup and compare them to the sets of catalytic residues predicted for the sg proteins. we demonstrate based on these computational methods that the majority of the putative annotations in the cs superfamily are likely incorrect. currently, biochemical assays are being used to test these predictions. preliminary biochemical results show that one sg protein, thermus thermophilus q sls _thet , classified as a probable enoyl-coa hydratase, possesses hydrolase activity as predicted by our methods. the outcomes of this project will be to successfully classify the biochemical functions of sg proteins based on their local structure at the predicted active sites and to provide a conceptual framework for the functional classification of the remaining sg proteins within the pdb. this work is supported by nsf-che- . directly observing the synergistic dynamics in f-actin and microtubule assembly jun zhang , deqiang wang the department of cell biology and genetics, key laboratory of molecular biology on infectious disease although important in cellular activities, little attention was paid to the synergistic effects of actin and microtubule cytoskeleton assembly. with the time-lapse atomic force microscope (tl-afm), we directly observed the large-scale dynamic structure of actin filaments formed in the presence or absence of microtubulin in solution. in absence of microtubulin, the g-actin could be polymerized into ordered filamentous structures with different diameter from the slimmest filament of single f-actin to giant filament in tree-like branched aggregates. the polymerized actin filaments, to which our most intense attention was attracted, was discretely arranged and showed obvious polymorphism in structures completely distinct from those in the presence of microtubulin. the supra-molecular complex structures of the latter were mainly composed of single f-actin and/or multifilaments clearly consisting of several single f-actin and regularly cross-linked with the assembled microtubular bundles. the experimental results demonstrated that the f-actin dynamics could be coordinated by microtubule assembly. further analyses implied that the interactions between f-actin and microtubule could prevent the emergence of structural polymorphism of f-actin alone, and give rise to organization of specific complex structures instead. it was suggested that dynamic synergy between the f-actin and microtubule would be implicated in living cells. the adaptor protein - - is found in a diverse range of pathologically relevant protein-protein interactions (ppis). as - - is a hub protein with very diverse interactions, it is able to influence the intracellular localization of their binding partners and they are key regulators of signal transduction processes as well as regulators of cell cycle functions.nevertheless, there are only few examples of - - acting extracellularly. one of the extracellular targets for - - is aminopeptidase n (apn). apn is an extracellular trans-membrane enzyme that acts as a receptor for - - . binding to apn, - - excreted by keratinocytes can upregulate the excretion of matrix metalloproteinase- (mmp ) in fibroblasts. mmp , by breaking down collagens, is key in the remodeling of the extracellular matrix. modulation of the - - /apn interaction thereby may play a crucial role in the fundamental understanding and ultimately treatment of wound healing, respiratory diseases and tumor growth. in the eukaryotic cell, the - - dimer operates as an adapter platform for binding partners. a wide range of classes of (small) molecules, natural products and peptides has been used to modulate the ppis, providing either stabilization or inhibition of the interactions of - - with its binding partner. binding partner fragments or peptides are known to bind to the - - binding groove via arecognition motif containing a phosphorylated serine or threonine. making use of the dimeric structure of - - , novel small-molecule inhibitors may be tethered to exploit the bivalent effect. from a large virtual screening and experimental validation, a scaffold containing a phenyl phosphonic moiety was identified, showing inhibitory properties for - - ppis. potent derivatives of this scaffold were bridged by polyethylene glycol (peg) linkers of varying lengths, thereby facilitating the compound to reach both binding sites of the - - dimer and concurrently increasing the compound's solubility in aqueous solution. similar bivalent inhibitors have been proven to synergistically increase their efficacy. biophysical evaluation by means of fluorescence polarization (fp) inhibition competition assays, revealed an increase of the half maximal inhibitory concentration (ic ) from approximately lm for the monomeric phenyl phosphonate to approximately . lm for the bivalent inhibitor with a Å linker. this demonstrates a -fold increase of inhibitory effect towards - - and its binding partner peptide mimic. extensive thermodynamic, kinetic and structural analysis of the interaction is in progress.phosphonic moieties have been shown to pass the cell membrane poorly, due to their highly charged character. by being able to specifically inhibit the extracellular interaction between - - and apn, these inhibitors are prevented from interfering with the extensive intracellular - - interactome. hence, these bivalent phenyl phosphonate inhibitors provide a promising strategy towards extracellular application. the mre complex is an oligomeric assembly comprising of dimmers of mre and rad proteins in archea and additionally nbs subunit present in eukaryote. it is the central player in the dna damage response -a functional network comprising dna damage sensing, signal transduction, cell cycle regulation and dna double strand breaks (dsbs) repair [ ] . recent structural studies revealed that rad hinge domain is rather a short kink in the coiled-coil region and adopts unusual dimerization mode by intermolecular coordination of zn(ii) and formation of so-called zinc hook domain [ ] . to date, very limited structural data on the zinc hook domain have been reported, the only known structure was resolved for rad homologue from hyperthermophilic archaeon -p. furiosus. unusual zn(ii) coordination mode in zinc hook domain raises question of how zinc hook domain assembles to form interprotein zinc binding site with sufficient stability to function at low intracellular free zn(ii) concentrations [ ] . our study on minimal zinc hook domain fragment ( aa) indicated low femtomolar affinity towards zn(ii) [ ] . extended zinc hook domain fragment ( aa) reveals even zeptomolar affinity. therefore, our main goal was to probe the thermodynamic and structural effects that are hidden in the small interprotein interface and are responsible for the dimerization of the large and critical protein machinery. probing of those effects was achieved by detailed biophysical characterizations (including potentiometry, nmr, hdx ms and cd spectroscopy) of protein fragments of zinc hook domains with a number of point mutations. we showed that extremely high stability of zinc hook domain from p. furiosus is achieved by the formation of hydrogen bond network in b-hairpins and interprotein hydrophobic core. eindhoven university of technology dna-based molecular circuits have become a very attractive tool in molecular imaging, synthetic biology, molecular diagnostics and biomolecular computing. the highly modular and predictable nature of watson-crick base pairing allows the construction of complex circuits using a limited set of logic gates and building blocks. however, the lack of generic approaches to interface dna-based molecular circuits with protein activity limits their application in biomedicine and molecular diagnostics. here we present a new, highly modular approach to control the activity of a reporter enzyme based on the dna-directed assembly and disassembly of a complex between tem -b-lactamase and its inhibitor protein blip. both proteins are conjugated to a unique oligonucleotide, allowing the assembly of the enzyme-inhibitor pair and inhibition of enzyme activity by the addition of a complementary template strand. addition of an oligonucleotide that is complementary to a loop sequence in the template results in the formation of a rigid dsdna spacer that disrupts the enzyme-inhibitor complex, restoring enzyme activity. using this noncovalent approach allowed easy tuning of the template and target sequences with only a single set of oligonucleotide-functionalized enzyme and inhibitor. to show the modularity of the system, a panel of different template sequences were selected. only in the presence of their complementary viral dna sequences restoration of enzyme activity was observed. in addition to this excellent specificity the system showed to by higly sensitive towards its target, since the presence of as little as fmol of target resulted in an observable increase in enzyme activity. the use of a stable and well-characterized enzyme-inhibitor pair, complemented by the modular design of our reversible dna-directed protein switch make it an attractive system to implement in dna-based molecular circuits. several studies demonstrated important roles of human carbonic anhydrases (hcas) in a variety of physiological and pathological processes. consequently, in recent years the catalytically active hca isoforms have become an interesting target for the design of inhibitors with biomedical applications [ ] . derivatized sulfonamides of type r-so nh represent the class of ca inhibitors (cais) mostly used and best characterized. the large number of crystallographic studies so far available on these molecules clarified the main factors responsible for the binding of the sulfonamide moiety to the ca active site. in particular, it has been highlighted that even though these molecules generally behave as very potent cais, they do not show selectivity for the different isoforms. indeed, the sulfonamide moiety plays a predominant role in the interaction with the enzyme, while any change in the nature of the r substituent has generally a rather marginal effect on the enzyme-inhibitor affinity. these characteristics make difficult the design of sulfonamide derivatives selective for the different ca isoforms. consequently, much efforts were dedicated in last years to the development of new inhibitors that, although presenting lower affinity for the ca active site, would be able to be more selective toward the different isoforms. carboxylic acids have been recently investigated as cais, showing that these molecules can adopt different binding modes to the enzyme active site. in particular, they can coordinate directly to the zinc ion or be anchored to the zinc-bound water molecule. however, the structural reasons responsible of this peculiar behavior have not been clarified yet. in a general research project aimed at providing insights into the binding mode of these molecules to cas, we have undertaken the characterization of two carboxylic acids, namely an ortho-substituted benzoic acid [ ] and a saccharine derivative, by means of kinetic, crystallographic and theoretical studies. exploring the mechanism of fibril formation using fluorescently labelled human lysozyme variants ana bernardo gancedo 'exploring the mechanism of fibril formation using fluorescently labelled human lysozyme variants' human lysozyme is a widely characterised protein whose mutational variants misfold into fibrils that are associated with systemic amyloidosis ( ) . although the process of aggregation for human lysozyme has been well studied, the details of early events within this process are not fully characterised. single molecule fluorescence microscopy has been used to determine the oligomeric distributions present in the aggregation process of a number of disease-related intrinsic disordered proteins (idps) ( ) . recent advances in site-specific labelling of human lysozyme ( ) have made this protein amenable to these single molecule fluorescence studies. we have introduced alexa-fluorophores into the i t variant of human lysozyme and have demonstrated that the process of in vitro fibril formation is not significantly altered. using these fluorophore-labelled proteins we can apply single molecule fluorescence to study the early aggregation events within this system, allowing us to compare protein aggregation in a globular protein and with the aggregation process of idp's. abstract protein structure, folding and function, while specific interactions with lipid molecules can also contribute towards the biological activity of some membrane proteins. improving understanding of the interactions has resulted in the development of artificial lipid systems that allow the bilayer properties to be rationally manipulated in vitro to control protein behaviour. the bacterial transporter lacy is a well known integral membrane protein from the major facilitor superfamily, responsible for the protondriven uptake of d-lactose in e. coli. with a high resolution structure available and considerable understanding of mechanistic detail, and with observed changes to both structure and function in different bilayer environments, lacy is a good model system for examining the behaviour of a major class of membrane proteins in these lipid systems. purified lacy has been reconstituted into liposomes and droplet interface bilayer systems of varying lipid composition and the effect on protein function and bilayer properties examined. targeting abeta oligomers by trehalose-conjugated peptides: a molecular dynamics study alzheimer's disease (ad) is currently one of the most common and devastating forms of dementia correlated with beta-amyloid peptide (abeta) accumulation in human brain tissue [ , ] . inhibiting abeta selfoligomerization in brain tissue remains one of the main strategies to prevent or treat this disorder. as a consequence, in recent years much efforts have been spent in the understanding of the amyloid fibril growth process and its modulation by putative drug molecules. an interesting class of compounds able to prevent abeta fibrillogenesis, is represented by beta-sheet-breaker (bsb) peptides [ ] . although these molecules are thought to recognize in a self-complementary manner the abeta hydrophobic core region, however their precise mechanism of interaction is still unclear. in this context, we have studied the structural basis underlying the inhibitory effect of abeta( - ) fibrillogenesis explicated by two promising trehaloseconjugated bsb peptides (ac-lpffd-th (thct) and th-succinyl-lpffd-nh (thnt)) [ ] using an all-atom molecular dynamics (md) approach [ , ] . the pentameric nmr structure [ ] of abeta has been used to model amyloid protofibril, and the two protofibril ends have been investigated as putative binding sites. our simulations suggest that the interaction with the two protofibril ends occurs through different binding modes. in particular, binding on the odd edge (chain a) is guided by a well defined hydrophobic cleft, which is common to both ligands (thct and thnt). moreover, targeting chain a entails a significant structure destabilization leading to a partial loss of b structure and is an energetically favoured process, as assessed by mm/pbsa calculations. a significant contribution of the trehalose moiety to complexes stabilities emerged from our results. the basic structural unit of chromatin is the nucleosome, which is composed of histone proteins forming a scaffold with about base pairs of dna wrapped around. chromatin compacts eukaryotic genomes and regulates gene activity, which is mediated in part by posttranslational modifications (ptms) on the n-terminal tails of the histones. uncovering the detailed relationship between histone tail modifications and gene activity is a major topic of biomedical sciences and general techniques for generating nucleosomes with defined modification patterns in large numbers would greatly facilitate such investigations. to this end we are establishing a chemical toolbox for designer chromatin with defined histone ptm patterns. a protein semysinthesis approach is used that bases on "ligation-ready nucleosomes" with truncated histone h that can be ligated with the corresponding synthetic histone tail. we resorted to sortase-mediated ligation as chemoselective ligation method. here we report our recent developments in establishing the envisioned chemical toolbox for designer chromatin. evaluating cation-pi and pi-pi interaction in proteins using various biophysical methods in proteins the aromatic residues phenylalanine (phe), tyrosine (tyr), and tryptophan (trp) can be involved in aromatic interactions known as cation-pi and pi-pi interactions (dougherty ). compared to other non covalent interactions in proteins, like h-bonds, dipole-dipole, or van der waals interactions, relatively little is known about the pi-pi and the cation-pi interactions. the strength of both aromatic interactions is dependent on the pi-electron density in the aromatic residues. a lowering of electron density can be created by introducing strong electron-withdrawing substituents like fluorine atoms in the aromatic ring (dougherty ). in this way a nearly isosteric change in the aromatic system results in a marked change in electron density. substitution with methyl groups is known to slightly increase the electron density. the response to low cellular oxygen levels in humans and other animals is induced by the hypoxia inducible transcription factors (hifs). these transcription factors are regulated by hypoxia inducible factor prolyl hydroxylases (phds), which act as 'oxygen sensors' by hydroxylating hifs, thus leading to the proteomic degradation of the transcription factors. over the last years, there have been multiple reports that describe additional phd substrates other than hifs. among them are the large subunit of rna pol ii, several transcription factors, and components of signalling pathways. validating these reports is of major medicinal relevance given that phd inhibitors are now in the late stage phase clinical trials. in order to investigate the selectivity of phds, the reported proteins have been tested as substrates for hydroxylation by mass spectrometry, and as binders or competitors of the phds. initial work on peptides that contain the putative hydroxylation sites has indicated that the phds are much more selective for their well-established substrate hif. however, in ongoing work these initial results are going to be validated on protein level by co-expressing phds with the reported substrates. additionally, peptides of reported substrates were screened for their ability to alter the kinetics of hif-hydroxylation by phd . an inhibitory effect of at least two different peptides on phd was observed, suggesting that there is an interaction between the prolyl hydroxylase and these peptides. in order to investigate the mode of binding and inhibition, nmr studies have been carried out and binding of the two inhibitory peptides on phd has been shown. altogether, these results indicate that, although phds might be more selective for hif as a substrate as it was initially thought, the enzymatic activity of the prolyl hydroxylases is possibly influenced by a number of other proteins that can directly bind to phds. non-natural aminoacids via the mio-enzyme toolkit alina filip , judith h bartha-v ari , gergely b an oczy , l aszl o poppe , csaba paizs , florin-dan irimie biocatalysis and biotransformation research group, department of chemistry, ubb, department of organic chemistry and technology an attractive enzymatic route to enantiomerically pure to the highly valuable a-or b-aromatic amino acids involves the use of aromatic ammonia lyases (als) and aminomutases (ams). all these enzymes have in common an auto-catalically formed -methylene- , -dihydroimidazole- -one (mio) electrophilic prosthetic group, and show high structural and sequence similarities. the recent advances in improving the functional properties of these enzymes increased both their biocatalytic and therapeutic applications. we aimed to create a library of recombinant mio-enzymes consisting of the pals and pams with large substrate promiscuity in order to provide access to various non-natural aminoacids through enzymatic ammonia addition and/or ammonia elimination reactions of the substrate library already available in our researchgroup. the developed complementary substrate and enzyme library would provide the mio-enzyme toolkit useful for the synthesis of nonnatural aminoacids. the synthetic gene of the enzymes (pcpal, rtpal, avpal, papam) were cloned into pet b_j expression vector using xhoi and bpu i cloning sites. the plasmid dna was transformed to several e.coli host strains (rosetta, bl , origami ) in order to optimize the expression yields. the enzymes containing an n-terminal his -tag were purified with affinity chromatography, followed by ion-exchange or/and size-exclusion chromatography, obtaining pure and homogenous proteins, in their tetrameric, presumably native fold. the enzyme activity and the kinetic parameters of the purified enzymes was determined towards the natural substrate l-phenylalanine, as well as towards novel bulkier aromatic substrates (heteroaryl alanines, styryl alanines, biphenylalanines). furthermore to enhance their biocatalytic applicability we covalently immobilized the enzymes to carboxylated single-walled carbon nanotubes (swcnt cooh) using linkers with different lengths, and tested the activity and recycling of the immobilized enzyme. antibodies that bind protein antigens are indispensable tools in biochemical research and modern medicine. utilizing a phage display selection strategy, we have obtained synthetic antigen binders (sabs), based on a fab fragment of igg, to a wide array of proteins as distinct as membrane proteins, structural proteins, scaffold proteins and nuclear targets. here we demonstrate the applicability of the sabs towards the native, full-length proteins in cells. we show that the generated sabs are able to pull-down endogenous proteins from mammalian cell extracts along with their natural binding partners. we developed a method of utilizing our high affinity and specificity binders as fluorescently labeled tools to visualize target proteins in their native environment in the cells without the need of secondary antibodies or blocking reagents. our system also includes a method of efficient delivery of generated antibodies to living cells, where they can perform their function. the sabs have been successfully used for altering biological processes in a controllable manner. in vitro evolution from pluripotent peptide libraries with natural neurotoxin scaffolds to target receptors, proteases and trophic factors tai kubo , mohammed naimuddin , seigo ono national institute of advanced industrial science and technology (aist) in vitro evolution from pluripotent peptide libraries with natural neurotoxin scaffolds to target receptors, proteases and trophic factors small molecule natural products are precious resources for drug discovery. during millions of years of evolution, natural products must have been exposed to various selection pressures and have been refined in structure and function to obtain the present features. in some peptide neurotoxins, however, the basic molecular scaffold mainly configured by disulfide (s-s) bridges and/or alpha/beta structures, is strictly conserved within each family even under the evolution pressure. on the other hand the loop regions, which are not heavily involved in scaffold formation, are highly diverged. this mode of molecular evolution named 'accelerated evolution', is reasonable to quickly adapt to the vigorous change of the environment. the evolutionally selected scaffold is compact harboring both rigidity and flexibility in nature, and it may support a topology appropriate for target recognition and selective interaction. inspired by the system, we designed random peptide libraries from the peptide neurotoxins of the accelerated evolution. a three-finger ( f) shaped snake neurotoxin consists of huge family evolved by accelerated gene evolution. we prepared a f-peptide library by introducing random sequences in each fingertip. another random peptide library with an ick (inhibitor cystine knot) motif was prepared based on a neurotoxin gtx - from spider; originally identified as a t-type ca channel modulator. each library was subjected to in-vitro evolution directed to specific target molecules. for the f-peptide library cdna display method was applied to select binders. when interleukin- (il- ) receptors were targeted, the selected f peptides showed binding affinities (kd nm) comparable to the native ligand il- . when trypsin was targeted, peptides with serine protease inhibitor activities similar to sti and bpti (ki nm) were isolated. specific binders to a trophic factor vegf were also generated from the f library. to target membrane proteins, we developed a unique in-vitro evolution system, and named it as the periss (intra periplasm secretion and selection) method. in the system, target membrane proteins are expressed in inner membrane of e. coli and peptides are secreted to the periplasmic space, in between the inner and outer membranes; and the space is served for interaction and selection. the periss method enabled us to identify a peptide specific to muscarinic receptor m subtype from the ick peptide library. in conclusion, it was proved that the library designed from the scaffold of peptide toxin, which evolved in the mode of accelerated gene evolution, has pluripotency in target recognition, interaction and even bioactivity. phenylalanine ammonia lyase from petroselinum cripsum (pcpal) belongs to the class of enzymes containing -methylideneimidazole- -one (mio) as a prostetic group and it is responsible for the conversion of l-phenylalanine into trans-cinnamic acid. this reaction is reversibile under high ammonia concentration. we analyzed several factors that can influence the enantioselective synthesis of nitrophenylalanine mediated by whole cells as well as purified mio-containing and mio-less pcpals. first we investigated the behaviour of the enzymes depending on the ammonia concentration. we also inspected the influence of the ph on the pcpal catalyzed biotransformations. based on our results, we concluded that variation of ammonia concentration and the ph leads to decrease of enantioselectivity, suggesting that pcpal is able to catalyze the formation of both l-and d-enantiomers of electron-deficient structures. all microbial cellulase appears to have a conserved 'sg' amino acid sequence at an identical position in the n-terminal domain. the properties of the n-terminal amino acid sequence were also predicted computationally. this analysis showed that n-terminal sequence of the enzyme is unstable. the nterminal sequence also showed potential cleavage sites by different proteases which may contribute to its instability. the secondary structure analysis showed that the n-terminal sequence has % of the a.a. sequence in extended strand and % in random coil conformation. the n-terminal sequence was also analyzed for potential phosphorylation sites. while no potential serine and threonine sites were predicted, two tyrosine phosphorylation sites were predicted in the n-terminal sequence. the n-terminal sequence was also examined for the presence of kinase specific phosphorylation sites. the results showed the presence of one potential site which may be phosphorylated by pkc at position of the n-terminal sequence. the analysis for the prediction of the presence of oglcnac sites revealed that two such sites may potentially be present in the sequence. we have also predicted the ligand binding site in the n-terminal sequence of the protein. protein arginine methylation catalyzed by protein arginine methyltransferases (prmts), is a pivotal protein post-translational modification involved in a growing number of physiological and pathological processes including signal transduction, proliferation, differentiation and malignancy. prmt accounts for the majority of protein arginine methyltransferase activity in mammalian cells and, in consistence, a large amount of cellular substrates have been identified. several studies have reported that the activity of prmt changes upon stimulation in various cellular processes. in mammalian cells, prmt exists in a high molecular weight complex. the interacting partners of prmt , such as antiproliferative proteins btg and btg , protein phosphatase a, the orphan receptor tr , and ccr -associated factor (hcaf ) are shown to play a role in modulating the methyltransferase activity and the substrate selectivity of prmt . due to the pivotal roles of prmt in physiological and pathological conditions, intensive efforts have been put on the search of small synthetic chemical molecules which can efficiently modulate the activity of prmt for the potential development of therapeutics. in light of this, the intracellular small molecules that either transmit extracellular stimulation or act as cofactor to dictate the activity of prmts in cells are still poorly understood. our study focused on examining how cellular ions might affect the activity of prmt and found that divalent and monovalent ions differentially modulated the catalytic activity of prmt toward different substrates. oligomerisation properties of light-dependent protochlorophyllide oxidoreductase prothoracicotropic hormone (ptth) is one of the most important neuropeptide regulators for insect molting and metamorphosis. however, preparation of its recombinant protein has hardly been successful, because it is a homodimer protein with very complicated disulfide-bond structure. for example, silkworm ptth has three intramolecular disulfide bonds in its -residue polypeptide chain, and the two chains are further linked by an additional intermolecular disulfide bond to form the homomeric dimer. although the recombinant silkworm ptth was previously expressed in escherichia coli, the product was obtained only in precipitation fractions, and refolding of the precipitated protein provided the active dimer ptth in very poor yield. under such reductive conditions as in cytosol of the e. coli cells, formation of the correct disulfide-bond arrangement must be difficult. alternatively, for the heterologous expression of the silkworm ptth, we employed brevibacillus choshinensis (formally referred to as bacillus brevis), which has achieved good results in expression of various disulfide-bond-containing proteins. in this study, the silkworm ptth was expressed in the brevibacillus cells with an additional his -tag sequence at the c-terminus, for easier detection and purification. first of all, since the brevibacillus bacteria are equipped with a secretory system of the expressed proteins, a secretory signal sequence to be attached before the silkworm ptth was carefully selected. among four candidates in a commerciallyavailable kit, a signal sequence derived from an intrinsic cell-wall protein mwp gave better results in expression levels of the protein. second, incubation time of the cells was optimized, because an oligomerization state of the secreted ptth in the cell culture medium changed with the time. in the medium, various ptth oligomers including a monomer and a dimer were initially observed, but higher oligomers became a major portion of the secreted product after longer incubation than h. incubation for - h may be suitable for obtaining the native dimer form of the silkworm ptth. to remove the undesired monomer and higher oligomers, which mostly retained free sulfhydryl groups, the secreted proteins were treated with maleimide-peg -biotin. in the purification using a ni -nta column, the dimer of the his -tagged silkworm ptth was eluted with an imidazole gradient, separately ahead of other biotinylated proteins, probably due to interaction of the peg spacer with the ni -nta groups of the resin. after the reversed-phase hplc purification, the final product showed a single band on the nonreductive sds-page, and it had adequate ecdysone-releasing activity from isolated silkworm prothoracic glands. the brevibacillus bacteria are most promising host cells for the heterologous production of the insect ptth. role of the disulfide bridges in the transmembrane region of the insect prothoracicotropichormone receptor, torso torso is an insect cellular-membrane protein, which was recently identified as a receptor for prothoracicotropic hormone (ptth). although ptth is one of the important regulatory molecules in insect molting and metamorphosis, activation mechanism of torso by the ligand has not been elucidated yet. in this study, an oligomerization manner of the silkworm torso was examined, using heterologous expression in drosophila s cultured cells, because torso is a single-polypeptide receptor tyrosine kinase (rtk), and activation of such rtks is often triggered by the ligand-induced receptor dimerization on the cellular membrane. when activated with silkworm ptth, dimerization of the silkworm torso in the s cells was observed, using a cross-linking reagent bs , and the subsequent receptor autophosphorylation and downstream erk phosphorylation were also detected. surprisingly, however, the torso dimerization was revealed to occur even without the ligand stimulation, while the autophosphorylation and the erk phosphorylation were held in response to the stimulation. when fractionated by non-reductive sds-page, the silkworm torso showed an obvious dimer band, in addition to a faint monomer band, both with and without the ptth simulation, even though the receptor was not treated with the cross-linking reagent. this indicates that the torso protein is expressed originally as a disulfide-bond-linked dimer. in addition, by examining oligomerization states of several truncation and substitution mutants, cysteine residues in the transmembrane region were found to participate in the intermolecular disulfide bridges, linking the two receptor molecules in the dimer. when all of the three cysteines in the transmembrane region were replaced by phenylalanines, the disulfide-bond-linked torso dimerization was not observed, but spontaneous, ligand-independent association of the torso molecules was detected using the crosslinker bs . this spontaneous dimerization caused the apparent torso autophosphorylation, but it could not induce the downstream erk phosphorylation. consequently, without the intermolecular disulfide bridges, torso loses its responsiveness to the ptth stimulation. in conclusion, the disulfide bridges in the transmembrane region may play a role to preserve suitable relative position between the two torso molecules, which could induce ligand-dependent autophosphorylation leading to activation of the downstream signaling pathways in the cells. the yeast enzyme neutral trehalase (nth , ec . . . ) from saccharomyces cerevisiae hydrolyses the non-reducing disaccharide trehalose which serves as an energy source and a universal stress protectant in many different organisms. enzymatic activity of nth is enhanced by the yeast - - protein (bmh and bmh ) binding in a phosphorylation-dependent manner. nth activity is also regulated by ca binding to the ef-hand-like motif containing domain of nth [ ] .the native tbe page and analytical ultracentrifugation show that nth forms very stable complexes with bmh and bmh [ ] . to study the structure of nth alone and its complex with the - - protein we used circular dichroism, h/d exchange coupled to mass spectrometry, chemical cross-linking [ ] and small angle x-ray scattering (saxs) [ ] . at the same time protein crystallography of nth alone and its complex with bmh is performed.the low resolution structure of pnth :bmh protein complex revealed that binding of bmh induces a rearrangement of the whole nth molecule and that the region containing the ef-hand motif forms a separate domain which interacts with both bmh and catalytic domain of nth . we proved that integrity of the ef-hand motif is crucial for the bmh mediated activation of nth and ca binding. our data suggest that the ef hand-like motif functions as the intermediary through which bmh modulates the function of the catalytic domain of nth . these structural changes probably enable the substrate entry into the enzyme active site [ ] . our study of - - protein complex with the fully active enzyme nth offers a unique structural view of nth activation enabling us to better understand the role of the - - proteins in regulation of other enzymes. the assembly of self-regulating synthetic biochemical pathways in vitro has great potential as alternative catalysts for the high-yield production of low value/high volume commodity chemicals from biomass. high yields of low-value/high volume compounds that are required for economic viability is particularly difficult via traditional in vivo metabolic engineering of microbes due to competing biochemical pathways and toxicity. we have developed an alternative approach, called synthetic biochemistry, where the glycolysis pathway of central metabolism is reconstituted in vitro with an anabolic pathway that can produce useful compounds at high yield. in the specific synthetic biochemistry system described, reducing equivalents, atp, and carbon from glycolysis are funneled through the anabolic mevalonate pathway to produce the monoterpene limonene from glucose. the successful implementation of the in vitro pathway required development of a molecular purge-valve consisting of an nad and nadp specific reductase (ie wild-type and mutant pyruvate dehydrogenase), and nadh oxidase, noxe, to maintain proper nadp /nadph cofactor balance while allowing continuous carbon flux. we find that the purge-valve concept is readily transportable to other nad(p)h generating steps in central metabolism and can be used to convert glucose to limonene at high yield. chitinases (ec . . . ) are enzymes that randomly hydrolyze b- , glycosidic bonds of chitin and produce n-acetylchitooligosaccharide ((glcnac)n) that has various physiological functions such as immunostimulatory activity. most of fish takes crustacean such as shrimp and crab as food. therefore, the fish has chitinase in the stomach to chemically disrupt the chitinous envelope of crustacean. four chitinase isozymes ( - kda), pachia [ ] and pachib [ ] , and ptchia and ptchib, [ ] were purified from the stomach of silver croaker pennahia argentatus and threeline grunt parapristipoma trilineatum, by ammonium sulfate fractionation and column chromatographies, respectively. all the chitinases were stable and showed activity in the acidic ph range (ph - ). pachia and ptchia preferentially degraded the second glycosidic bond from the non-reducing end of (glcnac)n and pachib and ptchib had a preference for the third glycosidic bond of those. all the chitinases showed different substrate specificity toward insoluble long substrates. moreover, chitinase cdnas (pachi- and pachi- ) encoding pachia and pachib, and cdnas (ptchi- and ptchi- ) encoding ptchia and ptchib were obtained by cdna cloning using the rt-pcr and race method. the deduced amino acid sequences of all the chitinase cdnas contained n-terminal signal peptide, gh family catalytic domain, linker region, and chitin-binding domain. phylogenetic tree analysis of vertebrate chitinase revealed that fish stomach chitinases form unique chitinase isozyme groups, acidic fish chitinase- (afcase- ) including pachia and ptchia, and acidic fish chitinase- (afcase- ) including pachib and ptchib, which was different from an acidic mammalian chitinase (amcase) group. [ , ] the previously reported purified fish stomach chitinases [ ] can also be classified into two chitinase isozyme groups, afcase- and afcase- , by the n-terminal amino acid sequence. this study suggested that fish have excellent chitin degrading enzymatic system in which two different chitinases isozyme groups, afcase- and afcase- , with different degradation patterns are expressed in the stomach. recently, the enzymes produced by psychrophilic organisms have gained huge interest especially in the studies of temperature adaptation of the protein. previously, a cold-adapted yeast, glaciozyma antarctica pi was isolated from a marine environment in antarctica and the yeast was known to produce lipolytic and proteolytic enzymes. a gene encoding a unique recombinant bifunctional enzyme (lippi ) with cold active lipase with protease activity was successfully expressed, purified and characterized. temperature profile of the bifunctional lippi enzyme showed that the lipase functions optimally at c whereas the protease was more active at c. ph profile showed that both lippi lipase and protease were active at near neutral condition. activity of lippi lipase and protease were also activated in the presence of cacl but its protease counterpart seemed to be more active in the presence of zncl . effect of surfactants showed lippi lipase was activated by tween and sls and in contrast, lippi protease was almost deactivated in all surfactants tested. the presence of organic solvents did not affect both the lipase and protease activities. the lipase was more stable at solvents with higher log p value whereas the protease was slightly activated at low log p value particularly with dimethylsulfonyl. inhibitor studies revealed that lippi lipase was partially inhibited with edta and pmsf whereby the lippi protease was inhibited by pepstatin, edta and pmsf. lippi enzyme was successfully crystallized via vapour diffusion method. crystal of lippi enzyme was diffracted via synchrotron radiation. the three-dimensional structure of cold-adapted pi provided insight into cold adaptation and better understanding of the structural properties of lippi enzyme. the bifunctional properties of the enzyme could be potential candidate for low temperature industrial application. conformation-specific antibodies as enhancers and inhibitors of phosphatase activity of dep malgorzata nocula-lugowska , mateusz lugowski , anthony a. kossiakoff the university of chicago dep- (cd /ptp-h) is a transmembrane receptor-like protein tyrosine phosphatase (ptp) that has been implicated in the density-dependent regulation of cell growth, differentiation and transformation. it counteracts protein kinases by dephosphorylating a number of their substrates as well as the kinases themselves, thus potentially controlling the specificity of signals. for example egfr, vegfr , met, pdgf b receptor have been shown to be dephosphorylated by this phosphatase. dep- has been shown to act as a tumor suppressor and it has been proposed as a molecular target in antiangiogenesis therapy. as a result, both enhancers and inhibitors of dep- activity have the potential of elucidating pathways responsible for abnormal cell behavior. we generated synthetic antibodies against intracellular catalytic domain of dep- that act as modulators of the enzyme's phosphatase activity. by applying a combination of selection pressures an array of antibodies has been raised from phage display libraries of fab fragments which are capable of either enhancing or inhibiting dep- activity. in phosphatase assays with catalytic domain of dep- the antibodies demonstrate non-competitive or mixed kinetics. the crystal structure of dep- -inhibitor complex shows that this antibody binds to the part of the protein that is distant from the active site and acts by locking the enzyme in the nonnatural catalytically inactive state by hindering the closure of the wpd loop which is crucial for the reaction to occur. by contrast, as judged from the crystal structure of a complex of dep- with the antibody that enhances its phosphatase activity, this antibody seems to act by stabilizing the naturally found active state of dep- with wpd loop in the closed conformation. the antibodies are also able to recognize dep- in cells, as they stain dep- in immunofluorescence experiments. to test the applicability of raised antibodies in cells the activator was additionally used to pull down full-length endogenous dep- after being delivered to live cells. inhibition and enhancement of dep- activity by locking the enzyme in conformations which are either natural or imposed by allosteric binding of antibodies seems to be a mechanism that can be utilized to modulate activity of other tyrosine phosphatases. investigating acinetobacter baumannii pathogenesis: crystal structure of wbjb epimerase from a polysaccharide biosynthesis cluster oxygen homeostasis is regulated by hypoxia inducible factor, a transcription factor. when the oxygen level becomes too low (hypoxia), hypoxia-inducible-factor (hif- a) activates the expression of over a hundred genes, associated with angiogenesis, erythropoiesis, vegf (vascular endothelial growth factor), cell migration, and energy metabolism etc. hif- a cellular level is highly dependent on oxygen concentration and regulated by oxygen sensor enzyme, hif prolyl hydroxylase (phd plant sulphite reductase (sir) forms an electron transfer complex with ferredoxin (fd) for the reductive conversion of sulphite to sulphide. although previous studies have highlighted electrostatic interactions between oppositely-charged residues of the two proteins, detailed thermoenergetics of the intermolecular interaction for the complexation remains unknown. we herein carried out isothermal calorimetry of fd:sir complex formation at various nacl concentrations. driving force plot constructed from calorimetry showed that the complex was thermodynamically stabilized by both enthalpy and entropy through favourable electrostatic and non-electrostatic interactions. increasing nacl concentrations weakened interprotein affinity and contribution of the negative enthalpy changes became decreased, while no such significant decrease was found in the contribution of positive entropy changes. furthermore, a negative heat capacity change obtained from the enthalpy changes at distinct temperature indicated a contribution of hydrophobic interactions. these findings suggested that both electrostatic and nonelectrostatic interprotein interactions were energetically important for the complex formation. fddependent sir activity assay revealed a bell shaped activity curve with a maximum under a certain nacl concentration, while the methyl viologen-dependent assay of sir exhibited a profile of saturating curve, suggesting that an optimized interprotein interaction is a crucial factor in control of fd-dependent-sir activity. a residue-based nmr measurement of n-labeled fd upon complex formation with sir revealed that charged and non-charged residues were differentially contributed in the complex formation depending on nacl concentrations. we proposed that non-electrostatic forces were also critical for forming the fd:sir complex, and an optimized complex conformation for maximum enzymatic activity was achievable by a delicate balance among non-covalent intermolecular forces. these results may be extended for understanding of complexation between redox proteins containing biased charge clusters. ornithine transcarbamylase has a spatially extended active site as computationally predicted lisa ngu , kevin ramos , nicholas delateur , penny beuning , mary jo ondrechen understanding how an enzyme catalyzes a reaction is a fundamental problem in protein science. biochemical experimentation has revealed catalytic mechanisms of many enzymes; however these studies have focused almost exclusively on amino acid residues in direct contact with the reacting substrate molecule(s). here we report on the computational prediction and experimental verification of the importance of distal residues in enzyme catalysis, using e. coli ornithine transcarbamylase as an example. partial order optimum likelihood (pool), developed at northeastern university, is a machine learning technique that only requires the tertiary structure of a protein to predict important catalytic residues, based on computed, residue-specific electrostatic and chemical properties. pool has been shown to predict accurately the catalytic residues and to discern between compact and spatially extended active sites. dynamic conformational changes during catalysis and strong electrostatic interactions give rise to significant coupling between remote residues and the canonical active site residues of an enzyme. this suggests that at least some enzyme active sites are spatially extended, with second-and third-shell residues playing significant roles in catalysis. in this project, we focus on ornithine transcarbamylase (otc), for which dynamic processes are believed to play a role in its catalytic mechanism. otc is reported to undergo induced-fit conformational changes upon binding carbamoyl phosphate, which affects the subsequent binding of ornithine. residues predicted by pool to be catalytically important include five in direct contact with the substrate, r , h , d , c and r . pool also predicted remote residues to form a spatially extended, triple-layer active site. guided by computational predictions and using site-directed mutagenesis and kinetics assays of asp , his , glu and arg variants, we show that these pool-predicted remote residues, located in the second and third layers, are important for catalysis. alternative energy is a major focus of current research efforts. biodiesel, a mixture of fatty acid alkyl esters, is one of the most versatile alternative fuels currently in use. this is due to the fact that it is similar to gasoline and compatible with diesel engines found throughout the existing global infrastructure. biodiesel precursor lipids are abundant in cultivated feedstock organisms such as algae and bacteria. however, the standard process for converting oil to biodiesel is heat-intensive and requires complete removal of water, reducing the overall net energy gained in its production. our work constitutes an attempt to explore enzymatic synthesis of biodiesel from lipids such as those derived from emerging fuel crops. previous literature describes fatty acid alkyl ester formation in human patients with mrsa staphylococcus aureus wound lesions. these esters are formed by partially characterized esterase activity from an unidentified source. we have identified two mrsa enzymes responsible for this activity by using a combination of size exclusion chromatography, gas chromatography-mass spectrometry, and mass spectrometric protein sequencing. these two highly similar enzymes in the glycerol ester hydrolase (geh) family of proteins catalyze the synthesis of fatty acid alkyl esters in aqueous conditions at or near room temperature. we have demonstrated that other non-staphylococcal lipases do not exhibit this behavior. we have expressed these staphylococcal esterases in e. coli, and shown via gas chromatography that the expressed proteins catalyze the formation of fatty acid alkyl esters. based on sequence similarity to homologous proteins that have already been crystallized, we have predicted a structure for these enzymes and have engineered mutant fusions with higher rates of catalysis. our design hypothesis is that increased avidity for substrate molecules will yield a higher substrate concentration in the vicinity to the enzyme. to increase substrate concentration we have designed and expressed one of the enzymes as a chimeric fusion with the drosophila melanogaster alcohol-binding protein lush. gc-ms determination of biodiesel production rate indicates that the chimeric fusion has a lower-order rate constant with respect to ethanol. in other words, the fusion enzyme is less dependent on substrate concentration and is a superior catalyst at low ethanol concentrations. this result indicates that the rationally designed modification of binding avidity constitutes a potential avenue for improving the ability of enzymes to catalyze reactions with low-concentration or low-solubility substrates. functional elements of a human antizyme essential for binding and inhibiting human ornithine decarboxylase proteases are ubiquitous enzymes that catalyze the hydrolysis of peptide bonds within protein substrates; they have served as key model enzymes for studying the molecular basis for catalytic power and specificity. protease substrate specificity is most often defined in terms of linear sequence motifs that flank the cleavage site; however, the natural substrates of proteases are proteins with -dimensional shapes and complex conformational dynamics that are not well represented by -dimensional sequence alone. these structural and dynamical properties can impact recognition and binding of substrates by proteases, as well as the efficiency of catalysis itself. in this study, we explore the importance of substrate structure and dynamics for proteolysis using as our model the cleavage of the kunitz-bpti family of canonical serine protease inhibitors by mesotrypsin. bovine pancreatic trypsin inhibitor (bpti), an archetypal serine protease inhibitor of the kunitz family, has a high affinity interaction with trypsin, yet its peptide bond hydrolysis is many orders of magnitude slower than other peptide substrates. mesotrypsin, a trypsin variant, has been shown to hydrolyze kunitz family inhibitors at accelerated rates; this is especially true of human kunitz domain inhibitors. amyloid precursor protein inhibitor (appi) and amyloid precursor like protein- (aplp ), two human kunitz domain family members, are hydrolyzed by mesotrypsin several hundred times faster than bpti. here, we present a new, unpublished crystal structure of a cleavage intermediate aplp bound to mesotrypsin, refined to . Å resolution, revealing a dramatic substrate conformational change we hypothesize to be required during cleavage of a kunitz domain. using this structure along with published structures of appi and bpti complexes, we have modeled acyl-enzyme intermediates of mesotrypsin, and we have carried out molecular dynamic simulations that explore the transition of the initially formed native-like acyl-enzyme through the conformational transformation that allows the progression of the hydrolysis reaction. we further identify a specific hydrogen bond, present in bpti but not appi, which forms a stabilizing feature of the bpti scaffold. using site directed mutagenesis, we probe the contribution of this bond to the proteolytic stability of bpti. collectively our data for these highly structured substrates show that proteolysis rates are limited by a necessary conformational change in the substrate as the reaction progresses. rigid substrates possessing stabilizing features that render them highly resistant to this conformational change are proteolyzed more slowly than more flexible substrates of similar structure. lpmos are copper metalloenzymes that carry out the oxidative cleavage of the b- , -glycosidic bond, generating new chain ends that can subsequently be processed by cellulases, boosting the cellulose degradation. lpmos have a b-sandwich conformation with a flat binding surface, allowing for the enzyme to bind to crystalline cellulose. the cu ion, required for activity, is located in a so-called "histidine brace", in which the n-terminal histidine is highly conserved. regioselectivity according to the carbon atom being oxidized, lpmo types are identified: type and type oxidizing at the c and the c respectively, type lpmos oxidizing both the c and the c adjacent to the glycosidic linkage. we were able to express a type- lpmo (phanerochaete chrysosporium gh d) and a type- lpmo (trichoderma reesei cel a) in p. pastoris. this has proven to be very challenging, as lpmo activity requires a perfect cleavage of the signal sequence. after activity assays on pasc, characteristic hpaec-pad traces were obtained which will serve as a reference for engineering experiments. enzyme engineering using the dm database, a structure based multiple sequence alignment tool, it is possible to identify residues specifically conserved in subsets of protein sequences. by defining a subset for each lpmo type, we were able to identify residues contributing to regioselectivity. these positions are now being rationally engineered in subsequent rounds of mutagenesis, using trcel a as a template. the effect of the mutations will be determined by analyzing the hpaec-pad trace released from pasc. the main goal is to investigate the possibility of deleting the c specificity in a type lpmo. folding topology determines substrate binding order in the ribokinase superfamily alejandra herrera-morand e , victor castro-fern andez , madrid, españa ribokinase superfamily comprises three enzyme families: the adp-dependent sugar kinases family, the atpdependent coenzyme kinases family and the atp-dependent sugar kinases family. in all these families there is a large domain composed by a rossmann motif but only the atp-dependent enzymes have a b-meander motif in the c-terminal end. interestingly, these enzymes display an ordered kinetic mechanism where the substrate that will be phosphorylated binds first to the enzyme. the adp-dependent enzymes present a topological re-ordering of the secondary structural elements which produces an equivalent tertiary structure, which can be thought as a non-circular permutation (ncp) of the bmeander region. these enzymes also display an ordered kinetic mechanism but with an inversed order being the nucleotide the first substrate to bind to the enzyme. as this b-meander region of the proteins constitutes almost entirely the nucleotide binding site, and given that the permutation is the major structural difference between adp and atp-dependent kinases, it could the responsible for the nucleotide specificity. to test this hypothesis we introduce, by permutation, an atp-dependent topology in the homologous adp-dependent glucokinase from t. litoralis (pergk). size exclusion chromatography and circular dichroism spectra show that both the wild type and the permutated enzyme eluted as monomers with similar hydrodynamic behavior, and have the same secondary structure content. kinetic assays employing atp or adp as substrate demonstrate that even in the presence of mm atp, the pergk enzyme is not able to carry out the phosphoryl transfer. to test if the ncp has an impact in the kinetic constants and substrate binding order we determine the kinetic mechanism through classical protocols, involving initial velocity studies, product inhibition and dead end inhibitors. the results demonstrate that the pergk enzyme presents an altered substrate binding order compared to the wild type enzyme, where glucose was the first substrate to bind to the enzyme and glucose- -p the last product to be released. also, ligand-induced conformational changes were determined in the crystal structures. the apo, the enzyme-glucose and enzyme-glucose-adpbs structures were determined at . Å, . Å and . Å resolutions, respectively. structure analysis reveals that glucose binding provokes major conformational changes in the pergk enzyme, whereas adp binding does not cause further changes in the conformation of the protein. the results show that although the permutation has no effect on the nucleotide preference it provokes a change in the substrate binding order that correlates well with that those observed in the crystal structures. also, they demonstrate that during the evolutionary history of the ribokinase superfamily folding topology dictates the substrate binding order (fondecyt ). background: human ceruloplasmin (cp) is a circulating copper-containing glycoprotein produced in the liver and first described as a component of alpha -globulin fraction of human plasma. cp belongs to the multicopper oxidase family and it is nowadays regarded as a "moonlighting" protein, because it changes its function according to substrate, localization and expression. cp plays a key role in copper transport and iron metabolism and it is also a potent inhibitor of leukocyte myeloperoxidase (mpo) (kd nm), a major source of oxidants in vivo. the protein is extremely susceptible to proteolysis. in fact, cp is a structural homolog of coagulation factors v and viii, that are physiological substrates of thrombin (fiia). interestingly, thrombin participates in both haemostatic and inflammatory responses: in some focus of inflammation, such as rheumatoid arthritis (ra), the high activity of fiia has been documented. it was demonstrated that fiia can promote the chemotaxis of neutrophils and monocytes and their adhesion to endothelial cells, to increase vascular permeability. all these effect are mediated by par- interaction, that are abundantly expressed in inflamed rheumatoid synovial tissues. aims: in this study the interaction of cp with thrombin was investigated to confirm the participation of fiia in "spontaneous" proteolytic degradation of cp. in fact, in vivo the integrity of cp is essential for its role in the transport or metabolism of copper. results: our results indicated that thrombin cleaves cp in vitro at arg-ser and lys-val bonds, generating a nicked species that retains the native-like fold and the ferroxidase activity of the intact protein, whereas the mpo inhibitory function of cp is abrogated. analysis of the synovial fluid of ra patients reveals that cp is proteolytically degraded to a variable extent, with a fragmentation pattern similar to that observed with fiia in vitro, and that proteolysis is blocked by hirudin, a highly potent and specific thrombin inhibitor. we demonstrate that fiia has intrinsic affinity for cp (kd - nm), independently of proteolysis, and inhibits cp ferroxidase activity (ki nm). mapping of thrombin binding sites with specific exosite-directed ligands (i.e. hirugen, fibrinogen gamma-peptide) and thrombin analogues having the exosites variably compromised (i.e. prothrombin, prethrombin- , alpha-thrombin), reveals that the positively charged exosite-ii of thrombin binds to the negative upper region of cp, while the protease active site and exosite-i remain accessible. these results suggest that thrombin can exacerbate inflammation in ra by impairing via proteolysis the mpo inhibitory function of cp and by competitively inhibiting cp ferroxidase activity. an artificial pathway for isobutene production by direct fermentation: combining metabolic engineering and protein engineering benoit villiers , franc¸ois stricher the purpose of global bioenergies is to develop innovative metabolic pathways for the production of light olefins from renewable resources, by direct fermentation. light olefins (ethylene, propylene, linear butylene, isobutene and butadiene) are the core of the petrochemical industry. however, microorganisms do not naturally produce light olefins and no bioprocess to convert renewable resources to these molecules has been industrialized so far. global bioenergies has developed an artificial metabolic pathway including all the necessary enzymatic reactions from feedstock to isobutene. the metabolic route leading to isobutene can be divided in three parts, the first one being the use of natural reactions occurring in the host microorganism. second, heterologous natural reactions were introduced into the same host microorganism. finally, in contrast with most former approaches, non-naturally occurring reactions as enzymatic key steps were used, for example the decarboxylation of hydroxyisovaleric acid into isobutene. such non-natural critical steps were made possible by taking advantages of the natural catalytic and substrate promiscuity of exogenous enzymes. candidate enzymes are then evolved using systematic, random and semi-rational approaches in successive rounds in order to reach the desired catalytic efficiency. since all these reactions are enzymatic, isobutene can be obtained by direct fermentation, e.g. a process wherein all the chemical transformations are carried on by the host microorganism. the scale-up of this process began in november in a pilot plant installed in pomacle-bazancourt, france, with an annual capacity of tons of oxidation-grade isobutene. importantly, production of a volatile compound such as isobutene (and other light olefins) by direct fermentation presents two major advantages: first, the product is spontaneously removed from the culture broth, which alleviates the limitations linked with titer issues. second, the purification process is considerably easier and cheaper since no energy consuming methods such as distillation or phase separation are necessary to purify the end product. for the first time, batches of industrially produced isobutene from renewable resources have been obtained in the first half of . this isobutene has been in turn converted into isooctane, an additive currently used to improve gasoline quality, which could also be used as a standalone fuel. a demonstration plant is planned in leuna, germany, with an annual capacity of tons of polymer-grade isobutene and ibn-one, a joint venture with cristal union ( th european beet processor), has been formed to build and operate the first plant in france converting renewable resources into isobutene. finally, while the isobutene process is progressing towards industrial scale, global bioenergies is also developing new artificial metabolic pathways enabling direct bio-production of butadiene and propylene. the development of a coupled enzyme assay to detect isochorismate pyruvate lyase activity protein folding is typically defined in terms of the spatial arrangement of structural elements, i.e. helices, sheets and loops. we have, however, been developing an alternative and complementary paradigm based on conserved hydropathic interaction networks within proteins. these networks can be viewed as environments comprised of a mixture of polar and hydrophobic interaction fields, and may be the most important factor driving protein folding. this concept applies even to the lowest structural level within a protein: the sidechain conformations (or rotamers). exhaustive statistical analysis of existing crystallographic structures of proteins showed rotameric preferences and led to the creation of rotamer libraries frequently used in multiple aspects of structural biology, e.g., crystallography of relatively low-resolution structures, homology modeling and biomolecular nmr. however, little is actually known about the forces and factors driving the preference or suitability of one rotamer over another. in our study, tyrosine was analyzed since its sidechain has a comprehensive set of hydropathic properties that made it ideal as a proof of concept residue. construction of d hydropathic interaction maps of tyrosine residues in our dataset, reveals the environment around each, in terms of hydrophobic (p-p stacking, etc.) and polar (hydrogen bonding, etc.) interactions. after partitioning the tyrosines into backbonedependent bins, a map similarity metric based on the correlation coefficient was applied to each mapmap pair to build matrices suitable for clustering. notably, the first bin representing tyrosines, reduced to unique hydropathic environments with most diversity arising from favorable hydrophobic interactions with many different residue partner types. polar interactions for tyrosine include ubiquitous hydrogen bonding with the phenolic oh and somewhat surprisingly a handful of unique environments for the tyrosine backbone. all but one of the environments are dominated by a single rotamer, the exception being an environment defined by a paucity of interactions with the tyrosine ring and as a consequence its rotamer is indeterminate. this is consistent with it being composed of mostly surface residues. each tyrosine residue attempts to fulfill its hydropathic valences and thus, structural water molecules are seen in a variety of roles throughout these environments. alanine was analyzed using the same protocol as well. having the smallest sidechain (and small hydropathic interaction maps), alanine allowed us to investigate a significantly larger database, permitting us to examine the correlation between hydropathic maps and various structural features. in conclusion, the analysis of hydropathic environments strongly suggests that the orientation of a residue in a three-dimensional structure is a direct consequence of its hydropathic environment, which leads us to propose a new paradigm, interaction homology, as a key factor in protein structure. it is not the surrounding residues that direct sidechain conformations, but rather the hydropathic "field" of the surrounding atoms. folding studies of independent domains of lysine, arginine, ornithine binding protein (lao) protein folding problem has been addressed from the past years until nowadays, however, we still can not explain how proteins acquire their native structure from their amino acid sequence. different approaches has been taken in order to study protein folding, for example, the comparative study of folding mechanism between homologues proteins with high identity of sequence and structure, and the study of independent regions within a single protein. previously in our laboratory, thermodynamic and kinetic folding properties of lysine, ornithine, arginine binding protein (lao), a amino acid periplasmic binding protein (pbp), composed by two rossmann fold domains (one continuous and the other discontinuous) attached by a hinge region, has been studied. even there is a functional research about binding characteristics of histidine binding protei ns (his j) domains of when expressed independently (chu, b. ); there are no folding studies in these conditions for this or another pbps. it should be noted that his j shares % of sequence identity and tertiary structure (rmsd Å) with lao. in order to know the folding effect of encoding different domains in the same poly peptidic chain, as well as its influence in function, we are studying the thermodynamic and kinetic characteristics of folding of independently expressed lobes of lao, and comparing with those of native protein. by now, we expressed and purified the discontinuous domain. circular dichroism (cd) and fluorescence intensity spectra show that this independent domain has primary and tertiary structure. thermal denaturation has a single cooperative transition, which indicates this domain is folded. thermodynamic analysis of temperature and urea-induced experiments suggest that lao's folding characteristics are not just the addition of those from independent domains. furthermore, folding and refolding kinetics suggest the presence of a burst phase intermediate. a hypothesis to reconcile the physical and chemical unfolding of proteins a comprehensive view of protein folding is crucial for understanding how misfolding can cause neurodegenerative diseases and cancer. when using physical or chemical perturbations, nmr spectroscopy is a powerful tool to reveal a shift in the native conformation toward local intermediates that act as seeds for misfolding. high pressure (hp) or urea is commonly used to disturb folding species. pressure favors the reversible unfolding of proteins by causing changes in the volumetric properties of the proteinsolvent system. however, no mechanistic model has fully elucidated the effects of urea on structure unfolding, even though protein-urea interactions are considered to be crucial. here, we provide nmr spectroscopy and d reconstructions from x-ray scattering to develop the "push-and-pull" hypothesis, which helps to explain the initial mechanism of chemical unfolding in light of the physical events triggered by hp. in studying mpnep from moniliophthora perniciosa, we tracked two cooperative units using hp-nmr as mpnep moved uphill in the energy landscape; this process contrasts with the overall structural unfolding that occurs upon reaching a threshold concentration of urea. at subdenaturing concentrations of urea, we were able to trap a state in which urea is preferentially bound to the protein (as determined by nmr intensities and chemical shifts); this state is still folded and not additionally exposed to solvent [fluorescence and small-angle x-ray scattering (saxs)]. this state has a higher susceptibility to pressure denaturation (lower p / and larger dvu); thus, urea and hp share concomitant effects of urea binding and pulling and water-inducing pushing, respectively. these observations explain the differences between the molecular mechanisms that control the physical and chemical unfolding of proteins, thus opening up new possibilities for the study of protein folding and providing an interpretation of the nature of cooperativity in the folding and unfolding processes. zinc: a promoter or inhibitor for iapp aggregation? feng ding , praveen nedumpully-govindan zinc ions have been found to play an important and yet complex role in human islet amyloid polypeptide (hiapp) aggregation, which is associated with b-cell death in type-ii diabetes (t d). both concentration-dependent promotion and inhibition of iapp aggregation by zinc ions have been observed in vitro. similarly, at the population level, both positive and negative correlations were reported between the activity of a b-cell specific zinc transporter and t d risk. zinc ions are able to bind a single histidine in hiapp and coordinate the formation of zinc-bound hiapp oligomers. we hypothesize that the relative zinc/hiapp concentration determines the population of zinc-bound hiapp oligomers with different molecular weights. we have applied molecular dynamics (md) simulations to systematically study the structure and dynamics of a range of zinc-coordinated hiapp oligomers, including monomers, dimers, trimers, tetramers, and hexamers. our computational results suggest that different zinc-bound oligomers have distinct aggregation propensities. high-molecular weight oligomers ( peptides) have higher aggregation propensity than zinc-free and zinc-bound hiapp monomers at mm concentration in silico. therefore, our results provide a molecular insight into the complex role of direct zinc binding on hiapp aggregation. at low zinc/hiapp stoichiometry, zinc binding promotes aggregation. as the stoichiometry increases and zinc ions bind to single hiapp peptides, the aggregation of hiapp is inhibited due to electrostatic repulsion between the charged zinc ions. our computational study sheds light on the complex role of zinc on hiapp aggregation and t d development. biomolecules function in the densely crowded and highly heterogeneous cell, which is filled up to a volume of % with macromolecules [ ] . often, artificial macromolecular crowding agents are used to mimic these conditions in vitro and the excluded volume theory is applied to explain the observed effects [ ] . however, recent studies emphasize the role of further contributions aside from a pure volume effect including enthalpic and solvent effects [ , ] . we study cosolute effects at high molecular and macromolecular concentrations via a thermodynamic analysis of the thermal unfolding of ubiquitin in the presence of different concentrations of cosolutes (glucose, dextran, polyethylene glycol, potassium chloride) [ ] . in contrast to the excluded volume theory, we observed enthalpic stabilization and entropic destabilization forces for all tested cosolutes. the enthalpic stabilization mechanism of ubiquitin in macromolecular polysaccharide solutions of dextran was thereby similar to the effects observed in monomeric glucose. further, it remains unclear how such cosolutes reflect the physicochemical properties of the complex cell environment as a characterization of the in-cell crowding effect is lacking. thus, we developed a fret-based macromolecular crowding sensor to study the crowding effect in living cells [ ] . the averaged conformation of the sensor is similar to dilute aqueous buffer and cell lysate. we find that the in-cell crowding effect is distributed heterogeneously and can change significantly upon osmotic stress. the presented method allows to systematically study in-cell crowding effects and understand them as a modulator of biomolecular function. the stability of biomolecules under co-solvent conditions is dependent on the nature of the co-solvent [ ] . this can alter a protein's properties and structural features through biomolecular interactions between its functional groups and the co-solvent molecules. ionic liquids (ils) represent a rather diverse class of co-solvents. the design flexibility of these molten salts is an attractive feature, allowing the properties of the il to be tuned to meet the requirements of different applications [ ] . particularly, the modulation of reaction pathways between folding states, offering possibilities to control irreversibility in non-native protein aggregation [ ] . this has led us to investigate the impact of ils as co-solvents with the well-known protein denaturant urea. urea is considered to be a non-ionic chaotrope disturbing considerable the grid of hydrogen bonds with the protein backbone. urea interacts preferentially with the protein surface, mainly apolar residues and that dispersion, rather than electrostatic interactions, is the main energetic contribution to explain the stabilization of the unfolded state of the protein and the irreversibility of the unfolding process in the presence of urea [ ] . a large body of multidomain protein folding work has been devoted to study monomeric proteins. how do multidomain multimeric protein fold, avoiding accumulation of stable intermediate is yet to be studied in detail. our present study is focussed on understanding the folding and assembly of the domains of a homodimeric l-aspraginase from a hyperthermophile pyrococcus furiosus (pfa). each monomer of pfa consists of distinct n-and c-terminal domains (npfa and cpfa, respectively), connected by a linker. the folding mechanism of each domain with respect to full length protein was studied by mutating one out of two tryptophans, one in each domain. domains were purified and studied individually to obtain parallel account of the folding of each domain in isolation. subunit assembly was studied by analytical size exclusion chromatography (sec), multiangle light scattering and functional activity. through far uv cd, intrinsic trp fluorescence and sec, we demonstrated that domain folding and subunit association were intimately linked in full length pfa. interestingly, en route to its folding there was complete absence of hydrophobic intermediates as probed by ans fluorescence. folding of npfa was highly cooperative and, it provides interacting surfaces for cpfa to fold and also facilitates subunit assembly. the folding cooperativity of isolated domains was very less compared to the folding cooperativity of their full length counterparts, as indicated by equilibrium m values. to our surprise, during ph induced denaturation, at ph and , the dimer dissociates into highly hydrophobic folded monomers which readily underwent amyloidogenesis. we showed that at such extreme conditions, cooperativity in folding process in multidomain multimeric protein is not solely governed by the folding of individual domains, rather by concomitant folding and association of domains directly into a quaternary structure. in other case, where subunit folding occurred prior to association, protein readily underwent extensive aggregation. groel assisted folding of multiple recombinant proteins simultaneously over-expressed in e.coli megha goyal , tapan kumar chaudhuri aggregation prone recombinant proteins very often form inclusion bodies and also exhibits poor yield of functional protein during in vitro refolding process from chemically denatured form. bacterial chaperonin groel provides folding assistance to several proteins, when over-expressed with one of the recombinant proteins. there are instances that groel in presence of few other co-expressed chaperones like dnaj, dnak etc provides better yield of folded protein during homologous and heterologous expression. considering the ongoing events in the cells, it is known that molecular chaperone groel assists in the folding of various proteins in the cytoplasm. hence attempt to fold multiple recombinant proteins over-expressing simultaneously with the co-expression of chaperones can be worth trying. this approach may cut down various complexities in the functional recombinant protein preparation, including time and effective cost. keeping this view in mind, folding of two simultaneously expressed aggregation prone proteins, kda e.coli maltodextrin glucosidase (malz) and kda yeast mitochondrial aconitase have been investigated with the co-expression of groel and groes in e.coli cytosol. it has been previously reported that both the chosen proteins undergo co-expressed groel-groes assisted folding in e.coli cytosol, when they over-express alone. in this study we have optimized the overexpression of malz and aconitase simultaneously in e.coli. further optimisation was carried out to coexpress groel along with malz and aconitase. based on the basic philosophy that soluble protein mainly contains folded fraction, the event of groel/es assisted folding of simultaneously overexpressed proteins, malz and aconitase was monitored through the attainment of soluble proteins under various sets of conditions such as temperature. the major outcome of the present study is that, with the groel-groes assistance, the yield of soluble proteins (malz and aconitase) together constitutes higher percentage of folded protein in contrast to the percent yield when a single protein was overexpressed. significance of this type of study relies on the fact that the cells can over-produce higher amount of recombinant proteins, when multiple over-expression takes place. not only pushing up cell's capability of over-expression, co-expression of groel and groes efficiently assists in the folding of multiple proteins simultaneously over-expressed in e.coli. amyloid fibrils associated with serious diseases including alzheimer's, parkinson's, and prion diseases promoted the challenge of studying protein misfolding, leading to the development of amyloid structural biology. amyloid fibrils form in supersaturated solutions via a nucleation and growth mechanism. although the structural features of amyloid fibrils have become increasingly clearer, knowledge on the thermodynamics of fibrillation is limited. furthermore, protein aggregation is not a target of calorimetry, one of the most powerful approaches used to study proteins. here, with b -microglobulin, a protein responsible for dialysis-related amyloidosis, we show direct heat measurements of the formation of amyloid fibrils using isothermal titration calorimetry (itc). the spontaneous fibrillation after a lag phase was accompanied by exothermic heat. the thermodynamic parameters of fibrillation obtained under various protein concentrations and temperatures were consistent with the main-chain dominated structural model of fibrils, in which overall packing was less than that of the native structures. we also characterized the thermodynamics of amorphous aggregation, enabling the comparison of protein folding, amyloid fibrillation, and amorphous aggregation. in order to obtain general thermodynamic properties of protein aggregations, we further investigated aggregation of glucagon and insulin, two of the most famous amyloidogenic peptide hormones, using itc. we also observed characteristic heat of spontaneous amyloid fibrillation of both proteins after a lag time. taken all together, we showed that thermodynamic studies on amyloid fibrillation and amorphous aggregation were indeed possible by means of itc-based qualitative and quantitative calorimetric analyses. itc will become a promising approach for clarifying the thermodynamic properties of protein aggregates. the more case studies are required toward the establishment of thermodynamics of protein misfolding and aggregation when hydrophobic proteins are, for any reason, exposed to the cytosol they are rapidly captured by protective complexes which shield them from the aqueous surroundings and decide their fate (by either targeting them to their correct membrane homes or marking them for degradation by the ubiquitin/proteasome system). the bag holdase is a heterotrimeric protein complex, comprising bag , ubl a and trc , which works closely with the cochaperone sgta to triage hydrophobic proteins and pass them along the appropriate pathway. sgta also interacts with viral proteins and hormone receptors and is upregulated in numerous cancer types. these functions require further investigation to determine the scope of sgta as a therapeutic target. our lab has solved the solution structure of the n-terminal dimerization domain of sgta and characterised its interaction with two different ubiquitin-like (ubl) domains in the bag holdase (one from ubl a and the other from bag itself) using nmr chemical shift perturbation data and other biophysical techniques including isothermal titration calorimetry and microscale thermophoresis. at this meeting i will report on the progress we have made in structurally characterising further key players that participate in this quality control, with the aim of clarifying the intricate network of molecular interactions that governs these processes in health and disease. ensemble, ribbon and electrostatics spacefill views of the sgta dimerization domain structure. the final panel shows the structure overlaid with its yeast homologue. alpha synuclein is a small protein ( kda) expressed at high levels in dopaminergic neurons. fibrillar aggregates of a-synuclein inside the dopaminergic neuron are the major components of lewy bodies and lewy neuritis inclusion, which are considered as potential hallmark of parkinson's disease (pd). both in vitro as well as in vivo studies suggest that the soluble, oligomeric forms of a-syn are the more potent neurotoxic species, responsible for neuronal injury and death in pd. therefore, molecules that inhibit the toxicity of oligomers either by reducing their formation or by converting their more toxic oligomeric state to less-toxic fibrillar state would be effective agents for the drug development against pd. curcumin is one of the asian food ingredients which has shown a potential role as therapeutic agent against many neurological disorders including pd. however, the instability and low solubility makes it less attractive for use as potential therapeutic agent. the present work focuses on screening of the compounds similar to curcumin but having better effects on the morphology and toxicity of oligomeric and fibrillar assemblies of a-syn, which could be used as therapeutic agent preferentially over the naturally occurring curcumin. we synthesized and analyzed the effects of nine compounds, which are structurally similar to curcumin, on different stages of a-syn amyloid aggregation. here, we showed that curcumin and its analogs accelerate a-syn aggregation to produce morphologically different amyloid fibrils in vitro. however, there is no significant effect of curcumin and its analogs on the secondary structure of preformed a-syn fibrils. furthermore, these curcumin analogs showed differential binding affinities with the preformed a-syn aggregates, possibly due to difference in their chemical structures. the present data suggest the promising role of curcumin analogs in the treatment of a-synucleinopathy disorders. in vitro folding mechanisms determine the forces applied during co-translational folding there is currently much debate as to whether experiments conducted in vitro describe the folding of proteins in vivo. in particular, it is often suggested that the co-translational folding of nascent protein chains is dominated by the presence of the ribosome and associated chaperones, and that folding mechanisms will be affected by the vectorial nature of translation. here we use an arrest peptide assay to investigate the co-translational folding of a number of all-a spectrin domains that exhibit a range of thermodynamic stabilities and in vitro folding rates. our unexpected finding is that that the force exerted on the ribosome by these domains is not related to either the thermodynamic stability of the domain, or to the folding (loading) rate, but rather to the in vitro folding mechanism. we infer that the in vitro folding mechanisms of these domains are unaffected by the presence of the ribosome -even when part of the nascent chain is retained within the ribosome exit tunnel. there has been much work to date investigating the intermediates present in stalled translation complexes -but now, for the first time, we can begin to directly explore the rate limiting transition state in the co-translational folding of homologous proteins. can the structure of a protein (h . ) depend on the treatment of a solvent medium (explicit vs effective) in a coarse-grained computer simulation? ras pandey , barry farmer university of southern mississippi, air force research laboratory solvent medium plays a critical role in orchestrating the structure and dynamics of a protein. in computer simulation modeling of protein structure in a solvent medium, explicit, implicit, effectivemedium, approaches are often adopted to incorporate the effects of solvation. because of the complexity in incorporating all atomic and molecular details, the multiple components, reaching the large-scale, etc. implicit solvent or effective medium approach is generally more viable than the explicit solvent methods. some of the pertinent characteristics such as excluded volume of the solvent constituents, its concentration, and the underlying fluctuations which may be important in probing some issues are generally ignored in effective medium or implicit solvent approaches. using a coarse-grained approach, we investigate the structure and dynamics of a protein (a histone, h . ) in the presence of both effective as well as explicit solvent media over a range of temperatures with the monte carlo simulations. the protein is represented by a coarse-grained chain of residues whose interactions are described by knowledge-based residue-residue and hydropathy-index-based residuesolvent interactions. in effective medium approach, each empty lattice site around the protein structure acts as a solvent. only a fraction of lattice sites are occupied by mobile solvent constituents along with the protein chain in explicit solvent medium. large scale simulations are performed to analyze the structure of the protein for a range of residue-solvent interactions and temperature in both explicit and effective solvent media. we study a number of local (e.g. solvation and mobility profiles) and global (radius of gyration and structure factor) physical quantities as a function of temperature. we find that the response of the radius of gyration of the protein in explicit solvent is different from that in effective medium solvent. thus, the presence of fluctuations in explicit solvent approach have considerable effects on the structure and dynamics of protein h . . differences due to type of solvent on the response of some of these quantities as a function of temperature as well as general similarities will be presented. single-molecule vectorial folding and unfolding through membrane pores david protein folding and unfolding in vivo is frequently vectorial. for example, proteins are synthesized at the ribosome and emerge n-terminal first. as the polypeptide chain emerges from a nm wide pore is free to fold, interact with partners or misfold . in another example, proteins are unfolded at the proteasome by pulling from either the n or c terminus against a - nm wide pore, applying a tension on the residues surrounding the terminus of the protein . under this conditions, proteins may behave differently than when unfolded/refolded with temperature or urea. this may have important implications, as protein folding and unfolding in vivo is related to both function and disease. we noticed that vectorial folding is inherently linked to nanometer size pores. making use of nanopore technology we developed a method to monitor protein unfolding during membrane translocation at the single-molecule level . briefly, an oligonucleotide attached at either end of a protein threads a single protein nanopore inserted in a lipid membrane. in response to an applied membrane potential, the oligonucleotide pulls the protein through the pore and as it is forced to translocate it unfolds. analysing the ionic current we obtain the unfolding pathway and information on the polypeptide sequence. this methodology has shown that proteins unfold with different kinetics when pulled from one terminus or the other . remarkably, it is also possible to say whether the protein has been phosphorylated or not, and where . we have recently advanced our model system to study protein folding after translocation at the singlemolecule level . a single-protein molecule was translocated through a pore and forced to translocate back at predetermined times. we measured the stability of the refolded state at different times and we obtained the vectorial folding pathway of the protein. further, we observed that the protein was capable of co-translocational folding and that this premature folding contributed to the complete translocation of the protein. our results show that nanopore technology applied to proteins can be used to describe the vectorial folding and unfolding of proteins, providing insight to how these processes may work in vivo. further, single-molecule protein sequencing is a possibility that could revolutionise our knowledge on biological processes. thermodynamics studies of oligomeric proteins, which are the dominant protein natural form, have been often hampered because irreversible aggregation and/or slow reactions are common. there is not a single report on the reversible equilibrium thermal unfolding of proteins composed by (b/a) barrel subunits, albeit this "tim barrel" topology is one of the most abundant and versatile in nature. the eponymous tim barrel, triosephosphate isomerase (tim) is a ubiquitous glycolytic enzyme that catalyzes the isomerization of glyceraldehyde- -phosphate and dihydroxyacetone phosphate. the unfolding of several tims, mainly of eukaryotic organisms, has been extensively studied. regarding thermal unfolding, eighteen tims, mainly from eukaryotes, as diverse as amoebozoa, euglenozoa, ascomycota and chordata, have been studied. even though a full thermodynamic characterization has been hampered by irreversible aggregation and/or the presence of hysteresis in all of them, the activation parameters that describe the kinetic control of five eukaryotic tims have been reported. we characterized the structure, catalytic properties, association state and temperature-induced unfolding of the eponymous tim barrel, triosephosphate isomerase (tim), belonging to five species representative of different bacterial taxa: deinococcus radiodurans (drtim), nostoc punctiforme (nptim), gemmata obscuriglobus (gotim), clostridium perfringens (cptim) and streptomyces coelicolor (sctim). irreversibility and kinetic control were observed in the thermal unfolding of nptim and gotim, while for drtim, sctim and cptim, the thermal unfolding was found to follow a two-state equilibrium reversible process, a behavior not observed previously for others tims. shifts in the global stability curves of these three proteins are related to organismal temperature range of optimal growth and modulated by variations in maximum stability temperature and in the enthalpy change at that temperature. reversibility appears to correlate with low isoelectric point, the absence of residual structure in the unfolded state, small cavity volume in the native state structure, low conformational stability and a low melting temperature. furthermore, the strong coupling between dimer dissociation and monomer unfolding may reduce the possibility of aggregation and favor reversibility. it appears that there is a delicate balance between several contributions whose concerted interplay is necessary to achieve thermal reversibility in oligomeric enzymes. furthermore, the finding that the three reversible proteins come from organisms from different phyla suggests that unfolding reversibility may be more common than what is currently known supported by a critical step in the late phase of human immunodeficiency virus type (hiv- ) infection is targeting of the virally encoded gag proteins to the plasma membrane (pm) for assembly. prior to assembly, the hiv- gag polyprotein adopts a compact "folded over" conformation and exists in the monomeric or low-order oligomeric states. whereas it is established that the nucleocapsid domain of gag specifically recognizes motifs in the viral rna genome for packaging, there is compelling evidence that the myristoylated matrix (ma) domain also binds to cellular rna to prevent premature gag targeting to intracellular membranes. upon transport of gag to the pm, the interaction of ma with rna is exchanged for an interaction of ma with pm components. this molecular switch induces an extended conformation of gag, leading to formation of high-order gag oligomers on the pm. because gag is anchored and therefore captured by its interaction with the available phospholipids, the intracellular targeting of gag is likely to be determined by the relative strength of its interaction with the dominant lipids composing each membrane subcompartment. the key to understanding this essential molecular switch is elucidating at the molecular level the interaction of ma with specific pm components. for over two decades, biochemical, in vivo, in vitro and genetic studies have focused on factors that modulate binding of retroviral gag proteins to membranes but only recently the structural and molecular determinants of gag assembly have begun to emerge. in addition to the electrostatic interactions between a highly conserved basic region of ma and acidic phospholipids, it is now believed that the hydrophobicity of the membrane interior represented by the acyl chains and cholesterol also play important roles. we employ nmr methods to elucidate the molecular determinants of gag binding to the membrane. our structural studies revealed that phosphatidylinositol- , -bisphosphate (pi ( the production of functionally antibodies depends on the transition of immature b cells to mature plasma cells and is tightly linked to several "quality control" check points. during b cell development, the pre-b cell receptor (pre-bcr) is the first checkpoint which determines the viability and proliferation of the pre-b cell. the pre-bcr is composed of an immunoglobulin (ig) heavy chain molecule associated with an ig light chain-like molecule called the surrogate light chain (slc). the slc is composed by two proteins k and vpreb which possess a unique region at the n-or c-terminus, respectively. vpreb lacks a b-strand which is provided by the k protein allowing the non-covalent interaction essential for formation of the slc heterodimer. our understandings of the molecular mechanism of slc function and assembly are still at an early stage. in particular, we do not know how the slc associates and forms the pre-bcr for the selection of all heavy chains (hcs). our study focuses on dissecting the "fab fragment" of the pre-bcr to study the effect of the unexpected structural features of the slc to gain insight in hc selection. the analysis of the assembly of the slc revealed a significant difference between the single domains and the complexes in terms of stability and assembly. the folding behavior of the ch domain in the presence of the slc is key for the first quality control mechanism in the endoplasmic reticulum (er) prior to surface expression. our results show that the slc interacts with ch domain in a similar manner to the cl domain. thus, the folding of the naturally disordered ch domain upon interaction with the slc releases the hc retention in the er by bip. taken together, our study provides new insights into the folding and assembly of the "fab fragment" of the pre-bcr and paves the way for a detailed mechanistic understanding of hcs selection by the unique slc. though the - (sfgailss) region of human islet amyloid polypeptide (hiapp) has long been known to be crucial for amyloid fiber formation, lack of b-ordering of this region in structures of the final fiber as determined by both nma and x-ray has been puzzling. new evidence now suggests that the fgail region forms ordered b structures only in early intermediates. we present new dir studies on the fgail region of hiapp, with uniformly c o labeled amides, along with spectral and kinetic modelling. evolution of the peak frequency and d lineshape of the labeled region clearly present a transition from random coil to a stable b sheet, a conclusion which is substantiated by simulation of the d ir spectra. as determined from kinetic modeling, the fgail b-sheet creates a free energy barrier that is the cause of the lag phase during aggregation. these findings help to rationalize a broad range of previous fragment and mutation studies as well as provide a mechanism for fiber formation that has self-consistent kinetics and structures. the temperature dependence of protein stability in living cells studies addressing the consequence of crowding that exist in the interior of cells have reached an interesting stage. experimental data so far, predominantly from, small to medium sized proteins are indicating that, in general, natively folded proteins including, intrinsically disordered, gain structure and stability under conditions mimicking cell interior. however, on the other hand, a few studies on small proteins indicate destabilization of the native state. in very few instances, crowding resulted in compaction and aggregation of the unfolded and partially folded states. experimental data on the consequences of cell-like crowding situation on relatively large proteins with complex folding free energy landscape are absent. alpha subunit of tryptophan synthase, a kda tim barrel protein, provides a unique opportunity to address the consequence of crowding on the structure and stability of the native state and also on a partially folded state stable equilibrium intermediate populated in its (un)folding reactions. in the presence of increasing amounts the most commonly used crowding agent, ficoll- , a non-monotonous increase in the far uv-cd is observed for the native state. a steady increase up to mg/ml ficoll followed by a decrease in far-uv cd region is observed, indicating loss of structure at increased concentrations of the crowding agent. h- n hsqc nmr and fluorescence (fl) spectra confirm the of loss of structure at higher concentrations of ficoll- . loss of native base line in the urea induced unfolding reaction monitored by cd and fl clearly confirms the destabilization of the native state. similar to the structural changes observed for the native state, for the equilibrium intermediate state maximally populated at m urea also, non-monotonous changes in the far uv cd and fluorescence spectra are observed. the highly populated equilibrium intermediate shows an initial steady increase in the far uv cd signal followed by a sudden decrease. our results suggest that the structure of both native and partially folded states may be affected under crowding conditions. alpha- antitrypsin (aat) is a -kda serine protein inhibitor (serpin), which acts as an inhibitor of neutrophil elastase within the lungs. during inhibition, the protein undergoes a dramatic conformational change in which its exposed reactive centre loop (rcl) is cleaved and inserts into the central a-sheet as an extra beta-strand. this highly dynamic protein is also susceptible to mutations, resulting in misfolding and the accumulation of ordered polymers as intracellular inclusions within the endoplasmic reticulum of hepatocytes, where aat is synthesized. despite much knowledge of the folding and misfolding properties of aat as an isolated protein, very little is understood of how aat acquires its structure during biosynthesis. like all proteins, the biosynthesis of aat takes place on the ribosome, and protein folding occurs in a co-translational manner as the nascent polypeptide chain emerges from the ribosome's exit tunnel. this study aims to develop the biochemical and nmr structural strategies to characterize the co-translational folding characteristics of aat as it is being synthesized on the ribosome. for these studies, we have designed a series of secm-stalled ribosome nascent chain complexes (rnc) of aat of different lengths, which mimics the "snapshots" of the protein synthesis, capturing the folding process of the nascent chain during its emergence from the ribosome. using this library, we have recently developed a strategy to produce large quantities of the rncs both in vitro and in vivo within e. coli, a prerequisite for detailed biochemical and structural studies. using the aat-rncs, we are developing a suite of biochemical strategies to probe the capacity for aat nascent chains to adopt native structure on the ribosome. we have combined protease inhibition assays, western blot and native-page analysis to demonstrate that aat can fold while bound to the ribosome. in addition, we have employed a cysteine-based modification "pegylation" assay to probe lowresolution structural information of aat-rnc and this will guide our structural studies by nmr spectroscopy to provide a detailed understanding of aat folding on the ribosome at high resolution. thermodynamic properties of proteins vary with the environmental solvent condition (temperature, ions, ph, denaturants, etc.). although the effect of each environmental factor on proteins has been well studied, the complex effect of more than two environmental factors was not studied thoroughly. in this study, we investigate the simultaneous effect of urea denaturation (disruption of non-covalent bonds in proteins) and acid denaturation (titration of protein residues) on the nature of the folding transition for cyu protein. we performed the molecular dynamics simulations of bbl (pdb code: cyu) protein in various urea concentration at k. we calculated ph-dependent free energy landscape using the extended munoz-eaton model and described the phase diagram for the folding transition of bbl at various ph value and urea concentration. we mapped out the phase diagram of the folding transition of cyu, which clarifies the condition with which it undergoes the cooperative folding transition or the barrierless folding transition. biophysical analysis of partially folded states of myoglobin in presence of , , -trifluoroethanol paurnima talele , nand kishore the protein folding process involves one or more distinct populated intermediates. one such partially folded structure of particular importance observed during protein folding pathway is molten globule state. the properties of a molten globule state are intermediate between those of native and unfolded protein molecules. the importance of studying equilibrium molten globule is in its greater stability and flexible structure which has been shown to bind a variety of substrates and play a definite role in certain human diseases via aggregation, misfolding or some other mechanism. a protein must assume a stable and precisely ordered conformation to perform its biological function properly. the stability of a protein under specific conditions depends on its interactions with the solvent environment. therefore it is essential to understand protein folding intermediates, protein solvent interactions and protein stabilization. we have made attempts to thoroughly investigate the formation of stable molten globule state of the protein induced by alcohol using combination of calorimetric and spectroscopic techniques. the presentation will cover the topic on biophysical studies on partially folded states of myoglobin in presence of , , -trifluoroethanol. the thermal denaturation of myoglobin was studied in the presence of , , -trifluoroethanol (tfe) at various ph values using differential scanning calorimetry and uv-visible spectroscopy. the most obvious effect of tfe was lowering of the transition temperature with increasing concentration of tfe up to . mol•dm- , beyond which no thermal transitions were observed. the conformation of the protein was analyzed by a combination of fluorescence and circular dichroism measurements. at ph . and . , partially folded states of myoglobin were confirmed by cd spectroscopy. quantitative binding of ans to the tfe induced molten globule state of myoglobin was studied by using isothermal titration calorimetry (itc). the results enable quantitative estimation of the binding strength of ans with the molten globule state of myoglobin along with the enthalpic and entropic contributions to the binding process. the results also suggest occurrence of common structural features of the molten globule states of proteins offering two types of binding sites to ans molecules which has been widely used as a fluorescence probe to characterize partially folded states of proteins. modules. each cbr comprises a b-hairpin core followed by a short linker sequence. choline molecules are bound between two consecutive repeats through hydrophobic and cation-p interactions with aromatic side chains. apart from its biotechnological applications as an affinity tag for protein immobilization and purification, clyta is useful as a model for understanding the folding and stability of repeat proteins. in this sense, we proposed to get minimal peptides encompassing the sequence of a single cbr or even only its b-hairpin core able to maintain the native fold and the ability to bind choline. to that end, we first proceeded to analyze the peptide comprising the third b-hairpin core, denoted as clyt . based on cd and nmr data we demonstrate that the peptide clyt conserves its native bhairpin structure in aqueous solution, but forms a stable, amphipathic a-helix in detergent micelles and as well as in small lipid vesicles [ ] . considering the great differences in the distribution of hydrophobic and polar side chains shown by clyt b-hairpin and a-helix, we propose that amphipathic structures are stabilized in micelles or lipid vesicles. this "dual" behavior is the only up-to-now reported case of a micelle-induced conformational transition between two ordered peptide structures. to check whether other cbr repeats also undertake b-hairpin to a-helix transition in the presence of micelles, so that it represents a general tendency ascribed to all pneumococcal choline-binding modules, we will show new experimental evidences based on cd and nmr structural studies on peptides derived from the bhairpin cores of other clyta repeats, as well as in modified clyt peptides. continuing our studies of the effect of like-charged residues on protein-folding mechanisms, in this work, we investigated, by means of nmr spectroscopy and molecular-dynamics simulations, two short fragments of the human pin ww domain [hpin ( - ); hpin ( - )] and one single point mutation system derived from hpin ( - ) in which the original charged residues were replaced with non-polar alanine residues. results, for both original peptide fragments of hpin demonstrate the presence of ensembles of structures with a tendency to form a b-chain reversal. understanding the biology of huntington's disease via the pathogenic huntingtin monomer huntington's disease (hd) is caused by an abnormal extension of the polyglutamine (polyq) region within exon of the protein huntingtin from typically glutamines to over . disease onset correlates with the huntingtin misfolding and causing the formation of aggregates, however recent studies have postulated that pathogenic huntingtin monomer may form compact structures that are responsible for neuronal toxicity in hd. we sought to examine the conformation of huntingtin monomers, how polyq sequence length affects monomer structure and which protein-binding partners in the cell may exert a gain-of-toxic mechanism in pathology. hydrogen-deuterium exchange mass spectrometry was used to measure the degree of structure in both non-pathogenic ( q) and pathogenic ( q) huntingtin, with results showing that both forms exchanged % of potential nh hydrogen bond donors within seconds (n ), with little to no further exchange over the following ten minutes. this result suggested that the pathogenic conformations are not stabilized by slow exchanging hydrogen bonds. binding partners to the monomer were assessed in neuro a cell culture by immunoprecipitation and quantitative ms/ ms proteomics approaches after depletion of aggregates by pelleting. proteins that more prevalently co-precipitated with pathogenic huntingtin included fused in sarcoma (fus), glycine-trna ligase (gars), peroxiredoxin (prdx ), phosphatidylethanolamine-binding protein (pebp /rkip), and histone subunit hist h a, all of which were significantly enriched by two-fold or greater. rna-seq analysis indicated that none of these proteins had altered expression levels, suggesting that the binding interactions are not due to changes in background abundance. overall we found that the conformational differences are subtle, yet are sufficient to generate several specific proteome interactions that offer clues to a toxic gain-of-function mechanism in pathology. work is ongoing to probe the more subtle changes in conformation and the importance of these interactors to mediating mechanisms of dysfunction. hereditary tyrosinemia type i is an autosomal recesive disorder caused by deficiency of fumarylacetoacetate hydrolase (fah) enzyme. deficiency of fah leads to cellular accumulation of toxic metabolites which include mainly, succinylacetone (sa), maleylacetoacetate (maa) and fumarylacetoacetate (faa) in many body tissues. fah is mainly expressed in hepatocytes and renal proximal tubular epithelium. therefore, liver and kidney are the two primary organs affected by this disorder, and development of hepatocellular carcinoma is the major symptom. missense mutations leads to a loss of enzymatic efficiency which, in a high number of mutations, correlates with loss of kinetic and thermodynamic stability of the enzyme. in our ongoing project, we are trying to elucidate the molecular basis of tyrosinemia by means of biophisical and structural characterization of fah wild type along with its mutations. this knowledge should help us design new therapies based on the identification of pharmacological chaperones that could restore the altered enzymatic stability of the enzyme. human fah wild type and selected mutants were synthesized and inserted in an expression vector for e. coli. the proteins were purified in a fplc and, their thermodynamic and kinetic stability investigated using circular dichroism. our preliminary results confirm the loss of termodinamic stability of different mutants and its variability compared to wild type protein. repulsion between net charges of subunits during ferritin assembly daisuke sato , hideaki ohtomo , atsushi kurobe , satsuki takebe , yoshiteru yamada , kazuo fujiwara , masamichi ikeguchi department of bioinformatics, graduate school of engineering, soka university, jasri/spring- the organisms have a lot of spherical shell-shaped supermolecules consisting of identical or distinct subunits (e.g., ferritin, virus capsid, lumazine synthase and encapsulin). such multimeric proteins spontaneously assemble into their native structures from the subunits to acquire the specific functions. however, the assembly mechanism of such supermolecules has not been understood in detail. hence, to investigate the assembly mechanism is biologically important. escherichia coli non-heme ferritin (ftn) consists of identical subunits, which are assembled into a spherical shell-shape with / / symmetry. ftn is able to store iron inside cavity. the subunit includes a-d helices forming -helix bundle, a long bc-loop between b and c-helices and a short e-helix at the c-terminal. ftn dissociates into dimers at acidic ph. the dimer was shown to maintain the native-like secondary and tertiary structures by circular dichroism spectra and small angle x-ray scattering (saxs). the acid-dissociated ftn is able to reassemble into the native structure when ph increases. to clarify ftn assembly mechanism, we performed the stopped-flow time-resolved saxs (tr-saxs) experiments. the saxs profiles could be acquired every ms after the initiation of reassembly. the initial velocity calculated from the forward scattering intensity increment was proportional to the square of the protein concentration, implying that the reaction is second-order. we propose the sequential bimolecular reaction, in which two dimers bind to form tetramer, then another dimer attaches to the tetramer to form a hexamer, and so on. the assembly rate depended on ph and ion strength, indicating that the electrostatic interaction plays an important role in the assembly reaction. the assembly rate decreased with increasing ph in the range from . to . and increased with increasing nacl concentration. this indicates that there are repulsive electrostatic interactions between assembly units and that they increases with increasing ph from . to . . a possible interaction is the repulsion between net charges of dimers since pi of ftn is expected to be . . to test this possibility, we made several mutants with different net charges. as mutational sites, we selected charged residues that are far from the subunit interface. selected sites were glu , glu , glu , glu and glu . we constructed the mutants with one, two, three or four glu -> gln substitutions of selected sites. the structures of those mutants were similar to that of wild-type ftn. if aforementioned hypothesis is correct, the assembly rate is expected to increase with increasing the number of substitution. the result agreed well with this expectation and strongly suggested that the electrostatic repulsion between dimers is an important factor determining the assembly rate of ftn. improved modeling of protein unfolding rates and pathways through solvation and modeling of beta-barrels benjamin walcott , , lu ıs garreta , christopher bystroff , , department of biology, rensselaer polytechnic institute, center for biotechnology and interdisciplinary studies, department of computer science, universdad del valle, department of computer science, rensselaer polytechnic intitute an understanding of the folding and unfolding pathways of proteins is integral to improving our ability to associate the structural impact of point mutations and disease etiology. information gained here can also be used for protein structure prediction and design. to model unfolding pathways in proteins we utilize a computational method called geofold. this approach uses recursive hierarchical partitioning of protein structure and finite elements simulation. geofold considers three types of partitioning operations: translational motion (break), single point revolute joints (pivot), and rotation around two points (hinge). from these operations, a directed acyclic graph (dag) is constructed where nodes correspond to the substructures created by these operations and the edges represent the operations. for each operation in the dag, its dissociation and reassociation rates are determined as a function of solventaccessible surface area, hydrogen bonds, voids, and conformational entropy. finite element simulations are carried out to simulate the kinetics of unfolding. this model accurately predicts changes in unfolding pathways due to disulfides in a four-protein case-study, but it fails to produce a realistic pathway for b-barrel proteins such as green fluorescent protein (gfp). to better model these barrel proteins, a new partitioning operation is introduced involving the breaking of all contacts between an adjacent set of b-strands, called a seam. in addition, to improve the accuracy of kinetic modeling, several updates have been made to the energy function, including an improved solvation model and a contact-orderbased estimation of the reassociation rates. the predicted unfolding rates and pathways using this improved geofold are compared with experimentally measured values in kineticdb for proteins with multi-state unfolding kinetics, point mutations, circular permutations, and engineered disulfides. the presence of multiple domains in a protein can result in the formation of partially folded intermediates, leading to increased aggregation propensity. this can be reduced by cooperative, all-or-nothing folding of the multi-domain protein. in good agreement with ensemble folding experiments, a coarsegrained structure-based model of e. coli adenylate kinase (ake) folds cooperatively. ake has three domains, nmp, lid and core. we examine the role of the interfaces between these domains in facilitating folding cooperativity in ake. mutants in which these interfaces are deleted exhibit similar folding cooperativities as wild-type ake. on closer inspection, we observe that unlike a typical multi-domain protein in which one domain is singly-linked to its adjacent domain, nmp and lid are inserted into core, i.e. they are both connected to core by two linkers each. we create circular permutants of ake in which the inserted domains are converted to singly-linked domains, and find that they fold less cooperatively than wild-type ake. domain insertion in wild-type ake facilitates folding cooperativity even when the inserted domains have lower stabilities. the n-and c-termini of nmp and lid are constrained upon the folding of core and this facilitates their folding. thus, nmp and lid which undergo large conformational changes during catalysis can be smaller with fewer stabilizing interactions. in addition, inter-domain interactions need not be optimized for folding, and can be tuned for substrate binding, conformational transition and catalysis. analysis of protein domains using structural bioinformatics suggests several examples of multi-domain proteins in which domain insertion is likely to facilitate folding cooperativity. tuning cooperativity on the free energy landscape of protein folding pooja malhotra , jayant udgaonkar national centre for biological sciences, tata institute of fundamental research the mechanism by which a protein explores the free energy landscape during a folding or unfolding reaction is poorly understood. determining whether these reactions are slowed down by a continuum of small ( kbt) free energy barriers or by a few large (> kbt) free energy barriers is a major challenge. in this study the free energy landscape accessible to a small protein monellin is characterized under native-like conditions using hydrogen exchange in conjunction with mass spectrometry. cooperative and noncooperative opening processes could be directly distinguished from the mass distributions obtained in the ex limit. under native conditions, where the native state is maximally stable, the unfolded state is transiently sampled in an entirely non-cooperative and gradual manner. under conditions which stabilize the unfolded state or destabilize the native state of the protein, the slowest structure opening event becomes cooperative. the present study provides an understanding of the relationship between stability and folding cooperativity. it suggests that the cooperative transitions observed in unfolding reactions maybe a consequence of the changes in the stabilities of the unfolded state and the transition state. it also provides rare experimental evidence for a gradual unfolding transition on a very slow timescale. role of electrostatic repulsion between unique arginine residues on the assembly of a trimeric autotransporter translocator domain eriko aoki , kazuo fujiwara , masamichi ikeguchi haemophilus influenzae adhesin (hia) belongs to the trimeric autotransporter family. the autotransporter consists of an n-terminal signal peptide, an internal passenger domain and a c-terminal translocator domain. the signal peptide directs to export across the inner membrane via the sec system and is cleaved, the passenger domain is a virulence factor, and the translocator domain (hiat) is embedded in the outer membrane. the crystal structure of hia translocator domain (hiat) has shown that hiat forms a transmembrane b-barrel of b-strands, four of which are provided from each subunit. the b-barrel has a pore that is traversed by three a-helices, one of which is provided from each subunit. the protein has a unique arginine residue at . arg side chains from three subunits protrude from the b-strand toward the center of the barrel and are close to each other. these residues seem to have an unfavorable electrostatic effect on the assembly and decrease the trimer stability. to investigate the role of this residue on the trimer assembly and stability of hiat, we replaced this arginine with the neutral amino acid, methionine (r m) or the positively charged residue, lysine (r k), and properties of these mutants were investigated. hiat and two mutants were dissociated by formic-acid treatment, and they were able to reassemble in the presence of the detergent. to measure the time course of trimer reassembly, amounts of reassembled trimer and monomer were quantified by sds-page at different assembly times. although the neutralized mutation increased the rate of reassembly, the final amount of reassembled trimer decreased, especially at higher protein concentration. these suggest that the neutralized mutation cause the incorrect oligomer formation. the far-uv cd spectrum of reassembled wt hiat was nearly identical with that of the native wt hiat. however, the spectrum of the reassembled r m mutant was more intense that of the native r m mutant, although the proportion of trimer was much lower than that of the wt hiat. this suggests that the incorrect oligomer has a secondary structure different from the wt hiat. r k mutant showed assembly properties similar to those of the wt hiat. therefore, the repulsion between positively charged residues seems to be important for preventing hiat from misassembly. similar proximity of arginine residues is observed for hiv capsid protein, carboxysome shell protein, lumazine synthase and so on. the electrostatic repulsion between arginine residues may be a general mechanism for protein assembly. department of veterinary pathobiology, kagoshima university, institute for food sciences, hirosaki university, faculty of fisheries, kagoshima university, department of veterinary histopathology, kagoshima university, veterinary clinical training center, kagoshima university, department of veterinary anatomy, kagoshima university, sakamoto kurozu inc., the united graduate school of agricultural sciences, kagoshima university kurozu is a traditional japanese rice vinegar. during fermentation and aging of the kurozu liquid in an earthenware jar over year, solid residue called kurozu moromi is produced. in the present study, we evaluated whether concentrated kurozu or kurozu moromi could ameliorate cognitive dysfunction in the senescence accelerated p mouse. senescence accelerated p mice were fed . % (w/w) concentrated kurozu or . % (w/w) kurozu moromi for or weeks. kurozu suppressed cognitive dysfunction and amyloid accumulation in the brain, while kurozu moromi showed a tendency to ameliorate cognitive dysfunction, but the effect was not significant. we hypothesize the effect is caused by the antioxidant effect of concentrated kurozu, however, the level of lipid peroxidation in the brain did not differ in senescence accelerated p mice. dna microarray analysis indicated that concentrated kurozu increased hspa a mrna expression, a protein that prevents protein misfolding and aggregation. the increase in hspa a expression by kurozu was confirmed using quantitative real-time pcr and immunoblotting methods. therefore, the suppression of amyloid accumulation by concentrated kurozu may be associated with hspa a induction. however, concentrated kurozu could not increase hspa a expression in mouse primary neurons, suggesting it may not directly affect neurons. young-ho lee although amyloid fibrils are associated with a number of pathologies, their conformational stability remains largely unclear. we herein investigated the thermal stability of various amyloid fibrils. a-synuclein fibrils, freshly prepared at c at neutral ph, cold-denatured to monomers at - c and heat-denatured at - c. meanwhile, the fibrils of b -microglobulin, alzheimer's ab - /ab - peptides, and insulin exhibited only heat denaturation, although they showed a decrease in conformational stability at low temperature in the presence of chemical denaturants. a comparison of structural parameters with positive enthalpy and heat capacity changes which showed opposite signs to protein folding suggested that the burial of charged residues in the fibril cores contributed to the cold denaturation of a-synuclein fibrils. reinforced electrostatic repulsion at low temperatures may promote cold denaturation, leading to a unique thermodynamic property of amyloid fibrils. we propose that although cold-denaturation is common to both native proteins and misfolded fibrillar states, the main-chain dominated amyloid structures may explain amyloid-specific cold denaturation due to the unfavorable burial of charged side-chains in fibril cores. key structural differences between tbtim and tctim revealed by thermal unfolding molecular dynamics simulations angel piñeiro , miguel costas , andrea guti errez-quezada dept of applied physics, university of santiago de compostela, lab. of biophys. chem., dept of physical chemistry, fac. of chemistry, unam the thermal unfolding pattern obtained by differential scanning calorimetry for trypanosoma cruzi and trypanosoma brucei triosephosphate isomerase (tcim and tctim) are significantly different although the crystal structure of both proteins is almost indistinguishable and the sequences are highly homogolous. in order to explain these differences at molecular level a set of molecular dynamics simulations were performed at different temperatures between and k. the obtained trajectories were analyzed in detail and the residues that showed to be key in the unfolding pathway of each species were identified. a set of residues that behave significantly different between both proteins were selected and proposed for mutations. the general aim is to identify the minimum amount of residue mutations that allow providing tbtim with the behaviour of tctim and vice versa. experimental complementary work is also being performed on the same protein. repositioning som as a potent inhibitor of transthyretin amyloidogenesis and its associated cellular toxicity salvador ventura , ricardo sant'anna , maria ros ario almeida , nat alia reixach , raul insa , adrian velazquez-campoy , david reverter , n uria reig universitat aut onoma de barcelona, instituto de biologia molecular e celular, icbas, the scripps research institute, som-biotech, universidad de zaragoza transthyretin (ttr) is a plasma homotetrameric protein implicated in fatal amyloidosis. ttr tetramer dissociation precedes pathological ttr aggregation. despite ttr stabilizers are promising drugs to treat ttr amyloidoses, none of them is approved by the food and drug administration (fda). repositioning existing drugs for new indications is becoming increasingly important in drug development. here, we repurposed som , an fda-approved molecule for neurodegenerative diseases, as a very potent ttr aggregation inhibitor. som binds specifically to ttr in human plasma, stabilizes the tetramer in vivo and inhibits ttr cytotoxicity. in contrast to most ttr stabilizers, it exhibits high affinity for both ttr thyroxine -binding sites. the crystal structure of som -bound ttr explains why this molecule is a better amyloid inhibitor than tafamidis, so far the only drug in the market to treat the ttr amyloidoses. overall, som , already in clinical trials, is a strong candidate for therapeutic intervention in these diseases. neurometals as modulators of protein aggregation in neurodegenerative diseases s onia s. leal , joana s. crist ovão , cl audio m. gomes protein misfolding and aggregation is a hallmark across neurodegenerative diseases such as alzheimer's disease and amyotrophic lateral sclerosis (als). since these diseases are mostly sporadic, the formation of protein amyloids in the nervous system depends of chemical and biological triggers within the neuronal environment, such as metal ions [ ] . in this communication i will overview the metallobiology of neuronal calcium, zinc and copper, which are key players in brain function and have altered homeostasis in most neurodegenerative conditions. our recent work will illustrate how this allows establishing molecular mechanisms in neurodegenerative diseases [ ] [ ] [ ] [ ] [ ] . in the pursuit of this goal, in the last years we have been investigating superoxide dismutase (sod ), a cu/zn metalloenzyme that aggregates in the fatal neurodegenerative disorder als, as a model. in sod -als cases, this ubiquitous protein selectively aggregates in motor neurons, implicating a local biochemical factor in the process: interestingly, zn and ca levels are upregulated in the spinal and brain stem motor neurons of als patients, and increased ca triggers multiple pathophysiological processes which include direct effects on the sod aggregation cascade [ , ] . recently we established that calcium ions promote sod aggregation into non-fibrillar amyloid, suggesting a link to toxic effects of calcium overload in als [ ] . we showed that under physiological conditions, ca induces conformational changes on sod that increase sod b-sheet content and decrease sod critical concentration and nucleation time during aggregation kinetics. we also observed that calcium diverts sod aggregation from fibrils towards amorphous aggregates. interestingly, the same heterogeneity of conformations is found in als-derived protein inclusions. we thus hypothesized that transient variations and dysregulation of cellular ca and zn levels contribute to the formation of sod aggregates in als patients [ , ] . in a follow up study we combined experimental and computational approaches to show that the most frequent ligands for ca are negatively-charged gatekeeper residues located in boundary positions with respect to segments highly prone to edge-to-edge aggregation. calcium interactions thus diminish gatekeeping roles by shielding repulsive interactions via stacking between aggregating b-sheets, partly blocking fibril formation and promoting amyloidogenic oligomers such as those found in als inclusions. interestingly, many fals mutations occur at these positions, disclosing how ca interactions recreate effects similar to those of genetic defects, a finding with relevance to understand sporadic als pathomechanisms [ ] . the amino acid proline is well-known by its disorder promoting and helix breaking properties. prolines can be accommodated within transmembrane (tm) alpha-helices and participate in important biological tasks like signal transduction, ligand binding and helix-helix packing. x-ray crystallography and nmr indicate that proline residues in membrane proteins induce distortions of the helix geometry to different extents ranging from small bends to severe kinks. however, such studies provide essentially a static snapshot of membrane-embedded helices. therefore, the link between proline dynamics and function is not completely understood. in this work we have used singlemolecule f€ orster resonance energy transfer (smfret) and fluorescence correlation spectroscopy (fcs) to probe the structure and dynamics of the tm domain of human glycophorin a (gpa), a widely used model membrane protein for oligomerization studies. a fluorescent dye pair has been attached to both ends of the membrane-spanning region of gpa, which allowed monitoring the average distance and distance fluctuations between the attachment points. site-specifically double-labeled gpa has been reconstituted into two membrane-mimetic systems: sds micelles and phospholipid bilayers assembled into nanodiscs. using proline-scanning mutagenesis we have systematically evaluated the impact of proline residues in different positions along the membrane normal on transmembrane helix length and lateral packing. furthermore, we have investigated the distance distribution in tm helices containing native prolines, namely the insulin receptor and the nesprin protein. our results shed light into the relation between proline dynamics and the folding and function of tm helices. thermodynamic contributions of specific mutations of l e protein in the rna: protein interface region measured by analytical ultracentrifugation and gel shift assay bashkim kokona , , sara kim , margaret patchin , britt benner , susan white in saccharomyces cerevisiae, ribosomal protein l e acts as an autoregulator by inhibiting the splicing of its pre-mrna and translation of its mrna. the l e protein-rna binding site has been previously studied, revealing a rna kink-turn motif, which is characterized by a sharp bend in the phosphodiester backbone due to unpaired nucleotides and internal tertiary interactions. l e structural flexibility at the rna-binding interface makes such interaction an excellent model to explore the energetics of rna protein binding. we made l e k a, f a, and f w mutants to quantify the thermodynamic contributions of such interactions to the protein-rna complex. we used analytical ultracentrifugation sedimentation equilibrium (se) and sedimentation velocity (sv) to investigate conformational changes and protein-rna binding free energy changes due to mutations. our computed changes of binding free energy based on the sedimentation equilibrium experiments were consistent with the gel shift assay results. in addition, sedimentation velocity experiments on the l e wild type indicate that protein-rna interaction is highly dynamic and involves conformational changes of the kink-turn rna induced by l e protein. our results provide new insights on understanding the binding between ribosomal proteins and their rna molecules counterpart, which can be used to complement the x-ray structure. role of a non-native a-helix in the folding of equine b-lactoglobulin takahiro okabe , toshiaki miyajima , kanako nakagawa , seiichi tsukamoto , kazuo fujiwara , masamichi ikeguchi equine b-lactoglobulin is a small globular protein ( residues). although elg adopts a predominantly b-sheet structure consisting of nine anti-parallel b-strands (a-i) and one major a-helix in the native state, it has been shown that a non-native a-helical intermediate accumulates during the burstphase of folding reaction from the unfolded state in the concentrated denaturant. to ask whether the non-native helix formation is important for acquiring the native b-sheet structure, we determined first where the non-native a-helix is formed. a stable analogue of the burst-phase folding intermediate was observed at acid ph (a state). the amide hydrogen exchange experiment and proline-scanning mutagenesis experiment have shown that the non-native a-helix is formed at the region corresponding to the h strand in the a state. to investigate the role of this non-native a-helix on refolding reaction of elg, we constructed several mutant proteins, which were designed to destabilize the nonnative a-helix in the folding intermediate without perturbation on the native structure. a mutant, a t, fulfilled this requirement, that is, a t showed a native structure similar to that of the wildtype protein, and largely reduced cd intensity in the a state. then, the refolding kinetics were investigated by the cd and fluorescence stopped-flow method. a t mutation resulted in reduction of the burst-phase cd intensity, which confirmed that the non-native a-helix is formed around the h strand region. subsequent to the burst-phase, four kinetic phases were observed for a t and the wildtype protein. importantly, the folding rate constants of the four kinetic phases were similar between both proteins. furthermore, interrupted refolding experiments demonstrated that the native state was formed in the two parallel pathways in the two slower phases of the four kinetic phases. the relative amplitudes of the two pathways were similar between a t and the wild-type protein. these results clearly showed that the formation of the non-native helix has little effect on the folding rates and pathways, and suggested that the non-native helix formation may not be a severe kinetic trap for protein folding reaction. impact of the chaperonin cct in a-synuclein(a t) amyloid fibrils assembly ahudrey leal_quintero , javier martinez-sabando , jose mar ıa valpuesta , begoña sot centro nacional de biotecnolog ıa (cnb/csic)., centro nacional de biotecnolog ıa (cnb/csic)., centro nacional de biotecnolog ıa (cnb/csic)., centro nacional de biotecnolog ıa (cnb/csic) and fundaci on imdea-nanociencia cct is a eukaryotic chaperonin that uses atp hydrolysis to encapsulate and fold nascent protein chains. moreover, it has recently been shown that cct is able to inhibit amyloid fibers assembly and toxicity of the polyq extended mutant of huntingtin, the protein responsible of huntington disease. although this opens the possibility of cct being also able to modulate other amyloidopathies, this has not addressed yet. the work presented here intends to determine the effect of cct in the amyloid fibers assembly of a-synuclein(a t), one of the mutants responsible of parkinson disease. it is demonstrated that cct is able to inhibit a-synuclein(a t) fibrillation in a nucleotide independent way, suggesting that this effect is based on binding rather than on active folding. furthermore, using deletion mutants and assaying the interaction of cct with monomers, soluble oligomers and fibres, it has been possible to unravel the mechanism of this inhibition: cct interferes with fibers assembly by interacting with a-synuclein(a t) nac domain once soluble oligomers are formed, thus blocking the reaction before the fibers start to grow. amyloid-like aggregation of nucleophosmin regions associated with acute myeloid leukemia mutations daniela marasco , concetta di natale , valentina punzo , domenico riccardi , pasqualina scognamiglio , roberta cascella , cristina cecchi , fabrizio chiti , marilisa leone , luigi vitagliano department of pharmacy, cirpeb: centro interuniversitario di ricerca sui pepti, section of biochemistry, department of biomedical experimental and clinical scie, institute of biostructures and bioimaging nucleophosmin (npm ) is a multifunctional protein involved in a variety of biological processes and implicated in the pathogenesis of several human malignancies. npm has been identified as the most frequently mutated gene in acute myeloid leukemia (aml) patients, accounting for approximately % of cases ( ). the most frequent human npm mutations lead to variants with altered c-terminal sequences of the c-terminal domain (ctd) that, in its wild form, folds as a three helix bundle. aml modifications lead to (a) an unfolding of the ctd in the mutated protein and (b) its accumulation in the cytoplasm due to the loss of nuclear localization sequences with mutations of trp (mut e) and also of trp (mut a) ( ) . to gain insights into the role of isolated fragments in npm activities we dissected the ctd in its helical fragments. here we describe the unexpected structural behavior of the fragments corresponding to the helices h and h in both wild-type and aml-mutated variants. h region shows a remarkable tendency to form amyloid-like assemblies while only the muta sequence of h region is endowed with and b-sheet structure, under physiological conditions, as shown by circular dichroism, thioflavin t and dynamic light scattering. the aggregates of h , are also toxic to neuroblastoma cells, as determined by using the mtt reduction and ca influx assays ( ) . furthermore the effects of the local context on the different tendencies to aggregate of h and h were investigated and appeared to influence for the aggregation propensity of the entire ctd. since in aml mutants the ctd is not properly folded, we hypothesize that the aggregation propensity of npm regions may be implicated in aml etiology. these findings have implications to elucidate the pathogenesis of aml caused by npm mutants and aggregation phenomena should be seriously considered in studies aimed at unveiling the molecular mechanisms of this pathology. we report a resume of our study regarding the effects of microwaves in the range - mhz on a typical protein, myglobin. previous literature have concerned the effects on living and in vitro organic systems induced by high frequencies electromagnetic fields. we have focused our attention on a typical protein, myoglobin, because proteins are the simplest organic systems that are fundamentals in organic functions of livings. myoglobin is a protein found mainly in muscle tissue of vertebrates, consisting of a single protein chain with amino acids and one heme group that stores oxygen in the muscle cells. the physiological importance of myoglobin is mainly related to its ability to bind molecular oxygen. in particular, we focused our attention on the secondary structure of this protein in order to highlight whether exposure to microwaves unfold the protein producing transitions from a-helix component to b-sheet features. to this aim fourier transform infrared (ftir) spectroscopy have been used. the importance of this study is related to previous literature which indicated that transition from a-helix to b-sheet structure in a protein can be responsible for aggregation mechanisms that can lead to neurotoxicity and neurodegenerative disorders that can be considered as the first step to some pathologies [ ] [ ] [ ] . the aggregates consist of fibers containing unfolded proteins with a prevalent b-sheet structure termed amyloid [ ] . in our studies myoglobin in deuterium oxide (d o) solution was exposed for h to mobile phone microwaves at and mhz at a power density of w/m . ftir spectra were recorded by a spectrometer vertex v from bruker optics, following the protocol accurately described in [ ] [ ] [ ] . ftir spectroscopy analysis evidenced an increase in intensity of b-sheet structures and a significant shift to lower frequencies of about . cm- of the amide i vibration after exposure [ , ] . these results led to conclude that mobile phone microwaves induce proteins unfolding and formation of aggregates [ , ] . membrane proteins play a vital role in many biological processes, and yet remain poorly understood as they are frequently unstable in vitro. the goal of this project is to investigate the insertion and folding of membrane proteins into lipid bilayers, using a cell free expression system. we have used both e.colibased cell extracts (s ), and commercial translation systems (purexpress) in combination with synthetic liposomes of defined lipid composition. these studies will aid understanding of cooperative folding, folding intermediates, and the effects of the lipid bilayer on folding and insertion. model e.coli proteins have been investigated, as they can offer important insights into other proteins, and thus facilitate the further study of more biologically relevant proteins. it has been found that the rhomboid protease glpg spontaneously inserts into liposomes without the aid of an insertase such as secyeg. this spontaneously inserted glpg is functional, and is able to cleave bodipy-labeled casein, yielding a fluorescent product. the major facilitator superfamily (mfs) transport proteins lacy, galp and glpt have also been found to insert spontaneously into liposomes. it has been shown that the lipid composition of the liposomes has an effect on the amount of protein inserted into the bilayer, with all proteins tested to date preferring liposomes containing at least mol% dopg. ongoing and future work will involve the use of rare codons to alter the rate of translation, to investigate the effect this has on the final folded structure of the protein. preliminary work is also currently being done into whether the two domains of the mfs family transporters fold cooperatively or independently, thus aiding understanding into the folding and stability of membrane transport proteins. frederic greco , audrey toinon , nadege moreno , marie claire nicola€ ı rabies remains an important worldwide health problem that causes a fatal encephalomyelitis [ ] . currently, rabies in humans is under control in europe and north america following the use of efficient vaccines for dogs and wild animals. however, it still kills more than , people every year mainly in africa and asia [ ] . human vaccination prevents infection with very high efficacy. the vaccine contains an inactivated rabv produced on vero cells. rabv is an enveloped, negative single stranded rna virus which encodes five proteins, namely the nucleoprotein (n), the phosphoprotein (p), the matrix protein (m), the glycoprotein (g), and the viral rna polymerase (l) [ ] . the viral envelope is covered by trimer spikes of g-glycoprotein which is the most significant surface antigen for generating virus-neutralizing antibodies. here we illustrate the use of dsc (differential scanning calorimetry) to identify structural domains or proteins involved in thermal transitions. the dsc thermogram for intact beta-propiolactone inactivated rabv samples in pbs buffer reveals two major thermal transitions with a tm respectively at c and c. we have initially focused our investigations on one of the major proteins encode in rabv, glycoprotein g [ ] . glycoprotein g contains disulfide bridges on the ectodomain [ ] , is sensitive to bromelain cleavage [ ] and shows reversible conformation changes at low ph [ ] . considering these characteristics, our results provide evidence on the identity of one thermal transition observed by dsc. keywords: rabies virus, differential scanning calorimetry, protein unfolding domain swapping of the dna-binding domain of human foxp is facilitated by its low folding stability exequiel medina, sandro l. valenzuela, crist obal c ordova, c esar a. ram ırez-sarmiento and jorge babul departamento de biolog ıa, facultad de ciencias, universidad de chile, santiago, chile protein folding and dimerization (or oligomerization) are biologically relevant processes when reaching the quaternary structure is required for function. proteins that form dimers by exchanging segments or domains of their tertiary structure with another subunit, the so-called domain swapping phenomenon, are examples where folding and dimerization are tightly concerted processes. previous studies on domain swapping proteins, such as p suc and diphtheria toxin, have shown that, in general, a high kinetic barrier separates monomers and domain swapped dimers, and that this barrier can be lowered by promoting protein unfolding and refolding at high protein concentrations, thus favoring the swapped oligomer. recent crystal structures of the dna-binding domain of several human forkhead box (fox) proteins have shown that the p subfamily of these transcription factors (foxp) can form swapped dimers. the human foxp proteins are interesting models of domain swapping, because mutations of the dna-binding domain of these proteins are linked to diverse inherited disorders in humans, such as ipex and language deficits, and some of these mutations are located in the hinge region that connects the exchanged segment with the rest of the protein. moreover, foxp and foxp have been described to reach monomer-dimer equilibrium in solution after hours of incubation, suggesting that a low kinetic barrier separates both species. using foxp as a model of domain swapping, we analyzed the temperature and protein concentration effects on the dimer dissociation, obtaining the free energy change and enthalpy of the process by van't hoff analysis (dh of . kcal•mol- , ds of . kcal•mol- •k- and dg at c of . kcal•mol- ). these results indicate that the monomer-monomer association is an example of an enthalpy-driven process. to understand how foxp domains swap without protein unfolding, we performed equilibrium unfolding experiments using gndhcl as denaturant, showing that the wild-type protein has a low stability (dgu kcal•mol- , cm . m at c), in contrast to other domain swapping proteins with high kinetic barriers. we further explore the domain swapping mechanism of foxp through biased targeted molecular dynamics simulations, showing that the exchange process can occur by specific local destabilization and unfolding of the hinge region and helix h . to further corroborate that the low stability of wild-type foxp facilitates its domain swapping, we engineered a monomeric version of foxp through a single-point mutation in the hinge region, which has been previously described in the literature, and used this protein to visualize the effect of monomer stability in the dimer formation. comparison of the folding stability of the monomeric mutant a p and wild-type foxp shows that ddgu (mutant-wild-type) is . kcal/mol, concluding that the ability of foxp to domain swap rapidly can be explained through its low monomer stability and local unfolding of the exchange region. funding: fondecyt and . determining the coupled interactions that stabilize the structural framework of the ß-propeller fold loretta au , david green , , department of statistics, the university of chicago, department of applied mathematics and statistics, stony brook university, graduate program in biochemistry and structural biology, stony brook university, laufer center of physical and quantitative biology, stony brook university b-propeller proteins are a highly evolved family of repeat proteins that are involved in several biological pathways, such as signal transduction, cell-cycle modulation and transcription regulation, through interactions with diverse binding partners, despite having a similar fold. as for all repeat protein families, there is a consistent pattern in secondary structure for each repetitive region, in addition to the entire family. typically, four to ten propeller blades (each containing four anti-parallel b-sheets) are arranged in a toroidal shape, thus providing a large binding surface for ligands or other proteins. about % of known proteins adopt this distinctive fold, and although the requirements for tertiary structure and protein function are fundamentally encoded in primary structure, this relationship is not fully understood, and addressing it could provide insight on why the b-propeller fold is common. many techniques in comparative sequence analysis can successfully identify amino-acid conservation between closely related proteins, but molecular interactions between amino acids are often neglected, and further experimentation is still needed to determine the reasons underlying conservation. to explore how primary structure can dictate fold and function, we devised a computational approach to perform large-scale mutagenesis, by adapting the dead-end elimination and a* search algorithms (dee/a*), and also leveraged the structural conservation of each repeating region to understand how sequence variation influences protein fitness, defined here as a combination of stabilizing and binding interactions. dee/a* can evaluate low-energy protein sequences and their corresponding three-dimensional structures, and we used the bsubunit of a g-protein heterotrimer (pdb: gp , gia b g ) as a model system to demonstrate: ( ) how the multiple roles of individual amino acids in protein fitness can be deconvolved, and ( ) how epistatic interactions between them can contribute to structural stability. in doing so, we were able to identify important patterns in sequence complementarity between repeating regions that cannot be found using sequencebased methods alone. these results suggest that computational approaches can be used to determine important protein interactions, and help elucidate the prevalence of b-propeller proteins in biology. temperature induced conformational changes of the villin headpiece miniprotein stanislaw oldziej , wioletta _ zmudzi nska , anna hałabis the c-terminal subdomain of the actin-binding protein villin called hp (villin headpiece) has been used as a model protein in a number of studies of protein folding kinetics and protein folding mechanism [ , ] . the hp is a residue miniprotein with an alpha-helix bundle three-dimensional fold. the goal of our work was to determine conformational ensemble of polypeptide chain of the investigated miniprotein at a wide range of temperatures to get detailed information about how protein structure is influenced by temperature. d nmr spectra of the title miniprotein were registered at , and k. the three-dimensional structure of the hp based on restraints derived from nmr spectra registered at k is almost identical with structure deposited in the pdb database in the record f k [ ] . at higher temperatures ( and k) the general shape of the protein remains unchanged, with well packed hydrophobic core. however, with temperature increase alpha-helices start to melt. at k structure of the protein remains compact and in general shape similar to structure observed at k, but none of the alpha-helices could be observed. results obtained for hp protein are in agreement with previous observation for the trp-cage miniprotein [ ] , that with temperature increase regular secondary structure elements melt first before the break-up of the hydrophobic core of the protein. biological membranes provide a selective and chemically sealed barrier for cells. transport of ions and small molecules across the membrane is mediated by transporter proteins and the breakdown of a cell's ability to produce functionally folded membrane transport proteins can lead to dysfunction and has been implicated in many diseases . however little is known about the processes that govern the misfolding of a-helical integral membrane proteins, taking into account that these proteins fold and maintain functional structures within membranes of various organelles. the neurotransmitter sodium symporter (nss) protein family is an example of a-helical transporter proteins. the nss family encompasses a wide range of prokaryotic and eukaryotic ion-coupled transporters that regulate the transport of neurotransmitter molecules whose dysfunction has been implicated in multiple diseases and disor-ders . we have investigated the folding processes of prokaryotic homologue of the nss family leut responsible for the transport of neurotransmitters and amino acids to the sodium electrochemical gradient. previously folding processes of membrane transporters have mainly been characterised within detergent micelles. however, detergent micelles are not an accurate depiction of the environment of the membrane bilayer, with this in mind we have also attempted to investigate folding processes within a bilayer pd- nmr investigation of ph-induced unfolding of b domain of an escherichia coli mannitol transporter ii mannitol in the bacterial phosphotransferase system kim gowoon , yu taekyung , suh jeongyong the bacterial phosphotransferase system (pts) mediates sugar phosphorylation and translocation across the cytoplasmic membrane. cytoplasmic b domain (iib mtl) of the mannitol transporter enzyme ii mannitol, a pts family protein, delivers a phosphoryl group from a domain to an incoming mannitol that is translocated across the membrane. iib mtl is comprised of a four-stranded ß-sheet and three helices, representing a characteristic rossmann fold. we found that the iib mtl of escherichia coli unfolded at a mildly acidic condition. we made iib mtl mutants to investigate the mechanism of the ph-induced unfolding using nmr spectroscopy. we monitored backbone amide groups and side chain imidazole groups of histidine residues using d hsqc nmr, and pointed out a potential histidine residue that might be responsible for the unfolding. histidine residues may be generally important to the folding stability in response to environmental ph changes. can site-directed mutagenesis shed light on the refolding pattern of human glucose -phosphate dehydrogenase (g pd)? nurriza ab latif , , paul engel conway institute, univerversity college dublin, faculty of biosciences and medical engineering, universiti teknologi malaysia human glucose -phosphate dehydrogenase (g pd) is the first enzyme involved in the pentose phosphate pathway (ppp). this oligomeric enzyme catalyses the reaction of glucose -phosphate to form phosphogluconolactone with concomitant reduction of nadp to nadph. in erythrocytes nadph is important mainly for protection against oxidative stress. in connection with its role as the sole source of nadph, g pd deficiency commonly causes haemolytic disease and is known as the most common human enzyme deficiency globally. protein folding problems and instability are believed to be the major defects in the deficient enzymes. in this study, we employed site directed mutagenesis with hope to give more information on the role of -sh groups in the refolding of human g pd. two mutants were created: ) one in which all cys residues were replaced by ser and ) one in which only c and c were retained. the refolding of recombinant human g pd has been studied primarily by measuring the enzyme activity after refolding. we also used a combination of intrinsic protein fluorescence, ans ( -anilino- -naphthalenesulphonic acid) binding and limited proteolysis to look at the conformational change during the refolding. the results showed that gdnhcl-denatured recombinant human g pd wild type could be refolded and reactivated by rapid dilution technique. even though, as recombinants in e. coli, the mutants were well expressed and active, they remained inactive after attempts were made to refold them in vitro. the methods we applied may have provided some insights on the refolding pattern of this oligomeric protein, albeit qualitatively rather than quantitatively. a single aromatic core mutation converts a designed 'primitive' protein from halophile to mesophile folding connie tenorio , liam longo , ozan s. kumru , c. russell middaugh , michael blaber department of biomedical sciences, florida state university, department of pharmaceutical chemistry, university of kansas experiments in prebiotic protein design suggest that the origin of folded proteins may have favored halophile conditions. these results are consistent with salt induced peptide formation which shows that polymerization of amino acids is also promoted by high salt concentrations. as a result of various origin of life studies, a consensus on which amino acids likely populated early earth has emerged. these residues were synthesized by abiotic chemical and physical processes from molecules present in the surrounding environment. the properties of the consensus set of common prebiotic amino acids (a,d,e,g,i,l,p,s,t,v) are compatible with known features of halophile proteins, meaning these proteins are only stable in the presence of high salt concentrations. the halophile environment, thus, has a number of compelling aspects with regard to the origin of structured polypeptides. consequently, a proposed key step in evolution was, movement out of the halophile regime into a mesophile one commensurate with biosynthesis of "phase " amino acids -including the aromatic and basic amino acids. we tested the effects of aromatic residue addition to the core of a "primitive" designed protein enriched for the prebiotic amino acids (a, d, e, g, i, l, p, s, t, v) that required halophilic conditions for folding. the subsequent results show that the inclusion of just a single aromatic residue was sufficient for movement to a mesophile folding environment. thus, the inclusion of aromatic residues into the codon table could have conferred key stability to early proteins enabling adaptive radiation outside of a halophile environment. contact prediction methods that rely on sequence information alone, such as evfold, can be used for de novo d structure prediction and identification of functionally important residues in proteins. large multiple sequence alignments of protein families consisting of evolutionarily related and plausibly isostructural members reveal co-variation patterns that can be used to identify interactions between pairs of amino acids. we use a global probability model to disambiguate direct and indirect correlations. specifically, we use a maximum entropy approach called pseudo-likelihood maximization (plm) to distinguish causation (residue interactions) from correlation (correlated mutations) and compute evolutionary couplings (ecs). the inferred set of residue interactions can then be interpreted as physical contacts and used in de novo d structure prediction. furthermore, the interactions that are inferred can help guide experiments that measure the phenotypic consequences of protein substitutions, making the method useful for functional studies. the present work can be divided into three areas: (i) methodological improvements related to alignment, folding procedure, structure refinement and ranking; (ii) folding of proteins of known structure for benchmarking and prediction of proteins of unknown structure; and (iii) focused exploration of specific cases of interest. developing shuffle as a platform for expression and engineering of antibodies na ke , alana ali-reynolds , bryce causey , berkmen berkmen shuffle is a genetically engineered e.coli strain that allows disulfide bond formationin its cytoplasm with high fidelity. many proteins containing disulfide bonds have been successfully expressed in shuffle. in this study, we expressed, purified and characterized full-length monoclonal antibody igg in shuffle. for the first time, a fulllength igg can be functionally expressed in the cytoplasm compartment of an e.coli strain. in order to improve the folding and assembly of igg, we have investigated the expression of igg in various formats and vectors; we have co-expressed chaperones and other helper proteins with igg. several-fold increase in the yield of fulllength igg was observed. we characterized the shuffle produced igg and found it comparable to hybridoma produced igg. optimization of fermentation conditions for a large-scale production is in progress. we aim to develop shuffle as an easy, fast, robust platform for antibody engineering, screening and expression. experimental and computational studies of the effects of highly concentrated solutes on proteins: insights into the causes and consequences of quinary protein structure and cytoplasmic organization most studies of protein structure and function focus on pure, diluted samples; however, real-world biochemistry and typical biotechnological applications of proteins take place in complex media with very high concentrations of solutes ( - g/l) of varied size and chemical nature. on one side, this has recently fostered the study of proteins in vivo, in cell, or at least in media mimicking the native conditions. on the other hand, physical chemistry has for a long time studied the general effects of crowded and viscous conditions on proteins, looking mainly at coarse traits like diffusion and stability. but the general effects on traits relevant at atomic/residue resolutions have been less studied, and one fundamental issue remains unsolved: to what extent are proteins forced into interactions with highly concentrated solutes, and with what direct consequences? i will present here our ongoing efforts to dissect the fine effects of high solute concentrations and macromolecular crowding on proteins, based on nmr experiments and md simulations, two complementary techniques of high spatial and temporal resolutions. our results show that smaller solutes are prone to extensive interactions with proteins when at high concentrations while large solutes act chiefly through excluded-volume effects. overall, we observe location-specific perturbations of a protein's surface, its internal dynamics and internal dielectrics, and its hydration, all very dependently on the solute's size and chemical nature. our results support the growing notion that proteins should be studied in native-like media, adding that not only macromolecular crowders but also small molecules should be considered in these studies. last, the fact that high-concentration conditions affect far more than a protein's diffusion rate and stability suggests critical consequences of quinary protein structure and cytoplasmic organization on the regulation of proteins within cellular biochemistry. aldona jeli nska , anna lewandrowska , robert hołyst we developed an analytical technique for the study of interactions of ligands (e.g. cefaclor, etodolac, sulindac) with most abundant blood protein (e.g. bovine serum albumin) using the flow injection method. the experiments were conducted at high flow rates ( cm/s) in a long (> m), thin ( mm) and coiled capillaries. the compound of interest ( ml) was injected into carrier phase, which moved by the poisseule laminar flow. at the detection point we measure the concentration distribution of the analyte. the width of the final profile of the analyte concentration is inversely proportional to the effective diffusion coefficient of the analyte. from the differences between the widths of the concentration distribution of free and bound ligand we can determine value of the association constant. carbohydrate binding modules (cbms), which are defined as contiguous amino acid sequences within a carbohydrate-active enzyme, have been found in both hydrolytic and non-hydrolytic proteins and are classified into families, according to their primary structure similarity. the characterization of cbms by different methods has shown that these modules concentrate enzymes on the surface of polysaccharide substrates. it is thought that maintaining the enzyme in proximity with the substrate leads to more rapid degradation of the polysaccharide. therefore, the study of these kinds of modules or domains is relevant, since they are involved in multiple processes in organisms, like signaling, defense and metabolism; and some of them are involved in allergenic responses. in the present work we studied two different models: the first one is a cbm of the family from lactobacillus amylovorus (lacbm ) that binds starch these domains are present in a a-amylase like a repetitive tandem of five modules that are consecutive and do not present connectors. by means of itc and using a single recombinant lacbm domain we determined a ka . x m- for b-cyclodextrin and a ka . x m- for acyclodextrin. when the number of consecutive recombinant modules increased to three or five tandem modules, the ka values increased to m- ; however, these constants did not show an additive or a synergic effect. for these experiments we fitted the isotherms to different models and used different algorithms. additionally, we used circular dichroism in the uv-far region to determine if there existed conformational changes upon binding of the cyclodextrin molecules to the different tandem modules. we could only observe slight changes in a positive band centered around - nm, which has been explained in terms of p-p; interactions of the aromatic residues at the binding site. these cbms have been used as carriers for in vivo vaccine delivery and affinity tags. the second model is a hevein-like cbm of the family present in a chitinase-like protein from hevea brasiliensis (hbcbm ). hevein is a lectin from h. brasiliensis that shows a % identity with hbcbm . these cmbs are connected to the catalytic domain, in proteins such as chitinases, by a linker of approximately residues. in these experiments we used fluorescence techniques to determine the affinity constants for chitotriose. we previously reported a ka . x m- when using a hbcbm that has a met residue at the nterminal region. besides the aromatic residues at the binding site, the met residue also interacts with the ligand, as determined using crystallographic and docking techniques. the mutant hbcbm -r w that does not have the met residue showed a ka of . x m- with chitotriose, similar to the value reported for hevein using itc (ka . x m- ). interestingly, there exists an isoform of the hbcbm that has a connector between one cbm and a half cbm ( . xhbcbm ). this protein has a ka of . x m- with the same ligand. initiating vesicle formation at the golgi complex: auto-regulation and protein interactions govern the arf-gefs gea and gea margaret gustafson , j. chris fromme molecular decision-makers play critical roles in the effort to maintain efficient and accurate cellular functions. in the case of vesicular traffic at the golgi complex, the decision to initiate vesicle formation is made by a set of guanine nucleotide exchange factors (gefs) that activate the small gtpase arf , which is the master controller for the recruitment of cargos and coat proteins. saccharomyces cerevisiae possess three golgi arf-gefs, gea , gea , and sec , which work at distinct sub-compartments of the golgi to activate arf only when and where appropriate. in the case of sec at the trans-golgi network (tgn), this requires a positive feedback loop in which active arf relieves autoinhibition of sec , as well as recruitment to the golgi membrane and catalytic stimulation by signaling rab gtpases. we know far less about the decisionmaking process for gea and gea , which are responsible for retrograde traffic within the golgi and to the endoplasmic reticulum. i have found that both gea and gea can bind membranes weakly in vitro, an ability which is counteracted by their c-terminal hds domains. in addition, i have discovered membrane recruitment in vitro is aided by the rab gtpase ypt . however, these interactions cannot fully explain the distinct localization patterns of gea , gea , and sec , as all three have been shown to be recruited by ypt , which is found throughout the golgi. my work has revealed that in addition to the well-established distinct localization from sec , gea also occupies different golgi compartments from gea , so specific signals must exist which help the gefs decide where to go. my current efforts focus on understanding the roles of the other domains of gea and gea , identifying the signals which send them to different parts of the golgi, and unraveling the different roles they play in vesicle trafficking pathways. sequence variation in archaea through diversity-generating retroelements sumit handa , blair g paul , kharissa l shaw , david l valentine , partho ghosh department of chemistry and biochemistry, university of california san diego, marine science institute, university of california protein diversification is an essential tool for the survival and evolution for various species. diversitygenerating retroelements (dgr) in bacteria is known to generate massive variation in dna through an error prone reverse transcriptase and retrohoming, which leads to variation in protein sequence. recent discovery of dgrs in intraterrestrial archaeal systems have opened an opportunity to study this massive sequence variation in third domain of life (paul bg, et al. nat. comm.) here, we present the first crystal structure of variable protein from archaea with ligand-binding pocket is surface exposed. also, it has conserved c-type lectin (clec) fold, as shown by previous work on variable proteins, major tropism determinant (mtd) and treponema variable protein a (tvpa) which bind ligands through the clec fold. despite weak sequence identities ( - %) among these variable proteins, clec fold was found to be conserved. this variable ligand-binding site for archaea variable proteins can potentially generate variants. protein synthesis is a dynamic process mediated by a variety of proteins and enzymes. recent studies have shown that hydroxylation is a key post-translational modification involved in translation termination. in particular, the fe(ii)-and -oxoglutarate-dependent oxygenase, jumonji domaincontaining (jmjd ), regulates translation termination via the carbon hydroxylation of an invariant lysine residue, k , of the eukaryotic release factor, erf . in eukaryotes, translation termination is mediated by a release factor complex that includes erf . erf is comprised of three domains, and it is responsible for recognizing stop codons in mrna transcripts before triggering polypeptide release from the ribosome. the lysine residue hydroxylated by jmjd falls within the n-terminal domain and more specifically within the highly conserved niks motif. this motif has been identified by cross-linking and mutagenesis studies to play an essential role in stop codon recognition. while hydroxylation of k by jmjd has been found to increase translational termination efficiency, the exact molecular mechanism by which hydroxylation influences termination remains unclear. this work aims to understand how hydroxylation of erf affects translation termination by exploring the effect of hydroxylation on the structure, dynamics, stability, and binding of the n-terminal domain of erf (erf -n) using mass spectrometry, protein nmr spectroscopy, circular dichroism and differential scanning fluorimetry. in our efforts to understand the effect of hydroxylation, an additional jmjd -catalyzed modification, characterized by a da mass shift on k , was identified in vitro. the effect of this modification on erf was similarly explored. our findings suggest that hydroxylation has no effect on the in-solution nmr structure of erf -n, which experiences chemical shift changes localized to the target lysine residue. correspondingly, there are no significant differences in secondary structure content between wild type and hydroxylated erf -n. hydroxylation was also found to have no effect on protein stability or dynamics. interestingly however, the da modification appears to cause more significant chemical shift changes dispersed beyond the niks motif. this suggests a more global effect on the in-solution nmr structure despite the little differences observed in protein dynamics and secondary structure content. the da modification was also found to have a destabilizing effect on erf -n. neither hydroxylated nor da modified erf -n exhibited differences in rrna binding. while hydroxylation of erf was found to have little effect on protein structure, dynamics, stability, or binding, the da modification has marked effects on protein structure and stability. such differences suggest that this modification has the potential to play an important role in translation. functional and structural analysis of a gh ß-n-acetylglucosaminidase from the marine bacterium vibrio harveyi piyanat meekrathok , arthur t. porfetye , marco b€ urger , ingrid r. vetter , wipa suginta biochemistry-electrochemistry research unit, suranaree university of technology, max planck institute of molecular physiology vibrio harveyi b-n-acetylglucosaminidase (so-called vhglcnacase) is a new member of the gh glycoside hydrolase family responsible for the complete degradation of chitin fragments, with nacetylglucosamine (glcnac) monomers as the final products. however, the d structure of glcnacase is still unknown. in this study, crystal structure and function of glcnacase were investigated based on protein crystallography. size-exclusion chromatography and the native-page were employed to verify the protein state of glcnacase in a native form and the acidic active-site residues were mutated using sitedirected mutagenesis method. the effects of mutations on the binding and hydrolytic activities were studied by enzyme kinetics. to provide a structural basis of glcnacase, the wild-type enzyme was crystalized at k using a solution containing . m sodium acetate ph . and . m sodium malonate and recorded x-ray data. the wild-type enzyme was crystallized within days in the monoclinic crystal form, belonging to space group p , with unit-cell parameters a . , b . , c . Å. the crystal structures of v. harveyi glcnacase were solved and refined to highest resolution of . Å. structural investigation revealed that glcnacase comprises three distinct domains, designated as the n-terminal carbohydrate-binding domain, the a b topology domain and the tim-barrel catalytic domain. the substrate binding groove of glcnacase is a small pocket, which is suitable to accommodate a shortchain chitooligosaccharide. kinetic analysis revealed that a group of the adjacent d -h -e showed a significantly decreased activity as compared with the wild-type enzyme, and these residues might be important for enzyme catalysis. silencing the molecular timekeeper in human cancer alicia michael , stacy harvey , patrick sammons , amanda anderson , hema kopalle , alison banham , carrie partch university of california -santa cruz, university of oxford the circadian clock coordinates temporal control of physiology by regulating the expression of at least % of the genome on a daily basis. disruption of circadian rhythms through environmental stimuli (e.g. light at night) or genetic means can lead to the onset of diseases such as: diabetes, cardiovascular disease, premature aging and cancer. - the circadian clock orchestrates global changes in transcriptional regulation via the bhlh-pas transcription factor clock:bmal . pathways driven by other bhlh-pas transcription factors have a homologous repressor that modulates activity on a tissue-specific basis, but none have been identified for clock:bmal . we discovered that the cancer/testis antigen pasd fulfills this role to suppress circadian rhythms. pasd is evolutionarily related to clock and interacts with the clock:bmal complex to repress transcriptional activation. furthermore, deletion of one region, highly conserved with clock exon , alleviates repression by pasd to suggest that it utilizes molecular mimicry to interfere with clock:bmal function. structural and biochemical studies of the direct interaction of pasd with the clock:bmal complex using recombinant protein expression and biophysical techniques are currently underway. as a cancer/testis antigen, expression of pasd is natively restricted to gametogenic tissues but can be upregulated in somatic tissues as a consequence of oncogenic transformation. reducing pasd in human cancer cells significantly increases the amplitude of transcriptional oscillations to generate more robust circadian rhythms. our work suggests that mechanisms to suppress circadian cycling can be hard-wired in a tissue-specific manner and our data show that they can be co-opted in cancer cells to attenuate clock function. the scaffolding protein iqgap participates in various cellular functions such as cell-cell adhesion, cell polarization and migration, neuronal motility, and tumor cell invasion by binding to target proteins, including rac and cdc , two members of the rho family. to better understand the molecular basis of these interactions, we utilized in this study a novel time-resolved fluorescence spectroscopy to determine individual rate constants for iqgap interaction with fourteen different rho proteins. the results indicated that iqgap binds among rho proteins selectively to rac-and cdc -like proteins only in a gtp-dependent manner. moreover, the interaction of rho proteins with the c-terminal half of iqgap (grd-c), shorter fragment contains grd-gbd, only the grd and also grd-gbd with single and double phosphomimetic mutations s e and s d was performed. obtained results showed that, when both grd and gbd are existing, fluorescence changes is detected but for grd alone or in the case of s d or s e/s d no change was observed, suggesting that gbd and specifically, cysteine is critical for this interaction. furthermore, fluorescence polarization results showed that the grd-c interact with cdc and rac but not with rhoa, and interestingly the grd domain showed similar behavior, but with to folds lower affinity as compared with the grd-c. consistent with this, a gdp-bound form of cdc showed interaction with both grd and the grd-c in quiet comparable affinities. at last, competition experiments utilizing interacting partners of rac , e.g. tiam , p rhogap, plexin-b , p phox, pak and rhogdia, along with structural analysis, revealed two negative charged areas on the surface of rho-and rnd-like proteins, which might explain their inaccessible interaction with iqgap . the overlapping binding site of cdc and rac on the surface of iqgap together with the kinetic details of the selective interaction of iqgap with rac-and cdc -like proteins suggests that these interactions are most likely mediated via the same mechanism. ing dimerizes through its n-terminal domain, with a symmetric antiparallel coiled-coil structure , making it a bivalent reader of the h k me mark. ing is highly homologous with ing , but forms part of a different histone acetyl transferase complex . here, we show that ing is also a dimer and thus a bivalent reader of the h k me mark. however, the crystal structure of the n-terminal domain of ing shows an asymmetric dimer, different from the homologous ing domain. our nmr data (backbone assignment and paramagnetic relaxation effects) and saxs data indicate that the structure of the n-terminal domain of ing in solution is similar to ing , suggesting that the crystal structure of ing is likely a crystallization artifact. three point mutations in the n-terminal domain of ing have been described in oral squamous cell carcinoma: q r, i v, and c r . we have found that the n-terminal domains of the three mutants are dimeric coiled-coils but with different stability, as measured by thermal denaturation. while the q r mutant is as stable as the wild type, the i v and c r mutants are strongly destabilized, suggesting a role in cancer development at least for these two mutants. efforts so far, to combat alzheimer's disease (ad) have focused predominantly on inhibiting the activity of enzyme(s) that are responsible for the production of the main causative beta amyloid forming peptide. however, the inherent complexity associated with the network of pathways leading to the progress of the disease may involve additional targets for designing effective therapies. recent experimental findings have identified abelson's tyrosine kinase (c-abl), a non-receptor kinase involved in a variety of cellular functions as a new target for ad. in the present study we employed energy optimized multiple pharmacophore modeling strategy from multiple c-abl structures bound with ligands in the inactive atp binding conformation. virtual screening followed by docking of molecules from chembridge_cns database, and maybridge databases resulted in the identification of best scoring molecules. based on docking score and selectivity assessment and druggability parameters, four out of the molecules are predicted to show increased specificity for c-abl in comparison to closely related kinases. given the implied role of c-abl not only in ad but in parkinson's disease, the identified compounds may serve as leads to be developed as effective neurotherapeutics. rafael palomino , glenn millhauser , pietro sanna university of california santa cruz, the scripps research institute the central melanocortin system is recognized as a key regulator of energy balance and appetite. the hypothalamic melanocortin receptor, mc r, is a g-protein coupled receptor that is antagonized by the peptide ligand, agouti-related peptide (agrp), leading to increased feeding and weight gain. while much research has gone into how this ligand exerts its effects at the receptor, less is known regarding nonmelanocortin components of the pathway. syndecan- , a heparan sulfate proteoglycan, has previously been implicated in potentiating agrp antagonism, however details of this interaction are unclear. this work aims to investigate the role of syndecans at both a molecular level and in vivo. we hypothesize that agrp binds the glycosaminoglycan (gag) components of syndecans, and that this interaction increases the local concentration of the peptide near mc r. furthermore, we have previously shown that designed positive charge mutations to agrp lead to increased in vivo efficacy that is independent of mc r activity, and we hypothesize that this is due to greater affinity for the negatively charged gags. using isothermal titration calorimetry we have shown tight binding between agrp and heparan sulfate, the major gag component of syndecan- , and this affinity is strengthened by additional peptide positive charge. through nmr, we see that both positively charged and polar residues are necessary for binding various heparan sulfate polymers. these data implicate a specific region of agrp that is not required for mc r binding as being necessary in its role as a heparan sulfate binding protein. expanding on these findings, we are now using a syndecan knockout mouse line to explore the mechanism of differential feeding in our designed mutants. preliminary results indicate a reduction in weight gain in knockouts compared to their wildtype littermates post peptide administration. collectively, these data show that the physiologically relevant form of agrp, previously considered unable to interact with syndecans, is indeed a heparan sulfate binding protein. furthermore, our designed mutants have differential affinities for gags, with increased affinity correlating to increased feeding potency. finally, as the mc r pathway is thought to be a viable target for wasting disorders such as cachexia, we are interested in leveraging this data to improve the potency and stability of our designed agrp mutants. taken together, this work aims to develop new insights and probe the therapeutic potential of a critical metabolic pathway. evidence of a proteolytic phenomenon in the starch binding domain of the a-amylase from lactobacillus amylovorus zaira esmeralda s anchez cuapio , alejandra hern andez santoyo , sergio s anchez esquivel , romina rodr ıguez sanoja instituto de investigaciones biom edicas, universidad nacional aut onoma de m exico, instituto de qu ımica, universidad nacional aut onoma de m exico a-amylases are glycoside-hydrolases that catalyze the hydrolysis of internal a- , glycosidic bonds in starch and glycogen generating smaller oligosaccharides ( ). these multidomain proteins contain a catalytic barrel (b/a) and, in some cases, one or more non-catalytic domains whose function is generally described as carbohydrate binding module (cbm) and particularly as starch-binding domains (sbd). the sbd can bind granular starch increasing the local concentration of substrate at the active site of the enzyme and may also disrupt the structure of the starch surface ( ) . the a-amylase from lactobacillus amylovorus has a structure that consists of a catalytic domain (cd) and an unusual carboxy-terminal starch-binding domain with identical cbms (belonging to family ) in tandem ( ). each repeat acts as an independent fixing module with an additive or synergic effect between the units ( ). when we stored pure sbd from l. amylovorus we found multiple forms of low molecular weight with a constant pattern, which does not correspond to random degradation. interestingly, when the protein is stored at ph close to and edta is added, such proteolysis appears to decrease. so far, there is little information about the proteolytic process of amylases and the nature of it. here we show that divalent ions induce a proteolytic cleavage of the sbd, raising the possibility of an autoproteolytic activity. acknowledgments: this work is supported by grants papiit in - and conacyt . s anchez cuapio z is supported by a personal grant from consejo nacional de ciencia y tecnolog ıa, m exico. unnatural amino acid and related methods provided a special mechanism to implement site-specific spectroscopy active probe incorporation in a specific membrane protein in cells. the site specific incorporation resulted in a single signal during acquisition, resulting in unambiguous signal assignment. the protein specific labeling makes it possible for in situ membrane protein analysis using nmr or fluorescence detection. the f containing unnatural amino acid incorporation has been applied for dynamic studies of transporters in native lipid membrane, and the phosphorylation quantification analysis for tyrosine kinase in native lipid membrane with the aid of lipodisc. the fluorescent unnatural amino acid incorporation enabled the site-specific channel responses analysis upon ligand binding in a single cell. heather wiebe , noham weinberg , department of chemistry, simon fraser university, department of chemistry, university of the fraser valley the mechanism by which conformational changes, particularly folding and unfolding, occur in proteins and other biopolymers has been widely discussed in the literature. molecular dynamics (md) simulations of protein folding present a formidable challenge since these conformational changes occur on a time scale much longer than what can be afforded at the current level of computational technology. transition state (ts) theory offers a more economic description of kinetic properties of a reaction system by relating them to the properties of the ts, or for flexible systems, the ts ensemble (tse). the application of ts theory to protein folding is limited by ambiguity in the definition of the tse, although the experimentally observed first-order kinetics for folding of small single-domain proteins lends itself to interpretation by this theory. the pressure dependences of the folding rate constant can be used to obtain activation energies and activation volumes, which are rationalized as the properties of the folding tse. the large amount of activation volume data in the literature has gone largely uninterpreted at the quantitative level. we propose to utilize this data in conjunction with md-calculated volumetric properties to identify the tse for protein folding. the effect of pressure on reaction rates is expressed in terms of logarithmic pressure derivatives, known as activation volumes. according to ts theory, activation volumes can be identified as the difference in volume between the ts and reactant species: activation volumes dv ‡ have been experimentally determined for the folding of several proteins. the concept of activation volume can be extended to that of a volume profile, dv(y), which describes how the volume of a system changes along reaction coordinate y. if the position y ‡ of the ts along the reaction coordinate is unknown, it can be found by locating dv ‡ on the volume profile: such volume profiles can be built using our recently developed md-based displacement volume method.* using this method, volumes of single molecules can be calculated by taking the difference between the volume of pure solvent and solvent containing the desired solute. this method takes into account the strength and type of solvent-solute interactions as well as the geometrical configuration of the solute. in this work, we present the successful application of this method to several conformationally flexible systems. structure of the p paf/pcna complex and implications for clamp sliding on the dna during replication and repair to the canonical pip-box binding groove on the pcna front face. in contrast to other pcna interacting proteins, however, p paf also contacts the inside of, and passes through, the pcna ring. the mostly disordered p paf chain termini thus emerge at opposite faces of the ring, but remain protected from degradation by the s core proteasome. we also unveil a novel dna binding activity of p paf, both free and bound to pcna, which is mainly mediated by its conserved histone-like n-terminal tail. molecular modeling shows that a ternary complex with a duplex dna inside the pcna ring is energetically feasible and our electron micrographs show increased density inside the ring. we propose that p paf acts as a flexible drag that regulates pcna sliding along the dna, and may facilitate the switch from replicative to translesion synthesis polymerase binding upon dna damage. acknowledgements: this work has been mainly sponsored by mineco grant ctq - and juan de la cierva- contract to alfredo de biasio. metabolic syndrome (mets) is one of the leading causes of the death worldwide; however, exact pathophysiological mechanisms of mets remain largely unknown. growing evidence suggests that the increased availability of glucocorticoids at the tissue level play an important in mets development. one of the major determinants of glucocorticoid local action seems to be the enzyme b-hydroxysteroid dehydrogenase ( b-hsd ). this enzyme is a well-known member of the short-chain dehydrogenase/reductase (sdr) superfamily. it is an important carbonyl reducing enzyme that, besides its role fine-tuning of glucocorticoids actions, is involved in the biotransformation of drugs and in the development of lung cancer through metabolism of the tobacco specific carcinogen nnk. the phylogenetically closest relative of b-hsd is dhrs enzyme from the same superfamily. unlike b-hsd , dhrs is poorly characterized however it can be supposed at least partially overlapping function to b-hsd . moreover its possible association with similar pathological conditions in human as b-hsd has already been indicated by several studies. the aim of this study is the basic biochemical characterization of dhrs . the enzyme is a member of cluster of "classical" sdr; such members are considered to be retinoid and steroid metabolizing enzymes, so characterization the enzyme was based on this assumption. dhrs was prepared in recombinant form in the sf cell line. it was proved that this enzymes is an integral membrane-bound enzyme localized in the endoplasmic reticulum with luminal orientation, similarly to b-hsd . known substrates of b-hsd and related enzymes were tested also as substrates of dhrs . it was proved that dhrs is nadph-dependent reductase with important substrates as steroid hormones cortisone and androstene- , -dione, all-transretinal and also xenobiotics as , -naphtoquinone or carcinogen nnk at least in vitro. for better understanding of the catalytic function of dhrs its structural model was prepared and it is used also for the identification of additional substrates by ligand virtual screening. dhrs enzyme is expressed in several human tissues as adrenals, liver, prostate, small intestine and kidney. these brand new initial results point to the possible involvement of dhrs in important cellular processes that deserve further investigation. these results will lay the foundation for an understanding of dhrs role in human physiology resp. pathophysiology. this project was supported by grant agency of charles university ( /c/ ) and unce / ). structure-based functional identification of helicobacter pylori hp as a nuclease with both dna nicking and rnase activities bong-jin lee , ki-young lee hp is a conserved, uncharacterized protein from helicobacter pylori. here, we determined the solution structure of hp using three-dimensional nuclear magnetic resonance (nmr) spectroscopy, revealing that this protein is structurally most similar to a small muts-related (smr) domain that exhibits nicking endonuclease activity. we also demonstrated for the first time that hp is a nicking endonuclease and a purine-specific ribonuclease through gel electrophoresis and fluorescence spectroscopy. the nuclease activities for dna and rna were maximally increased by mn( ) and mg( ) ions, respectively, and decreased by cu ( ) ions. using nmr chemical shift perturbations, the metal and nucleotide binding sites of hp were determined to be spatially divided but close to each other. the lysine residues (lys , lys and lys ) are clustered and form the nucleotide binding site. moreover, site-directed mutagenesis was used to define the catalytic active site of hp , revealing that this site contains two acidic residues, asp and glu , in the metal binding site. the nucleotide binding and active sites are not conserved in the structural homologues of hp . this study will contribute to improving our understanding of the structure and functionality of a wide spectrum of nucleases. high-fidelity recombinant protein production in a silkworm bioreactor sungjo park , in-wook hwang , tatsuya kato , enoch park , andre terzic center for regenerative medicine, mayo clinic, laboratory of biotechnology, shizuoka university the domesticated silkworm, bombyx mori, is an attractive host naturally equipped with a proficient posttranslational modification machinery adequate to fulfill stringent demands of authentic recombinant protein production. silkworm-based protein expression has originally relied on a prototype baculovirus vector system that employs silkworm as a bioreactor in place of more traditional cell lines. recent development of the silkworm trophic b. mori nucleopolyhedrovirus (bmnpv) bacmid launches a second generation of silkworm-based protein production technology. introducing the recombinant bacmid dna into silkworms expedites heterologous protein expression by eliminating prior virus construction and amplification steps. salient examples of heterologous eukaryotic proteins produced in silkworms are acetyl-coa carboxylase , malonyl-coa decarboxylase, spot /mig heterodimer and a , -sialyltransferase with consistent high levels of protein expression. thus, equipped with a fail-safe post-translational modification machinery, eukaryotic proteins are readily bioengineered using a silkworm-based protein expression platform. studies exploring potential applications of synthetic antifreeze proteins in the frozen food industry ho zee (charles) kong , conrad perera , ivanhoe leung , nazimah hamid , viji sarojini school of chemical sciences, the university of auckland., school of applied sciences, auckland university of technology in nature, certain species of plants, insects and fish produce a group of antifreeze glycoproteins and polypeptides which enable them to survive the freezing temperatures of their natural habitat. these naturally occurring antifreeze proteins (afps) were first discovered in polar fishes such as antarctic notothenioids and winter flounder. these afps have the ability to bind to ice crystals and restrict their size and morphology; decrease the freezing point of water and inhibit the ice recrystallization processes. ice crystal formation is of primary concern to the frozen food industry, as ice crystal formation during freezing can be disruptive to and cause damage to the cellular structures in food. the unique properties of afps can be developed into a potential solution to minimize freeze-thaw damage to frozen food. a number of tailor made synthetic analogues based on the naturally occurring afps were successfully designed and synthesized. antifreeze activity studies of the afps were carried out using the clifton nanoliter osmometer attached with a microscope. the afps exhibited thermal hysteresis as well as modification of ice crystal morphology, confirming their antifreeze activity in vitro. the ability of these synthetic afps in preserving the texture and structure of frozen food was evaluated using the techniques of scanning electron microscopy. the afps showed great potential to preserve the cellular structures of frozen food samples during freeze-thaw process. additionally, secondary structure analysis of the afps was carried out using circular dichroism. this presentation will summarize our current results on the design, synthesis and anti-freeze activity analysis of the synthetic afps. invasive fungal infections remain a leading cause of death in immunocompromised patients. current antifungal agents have a host of issues including limited efficacy, host toxicity and an alarming increase in resistance. current research in our laboratories is focused on targeting the calcineurin signaling pathway that has been shown to be required for fungal pathogenesis. calcineurin is a highly conserved serine-threonine-specific ca -calmodulin-activated phosphatase important in mediating fungal pathogenesis and stress responses. it is a key regulator of a signal transduction network required for survival of the most common pathogenic fungi in humans, making it an ideal target for fungal drug development. calcineurin is a heterodimer of a catalytic (a) and regulatory (b) subunit. phosphatase activity requires association of the two subunits. calcineurin is also the target of the immunosuppressant fk , which functions as an inhibitor by first complexing with the peptidyl-prolyl cis-trans isomerase immunophilin, fkbp . the fkbp -fk complex subsequently binds to calcineurin in a groove between the a and b subunits and inhibits its activity. although fungal calcineurins are targeted by fk , it also targets mammalian calcineurin and is thus immunosuppressive in the host. in order to improve therapeutic efficacy, we have undertaken a unique effort that utilizes both structural biology and molecular mycology in an effort to overcome the fungal versus human specificity barrier. the nmr studies to be presented here have been focused on determining the resonance assignments and solution structures for the fkbp proteins from the pathogenic fungi candida albicans, candida glabrata and aspergillus fumigatus. notably, the x-ray crystallography structures of the wild-type candida albicans and aspergillus fumigatus fkbp proteins revealed an intriguing intermolecular interaction involving four residues in the 's loop including pro (in c. albicans) and pro (in a. fumigatus) which are stabilized in the cis conformation. these data suggest that the protein might use itself as an enzyme substrate. in efforts to establish if this interaction remains in a solution environment, we have determined the nmr structure and measured the t relaxation rates for the wild-type a. fumigatus fkbp protein and for the p g mutant variant that adopts a dramatically different orientation of the 's loop and does not form an intermolecular interaction in the crystal structure. the nmr chemical shift data indicate that, while the remainder of the protein structure remains unchanged, the 's loops in the two variants are indeed different. in addition, the t relaxation rates of the residues in this region are dramatically dissimilar in the two variants, but remain identical throughout the rest of the protein. we have also begun inhibitor binding studies of all of the fkbp proteins from each of the pathogens by titrating the fk inhibitor into native and mutant fkbp proteins in order to examine conformational changes associated in the protein upon complex formation. using this approach we plan to determine the relative kd values for binding of each inhibitor to the fkbp protein from each pathogen for comparison of binding proclivities. lupin (lupinus angustifolius l.) b-conglutin proteins: structure functional features, catalytic mechanism modeling and cross-allergenicity identification using protein threading and molecular docking methods lupin is an important pulse, which displays a wide range of benefits in agriculture, particularly these involved in possible plant pathogen suppression. furthermore, lupin seed proteins promote different positive health aspects, preventing cardiovascular disease, and reduction of glucose and cholesterol blood levels. "sweet lupine" seeds seem to be promising as a source of innovative food ingredients due to averaged protein content similar to soybean and an adequate composition of essential amino acids. thus, lupin seeds may be important source of proteins for human and animal consumption. however, and as drawback feature, the number of allergic people to lupin seed proteins is rising, becoming a serious and a growing problem in the western world, because of the rapid introduction of lupin seeds as new ingredients in traditional and novel foods. the goals of this study are the characterization the structure-functional properties of lupinus angustifolius l or narrow leafed lupin (nll) b-conglutin proteins, with a focus in its catalytic mechanism, and its molecular cross-allergenicity with other legumes, i.e. peanut, by extensive analysis using different computer-aided molecular approaches covering (i) physicochemical properties and functional-regulatory motifs, (ii) sequence analysis, -d and d structural (threading) modeling comparative study and molecular docking, (iii) conservational and evolutionary analysis, (iv) catalytic mechanism modeling, and (v) sequence, structure-docking based b-cell epitopes prediction, while t-cell epitopes were predicted by inhibitory concentration and binding score methods. b-conglutins (vicilin-like or s proteins) are seed proteins typically found in reserve tissues (endosperm and cotyledon). they belong to the cupin superfamily of proteins, containing a globular domain constituted by a conserved b-barrel. two barrels were found in all b-conglutin protein isoforms and an additional mobile n-terminal arm constituted bye a-helices. molecular modeling analysis has shown that one of this barrel contain a semi-conserved metal binding motive (hyx. . .r), typically found in oxalate oxidase (oxox) enzymes. interestingly, our results revealed considerable structural differences between b-conglutin isoforms, particularly affecting -d elements (loops and coils), and numerous micro-heterogeneities are present in fundamental residues directly involved in epitopes variability, which might be a major contributor to the observed differences in cross-reactivity among legumes. we also identified multiple forms of b-conglutins polypeptides ranging from - kda, with ige-binding characteristics in atopic patients. thus, b-conglutins might be considered as major allergen in different species of lupin, including the "sweet lupin" group, since several of these polypeptides were recognized by human iges, having the potential to trigger an immune response leading to allergy symptoms. influenza virus is one of the most prevalent pathogens causing respiratory illness which often leads to serious post influenza complications such as pneumonia and myocarditis. some viruses, as the avian influenza h n , are especially dangerous and draw special attention of who. this highly pathogenic virus spreads quickly among domestic poultry and wild birds resulting in high mortality. what is more distressing, the h n virus may be transmitted to humans. because of antigenic drift it is impossible to deliver an effective vaccine against all subtypes of the h n virus. moreover, traditional egg-based production of influenza vaccines is time-and cost-consuming, what makes it inadequate in case of a pandemic. hence, we have developed an efficient production process of influenza vaccine based on a recombinant hemagglutinin antigen (rha). recombinant vaccines underlay strict regulations and quality requirements. the purpose of this work was to develop a battery of analytical methods that allow to evaluate key quality attributes of rha on each stage of production. at first, we have focused on rha structure as a crucial issue for its activity. the primary structure of rha was confirmed by peptide mapping and tof/tof fragmentation (hplc, maldi tof/tof). furthermore, ftir analysis was used to evaluate the secondary structure of the protein. the disulfide bonds, which stabilize the tertiary structure, were assigned by peptide mapping. additionally, free thiols were measured using ellman's reagent. moreover, we have employed rp-hplc, sec-mals and dls to explore oligomerization of rha. these techniques appeared to be useful not only to confirm existence of native oligomers, but also to find and discard misfolded fraction, aggregates and truncated forms. in addition, two analytical methods (rp-hplc and cge) were developed to assess the purity of rha as required by ich guidelines. we also have determined isoelectric point and heterogeneity of rha by cief. afterward, developed methods were applied in the stability studies that provide a valuable insight into a chemical degradation process and conformational changes of rha during storage. this work was supported by innovative economy operational program, grant no. wnd-poig. . . - - / - as a part of project "centre of medicinal product biotechnology. package of innovative biopharmaceuticals for human and animal therapy and prophylactics." muscle cell atrophy via hsp gene silencing was counteracted by celastrol-mediated hsp overexpression molecular chaperone heat shock proteins (hsp) are known to assist protein quality control under various stresses. although overexpression of hsp was found to promote muscle mass retention in an unloading state, it is unclear whether muscle atrophy is induced by suppression of hsp expression and is counteracted by active hsp overexpression. in this study, we pre-treated hsp sirna to rat l cells for the hsp gene-silencing, and determined myotube diameter, hsp expression and anabolic and catabolic signaling activities in the absence or presence of triterpene celastrol (cel), the hsp inducer. relative to a negative control (nc), muscle cell diameter was reduced by % in the sirna-treated group, increased . -fold in the cel-treated group and remained at the size of nc in the sirna cel group. hsp expression was decreased % by sirna whereas the level was increased -to -fold in the cel and sirna cel groups. expression of foxo and atrogin- was increased . -to . -fold by sirna, which was abolished by cel treatment. finally, phosphorylation of akt , s k and erk / was not affected by sirna, but was elevated -to -fold in the cel and sirna cel groups. these results suggest that hsp downregulation by hsp gene-silencing led to muscle cell atrophy principally via elevation of catabolic activities. such anti-atrophic effect was counteracted by cel-mediated hsp overexpression. the centers for disease control and prevention report that at least million people in the united states will become ill due to antibiotic resistant pathogens leading to , deaths each year. in order to circumvent these resistance mechanisms, it is essential to quantitatively understand how the function of the protein(s) involved relates directly to resistance. integral membrane efflux pumps are known determinants of single-drug and multi-drug resistance in a wide variety of pathogenic organisms. these transporters are proteins whose characterization typically requires reconstitution in an artificial membrane. subsequently, these important proteins are difficult to characterize by traditional in vitro studies. my project aims to determine the physicochemical parameters of the efflux pump tetb utilizing molecular biology and mathematical modeling. tetb is composed of transmembrane (tm) alpha-helices and is found within the inner membrane of gram-negative bacteria. this protein allows for the efflux of tetracycline (tet), doxycycline (dox), and minocycline (mcn) antibiotics from the cytoplasm into the periplasm. these tetracyclines are a bacteriostatic class of antibiotics that inhibit protein synthesis by binding to the s ribosomal, therefore, blocking the binding of aminoacyl-trna. for cells grown in tetracyclines, the efflux mechanism of tetb decreases the cytosolic antibiotic concentration allowing for the rate of protein translation to increase. i have inserted a tet(b) expression system into the chromosome of an escherichia coli lab strain and have determined its growth profile under various concentrations of tet, mcn, and dox using a high-throughput -well plate format. the growth rate profiles correlate with tetb pumping rates for each drug. tetb more readily pumps out tet compared with dox and mcn and we observe that cells expressing tetb can grow at higher tet concentrations compared with dox and mcn. the shapes of the growth rate profiles produced in the different drugs give insight into the physicochemical mechanism of tetb. we have built a preliminary mathematical model that can simulate these growth profiles and predict efflux pump physicochemical parameters. we are currently working on understanding how efflux expression effects bacterial growth by testing ribosome binding site (rbs) sequences of varying strengths in our tet(b) expression system. future work is geared toward modeling more complex efflux pumps such as the tripartite pumps which traverse both bacterial membranes and cause multi-drug resistance. collectively, this project aims to build an in vivo system which will allow for the characterization of a variety of efflux pumps without the arduous tasks of protein purification and subsequent reconstitution. ( ) identified a small transmembrane region of both kcne and kcne that are essential for their unique modulation of the kcnq channel. by swapping a triplet motif in the transmembrane region of kcne and kcne , we can flip the primary function of these two proteins. while the key for kcne and kcne 's unique modulating is believed to lie in this triplet motif, the mechanism and structural changes involved in this modulation is not fully understood. by using nmr spectroscopy, biochemical studies, and computational docking, we aim to look at the structural and conformational differences between kcne and the triple mutant kcne substituted with the three essential kcne residues. we have expressed and purified n-labled kcne triple-mutant in sufficient quantities for nmr studies in lmpg detergent micelles and other membrane mimetics, and we have collected d nmr spectra using a trosy-based pulse sequence. partial backbone assignments of kcne triple mutant have been determined by aligning and transfer assignments of the wt kcne previous determined in our lab. with the structure of kcne triple mutant determined, we aim to computationally dock the triple mutant into a model of the full-length kcnq channel in the open and closed state. lastly, we will compare the known structure of kcne docked to a model of kcnq to that of the kcne triple mutant to determine key interactions, significant structural and conformational changes, and how the triple motif region gives rise to its specific structural and functional differences. with this information, we can begin to understand the mechanism of the functional diversity of the kcne family on kcnq potassium channel. biochemical characterization of brassica napus diacylglycerol acyltransferase and its regulatory domain .a) expressed in saccharomyces cerevisiae. purified bnadgat in n-dodecyl-b-d-maltopyranoside (ddm) micelles behaves as dimers, which can associate further to form tetramers. the acyl donor preference of the major dimeric form with sn- , -diolein as acceptor follows the following order: a-linolenoyl-coa > oleoyl-coa palmitoyl-coa > linoleoyl-coa > stearoyl-coa. the first residues of bnac.dg-at .a corresponding to a soluble regulatory region was expressed in escherichia coli and purified. truncation of this soluble domain reveals that the dimeric interface is located within residues - , while the first residues allow formation of tetramers. this n-terminal region was implicated as an allosteric exosite for acyl-coas as revealed by previous lipidex- binding studies. in the current study, circular dichroism spectroscopy and isothermal titration calorimetry were used to probe the binding kinetics and thermodynamics. dgat appears to shift between two oligomerization states, a phenomenon that may be related to regulation of enzyme activity and mediated by the n-terminal domain. alteration of lysine and arginine content as a strategy to modify such an interaction was found to increase the activity of rdrp in vitro. further, deletion of c terminal amino acid residues also resulted in increase in the polymerase activity that was comparable to the full length rdrp-p complex. it was proposed that the conserved c terminal disordered domain of rdrp was responsible for interaction with p and modulation of the activity. in the present study, role of the c terminal disordered domain was further investigated by determining the oligomeric status of the complex and the c terminal deletion mutants of rdrp and also by quantitating the rdrp-p interaction using surface plasmon resonance. size exclusion chromatography revealed that rdrp eluted in the void volume of the column whereas a significant fraction of the rdrp-p complex eluted at a position corresponding to the size of the : complex of rdrp and p ( kda). activity measurements indicated that the heterodimeric complex was more active than the aggregate eluting in the void fraction. interestingly, the c terminal deletion mutants of rdrp (c del & c del rdrp) were also found to be less aggregated as compared to full length rdrp and some of the protein eluted at a position corresponding to the respective monomers. these monomers were also more active than the aggregate fractions. these results demonstrate that the increase in activity observed either upon interaction with p or deletion of the c terminal domain could be due to the change in the oligomeric state of rdrp. in order to further analyze the interaction of rdrp with p surface plasmon resonance was used. rdrp and its deletion mutants were immobilized on biacore sensor surface and p protein was used as an analyte. full length rdrp and c del rdrp were shown to interact with p with kd values of . and um respectively. however, c del and c del rdrp did not show any binding with p . these results suggest that the region - from the c terminus of rdrp is essential for the interaction with p . further, the c del rdrp was inactive although c del rdrp continued to be active suggesting that residues - from the c terminus are crucial for rdrp activity. further studies are in progress to identify the residues within these motifs that may be essential for the activity or interaction with p . aggregation of androgen receptor in spinal bulbar muscular atrophy is a multistep process spinal bulbar muscular atrophy (sbma) is a member of the polyglutamine (polyq) expansion diseases, like huntington disease, and it is caused by a genetic expansion of the polycag tract in exon of androgen receptor (ar) that codes for the polyq region. sbma is a late onset disease, which involves a progressive degeneration of the motor neurons and consequent muscular atrophy. there is still no treatment available for this disease. ar is a nuclear receptor that responds to testosterone and that regulates the expression of the masculine phenotype. it is composed of an intrinsically disordered nterminal domain (ntd) that bears the polyq tract, a dna binding domain and a ligand binding domain. aggregates of ar protein with an extended polyq are observed in the motor neurons of sbma patients. in vitro studies showed that aggregation of androgen receptor takes place only in presence of testosterone and that the cleavage of the protein by caspase is a crucial event for cytotoxicity. however, there is no clear knowledge of the mechanism of aggregation, for this protein. an increasing body of evidence supports the hypothesis that the aggregation of these proteins is controlled by regions flanking the polyq tract, by regulating the rate of aggregation depending on their secondary structure. we have applied nuclear magnetic resonance (nmr) and circular dichroism for generating information on the secondary structure of the n-terminal cleavage product of ar by caspase and we have studied its aggregation with a set of biophysical methods, like dynamic light scattering, an hplc sedimentation assay and transmission electron microscopy. we have found that the polyq tract of ar presents a high degree of helicity. we attribute this conformation to the n-terminal flanking region, characterized by high helicity and we have tested this hypothesis by performing mutations. we have also observed that the rate of the first step of oligomerization is not dependent on the number of glutamine repeats, but instead is due to self interactions of a region n-terminal to and far from the polyq. its progression to fibril is dependent to the number of glutamines in the tract. we have therefore identified two steps in the aggregation process of ar, where a motif far from the polyq at its n-terminal drives the early oligomerization, followed by the interaction of the polyq chains that stabilize it and determine the progression to fibrils. these findings shed a light for possible interventions on the ar oligomerization process, thus suggesting a different strategy to study the onset of the disease in sbma patients. destabilizing the transient helical conformation of islet amyloid polypeptide hastens peptide self-assembly and potentiates cytotoxicity carole anne de carufel , phuong trang nguyen , alexandre arnold , isabelle marcotte , steve bourgault amyloidogenic polypeptides can be divided into two different structural classes: those that are intrinsically disordered and those that show a well-defined structure in their monomeric soluble state. natively folded proteins, such as transthyretin, have to unfold (or misfold), at least partially, to form amyloids. in contrast, intrinsically disordered polypeptides, such as the islet amyloid polypeptides (iapp) and abeta peptide, need to undergo conformational rearrangements allowing the formation of locally ordered structure(s) to initiate the amyloidogenic process. studies have shown that iapp and abeta adopt an alphahelix conformation in the initial steps of amyloidogenesis. this intermediate is believed to be on-pathway to fibril formation, although this hypothesis is still the matter of debate. in this study, we designed human iapp (hiapp) derivatives in which alpha-helix destabilizing substitutions were incorporated into the putative helical segment of iapp to probe the initial structural event in amyloid formation. using trifluoroethanol titration, we observed by cd spectroscopy that strategic incorporation of d-amino acids at positions and leads to an iapp derivative (diapp) that cannot fold into a helix. in homogeneous solution, hiapp and diapp show similar kinetics of fibrillization, as measured by thioflavin t fluorescence. although their amyloid fibrils display different characteristics by afm, iapp and diapp are able to self-associate to form amyloids when mixed together and when seeded with one another. studies in heterogeneous environment, notably in presence of glycosaminoglycans and model membranes of dopc/dopg ( : ), showed a helical intermediate for hiapp while only a beta-sheet secondary structure was apparent for diapp. while the rate of amyloid fibril formation was increased for both peptides, diapp was drastically affected by these anionic biomolecules with an absence of lag phase. the incapacity of adopting a transient helical conformation accentuates cell toxicity, supported by the caspase / activation level and the increase in intracellular calcium level. overall, this study indicates that the helical intermediate is offpathway to iapp amyloid formation and offers novel mechanistic insights for the development of molecular identities modulating peptide self-assembly and iapp-induced cytotoxicity. for an organism to survive, its proteins must adopt complex conformations in a challenging environment where macromolecular crowding can derail even robust biological pathways. the situation is perilous: many diseases arise from improper folding of just a single protein. to cope, cells employ a repertoire of molecular chaperones and remodeling factors that usher unfolded proteins into active conformations, sequester them, or target them for degradation. yet, not all aggregated proteins are the result of mis-folding. yeast prions are self-templating protein-based mechanisms of inheritance that rely upon chaperones for their propagation. the best studied of these is the prion domain (nm) of sup , which forms an amyloid that can adopt several distinct conformations (strains) that produce distinct phenotypes. using genetic, biochemical, spectroscopic, and solid state nmr techniques, we investigated the structural and dynamic underpinnings of sup amyloids and found that prion strains differ in both their atomic structure as well as their dynamic motions. interestingly, these mobility differences correlate with differences in the interaction with molecular chaperones in vivo. limitations on the specificity and sensitivity of biophysical techniques typically restrict structural investigations to purified systems at concentrations that are orders of magnitude above endogenous levels. therefore, i developed an approach to apply a sensitivity-enhancement technique for nmr, dynamic nuclear polarization (dnp), to investigate interactions between sup and molecular chaperones at endogenous concentrations in their native environments. critically, i found that the cellular environment induced structural changes in a region of sup that is intrinsically disordered in purified samples but known genetically to influence prion propagation from one generation to the next. this approach enables structural and mechanistic investigation of proteins in biologically relevant contexts. genetic instability within regions encoding repetitive proteins as a driver of adaptation stephen fuchs more than ten percent of all eukaryotic proteins contain within them a region of repetitive amino acid sequence. these repetitive domains range from short stretches of a single amino acid to multiple copies of longer, heterogeneous amino acid sequences and generally show lack of defined structure. they play diverse roles in cells including acting as structural proteins, promoting cell-cell interactions, and mediating the assembly of molecular machines. tandem repeat proteins are known to be variable in length within cellular populations although the mechanisms dictating this variability have not been elucidated. here we describe work uncovering specific features within the coding sequences of repetitive proteins that contribute to tandem repeat instability in yeast. furthermore, we demonstrate that cells will expand and/or contract repetitive regions in order to adapt to environmental stresses and describe a role for dna repair proteins in this process. lastly, we demonstrate how these mechanisms are likely conserved in higher eukaryotes, including humans. this study uncovers the molecular basis for an important aspect of natural protein evolution and describes a novel mechanism for adaptation in response to environmental changes. a proline-tryptophan turn in the intrinsically disordered domain of ns a protein is essential for hepatitis hepatitis c virus (hcv) nonstructural protein a (ns a) and its interaction with the human chaperone cyclophilin a (cypa), a peptidyl-prolyl cis-trans isomerase (ppiase), are both targets for highly potent and promising antiviral drugs that are in late stage of clinical development [ , ] . despite its high interest in the development of drugs to counteract the worldwide hcv burden, ns a is still an enigmatic multifunctional protein poorly characterized at the molecular level. ns a is required for hcv rna replication and is involved in viral particles formation and regulation of host pathways. thus far, no enzymatic activity or precise molecular function has been ascribed to ns a that is composed of a highly structured domain (-d ), as well as two intrinsically disordered domains (-d ) and (-d ). ns a-d structure has been solved by x-ray crystallography and ns a-d and -d have been characterized by nmr spectroscopy. these two last domains do not adopt a stable d structure but rather exist as an ensemble of highly dynamic conformers. using nmr spectroscopy, hcv ns a-d has been shown to establish a direct interaction with the human cypa and to be a substrate for the enzymatic ppiase activity of cypa [ ] . the cypa interaction site in ns a-d is composed of nearly residues that correspond to the most conserved region of the domain, with proline residues being strictly conserved among all hcv genotypes. whereas ns a-d is mainly disordered, some of its nmr resonances, corresponding to residues in the cypa binding site, display unexpected h and n nmr chemical shifts for an intrinsically disordered domain. thus we have further characterized this region by nmr spectroscopy. a short structural motif in the disordered ns a-d has been identified and we solved its nmr structure. in a cellular assay, we showed that this structural motif, a minimal pro -trp turn, is essential for hcv rna replication. we demonstrated that this pro-trp (pw) turn is required for proper interaction with the host cypa and influenced its enzymatic ppiase activity on residue p of ns a-d . this work provides a molecular basis for further understanding of the function of the intrinsically disordered domain of hcv ns a protein. in addition, our work highlights how very small structural motifs present in intrinsically disordered proteins can exert a specific function. [ ] [ ] . this -residue peptide also shows toxicity towards mammalian cells but at higher concentrations, suggesting its possible usefulness as a treatment for trypanosomiasis. here we present the peptide's relative cytotoxicity for bloodstream and procyclic forms of t. brucei and for mammalian cells, the fate of the peptide in t. brucei using fluorescently-labelled bt- , and its three dimensional structure using nmr spectroscopy.minimum inhibitory assays confirmed the peptide's selective toxicity towards both bloodstream and procyclic forms of t. brucei, demonstrating its potential to serve as a starting point for a trypanocidal drug. fluorescence spectrophotometric experiments, carried out using fluorescein labelled bt- , show that the peptide is released from the external surface of the parasite into the suspending medium under de-energized conditions but retained in energized cells. heteronuclear and homonuclear biomolecular nmr experiments (tocsy, noesy, h- c-hsqc, h- n-hsqc, etc) folowed by structural calculations (chemical-shift based as well as simulated annealing techniques) in the free state indicate that this peptide is mostly unstructured in aqueous solution, suggesting that there is a major conformational change upon binding to t. brucei that is required for uptake. we suggest that the evolutionary pressure that selected for the intrinsically disordered structure of this peptide was the advantage it conferred upon the host to bind to many different surface structures throughout the microbiological world. physikalische biologie, heinrich heine university, structural biochemistry (ics- ), research centre j€ ulich, chemistry and biotechnology, swedish university of agricultural sciences (slu) the misfolding and amyloid formation of proteins featuring intrinsically disordered regions is a pathological hallmark of several neurodegenerative diseases, including alzheimer's disease and parkinson's disease. engineered binding proteins targeting amyloidogenic proteins aid in the elucidation of the aggregation mechanism and suggest therapeutic strategies. we have constructed phage display libraries enriched in binders to amyloidogenic intrinsically disordered proteins, using zab , a protein with high affinity for the amyloid-beta peptide, as a scaffold. binding proteins selected from these libraries are termed beta-wrapins (beta-wrap proteins). the beta-wrapins as and hi exhibit nanomolar affinity for monomeric alpha-synuclein or islet amyloid polypeptide, respectively. as and hi potently inhibit in vitro amyloid formation and toxicity at substoichiometric concentration ratios, indicating that they interfere with the nucleation and/or elongation of amyloid fibrils. the nmr structures of the betawrapin:target complexes reveal beta-hairpin motifs in alpha-synuclein and islet amyloid polypeptide which are stabilized by coupled folding and binding. in the case of alpha-synuclein, the beta-hairpin is formed in the sequence region - which contains the beta-strand segments b and b of amyloid fibril models and most disease-related mutations. we show by disulfide engineering, biophysical techniques, and cell viability assays that intramolecular tertiary interactions between the b and b segments of alpha-synuclein interfere with its aggregation, and moreover inhibit aggregation of amyloid-beta peptide and islet amyloid polypeptide. our results reveal a common preference of different amyloidogenic proteins for formation of beta-hairpin motifs and demonstrate a critical role of hairpin conformers in the control of amyloid formation. interaction profiling through proteomic peptide phage display cecilia blikstad , moon-hyeong seo , norman davey , roland arnold , sachdev s sidhu , philip m kim , ylva ivarsson department of chemistry -bmc, donnelly centre a considerable part of the human proteome is intrinsically disordered. the disordered regions are enriched in short motifs serving as docking sites for peptide binding domains. domain-motif interactions are crucial for the wiring of signaling pathways. these interactions are typically transient and difficult to capture through most conventional high-throughput methods. we therefore developed a novel approach for the large-scale profiling of domain-motifs interactions called proteomic peptide phage display (prop-pd) ( ). in prop-pd we combine bioinformatics, oligonucleotide arrays, peptide phage display and next-generation sequencing. this allows the interrogation of domain-motif interactions on a proteome-wide scale and the de novo motif discovery.in our pilot experiment we generated two distinct phage libraries, one displaying all human c-terminal sequences and one displaying c-termini of known virus proteins. we used the prop-pd libraries to identify interactions of human postsynaptic density /discs large/zonula occludens- (pdz) domains. we successfully identified novel pdz domain interactions of potential relevance to cellular signaling pathways and validated a subset of interactions with a high success rate. recently, we created a prop-pd library that displays peptides representing the disordered regions of the human proteome. we validate our disorderome library against a range of peptide binding domains, which provides novel insights into their binding preferences and suggest interactions of potential biological relevance as will be presented here. prop-pd can be used to uncover protein-protein interactions of potential biological relevance in high-throughput experiments and provides information that is complementary to other methods. prop-pd is scalable and can be developed to any target proteome of interest. phosducin is a kda phosphoprotein that regulates visual signal transduction by interacting with the gtbg; subunit of the retinal g-protein transducin. the function of pdc is regulated by phosphorylation at ser and ser in a process that involves the binding of phosphorylated pdc to the regulatory - - protein, but the molecular mechanism of the regulation by - - protein is still unknown. pdc was also suggested to be involved in transcriptional control, the regulation of transmission at the photoreceptorto-on-bipolar cell synapse, and the regulation of the sympathetic activity and blood pressure [ ] [ ] [ ] . here, the solution structure of pdc and its interaction with the - - protein were investigated using small angle x-ray scattering, circular dichroism, quenching of tryptophan fluorescence, analytical ultracentrifugation, hydrogen-deuterium exchange coupled to mass spectrometry and nuclear magnetic resonance. we show that the - - protein interacts with and sterically occludes both the n-and c-terminal gtbg binding interfaces of phosphorylated pdc, thus providing a mechanistic explanation for the - - depedent inhibition of pdc function. the - - protein dimer interacts with pdc using surfaces both inside and outside its central channel. the n-terminal domain of pdc, where both phosphorylation sites and the - - binding motifs are located, is intrinsically disordered protein which remains likely highly flexible when bound to - - indicating the fuzzy-like character of this complex. in addition, it has been speculated that the - - protein binding decreases the rate of pdc dephosphorylation after a light stimulus through its interaction with phosphorylated ser and ser , thus lengthening the time that pdc remains phosphorylated after a light exposure. pdc is dephosphorylated in vivo by protein phosphatases known to cause neurodegenerative disease in a polyglutamine-length dependent manner. despite intense study, the molecular basis of polyq toxicity in hd or any of the other diseases has only partially been elucidated and potential routes to therapeutic intervention are sparse. the use of genetically tractable model organisms to identify the cellular pathologies caused by mutant huntingtin expression is essential to our understanding of the disease pathology in humans. in eukaryotes, many of the protein folding homeostasis pathways are highly conserved and yeast cells expressing a glutamine-expanded fragment of huntingtin exon exhibit a polyq length-dependent toxicity that recapitulates many of the basic protein folding defects associated with polyq diseases in neurons. taking an unbiased approach, we screened an overexpression library of the entire yeast genome for suppressors and enhancers of polyq toxicity and identified seven proteins with prion-like, q-rich domains that are strong suppressors in yeast. intriguingly, the q-rich domains of these proteins, and several other q-rich domains, suppress toxicity when expressed in isolation. these suppressors are also efficacious in mammalian cells and, strikingly, one suppressor was independently shown to alleviate polyq-expanded ataxin- toxicity in a drosophila model. in yeast, the suppressors co-aggregated with an otherwise highly toxic glutamine expanded huntingtin exon protein (htt q), resulting in a non-toxic aggregate and eliminating populations of diffusible oligomeric species. using a transcriptional sensor for protein coaggregation, we determined that yeast and human proteins that normally co-aggregated with htt q did not co-aggregate with these hetero-aggregates. thus, these q-rich domains may suppress htt q toxicity by two complementary mechanisms: trapping potentially toxic oligomers in larger aggregates and by limiting the interactome of the larger htt q aggregates. structuring disorder: the case of the intrinsically disordered unique domain of c-src mariano maffei about two thirds of eukaryotic proteins contain large intrinsically disordered regions. they represent a change of paradigm from "structure-function" to "information-function" (uversky, ; babu et al., ). structured proteins are information rich, but the current challenge is to discover how information is stored in disordered protein. regulation of c-src activity, the first discovered oncoprotein, by its intrinsically disordered n-terminal region has been recently demonstrated (perez et al., ). functional studies have revealed that mutations in the ulbr cause strong phenotypes when introduced in fulllength c-src and expressed in xenopus laevis oocytes (perez et al., ) or in human sw colorectal cancer cells (unpublished). however, the connection with the classical regulatory mechanisms is still missing. c-src domain structure consists of four "src-homology" domains: sh , sh , sh and sh , arranged in this order from the n-terminus to the c-terminus, with the intrinsically disordered "unique" domain separating the sh and sh domains. classically, the sh and sh domains are involved in regulation and the sh domain is the membrane anchoring site. we will present our recent results showing that the unique domain is part of a long loop closed by the interaction of the sh and sh domains (maffei et al., ). the conformational freedom of this disordered region is further restricted through direct contacts between the rt-loop of the sh domain and, primarily, residues located within the recently discovered unique lipid binding region (ulbr). the interaction between the unique and sh domains is allosterically modulated by a poly-proline ligand binding to the canonical binding site of the sh domain (maffei et al., ) . these results demonstrate a direct connection between classical c-src regulation involving the sh domain and the new regulation mechanisms involving the intrinsically disordered regions and provide new evidence of the functional importance and the underlying mechanism behind regulation of signalling pathways by intrinsically disordered domains. in mammalian cells, the golgi reassembly and stacking proteins (grasp and grasp ) are involved in the stacking of golgi apparatus cisternae and in the formation of the golgi ribbon. since grasps have been identified in many organisms, other roles for grasps have already been pointed out, such as chaperoning and transport of other proteins, involvement in cell apoptosis, cell migration, unconventional secretion, and in mitosis. in saccharomyces cerevisiae, it is observed that only % of the golgi cisternae are in stacks and do not form ribbon structures. this build yeast contains a single grasp, called grh , that is analogue to grasp . the structural differences of the golgi apparatus and the functional repertoire of grasps suggest a structural dynamic of these proteins. here, we used a combination of biophysical/biochemical methods to investigate the behavior of grh . bioinformatics and circular dichroism (cd) analyses of grh indicated a high percentage of either flexible regions or extended loops. the partial unfolded grh structure in solution folded into more ordered structures under temperature increasing, dehydration onto a surface and nonaqueous solvents as reported also by cd. hydration of the dehydrated folded protein is a reversible process that is accompanied by unfolding. furthermore, grh showed slow migration in sds-page, high susceptibility to proteases and low cooperativity of the chemical-induced unfolding process. fluorescence of trp residues along with cd data showed grh preserves a considerable amount of residual secondary structure, and the unfolding transition monitored by trp presented higher cooperativity. another cooperative transition was also reported by the extrinsic hydrophobic fluorescence probe ans upon chemical denaturation. these set of experiments indicate that grh behaves as a protein containing intrinsically disordered regions (idrs), characterized by unstructured regions of high polypeptide mobility experiencing many conformations. these findings suggest that an idp-like behavior may be the solution found by nature to account for grh functional need for interactions with several different partners in the cell. conformational changes governing dengue virus capsid protein function and its inhibition by pep andr e f. abstract dengue virus (denv) infection affects millions of people and is becoming a major global disease for which there is no specific treatment available. the interaction of denv capsid (c) protein with host lipid droplets (lds) is essential for viral replication. pep - , a peptide designed based on a denv c intrinsically disordered conserved region, inhibits this crucial interaction. combining bioinformatics and biophysics we determined pep - structure and ability to bind different phospholipids, in the context of denv c function. pep - becomes a-helical upon binding to anionic phospholipids. structure prediction of denv c n-terminal intrinsically disordered region reveals orientations that alternatively shield or expose denv c hydrophobic pocket, supporting a novel autoinhibitory role for this region. these findings pave the way for similar studies to understand disordered proteins and improved peptidomimetics drug development strategies against flaviviruses. topics intrinsically disordered proteins protein-lipid interactions pf- developing mechanistic insight into modulators of tau aggregation eri nakatani-webster , hannah baughman , shaylin higgins , abhinav nath the pathological self-association of microtubule-associated protein tau is implicated in a range of neurodegenerative disorders collectively called tauopathies, perhaps the most prominent of which are alzheimer's disease (ad) and chronic traumatic encephalopathy (cte). tau aggregation in vitro shares many features in common with fibril formation by other amyloid-forming proteins: a nucleationdependent polymerization reaction progressing via oligomeric intermediates into b-sheet-rich fibrillar aggregates, characterized by a distinctive sigmoidal kinetic. over the years, many investigators have advanced our understanding of how these time-courses might best be characterized and interpreted. in particular, elegant analytical and numerical approaches have been developed that supersede the empirical sigmoidal equations typically used to fit fibril formation traces. these modern approaches have enabled more rigorous insight into the mechanism of amyloid formation, and into how small molecules, protein chaperones, and other binding partners can modulate the process. an understanding of a modulator's effects on amyloid formation mechanism is necessary in order for us to predict and engineer its effects on amyloid pathology in a biological context. a given modulator may affect rates of primary or secondary nucleation, elongation, or fibril fragmentation to different extents. each of these perturbations, individually or in combination, can alter the kinetics of aggregation, the final state of the amyloid fibrils, and the sampled ensemble of oligomeric intermediates. unfortunately, fitting of mechanistic models to amyloid formation kinetics is an example of an "ill-posed problem", in that dramatically different combinations of elementary parameters can nevertheless generate very similar sigmoidal kinetic traces. this has typically necessitated global analysis of amyloid kinetic traces collected over a broad range of protein concentrations -a substantial expenditure of time, effort and material that must then be repeated in the presence of a modulator in order to gain insight into its effects. we propose an alternative approach: to fit amyloid formation traces to a large distribution of parameter sets, and determine how various aggregation modulators affect the distribution of parameters. this socalled "parameter distribution analysis" enables the inference of mechanistic effects from measurements at a single protein concentration. parameter distribution analysis based on numerical modeling has been made tractable by advances in computer hardware and software, and can be easily extended to include additional mechanisms or phases relevant to a protein or modulator of interest. here, we illustrate how parameter distribution analysis, complemented by fluorescence correlation spectroscopy (fcs), electron microscopy (em) and other biochemical techniques, can shed light on fundamental aspects of tau amyloidogenesis. we examine the disparate effects that natural products, pharmacotherapies and protein chaperones can have on the mechanism of aggregation, and also discuss the effects of heparin (widely used as an inducer of tau aggregation). these insights demonstrate the value of parameter distribution analysis as applied to amyloid formation and other ill-posed biochemical problems. new insights into amyloidogenesis of tau protein induced by enantiomers of polyglutamic acid amyloidogenesis of tau protein leads to the formation of amyloid fibrils (ordered fibrillar protein aggregates) which are accumulated in neurons of central nervous system during the course of neurodegenerative diseases called tauopathies. studying tau (a typical intrinsically disordered protein) amyloidogenesis has been challenging for many reasons. positive charge on the tau molecule must be compensated (e.g. in the presence of polyanions) in order to initiate the process. heparin (glycosaminoglycan) has been the most intensively studied charge-compensating agent in this context. on the other hand induction of tau aggregation by polyglutamic acid is poorly characterized. mechanisms responsible for the propagation of tau conformations has become an interesting research objective. prion-like features of tau amyloid can be studied in vitro also in the seed-induced regime of aggregation. tau amyloid seeds can act as nuclei for amyloidogenesis. such seeds can be obtained by fragmentation of amyloid fibrils by means of sonication. given that amyloidogenesis can proceed through various assembly pathways resulting in distinct amyloid 'strains' (self-propagating structural variants of amyloid) we have used poly-l-glutamic acid (plga) and poly-d-glutamic acid (pdga) to direct tau onto different amyloidogenic pathways. we have hypothesized that the chirality of the inducers could lead to fibril polymorphism. in our studies, we have used a recombinant human n r tau isoform. we have been using transmission electron microscopy (tem), sedimentation and kinetic measurment. firstly, we have characterized unseeded plga-/pdga-induced tau aggregation to find out that corresponding kinetics were significantly different. secondly, we have used sonicated fibrils to characterize the kinetics of seeded processes. both plga-/pdga-induced amyloid seeds were able to efficiently seed tau aggregation in the presence of plga, whereas in the presence of pdga the aggregation was much less effective. surprisingly, we found that pdgainduced amyloid seeds were able to catalyze fibrillogenesis of tau more clearly in the presence of soluble plga than in the presence of pdga -the primary inducer. we could not induce aggregation of tau in the absence of polyglutamic acids which indicates that positive charge on tau molecules must be unconditionally compensated in order to promote amyloidogenesis. thirdly, using tem we have characterized different morphologies of tau amyloid fibrils generated in unseeded and seeded processes. finally, to further characterize properties of the fibrils we have performed sedimentation experiments. fibrils induced by plga, pdga and heparin revealed different sedimentation properties. heparin-induced fibrils underwent sedimentation more readily than pdga-induced fibrils, whereas plga-induced fibrils remained in the supernatant. these results indicate distinct physicochemical properties of these fibrils. we believe that our findings will contribute to the current understanding of the molecular dynamics of tau amyloidogenesis. self-organizing structures of alpha-synulceins and its aggregates by a coarse-grained monte carlo simulation ras pandey , peter mirau , barry farmer alpha-synuclein (asn) consisting of residues, an intrinsically disordered protein, is linked to such neurodegenerative diseases as parkinson's disease (pd) and alzheimer disease via toxic clumping into abstract amyloid fibrils. we investigate the structure and dynamics of an asn chain as a function of temperature by a coarse-grained approach where a residue is represented by a node. in our coarse-grained approach, a residue is represented by a node. the basic idea is borrowed from the 'united atom' approach in polymer chain modeling that has been used extensively where the benefits and pitfalls of the method is explored for decades. such coarse-grained method has also been used protein chain modeling in recent years (e.g. aip advances , ( )). although the atomic scale structural resolution is sacrificed its specificity is captured via a set of unique knowledge-based residue-residue interactions matrix (e.g. classic miyazawa-jernigan matrix, macromolecules , ( )). a number of local and global physical quantities are analyzed such as contact map, neighborhood and mobility profiles, mean square displacement of protein, its radius of gyration and the structure factor. based on the mobility profile, we are able to identify three distinct segment of asn along its contour, i.e. sluggish nterminal ( - ) and c-terminal ( - , least mobile) separated by the central region ( - ), the nonamyloid component (nac) with higher mobility. contact profile shows that the probability of intrachain residue aggregation (clumping) is higher in the n-terminal region than the c-terminal with least aggregation in the nac region. we find that the radius of gyration (rg) decays monotonically with the temperature, consistent with the finding of allison et al. (jacs, , ( ) ). from the detail analysis of the structure factor we are able to predict the variation of the spatial mass distribution with the temperature as the residues in asn chain organize and disperse by evaluating its effective dimension d. we find the protein conforms to a globular structure (d ) at the low temperatures and to a random coil (d ) at high temperatures which is consistent with the estimates of uversky et al. (j. biol. chem. , ( )). in addition, we provide the estimates of d ( d ) for the intermediate structures as the protein chain makes a transition from globular to random coil. questions under-investigation includes what are the effects of mutations (e.g. b-and g-synuclein), how does the structure of an isolated asn chain change in presence of many interacting protein chains, and how do they organize over the multiple length scales? attempts will be made to address some of these issues as the data become available. tear down the wall: dismantling the biofilm scaffold of e.coli cesyen cedeno , nani van gerven , wim jonckheere , imke van den broek , han remaut , peter tompa csga is the major subunit of the so-called curli fiber system. this is an amyloid structure formed in the outer membrane on e.coli and acts as a scaffold for the biochemical machinery/matrix in the extracellular milieu (biofilms). extracellular matrices of this nature are robust platforms helping bacteria colonization; in this context csga becomes a key target in order to break the architecture within bacterial biofilms. chaperones are molecular machines able to stabilize misfolding prone proteins or even retrieve proteins trapped in non-physiological states. here we show how erd acts as a molecular chaperone inhibiting the formation of csga amyloid fibers in vitro. this work illustrates an alternative approach towards biofilm treatment at a molecular level. coupled folding and binding of transcription factors sarah shammas , alexandra travis , jane clarke intrinsic protein disorder is ubiquitous in transcription, particularly within transcription factors, which frequently fold into structures upon binding to partner molecules (dna or protein). the coupled folding and binding reactions that take place between individual transcription factors and the key hub co-activator proteins are crucial in determining the expression profile of the cell, and hence its phenotype. these interactions have been well studied by structural and equilibrium methods. here we present mechanistic insights into the process, gained through complementary kinetics experiments, for the binding of five separate transcription factors to a single prototypical co-activator (cbp kix). the transcription factors investigated belong to cellular (cmyb, mll, creb, e a) and viral (htlv- blz) classes. these reactions are remarkably fast; after removing the effect of long-range electrostatic rate enhancement the association rate constant is still approximately x m- s- , which is just above the typically quoted upper limit for diffusion-limited reactions between pairs of proteins ( - m- s- ), and is also the highest such value we have found reported. this, combined with the apparent insensitivity of the association rate to residual structure within the unbound state, indicates that binding preceeds folding (induced fit mechanism). interactions between kix and its transcription factors are additionally modulated by allostery between its two binding sites. we investigate the basis for this, finding it to be mediated by changes in protein flexibility. alternative hit finding strategies for intrinsically disordered proteins, exemplified by forkheadbox transcription factors harm jan (arjan) snijder , maria saline , tomas jacso , frank janssen , mattias rohman , tyrrell norris astrazeneca r&d, discovery sciences, se- ,pepparedsleden forkhead box o (foxo) proteins are emerging as key transcription factors in insulin and glucose metabolism, regulation of immune responses, and to balance cell proliferation, apoptosis and senescence. foxo proteins are predicted to be intrinsically disordered proteins (idps); idps are largely unstructured and often function as hubs mediating multiple interactions. idps are considered to be largely evasive from classical small molecule interference and lead-generation approaches, as they lack defined binding pockets. the available methods for addressing these targets have been lagging behind and needs to be developed to assess tractability of this target class. here we have evaluated the tractability of fragment screening on various domains of a forkhead box o member. we could confirm the intrinsically disordered character of foxo and used nmr screening to identify fragments that interact with foxo. one of these fragments was subsequently confirmed as a direct foxo binder in d hsqc-nmr spectroscopy and this fragment showed an effect in a foxo reporter gene assay. these results demonstrate that fragment screening may be a valuable approach for intrinsically disordered proteins although challenges remain to expand these fragments into more potent hits in the absence of detailed structural data. the characterization of amyloid-beta peptide (abeta) oligomer samples is critical to advance in the field of alzheime rs disease (ad). here we report a critical evaluation of two methods used for this purpose, namely sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page), extensively used in the field, and electrospray ionization ion mobility coupled to mass spectrometry (esi-im-ms), an emerging technique with great potential for oligomer characterization. to evaluate their performance, we first obtained pure cross-linked abeta and abeta oligomers of specific order. analysis of these samples by sds-page revealed that sds affects the oligomerization state of abeta oligomers, thus providing flawed information on their order and distribution. in contrast, esi-im-ms provided accurate information, while also reported on the chemical modifications and on the structure of the oligomers. our findings have important implications as they challenge scientific paradigms in the ad field built upon the sds-page characterization of abeta oligomer samples. coarse-grained simulation of protein association: application to rate prediction and implication for association mechanisms yinghao wu , the kinetics of protein binding is of paramount importance for understanding cellular functions. for instance, the binding kinetics between membrane receptors and their ligands control the speed of signal transduction after cells are exposed to stimulation. the experimentally measured association rates of protein binding span ten orders of magnitude, a range that was divided into two regimes. it was proposed that a fast association regime is limited by protein diffusion, while the other side of the spectrum is controlled by conformational changes. consequently, all previous simulation methods neglected conformational changes when calculating the association rate of a diffusion-limited regime. however, the most updated theory of protein binding suggests that a protein remains in a pre-existing equilibrium of unbound conformations. binding shifts the equilibrium toward its bound state. this highlights the importance of conformational factors for regulating protein binding. enlightened by this conformational selection model, we hypothesize that the conformational flexibility of protein structures regulates association more widely than previously anticipated. we develop a new coarse-grained model to simulate the process of protein association via the kinetic monte carlo (kmc) algorithm. each residue in this model is represented by its ca atom and a side-chain functional site. a simple physically based potential is used to guide the relative diffusion of two interacting proteins. given the size of the simulation box and the length of the simulation, the association rate constant can be derived by counting the frequency of dimerization among a large number of simulation trajectories. we further designed a prediction strategy that accounts for both the conformational and energetic factors of binding. our method is able to predict rates of protein association that are highly correlated with experimentally measured values. due to the coarse-grained feature, our model was further applied to several special cases of protein association. in one example, we studied the binding kinetics of proteins with flexible linkers. the interaction between thrombin and its functional inhibitor, rhodniin, was used as a testing system. we captured the conformational changes of flexible linkers from the all-atom molecular dynamic simulations. we found that the association with full-length flexible rhodniin was faster than its two individual domains and that their dissociation was more difficult, supporting a "flycasting" mechanism in which partial structures of an intrinsic disordered protein (idp) dock to the target first, while the remaining segments undergo conformational searches and sequentially coalesce around the target. in another example, we studied the binding kinetics of membrane receptors from cellular interfaces. the interaction between membrane proteins cd and cd , cell adhesion molecules known to mediate the activation of t cells and natural killer cells, was used as a testing system. the diffusive properties of these proteins on lipid bilayer were captured from all-atom molecular dynamic simulations. we showed that both d and d association rates could be simulated quantitatively with our method. the calculated values were close to the experimental measurements. we also provided detailed analysis of how molecular diffusions and membrane fluctuations affected d association. pf- (un)structure-function relationships on the ureg enzyme in the nickel-dependent urease system barbara zambelli , francesco musiani , stefano ciurli urease is an essential enzyme for many pathogens and soil microorganisms. its activity relies on the presence of nickel in the active site ( ) . the incorporation of this metal ion into the enzyme requires the formation of a supra-molecular chaperone involving four accessory proteins, named ured, uref, ureg and uree. uree is a metallo-chaperone involved in nickel binding and delivery into the enzyme active site. ureg is a gtpase essential for providing energy to the process of nickel site assembly. uref and ured form a complex that regulates the gtpase activity of ureg. the present work focuses on ureg, which exists in solution as an ensemble of inter-converting conformations ( ) . this observation made this protein the firstly discovered natural enzyme with an intrinsically disordered behavior, possibly allowing it to interact with different protein partners, such as uree ( , ) and uref ( ) and cofactors, such as metal ions ( ), in the urease activation network. ureg folding was studied perturbing protein conformation with temperature and denaturants, and investigating its folding response using circular dichroism, nmr and fluorescence ( ). a combination of light scattering, calorimetry, mass spectrometry, and nmr spectroscopy shed light on the effect of metal ion binding onto the conformational equilibrium of ureg ensemble ( ) . the results suggest that metal binding and solution conditions modulate affect the protein-protein interactions and enzymatic activity of ureg. nuclear inclusion protein a-protease (nia-pro) is a protease involved in processing of pepper vein banding virus (pvbv) encoded polyprotein to generate various intermediates and mature proteins at different stages of the viral life-cycle. nia-pro has two domains-n-terminal viral protein genome linked (vpg) and the c-terminal protease domain (pro).vpg belongs to the group of proteins that are intrinsically disordered, but attain stable structures upon interaction with other globular proteins. such proteinprotein interactions have a regulatory role on the function of the interacting partners. previously, the influence of vpg domain on the activity of pro was studied and it was shown that there was a substantial increase in the protease activity upon interaction with vpg (both in cis and in trans). in the present investigation, several deletion mutants of vpg and nia were constructed with a view to delineate the domain of vpg involved in interaction with pro. it was observed that deletion of residues from nterminus of vpg resulted in a decrease in the activity of pro in cis and in trans probably because of the abrogation of interaction between the two domains. interaction studies using spr (surface plasmon resonance) and elisa confirmed that the n-terminal residues of vpg are important for interaction with pro. the n-terminal residues of vpg are a part of the disordered region of vpg and their deletion resulted in the change in the secondary structure of the vpg and its oligomeric state. the ser and trp residues of pro domain were shown to be important both for the interaction of the two domains and for the activity of protease by mutational analysis earlier. these residues were identified to be a part of wc loop (w -c ) which relay the conformational changes to the active site catalytic triad (his , asp and cys ) leading to activation. however, mutations of these residues did not completely abolish the protease activity as well as the interaction with vpg. therefore, in the present study h and h which are observed to interact with trp and c (via non-covalent interactions) were mutated to alanine and the h a and h a mutants showed a drastic reduction in the activity of protease. molecular dynamics simulations of the wild type pro and the mutants revealed that trp -his -his -cys interaction pathway of the wild type pro was disrupted in the mutants and additional residues were involved in the interaction pathway, such alterations in the network of interactions could be responsible for the loss of activity. however, a change in the oligomeric status of these mutants was also observed as compared to the wild-type pro, suggesting that these residues are important for both the structural and functional integrity of pro and its interaction with vpg. thus, these results provide a molecular insight into the vpg-pro interactions and the modulation of their structure and function upon mutation of residues that are part of the interaction interface. transthyretin (ttr) is one of many proteins that are capable of forming amyloid fibrils in vivo. this protein is associated with two distinct amyloidosis: familial cardiac amyloidosis (fca) that causes a restrictive cardiomyopathy and familial amyloid polyneuropathy (fap) that affect peripheral nerves, they are hereditary and caused by mutations in the ttr gene. the non mutated protein can also aggregate in cardiac tissue in advanced age patients. the diagnosis was established at university hospital since due to a collaborative between our group and the center of amyloidosis antônio rodrigues de mello (ceparm). the only mutation found in brazil was v m in patients diagnosed in france. our group discovered new mutation not described in brazil and a novel mutation not described yet a d. the diagnosed patients are registered in transthyretin amyloidosis outcomes survey (thaos). the novel mutation a d causes a severe restrictive cardiomyopathy that is certainly related to a higher profile of aggregation observed for this mutant if compared to others amyloidogenic mutants of ttr. structural predictions using a bioinformatics tool called foldx showed that the insertion of the mutation cause a electrostatic clash that facilitates the dissociation and aggregation of protein. this mutant was purified heterologously and biophysical studies revealed that this protein is a dimer and not a tetramer as commonly the ttr structure. the crystallographic structure indicates that this mutant is structurally identical to wild type. biophysical studies revealed that this protein is a dimer and not a tetramer as commonly the ttr structure. the thermodynamic stability of a d is lower than the wild type ttr. the aggregation profile showed us that this protein can aggregate in a higher manner and with a fast kinetic to that observed for others amyloidogenic mutants of ttr, forming fibers in two hours of aggregation. heterotetramers of a d and wt are able to aggregate in the same fiber structure. the analysis of interface interaction of this mutant using the pdbsum showed modifications in the profile of hydrogen bonds and non bonded contacts. in addition the oligomers of a d are toxic for primary culture of cardiomyocytes from murine heart. the amyloidogenic profile displayed by this new mutant can be directly correlated with the aggressiveness observed in the disease developed by the identified patient. furthermore the recent consolidation of ttr diagnosis in our university hospital led to the identification of the rare a d variant in a brazilian patient, suggesting that other new, uncharacterized mutants could be identified in the coming years. multiple cellular proteins interact with ledgf/p through a conserved unstructured consensus motif [ ] . the ledgf/p -mll -menin complex was structurally characterized, but only partially [ ] . using nmr spectroscopy, we identified and mapped a novel mll -ledgf/ p interface. colony forming assays in mll -af leukemic cells expressing mll interactiondefective ledgf/p mutants revealed that this additional interface is essential for leukemic transformation. interestingly, the newly defined interface overlaps with the binding site of known ledgf/p interactor, the hiv integrase [ ] . while the pathophysiological interactions of ledgf/p are intensively studied, its physiological role remains unclear. since ledgf/p contributes to hiv integration and leukemic transformation and has become a new therapeutic target for drug development, it is crucial to study its physiological interactions. in addition to hiv in and mll -menin, the ledgf/p integrase binding domain (ibd) also interacts with several other proteins [ , ] . our recent data (manuscript accepted in nat. commun.) revealed structural details of ledgf/p interactions with physiological binding partners. the interaction with the ledgf/p ibd is maintained by an intrinsically disordered ibd-binding motif (ibm) common to all known cellular partners. based on the knowledge of this motif, we identified and validated iws as a novel ledgf/p interaction partner. naturally occurring single mutants, i t, f i, w r and d h of lysozyme in human, have been known to form abnormal protein aggregates (amyloid fibrils) and to accumulate in several organs, including liver, spleen and kidney, resulting in familial systemic amyloidosis. these human pathogenic lysozyme variants are considered to raise subtle conformational changes compared to the wild type. here we examined the effects of the aberrant mutant lysozymes i t, f i,w r and d h, each of which possesses a point mutation in its molecule, on a cultured human cell line, hek , in which the genes were individually integrated and overexpressed. western blot analyses showed lesser amounts of these variant proteins in the medium compared to the wild type, but they were abundant in the cell pellets, indicating that the modified lysozyme proteins were scarcely secreted into the medium but were retained in the cells. immunocytochemistry revealed that these proteins resided in restricted regions which were stained by an endoplasmic reticulum (er) marker. moreover, the overexpression of the mutant lysozymes were accompanied by marked increases in xbp s and grp /bip, which are downstream agents of the ire _ signaling pathway responding to the unfolded protein response (upr) upon er stress.rnai for the mutant lysozymes' expression greatly suppressed the increases of these agents. next, we addressed the interaction between amyloidogenic lysozyme and grp /bip as the former proteins were obtained by immunoprecipitation with the latter protein as well as colocalization of both proteins in the er. lysozyme composes of a-domain rich in helices and b-domain rich in sheet. two helices of a and a in the n-terminal region arrange in parallel and face to face where hydrophobic amino acids at the f, l , l , l , l and l allocate with equal interval there. in the back of dock, there is a core region of amyloid fibril formation, of which the side chain of i is exposed on the protruding. probably, these hydrophobic amino acids might be crucial for lysozyme folding. although mutated lysozymes undergo folding by grp /bip in such environment, the dissociation of the grp from lysozyme by failure of folding is likely inhibited and both proteins remain bound to, resulting in staying to the er. a part of aberrant lysozymes seem to remain bound to grp /bip during folding and insolubilize with aggregation, thus accumulate in the er accompanied with er stress. lysozyme amyloidosis might be caused by long-term accumulation in the endoplasmic reticulum of the abnormal protein. structural characterization of toxic oligomers that are kinetically trapped during alpha-synuclein fibril formation the accumulation of abnormally aggregated proteins within the body is a common feature of several medical disorders, such as alzheimer's disease, parkinson's disease and diabetes mellitus type . while the specific protein found to be the major component of such deposits varies from one disease to another, the formation of the pathological aggregates seems to occur via a common process of misfolding and self-assembly of a normally soluble polypeptide chain into a series of oligomeric intermediates and, ultimately, into insoluble amyloid fibrils that accumulate within specific organs and tissues. increasing evidence indicates that certain oligomeric protein species generated during the self-assembly of specific proteins into ordered fibrillar aggregates can be highly cytotoxic and are likely to be key players in the initiation and spreading of neurodegenerative diseases. however, little detailed structural information is currently available for these oligomeric species due to their often transient nature and, more importantly, because of their variability in terms of size and structure. we report here the isolation and detailed characterization of an ensemble of stable toxic oligomers of alpha-synuclein, the protein whose deposition is the hallmark of parkinson's disease. by defining and minimizing the degree of heterogeneity of these isolated alpha-synuclein oligomers which have accumulated during the process of amyloid formation, we have identified distinct subgroups of oligomers and determined their structural properties and three-dimensional molecular architectures. this characterization has been achieved by the application of a set of complementary biophysical techniques, including a variety of spectroscopic techniques along with analytical ultracentrifugation, atomic force microscopy, and electron microscopy. although these oligomers exist in a range of sizes, with different extents and nature of beta-sheet content and exposed hydrophobicity, all the oligomeric subgroups possess hollow cylindrical architectures with marked similarities to amyloid fibrils. this suggests that these types of oligomers are kinetically trapped during protein self-assembly and that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their beta-sheet structures. our findings reveal the inherent multiplicity of pathways of protein misfolding and the key role the beta-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the rates of structural conversions, and thus the kinetic stabilities and pathological nature of different amyloid oligomers. the results of this study provide the basis for a more complete understanding of the nature of the self-assembly of polypeptides into beta-sheet rich amyloid aggregates, and potentially contributes to efforts to identify specific targets for drug discovery. fish otoliths and mammalian otoconia, biominerals composed of calcium carbonate and organic matrix, are involved in the functioning of the inner ear, the sensory organ that plays an important role in hearing and balance [ ] . however, their developmental origins, growth, and the role of the matrix, especially the protein component, are still poorly understood. it has been shown that proteins involved in the formation of biominerals are usually very acidic. they often belong to the group of intrinsically disordered proteins (idps), a class of proteins devoid of a rigid tertiary structure [ , ] . the shape and polymorph selection of calcium carbonate otolith in danio rerio is controlled by the starmaker (stm) protein [ ] . recently, a gene was identified encoding the starmaker-like (stm-l) protein from oryzias latipes, a putative homologue of stm. it has been suggested that stm-l has a similar function as stm, although there is no sequence similarity between stm and stm-l [ ] . several methods, such as size exclusion chromatography, cd spectroscopy and analytical ultracentrifugation demonstrated that stm-l is an coil-like idp, with the tendency to form locally ordered structures [ ] . because stm-l was suggested to play a crucial role in calcium carbonate mineralization, it is possible that calcium ions may influence its conformation, as was previously shown for stm [ ] . however, other ions may also be involved in this process. the aim of this study was to investigate the effect of mono and divalent metal ions on the conformation of stm-l. we used single molecule f€ orster resonance energy transfer (smfret) and fluorescence correlation spectroscopy (fcs), which have shown that calcium ions compacts the proteins most efficiently, followed by magnesium and the monovalent ions. the difference in the effect of monovalent and divalent ions on the protein dimensions is likely to result from the different properties of the ions, like charge density and radius. cd experiments have shown that a high excess of calcium ions caused the formation of ordered secondary structure in stm-l, which may be crucial for the formation of calcium carbonate crystals, when the ratio of building ions to protein is high. it has been demonstrated that dmp is proteolytically processed into fragments, including k n-terminal region and k c-terminal region. as many proteins characterized to be engaged in biomineralization, dmp and its fragments belong to the group of intrinsically disordered proteins (idps). it has been suggested that dmp and its fragments can take a part in otoconia mineralization, as the protein is present in mouse otoconia, but the role of dmp and its fragments in the mineralization of calcium carbonate has not been examined until now. to determine the influence of the dmp fragments for otoconia development, k dmp protein was expressed in bacterial expression system, purified and used in in vitro biomineralization test of calcium carbonate. in particular, immobilized metal anion affinity chromatography (imac) was applied as a first step of purification procedure. because of high content of acidic amino acids, ion exchange chromatography with a mono q column was used as a next step. the development of insects is regulated by the combined action of ecdysteroids and juvenile hormones (jh). pulses of -hydroxyecdysone ( e) initiate each step of metamorphosis, while jh modulates its action and prevents precocious differentiation. the biological and molecular mechanism of e action is well described. in contrary, the way of the jh activity is still poorly understood. in wilson and fabian [ ] reported that drosophila melanogaster mutants lacking met are resistant to toxic doses of jh and its analogue methoprene. it has been proved, that met binds jh at physiological conditions. therefore met is believed to be a putative jh receptor. met may also be involved in a cross-talk between two hormonal signalling pathways, involving e and jh. the detailed structure of met is still unknown. therefore our main aim is to characterize structural properties of met. in silico analysis performed on a full-length met suggested, that n-terminal part of met contains three conserved domains characteristic for bhlh-pas transcription factors, whereas c-terminal part is most probably unstructured. )). capitalizing on self-and cross-amyloid interactions, we designed highly effective, peptide-based inhibitors of amyloid self-assembly of abeta and iapp. due to their favourable properties the designed peptides are promising leads for targeting protein aggregation in ad, t d or both diseases while the inhibitor design strategy should be applicable to other amyloidogenic polypeptides and proteins as well. apoptosis, the process of programmed cell death, must be carefully regulated in multi-cellular organisms to ensure proper tissue homeostasis, embryonic development and immune system activity. the bcl- family of proteins regulates the activation of apoptosis through the mitochondria pathway. dynamic interactions between pro-and anti-apoptotic members of this family keep each other in check until the proper time to commit to apoptosis. the point of no return for this commitment is the permeabilization of the outer-mitochondrial membrane (omm). translocation of the pro apoptotic member, bax, from the cytosol to the mitochondria is the molecular signature of this event. molecular interactions and conformational changes associated with this event have been difficult to obtain due to challenges associated with taking subtle measurements in the complex environment of live cells. to circumvent these challenges, we developed a novel method to reliably detect f€ orster resonance energy transfer (fret) between pairs of fluorophores to identify intra-molecular conformational changes and inter-molecular contacts in bax as this translocation occurs in live cells. in the cytosol, our fret measurements indicated that the c-terminal helix is exposed instead of tucked away in the core of the protein. this coincided with measurements using fluorescence correlation spectroscopy (fcs) that showed that cytosolic bax diffuses much slower than expected, suggesting possible complex formation or transient membrane interaction. we propose that this exposed helix allows for this contact to occur. cross-linking the c-terminal helix (a ) to helix a reduced the instances of these interactions while at the same time yielded fret measurements that are consistent with the a helix tucked into the core of the protein. after translocation, our fret measurements showed that bax molecules form homo-oligomers in the mitochondria through two distinct interfaces involving the bh domain (helix a ) and the c-terminal helix. these findings provide insight into the molecular architecture that may involve possible contacts with other bcl- proteins to permeabilize the omm, which would also be necessary for the regulation of apoptosis. abstract spatial resolution is especially advantageous for bacterial cells because of their small sizes. in the past few years the spatial organization and dynamics of a variety of bacterial cellular structures and protein macromachineries have been revealed with unprecedented details. as the field matures, it is now time to focus on the functional aspect of the observed spatial organizations and dynamics. are they essential in carrying out a specific cellular function? do they play a regulatory role in controlling the on and off of a certain cellular process? in this work i will present a few examples from our laboratory that examine the spatial and functional organization of macromolecules involved in bacterial cell division. transcription factors (tf) exert their function by interacting with other proteins and binding to dna. the nucleus is a compartmentalized space, and the spatial organization of tfs and their partners represents other step of gene expression regulation. we used the glucocorticoid receptor (gr) as a model of tf's mechanism of action. gr is a ligand-activated tf with a relevant role in physiology and a great variety of effects. it can be recruited to specific response elements on dna or interact with other tfs. also, the activity of gr is modulated by different co-regulators, e.g. tif /grip . gr and tif do not distribute homogeneously within the nucleus but accumulate in distinctive clusters. the functional role of this particular intranuclear organization remains unknown. we used advanced fluorescence microscopy techniques to study the dynamics of gr and tif in the nucleus of living cells with high spatial and time resolution. gr and tif fused to fluorescent tags were transiently expressed in newborn hamster kidney (bhk) cells and visualized by a confocal microscope. fluorescence correlation spectroscopy (fcs) experiments were carried on to measure the intranuclear mobility of both proteins. the method is based on the analysis of fluorescence intensity fluctuations due to the movement of fluorescent molecules in and out the confocal volume. the data could be fitted with a model that considers a free diffusion of tif and gr in the nucleus and their binding to fixed targets. we also studied the dynamics of different gr mutants in the presence of different ligands and our results suggest that the binding depends on dna. both gr and tif autocorrelation curves reveal an increase in the bound population upon gr activation by its agonist dexamethasone (dex). a cross-correlation analysis showed that, as expected, dex-stimulus increases the population of gr-tif complexes. without hormone, gr shows a homogeneous distribution and tif forms large clusters in the nucleus. upon dex-binding, gr accumulates in the nucleus, is rapidly recruited to tif foci and there is an important re-distribution of both proteins, that co-localize in the same pattern of small intranuclear clusters. the dynamics of gr and tif molecules at these clusters were studied by performing orbital-scanning measurements, tracking the clusters position in silico and analyzing the intensity fluctuations of the clusters along time. a positive cross-correlation between both channels indicates that dex-bound gr and tif interact at these foci and dissociate from them forming tif -gr hetero-complexes. in conclusion, advanced fluorescence microscopy methods allowed obtaining a dynamical map of gr distribution and function in the nucleus of mammalian living cells. assembly of membrane pores as a mechanism for amyloid cytotoxicity by the bacterial prionoid repa-wh cristina fern andez , rafael n uñez-ramirez , mercedes jimenez , germ an rivas , rafael giraldo amyloid fibril formation is associated with human neurodegenerative diseases. prefibrillar oligomers formed during the fibril assembly process, rather than mature fibrils are known to be central to disease abstract and may be responsible for cell damage. a commonly proposed mechanism for the toxicity of small oligomers is their interaction with the lipid bilayer of cell membranes, leading to loss of membrane integrity [ ] . recent studies from our laboratory have shown that repa-wh , a winged-helix domain from a bacterial plasmid replication protein, can assemble into amyloid fibrils in vitro. when expressed in escherichia coli repa-wh functions as a cytotoxic protein that shares features with the mammalian amyloid proteinopathies. these features have proved repa-wh to be a suitable synthetic model system to study protein amyloidosis [ , , ] . in this work, using the repa-wh bacterial model system, we have studied the interaction between the protein and model membranes (large and giant unilamellar lipid vesicles, luvs, and guvs respectively). repa-wh shows association and aggregation to membranes composed of anionic phospholipids. protein association in guvs did not result in lysis of the vesicles, suggesting the assembly of discrete protein pores as the mechanism for repa-wh membrane damage. to investigate the formation of pores we analyzed by electron microscopy the aggregation of repa-wh in the presence of a pre-formed e. coli lipid monolayer. the em images show the presence of pore-like particles on the monolayer. amyloid pores formation explains the permeabilization effect of repa-wh in vesicle models and is in agreement with observations for human amyloidogenic proteins. the approaches presented here provide a deeper insight into amyloid cytotoxicity towards membranes and will make possible the assay of inhibitors and effectors of amyloidosis under controlled conditions. references: b -adrenergic receptor (b ar) is a member of g protein-coupled receptors, which represent the single largest family of cell surface receptors involved in signal transduction. b ar recognizes a variety of ligands and communicates with cytoplasmic g-proteins by transmitting signals through the cellular membrane. thus, investigation of communication pathways for b ar may give important insights for understanding its allosteric mechanisms and identifying new target sites for more specific and efficient drug molecules to be used in the treatment of pulmonary and cardiovascular disease. in this study, various conformations from ms molecular dynamics (md) simulations and available crystal structures of human b ar were investigated to reveal alternative signaling pathways between its extra and intracellular regions. specifically, shortest communication paths connecting key residues (more than Å apart) at the orthosteric ligand binding site (d , s , t , f , n ) to either l or s located near the g-protein binding site were investigated. the conformers from previous md simulations [ ] include the intracellular loop (icl ), which especially affects the transmembrane collective dynamics but is lacking in x-ray structures. the protein was described as a graph composed of nodes linked by edges. nodes were placed at the alpha-carbon atoms and the edges were calculated based on the number of atom-atom interactions within a cut-off distance . Å for each residue pair. twenty shortest pathways were revealed using k-shortest path algorithm [ ] on the coarse-grained network. our results indicated that distinct signaling paths progressed most frequently on tm but alternative paths were also present, which passed partially through tm , tm , tm or tm depending on the conformation. among the critical residues that transmitted the signal between distant sites, f and n were detected, whose functional roles were reported in previous experimental studies. pathway shifting was observed depending on the open-to-closed transition of icl during md simulations. the sulfonylurea receptor (sur ) is an atp binding cassette (abc) protein that forms the regulatory subunit in katp channels found in the pancreas and the brain. mgatp binding and hydrolysis at the two cytosolic nucleotide binding domains (nbd and nbd ) in sur control gating of the katp channel pore. , proper regulation of katp channel gating by sur is critical. over mutations that lead to diabetes, hyperinsulinism and developmental delay have been identified in different domains of sur , including the nbds. therefore, molecular-level understanding of the structure and function of the nbds is essential for designing improved treatments for sur-related diseases. here we present biophysical and biochemical studies aimed at understanding the effect of disease-causing mutations on the conformation and nucleotide binding of sur nbd . specifically, we are investigating sur nbd mutations that cause neonatal diabetes (r w and h t) or congenital hyperinsulinism (c d, g v, r g, r d and k t). our nuclear magnetic resonance (nmr) data shows that the hyperinsulinism mutation k t causes chemical shift changes throughout the spectrum of nbd , implying overall changes in protein conformation that may affect mgatp binding and inter-domain interactions with other domains in the sur protein. size-exclusion data show that the other hyperinsulinism mutations (c d, g v, r g, r d) produce mostly aggregated protein, likely as a result of misfolding of nbd . misfolding of nbd may be the underlying cause of reduced katp trafficking seen with these mutations and hence decreased katp channel gating observed in hyperinsulinism. in contrast to the k t mutations, the congenital diabetes-causing mutations (r w and h t) cause few nbd nmr spectral changes. however, the congenital diabetes mutation r w decreases the affinity of nbd for mgatp, which is unexpected for congenital diabetes mutations. our fluorescence, circular dichroism and microscale thermophoresis data corroborate the results that we have obtained by nmr spectroscopy. our data provide molecular-level details on the effects of disease causing mutations in human sur . egfr increased stability: rmsf of the ca atoms during the md simulations suggest that glycosylation is associated with dampened motions, suggesting that the glycans stabilize the structure. subdomain iii is the most stabilized while subdomain i is stabilized largely in the proximity of the ligand. both dimer interfaces including the dimerization arm from domain ii and the tip of domain iv fluctuate less upon glycosylation. hydrogen bonding; persistent interactions seen for protein-glycan: in the disaccharide-containing system, we observed three highly occupied hydrogen bonds between the glycans and domain iii and iv of egfr. hydrogen bonds of domain iii involve the residue asp in which a sidechain oxygen interacts with oxygen atoms of the n-acetylglucoseamine linked to asn . in domain iv a hydrogen bond is seen between the cys backbone amide and the oxygen atom of n-acetylglucosamine linked to asn . in the oligosaccharide-containing system hydrogen bonds observed between the glycan attached to asn and domain ii. these hydrogen bonds form between the gln sidechain oxygen atom and cys backbone oxygen atom and the mannose linked to asn . the reduction in the mobility of these amino acids suggests that hydrogen bonds impart stability to both the sugars and to the interacting egfr. insects possess a complement-like immune response utilizing thioester-containing proteins, or teps. the only arthropod tep of known structure is anopheles gambiae tep , which is a key component in the natural immunity of this mosquito to malaria parasites (genus plasmodium). unlike vertebrate complement factors, agtep does not contain an anaphylatoxin domain which acts to regulate a massive conformational change accompanying activation of the protein. the mechanism of agtep must therefore involve an alternative mechanism for allosteric regulation of thioester activation. in place of a small internal domain, a large, heterodimeric complex of two leucine-rich repeat (lrr) proteins, lrim and apl c, have been shown to specifically bind and stabilize the active conformation of agtep . i will present my group's most recent work in this area. we have shown that different alleles of tep , which are known to influence the vectoral capacity of wild mosquitoes, differ significantly in their susceptibility to thioester hydrolysis. allelic variation is centered on residues at the protein-protein interface within tep containing the thioester bond. the lrim /apl c heterodimer is shown to form an extended and flexible ensemble in solution. two closely-related genes to apl c, apl a and apl b, can also form a complex with lrim , and apl b lrr domain can form a homodimer. we propose that a flexible and heterogeneous group ensemble of lrim /apl dimers interact with the active conformation of tep , thereby producing an array of immune complexes to protect mosquitoes from a diverse set of pathogens. human flap endonuclease- (hfen ) is an essential metallo-nuclease involved in okazaki fragment maturation and long-patch base excision repair. during these processes, bifurcated nucleic acid intermediates with ssdna '-flaps are generated by polymerase strand displacement synthesis and then cleaved one nucleotide into the downstream duplex by fen to create a nicked-dna that is a suitable substrate for ligase. until recently, how hfen achieves tremendous catalytic power (rate enhancements > exp ) and exquisite selectivity for the scissile phosphate had been understood poorly ( ) . in , the grasby and tainer labs solved the structures of hfen in complex with product and substrate. this study revealed that scissile phosphate selectivity is largely due to the substrate dna undergoing a novel di-nucleotide unpairing (dnu), which places the scissile phosphate diester in contact with the requisite divalent metal ions. in addition, by comparing the structures of hfen alone ( ) and in complex with substrate and product dnas ( ), grasby and tainer proposed a model, whereby protein conformational changes occur upon binding substrate resulting in placement of key basic residues that position and/or electrophillically catalyse hydrolysis of the scissile phosphate diester. further work using a cd-based assay showed that metals are absolutely required for dnu, whereas the key basic residues in the active site are not. surprisingly, perturbations to the protein structure that are much more distant from the fen active site (i.e., helical cap) prevent dna unpairing, implying that the fen protein actively participates in the unpairing process ( , ); however, how it does remains a mystery. the maximal multiple turnover rate of hfen reaction is rate-limited by enzyme product release, whereas hfen kinetics under substrate-limiting conditions ([e]<[s]<km) suggest that the enzyme is approaching catalytic perfection (i.e., almost diffusion controlled - exp m- s- ) ( ) . previous work has shown that a paralogue of hfen (i.e., t fen ) is not ratelimited by chemistry under single turnover (st) conditions, but rather some physical limitation such as conformational change ( ) . to ascertain whether the protein and/or dna conformational changes of hfen mentioned above are rate limiting under st conditions, we have initiated kinetic and nmr studies to determine the role of conformational dynamics in the catalytic cycle of hfen . owing to the latest advance in the computational technologies of microprocessor, high-speed inter-processor connection, and parallelization algorithm, all-atom molecular dynamics (md) simulations of microsecond time scale are getting popular to study the pharmaceutical target proteins. an antibody binds to its antigen with structure changes. a small molecule moves around the target protein and finds an entrance to get into the binding site. these phenomena can be observed in microsecond simulations, but the vital issue is whether the adopted force field is enough accurate to correctly describe the phenomena. we are developing a force field (fuji) based on general amber atom types and amber van der waals parameters in order to describe arbitrary organic molecules in a unified manner including proteins and nucleic acids. we use the first principle theoretical method to refine the force field in contrast to common empirical fittings to experimental data. the dihedral torsion parameters of protein backbone were determined to agree with the torsion energy profiles calculated by high-level quantum mechanical theory for the model systems of protein backbone. conformational preferences of dipeptides in water were measured by vibrational spectroscopy. comparing with the experimental distribution of ramachandran angles of dipeptides, how accurately molecular calculations predict the conformation distribution of dipeptides were investigated for various force fields and semiempirical quantum methods. fuji force field gave the best prediction score among the various methods (tzanov et al, ) . in this work we examine dynamical structure behaviors of pharmaceutical target proteins by microsecond md simulations with fuji force field. epiregulin (epr) is a transmembrane protein with residues belonging to the epidermal growth factor family. mature epr ( residues) binds to epidermal growth factor receptor (egfr), which stimulates the proliferative signaling in cancer cells. our epr antibody has a proline at the residue in the third complementarity-determining region (cdr) of the heavy chain, which has cis peptide bond in free state and trans one in the bound state with epr according to x-ray crystallography analysis. in order to clarify the structure changes in binding we performed extensive md simulations of our antibody (fab; residues) and epr. the total simulation time was about microseconds. we also performed md simulations for a protein kinase and a small molecule. we show how the complex system reaches thermal equilibrium states in simulations from the stiff x-ray crystal structure. the calculations are compared with experiments such as x-ray crystal structures and thermodynamic quantities measured by isothermal titration calorimetry (itc) and surface plasmon resonance (spr). this research has been supported by mext spire supercomputational life science (hp , hp ) and first kodama project in japan. structure-based recombination of drug resistance enzymes: structural and functional tolerance to new dynamics in artificially-evolved enzymes our understanding of the contribution of protein dynamics to function is still emergent. in a protein engineering context, do we need to take into account the dynamics in order to maximize the fitness and function of the resulting proteins? using high resolution crystal structures, nmr relaxation dispersion and ms molecular dynamics simulations, we compare two naturally evolved homologous class a b-lactamases, tem- and pse- which share a high degree of structural and functional conservation. we observed a conservation of dynamics on a catalytically relevant timescale. this is consistent with dynamics being an evolutionarily conserved feature. however, laboratory-engineered chimeric enzymes obtained by recombination of the two homologs exhibit striking dynamic differences, despite the function and structure being conserved. the laboratory-engineered chimeras are thus functionally and structurally tolerant to modified dynamics on the timescale of the catalytic turnover. this tolerance of blactamases to dynamic changes could be linked to the high fitness of the naturally evolved proteins and implies that maintenance of native-like protein dynamics may not be essential when engineering functional proteins. conformations solution. an immense advantage of our recombinant system is the possibility to perturb it by introducing unnatural amino acids for subsequent labelling with fluorescent dyes or to omit subunits from the multisubunit rnap. comparing free, nucleic acid-and factor-bound rnap complexes we found that the position of the clamp is modulated during the transcription cycle in a fashion exceeding the simplified closed-open picture drawn from crystallographic studies. we show that the clamp adopts at least two distinct conformations in both initiation and elongation complexes. moreover, we were able to link conformational states to the catalytic activity of the rnap. transcription factors tfe and spt / , which both bind to the rnap clamp domain and stimulate the activity of the enzyme, shift the equilibria towards one of the main conformations suggesting that both prompt an allosteric switch that influences the structure of the rnap. hence, our data provide a mechanistic rationale for the function of these factors and provide new insights into the complex dynamic behaviour of the transcriptional machinery. solvent models for protein simulations -the good, the bad and the applications duy hua , amitava roy , he huang , carol post the solvent environment plays a crucial role in determining the structure, dynamics and function of a biomolecule. in order to utilize molecular dynamics (md) simulations to examine the structural changes abstract of biomolecules, it is imperative that the solvent environment be accurately modeled. implicit solvent models (isms), in which water molecules are absent and the solvent effect is estimated using an energy function, offer a low-cost approach to describe the solvent environment in md simulations. isms have been used for a variety of biological studies; yet, the performance of implicit solvation methods in capturing the structural characteristics of proteins has not been rigorously demonstrated. in this work, three isms, namely gbmv ii, facts, and scpism, were evaluated for their abilities to emulate the solvent environment and its effect on the structures, dynamics and electrostatic interac- thioredoxins (trx) are ubiquitous proteins that play a key role in redox state regulation of the cell. they interact with multiple targets in the cell. our group solved the structure of s. cerevisiae (ytrx ) and measured its dynamics in different timescales (pico to seconds). it is well established that the asp is the proton acceptor in the reduction process of the target protein. we proposed that asp also works to couple hydration and motion of the interacting loops. we studied conformational motions of the residues of water cavity and interacting loops. the water molecules in water cavity of the protein play a vital role in the redox activity of thioredoxin. the degree of mutational frustration explains in a great deal the multiple timescales observed in the protein dynamics in ytrx and the mutant d a. the ytrx displays a complex equilibrium between the ground state and several excited hydration states. this excited states are more permeable to water and it is key to understand the proton transfer and activation of cys . in this current study, we examined the mutant d a, and observed that this small hydrophobic residue induces conformational exchange in residues exposed to the water cavity, such as cys . along with molecular dynamics simulations and calculation of mutational frustration levels, we were able to describe conformational details of the water cavity. it is formed by three independent contiguous lobes which we called lobes a, b and c. lobe b is the central lobe that contains the catalytic important residues cys and asp . it closed upon mutation d a. lobe a and c remains open upon mutation, meaning that their hydration are independent. the nmr structures for the oxidized and reduced state of the mutant d a has been studied. in-vitro and in-silico studies of ligand binding to the nuclear receptor ppargamma using fret and md narutoshi kamiya , gert-jan bekker , takuma shiraki , haruki nakamura ipr, osaka university, kinki university the nuclear receptor ppargamma is a ligand-dependent transcription factor which regulates gene expression related to glucose homeostasis and insulin sensitization. it is a potential therapeutic target for metabolic syndrome, cancer, and inflammatory diseases. crystal structures have revealed that its ligands bind deep inside the pocket. however, how the ligand recognizes ppargamma and penetrates inside the pocket remains unclear. we have measured the binding kinetics using the fret method and have calculated potential dissociation paths via molecular dynamics (md) simulations. the binding kinetics were measured using fret between the ligand, c -bodipy, which is a fluorescent pigment structurally similar to one of its agonists -oxo-eicosatetraenoic acid, and cyan fluorescent protein (cfp) which is fused to ppargamma. fluorescence of c -bodipy is observed when it is close to cfp when cfp is excited by light at nm. the ligand was simultaneously added to the ppargamma-cfp solution using the stopped-flow method. time-resolved fluorescence signals of c -bodipy and cfp were also monitored. two time constants were observed by the fret experiment, which correspond with the landing and docking processes. we used our new md program, psygene-g [ ] , which utilizes the gpu for acceleration of the non-bonded interactions, such as electrostatic interaction which uses our original zero-dipole summation method [ , ] . previously we were able to attain similar thermodynamics with respect to the particle mesh ewald method in membrane proteins and dna-water-ion systems [ , ] and have applied it to the thermodynamic study of dynein [ ] . we have introduced random accelerated md (ramd) into the psygene-g program to predict a ligand's dissociation path from the initial crystal structure (pdb id: zk ). in ramd, an external random-directional constant force is added to the ligand. we executed -ramd simulations with different random seeds for the initial pull directions. twelve ramd trajectories managed to dissociate within ns, while the remaining simulations did not manage to disassociate within this time-frame. in all twelve cases the ligand went past r , which is located at the surface of ppargamma. interestingly, a somatic mutant (r h) is related to colon cancer. we concluded that r plays a role of a gate keeper to assist ligand binding. sumoylation is a post-translational modification involved in various cellular processes, such as nuclearcytosolic transport, transcriptional regulation, apoptosis, protein stability, response to stress, and progression through the cell cycle. sumoylation is a conjugation of glysine residue of sumo protein with lysine residue in target(rangap ) protein through the influence of enzyme e (ubc ) and e (ranbp ). sumo proteins modify the function of target protein in the cell. but the kinetic mechanism of sumoylation is not well-understand. we traced out the full kinetic process of sumoylation of the sumo -ubc -rangap -ranbp complex in atomic detail via molecular dynamic simulation. we uncovered the kinetics of sumoylation mechanism and verified the important residues which play a key role in the sumoylation process. the comparison of our results with the experimental one are also discussed. human potassium channel kcnq is expressed in several tissues including inner ear, heart muscle, lung, intestine, and stomach, each requiring a unique current profile for proper function. to tune its output current, kcnq complexes with several accessory proteins from the kcne family, each kcne family member modulating kcnq distinctly: kcne causes the channel to delay opening and become more conductive in the open state, kcne causes the channel to be constitutively conductive, and kcne closes the channel. the purpose of this study is to uncover the atomic-scale, mechanical mechanism of how kcne modulates kcnq . to do this we modeled the interaction between kcnq and kcne with a hybrid experimental-computational approach. our strategy is to determine the nmr structure of kcne alone in bilayer-mimicking bicelles, build a homology model of the open-state kcnq , and dock the structure of kcne onto the model of kcnq using electrophysiology-based restraints to validate and refine the resulting model complexes. paramagnetic relaxation enhancement, residual dipolar coupling and sequence analysis suggest the single span transmembrane helix of kcne is significantly curved. hydrogen-deuterium exchange, nuclear overhauser effect cross peaks, and amber simulation suggest kcne carries a significant amount of water in the c-terminal side of the transmembrane helix. the h- n two dimensional nuclear magnetic resonance spectra of a known phenotype-changing mutation in the center of the transmembrane helix, v t, suggests the helix adopts multiple conformations when the extra side-chain hydroxyl group is present. a docking funnel corroborates a binding pocket suggested by the in vivo electrophysiology data where kcne wedges between a helix near the potassium pore and two helices in the voltage sensitive element of the channel. within this pocket, we believe the conformational sampling and structural rigidity-or flexibility, in the context of the v t mutant-of the accessory protein kcne directly influence the modulation profile. watching conformational changes in proteins by molecular dynamics simulations kresten lindorff-larsen proteins are dynamical molecules and their ability to adopt alternative conformations is central to their biological function. examples include motions that underlie allosteric regulation or ligand binding, or protein dynamics in enzymes that can modulate the overall catalytic efficiency. protein motions can often be described as an exchange between a dominant, ground state structure and one or more minor states. the structural and biophysical properties of these transiently and sparsely populated states are, however, difficult to study, and an atomic-level description of those states is challenging. in an attempt to determine how well molecular dynamics simulations can capture slow, conformational changes in protein molecules we have studied two different protein systems which are known to undergo conformational exchange on the millisecond timescale, and for which structural information is available for both major and minor states. using enhanced-sampling all-atom, explicit-solvent molecular simulations, guided by structural information from x-ray crystallography and nmr, we show that current force fields and sampling methods allow us to sample experimentally-determined alternative conformations with surprisingly high accuracy. in particular, we find that we can reversible sample both the ground state and minor state, and that the simulations capture the structure of the minor states also. our simulations enable us to calculate the conformational free energy between the two states, and comparison with relaxation dispersion nmr experiments demonstrates a high accuracy. thus, we show for two distinct proteins that we can map the free energy landscapes and that both the structure and energetics are in excellent agreement with nmr experiments. our simulations provide insight into the structural and biophysical properties of transiently populated minor states, and help reinterpret previous experimental measurements. further, our results demonstrate that, at least in the two cases we have studied, modern simulation methods enable us to examine these otherwise "invisible" states of proteins and describe their structural, functional and thermodynamic properties. our results in this way help demonstrate how simulations can now add to our knowledge of transiently formed, "hidden" states of proteins. cell signaling is a fundamental process for all living organisms. g protein-coupled receptors (gpcrs) are a large and diverse group of transmembrane receptors which convert extracellular signals into intracellular responses primarily via coupling to heterotrimeric g proteins. in order to integrate the range of very diverse extracellular signals into a message the cell can recognize and respond to, conformational changes occur that rewire the interactions between the receptor and heterotrimer in a specific and coordinated manner. by interrogating the energetics of these interactions within the individual proteins and across protein-protein interfaces, a communication network between amino acids involved in conformational changes for signaling, is created. to construct this mapping of pairwise interaction energies in silico, we analyzed rhodopsin gpcr coupled to a gai b g heterotrimer. the structure of this g protein complex was modeled in the receptor-bound and unbound heterotrimeric states as well as the activated, monomeric ga(gtp) state. from these tertiary structural models, we computed the average pairwise residue-residue interactions and interface energies across ten models of each state using the rosetta modeling software suite. here we disseminate a comprehensive analysis of all critical interactions and create intra-protein network communication maps. these networks represent nodes of interaction necessary for g protein activation. structure and dynamics of the polymyxin-resistance-associated response regulator pmra in complex with the promoter dna yuan-chao lou , yi-fen kao , tsi-hsuan weng , yi-chuan li , chwan-deng hsiao , chinpan chen institute of biomedical sciences, academia sinica, institute of molecular biology, academia sinica pmra, an ompr/phob family response regulator (rr), takes part in the two-component system that manages genes for polymyxin resistance through a phosphorylation-dependent regulation. phosphorylation of ompr/phob rr induces the formation of a two-fold symmetric dimer in the n-terminal receive domain (rec), promoting c-terminal dna-binding domains (dbds) to recognize tandem repeat dna sequences on the promoter to elicit adaptive responses. recently, narayanan et al. presented the complex structure of active-like kdpe in complex with dna, which reveals a unique asymmetric rec-dbd interface that is necessary to form stable complexes for transcription activation. in this study, we report the . Å resolution crystal structure of bef -activated pmra in complex with promoter dna as well as its dynamics in solution by nmr. unlike the case of kdpe, pmra-dna complex structure reveals a different asymmetric rec-dbd interaction. interestingly, nmr assignments and dynamics study suggest that rec and dbd do not form stable contacts in solution; instead, domains tumble separately in the absence or presence of dna. relaxation dispersion experiments on methyl groups further show that several rec-dbd interfacial residues exhibit similar slow dynamics in the presence of dna. it is hence highly possible that the slow dynamics observed on these interfacial methyl groups are related to the formation of asymmetric rec-dbd interaction, which reduces the mobility of pmra-dna complex and promotes crystallization. in solution, two domains tumble independently and have diverse orientations, which together with the dbd-dbd interface may facilitate searching best interactions with rna polymerase holoenzyme for transcription activation. time-resolved x-ray observations of nano-scale protein assembly networks yufuku matsushita , hiroshi sekiguchi , noboru ohta , keigo ikezaki , yuji goto , yuji sasaki , the university of tokyo, graduate school of frontier science, advanced materials science, spring- , osaka university, institute for protein research protein aggregation and network formation in diverse protein species had been studying in various experimental approaches such as absorbance spectrophotometry, dynamical light scattering and x-ray scattering. however, these conventional methods are only able to observe averaged information on bulk conditions and also these techniques have a limitation of time resolved observations. recently, development of single molecular observation techniques are remarkable. diffracted x-ray tracking (dxt) is one of the notable single molecular observation methods to capture detailed intramolecular dynamics of an individual single protein molecule by the labeling x-ray method. in this study, we present an innovative method for observing the protein assembly networks of nano-scale size on the bulk solution using a dxt that possess pico-meter scale positional accuracy and micro-second time resolution. this method is founded by detecting angular rotational displacement of a coexisted and dispersed single gold nanocrystal (approximately nm) on target solution. for target sample, we choose a crystal precursor metastable state of mg/ml lysozyme solution (hen egg white lysozyme: . g/ mole on . m sodium acetate - w/v nacl, ph . buffer) and mg/ml of lysozyme solution such as stable condition. for reference sample, we prepare mg/ml and mg/ml of ribonuclease a solution (ribonuclease a (bovine): . g/mol, . m sodium acetate - w/v nacl, ph . buffer). the experiment was carried out at spring- bl xu. from dxt analysis, we obtained logarithmic rotational velocity distribution of mg/ml, mg/ml lysozyme solution and mg/ml, mg/ml of ribonuclease a solution during ms and we processed regression analysis of gaussian fitting in each distribution. from this result, mg/ml of lysozyme solution (stable) and mg/ml, mg/ml (reference) have definitely regressed by single peak normal gaussian distribution that are positional peaks at . mrad and . mrad and . mrad, respectively. in contrast, mg/ml of lysozyme solution consisted of double peak logarithmic distribution at . and . mrad. this peak separation tendency from dxt are typically observed in a supersaturated condition of the sodium acetate solution which coexisting nano-scale solute networks (dxt and small angle x-ray scattering (saxs) experiment). from this study, dxt measurement results and detailed analysis process for a protein solution such as crystal precursor metastable state of lysozyme solution are concerned that this technique is a powerful tool for observing nano-scale protein network's existence and its local dynamics in bulk solution. at the present time, we will demonstrate the detailed analysis and processing from dxt raw data and the conformity of the results from another experiment confirmation such as saxs and darkfield microscopy. finally, we present a detailed protein network model of lysozyme metastable solution from dxt result. increasing evidence suggests that conformational fluctuations experienced by proteins play a key role in promoting function. however, the experimentally derived dynamic behavior of discrete protein systems has yet to provide clear evidence on the evolutionary conservation of motional events between structural and functional protein homologues. in this work, we used a statistical coupling analysis (sca) approach to analyze the role of co-evolving amino acids in protein function and flexibility. sca facilitates the identification of distinct residue sectors and provides an understanding of the correlation between amino acid co-evolution and biological function. as a model system, we used the ribonuclease a (rnase a) family, which includes canonical members that are structurally similar, but perform diverse activities such as angiogenesis, anti-pathogenicity and neurotoxicity, while preserving the common ribonucleolytic function. analysis of sequences with sca . allowed the determination of five independent components (ics) corresponding to amino-acid sectors with distinct functional, structural and/ or dynamical roles within the family. while most ics correspond to residues critical for protein stability and catalytic function, ic correlates with residues experimentally determined to be highly dynamic on the catalytic timescale in several rnase a homologues, as we confirmed by nmr n-cpmg experiments. additionally, ic residues form a hydrogen-bonding network shown to allosterically modulate and coordinate catalytically productive motions. these results provide a direct observation between the role of residue sectors in stability and motions relevant to function in the rnase a family. this study highlights the role of concerted action of multiple residues towards a common function, which can guide experiments for engineering proteins to enhance function such as catalysis. halophilic archea are a type of extremophiles that thrive in hypersaline conditions. in order to avoid the osmotic shock, their cytoplasm is maintained with higher ionic strength up to - m. such a high ionic strength of intracellular environment protects the cells from desiccation or salting out. however, one wonders how such high salt concentrations keep the cell functional and active throughout all the cellular reactions (i.e. enzyme catalysis). the answer lies in the evolution of the halophiles that has left the halophilic proteins with a certain preference for amino acids composition [ ] (prefers more acidic residues and overall decrease in hydrophobic side-chains). it's also known that the salt concentration can affect the activity and the underlying mechanism [ ] of the halophilic enzymes. in order to further understand the haloadaptation, we have chosen to study a model system, dihydrofolate reductase (dhfr) from haloferax volcanii (hvdhfr). dhfr is a very well understood enzyme and known to bound variety of ligands that can modulate the dynamics and the energy landscapes of the enzyme [ ] . we have probed the ls-ms dynamics of hvdhfr bound to the cofactor nadph (holoenzyme) and other substrates (dhf, thf) using relaxation dispersion nmr. in order to understand the salt contribution to the dynamics and catalytic mechanism, the experiments were done in the increasing order of salt concentration, shedding light on the conformational ensembles involved in the catalytic mechanism. the nmr relaxation dispersion analysis of different enzyme complexes in the presence of m and m kcl has shown more conformational fluctuations than compared to the enzyme with m kcl. this result suggests that the enzyme preferably is highly active & samples more flexible conformers in the hypersaline environment. further, quantification of these conformers/invisible-states and their salt-dependence will provide new insights into the mechanism of the enzyme-catalysis. also, it will facilitate designing novel enzymes that can be used in the bioprocess industrial set-up under extreme conditions. . tadeo we have identified the extracellular protein hbps in the cellulose degrader streptomyces reticuli and shown that it specifically binds iron ions, heme and aquo-cobalamin. based on d crystal structures, structural alignments, sequence comparisons, mutagenesis, and comparative biochemical investigations, we identified the coordination sites for iron, heme and aquo-cobalamin, and binding kinetics were elucidated [ , ] . hbps interacts with the membrane-embedded sensor kinase sens. while under nonstressed conditions hbps inhibits sens autophosphorylation, under oxidative-stressing conditions activates it. sens in turn phosphorylates the response regulator senr that activates the transcription of anti-oxidative genes [ ] . we crystallized hbps and solved its d crystal structure that revealed an octomeric assembly which is required for interaction with sens [ ] . using mutagenesis, fret, cd spectroscopy, fluorescence spectroscopy and site-directed spin labelling combined with pulse electron paramagnetic resonance spectroscopy, we demonstrated that ironmediated oxidative stress induces both secondary structure and overall intrinsic conformational changes within hbps. we additionally showed that hbps is oxidatively modified, leading to the generation of highly reactive carbonyl groups and tyrosine-tyrosine bonds [ ] . we concluded that these molecular events are responsible for the hbps-mediated activation of the sensor kinase sens. this presentation will focus on the hbps structure and dynamics within the protein. indian institute of technology bombay for (fop-related protein of kda), a centrosomal protein, is conserved across all ciliated eukaryotes. it has been shown to be involved in ciliogenesis in rpe cells and in the s-phase progression in hela cells. it localizes to the pericentriolar satellite and at the centrosome. the localization of for at the centrosome has been found to be throughout the cell cycle. in paramecium, for is involved in docking of the basal body to the cell membrane and in the assembly of the transition zone. we found that for colocalizes with ciliated microtubules in nih t cells. since, the localization of for to the pericentriolar satellite is dependent on microtubules and the cilium itself is a microtubule-based structure; we sought to investigate the role of for in microtubule assembly. the over-expression of gfp-for in hela cells led to the depolymerization of microtubules while only the gfp expressing hela cells showed typical microtubule network. in addition, the microtubules of gfp-for expressing hela cells were found to be more sensitive towards nocodazole, a microtubule-depolymerizing agent, suggesting that for destabilizes microtubules. the effect of for on the polymerization of tubulin was monitored by light scattering, sedimentation assay and electron microscopy. in vitro, for inhibited the rate and extent of polymerization of tubulin. for example, , , and mm for inhibited the polymerization of purified tubulin by , , and %, respectively. further, the sedimentation assay suggested that for inhibited the polymerization of tubulin. in addition, electron microscopic analysis of the assembly mixture showed that for inhibited microtubule formation in vitro. the binding of for to purified tubulin was monitored by several complimentary techniques. using size exclusion chromatography, for was found to be co-eluted with tubulin indicating that for binds to tubulin. further, the interaction between for and tubulin was analyzed by surface plasma resonance technique and the result showed that for binds to tubulin with a modest affinity. the data together suggested that for regulates microtubule assembly through a direct interaction with tubulin. amide hydrogen exchange (hx) is widely used in protein biophysics even though our ignorance about the hx mechanism makes data interpretation imprecise. notably, the open exchange-competent conformational state has not been identified. based on analysis of an ultra-long molecular dynamics trajectory of the protein bpti, we propose that the open (o) states for amides that exchange by subglobal fluctuations are locally distorted conformations with two water molecules directly coordinated to the n-h group. the hx protection factors computed from the relative o-state populations agree well with experiment. the o states of different amides show little or no temporal correlation, even if adjacent residues unfold cooperatively. the mean residence time of the o state is ps for all examined amides, so the large variation in measured hx rate must be attributed to the opening frequency. a few amides gain solvent access via tunnels or pores penetrated by water chains including native internal water molecules, but most amides access solvent by more local structural distortions. in either case, we argue that an over-coordinated n-h group is necessary for efficient proton transfer by grotthuss-type structural diffusion. differences in redox reactions with nadp /h between ferredoxin-nadp oxidoreductases from bacillus subtilis and rhodopseudomonas palustris daisuke seo , hidehiro sakurai , pierre s etif , takeshi sakurai graduate school of natural science and technology, kanazawa univ., research institute for photobiological hydrogen production, kanagawa university, ferredoxin-nad(p) oxidoreductase (fnr) is a ubiquitous enzyme that catalyze the redox reaction between soluble small iron-sulfur protein ferredoxin and nadp /h. fnrs from photosynthetic green sulfur bacterium chlorobaculum tepidum (ctfnr), low-gc content gram-positive bacterium bacillus subtilis (bsfnr) and purple non-sulfur bacterium rhodopseudomonas palustris (rpfnr) are homodimeric proteins containing one fad prosthetic group per subunit. crystal structure analyses of ct-and bsfnrs have revealed their significant structural homology to nadph-thioredoxin reductase from eschelicia coli, which is distinct from the monomeric fnrs from plastids of higher plants, cyanobacteria, g-proteobacteria and putidaredoxin reductase. previous studies on steady-state reaction of bs-and rpfnrs with nadp /h by diaphorase assay have demonstrated that nadph oxidation rates of bsfnr and rpfnr were comparable to those of the fnrs from plastid and cyanobacteria. but affinities toward nadp /h and rates for oxidation of nadph differed significantly between bsfnr and rpfnr, despite their high amino acid sequence homology. in this work, we report pre-steady state reactions of bsfnr and rpfnr with nadp /h by a stopped-flow spectrophotometry. mixing oxidized bsfnr with nadph yielded a rapid formation of charge transfer complexes (ctcs) followed by a reduction of the enzyme. bsfnr was almost fully reduced at equilibrium. mixing photochemically reduced bsfnr with nadp also rapidly provided an absorption spectrum of ctc but followed reoxidation of reduced bsfnr was very slow. the amount of oxidized bsfnr after equilibrium depended on nadp concentration. kinetic analyses indicated that the rate-determining steps were the hydride-transfer reactions in both directions and the rate for the forward direction was much faster than that for the reverse direction. mixing oxidized rpfnr with nadph exhibited a rapid ctcs formation followed by a slower reduction of the enzyme. increase in nadph concentration reduced the observed rate, suggesting redox potential of rpfnr was similar to that of nadp /h couple in the presence of excess nadph. mixing photochemically reduced rpfnr with nadp rapidly provided ctcs. the decay corresponding to the reoxidation of reduced rpfnr with nadp involved two distinctive kinetic phases, which would be due to the similarity in hydride transfer rates of forward and reverse directions, and the presence of excess amount of nadp . kinetic analyses indicated that the rate-determining steps were the hydride-transfer reactions in both directions and the rate for the forward direction was close to that for the reverse direction. obtained data suggested bsfnr and rpfnr are optimized for different direction of the reaction and may play different physiological roles. stephan k€ ohler , , friederike schmid , giovanni settanni , institute of physics, johannes gutenberg university mainz, germany, graduate school materials science in mainz, fibrinogen is a serum multi-chain protein which, when activated, aggregates to form fibrin, one of the main components of a blood clot. fibrinolysis controls blood clot dissolution through the action of the enzyme plasmin, which cleaves fibrin at specific locations. although the main biochemical factors involved in fibrin formation and lysis have been identified, a clear mechanistic picture of how these processes take place is not available yet. this picture would be instrumental, for example, for the design of improved thrombolytic or anti-haemorrhagic strategies, as well as, materials with improved biocompatibility. here, we present extensive molecular dynamics simulations of the fibrinogen complex which reveal large bending motions centered at a hinge point in the coiled-coil region of the complex. this feature, likely conserved across vertebrates according to our analysis, suggests an explanation for the mechanism of exposure to lysis of the plasmin cleavage sites on fibrinogen coiled-coil region. it also explains the conformational variability of fibrinogen observed during its adsorption on inorganic surfaces and it is supposed to play a major role in the determination of the hydrodynamic properties of fibrinogen. in addition the simulations suggest how the dynamics of the d region of fibrinogen may contribute to the allosteric regulation of the blood coagulation cascade through a dynamic coupling between the a-and b-holes, important for fibrin polymerization, and the integrin binding site p . membrane curvature -the assembler of proteins mijo simunovic , , gregory voth , patricia bassereau chemistry department, the university of chicago, physico-chimie curie, institut curie many biological phenomena require the membrane to change its shape. this process is often mediated by curvature-generating proteins, most notably by those containing one of many bar domains. at the same time, membrane curvature controls the way proteins interact with one another and so it acts as a vital signaling mechanism in the cell. we combine theoretical modeling with microscopy imaging techniques to study the driving force underlying the reshaping of biological membranes induced by n-bar proteins. in particular, we employ a combination of coarse-grained molecular dynamics with field-theoretical simulation methods to study the assembly of proteins on the membrane at molecular resolution. this approach allowed us to elucidate the precise mechanism by which cell membranes rapidly and from large distances recruit proteins to membrane-reshaping sites. it also let us identify a surprising role of membrane tension in directing the strength and geometry of protein-protein interactions. by complementing our simulations with fluorescence and atomic force microscopies, we study the way molecular assembly of proteins affects the membrane morphology at a more macroscopic level. we demonstrated how largescale ordering of the proteins on the membrane is mediated by a strikingly long-range interaction that is driven by membrane fluctuations. in sum, our combined theoretical and experimental approach gives vital clues on how the mechanical properties of the membrane may regulate protein dynamics in living cells. adnan sljoka , alexandr bezginov kyoto university, university of toronto understanding how a protein functions depends in critical ways on predicting which parts are rigid and which are flexible. using rigidity-theoretical techniques, a number or programs such as first, proflex or abstract kinari can rapidly decompose a protein into flexible and rigid regions. in this study we extend this technique and develop a novel computational approach for detecting protein allosteric interactions. it is widely believed that the binding of a ligand at the allosteric site triggers a conformational change that is transmitted through the protein to cause a rearrangement and alteration of the shape of the active site. allostery was first described more than years ago; however, the underlying allosteric mechanism is still not well understood. we will introduce a rigidity-based allosteric mode of communication together with an algorithm which can detect transmission of rigidity and shape changes between two (or more) distant binding sites in allosteric proteins. our algorithm is also used to predict and identify regions in the protein that are critical for the coupled communication between distant sites (i.e. allosteric pathways). starting with a set of known gpcr -dimensional structures, we apply our methods and show how binding of an activating ligand (i.e. agonist) triggers small rigidity changes which propagate to the critical g-protein binding regions. in contrast, in the inactive gpcr structures no such rigidity allosteric communication is observed. detailed predictions and analysis on activated (agonist-bound) and inactive adenosine receptors is discussed and results are compared with experimental evidence. these results show that rigidity-based allosteric model and algorithm is a powerful new tool for detecting allostery in gpcrs. comparing the intrinsic dynamics of multiple proteins using elastic network models reveals global similarities based on their overall shape sandhya tiwari , , nathalie reuter , with increasing protein structural data, we find ourselves with the opportunity to study the dynamical similarities within large ensembles in order to understand the role of protein dynamics at various levels. is there evolutionary pressure to conserve dynamics? is this necessary to retain functionality, as it has been suggested? we performed strategic comparative analysis using the elastic network model, which has proven to be highly efficient and informative (fuglebakk et al., bmc bioinformatics, ) to investigate how the dynamics of related proteins change when their functional or oligomeric state shifts, and if it is conserved within protein families. in our work, we have found that proteins with different ligandbinding states, as in adenylate kinase (tiwari et al., bmc bioinformatics, ), or oligomeric states, as in the pyrr family (perica et al., science, ), can be classified based on the global similarity of their intrinsic dynamics. moreover, our recent analysis on functionally distinct protein families with the tim barrel fold indicates that the overall similarity in shape dictates similarity in intrinsic dynamics regardless of sequence and functional conservation or evolutionary links. we suggest that since shape dictates a large part of dynamical similarity in proteins, the changes in local flexibility play a strong role in differentiating various functional states. oxygen-affinity and cooperativity of hemoglobin introduction: the widely-held mechanism of allostery of hb has been that the changes from the r-quaternary/tertiary structures of oxy-hb to the t-quaternary/tertiary structures of deoxy-hb exert certain constraints on the coordination structure of the heme group, leading to a lower o -affinity of the hemes and thus that of hb [ ] . however, we found that there is no causal correlation between the static t/r-quaternary structures and the low/high o -affinities of hb, respectively [ ] . we explore an alternative mechanism of the regulation of the ligand-affinity and cooperativity in hb. results and discussion: the o -affinities of free fe[ii]-protoporphyrin ix-nitrogenous base complexes in organic solvents are very low (p > torr), whereas the apparent o -affinities of these metalloporphyrins, which are incorporated in apo-myoglobin, apo-hb, serum albumin, etc., increase substantially to p < - torr, though their coordination structures are apparently unchanged [ ] . such substantial increases in the apparent ligand-affinities of metalloporphyrin-containing proteins are accomplished by preventing/inteferring with the dissociation of the ligand by protein matrix, since the interior of globin is nearly fully packed by protein matrix. in hb, the dissociation process of the ligand proceeds through the "caged" state [ ] [ ] [ ] , which can be produced by cryogenic photolysis of the ligated-states at . k and in which the metal-ligand bond is broken and the un-bonded ligand is trapped near the bonding site within the globin moietiy. this "caged" state has spectral features distinct from those of either deoxyor ligated states of the respective hemoproteins. the apparent ligand-affinities of hb are regulated by heterotropic effectors without detectable changes in either static quaternary/tertiary structures of the globin moiety or the coordination/electronic structures of the metalloporphyrin moiety and thus the ligand-affinity of the metalloporphyrins themselves [ ] [ ] [ ] . the reduction of the apparent ligand-affinities of hb may be caused by increases in the migration rate of ligands through globin matrix from the "caged" state to solvent, resulting from the effector-linked, enhanced high-frequency thermal fluctuations which increase the transparency of the globin matrix toward small diatomic ligands [ ] [ ] [ ] . conclusion: the ligand-affinity of hb is regulated through protein dynamics by heterotropic effectors, rather than static quaternary/tertiary structural changes. thus, the "caged" state of hb acts as a critical transition state in regulation of the affinity for small diatomic ligands in hb [ ] . the role of metal ions in the regulation of life processes is extremely important. they act as signal transducers, protein configuration stabilizers, enzymatic cofactors, oxygen transport supporters and many others. for example, subtle perturbations in calcium homeostasis may lead to mental disabilities and are linked to diseases such as autism spectrum disorders (asd). in this study we focus on complex protein systems, mainly those present in the brain. we search for dimers mediated by the presence of metal ions, and determine the impact of the presence or absence of the latter on the structure and energetic properties of the complex in the protein-protein interface. we investigate ions' influence on the interface stability using classic molecular dynamics methods (md), including steered md. moreover, we apply a novel suite of enhanced md-based methods recently developed by our team (rydzewski & nowak) to explore ion diffusion pathways in protein fragments of the synapses. finally, we describe specific inter-protein ion binding motifs with the most important interactions, collating them with various structures deposited in the protein data bank [ ] . the binding of integrins to collagen plays a critical role in numerous cellular adhesion processes including platelet activation and aggregation, a key process in clot formation. collagen is an unusually shaped ligand, and its mechanism of recognition and role in selectivity and affinity are unique, and at this stage not well understood. the i-domain of the integrin protein binds to collagen specifically at multiple sites with variable affinities, however the molecular mechanism of integrin i-domain (ai) regulation remains unknown. using nmr, along with isothermal titration calorimetry, mutagenesis, and binding assays we are developing a novel integrated picture of the full recognition process of the integrin a i binding to collagen. the adhesion of the a b integrin receptors to collagen is cation-dependent with collagen binding a mg(ii) ion that is located at the top of the extracellular integrin a i-domain (a i). our results show evidence for a regulatory effect of the mg(ii) ion on a i affinity, by inducing allosteric ms-ms motions of residues distant from the binding site. we propose a novel model of a i recognition to collagen, comprising a two-step mechanism: a conformational selection step, induced by mg(ii) coordination, and an induced-fit step caused by collagen binding. hydrogen-deuterium exchange experiments show that the induced-fit step is facilitated by the reduced local stability of the c-terminus. we propose that the conformational selection step is the key factor that allows discrimination between high and low affinity collagen sequences. cytochromes p (cyp) are heme containing enzymes involved in the metabolism of endobiotics and xenobiotics, such as drugs or pollutants. [ ] in humans, cyps are attached to the biological membranes of endoplasmic reticulum or mitochondria by n-terminal transmembrane anchor and they are partially immersed by their catalytic domain to different level. [ ] generally, the composition of lipid membrane may significantly affect behavior of protein embedded in respective membrane e.g. the cholesterol in membrane alters membrane properties such as: thickening of the membrane, changing the stiffness or enhancing ordering of the membrane. furthermore, the increasing amount of cholesterol in membrane may also alter interaction with membrane proteins and affect solute partitioning between membrane and water molecules. [ ] cholesterol is also known to noncompetitively inhibit the most typical drugmetabolizing cyp -cyp a , [ ] however the mechanism was unknown. for this reason, we prepared the set of simulations of cyp a embedded in dopc lipid bilayers with various cholesterol concentrations ( , , , and % wt; figure ) and the ns long md simulations were carried out. md simulations showed the formation of funnel-like shape of the lipids close to the catalytic domain of cyp. in addition, the cholesterol molecules have tendency to accumulate in the vicinity of membrane-attached f/g loop. the catalytic domain sunk deeper into the membrane with cholesterol and also the number of amino acids in contact with membrane was bigger than in the pure dopc bilayer. in contrast, the presence of higher amount of cholesterol affected the pattern of channel opening effectively blocking the access to the active site from the membrane, which in turn may affect the substrate preferences and catalytic efficiency. [ ] finally, we study the effect of different lipid types on membrane-attached cyp a . anti-( -hydroxy- -nitrophenyl)acetyl (np) antibodies are one of the most widely analyzed type of antibodies, especially with respect to affinity maturation [ ] [ ] [ ] . affinity maturation is a process in which b cells produce antibodies with increased affinity for the antigen during the course of an immune response, and is like "evolution" in term of increasing antigen-binding affinity. during the course of affinity maturation, the structural dynamics of antibodies, which are closely correlated with the binding function, can change. to analyze the structural dynamics at atomic resolution and the single-molecule level, we tried to express and purify single-chain fv (scfv) antibodies against np. using scfv antibodies, we can also analyze the effects of key residues on affinity maturation via site-directed mutagenesis. as the first step, we have succeeded in generating a sufficient quantity and good quality of scfv of affinity-mature anti-np antibody, c , with a linker composed of four repeats of gggs. the scfv protein was expressed in the insoluble fraction of e. coli, and solubilized using m urea, followed by refolding by step-wise dialysis to decrease the urea concentration. the final step of purification using an antigen column indicated that approximately % of the solubilized protein was correctly refolded and possessed antigen-binding ability. the analytical ultracentrifugation (auc) analysis showed that the purified c scfv exists in the monomeric state with little oligomeric contamination. the secondary structure and thermal stability of c scfv were analyzed using circular dichroism (cd). the far-uv cd spectra of c scfv indicated typical b-sheet-rich structures. upon antigen binding, the far-uv cd spectrum remained unchanged, but the thermal stability increased by approximately oc. the antigen-binding function of c scfv was analyzed using a surface plasmon resonance (spr) biosensor, biacore. the binding affinity and kinetics of c scfv for np conjugated to bovine serum albumin immobilized on the sensor chip were similar to those of intact c . taken together, the results of auc, cd, and spr indicated that c scfv could be refolded successfully and would possess its functional structure. next, to analyze the structural dynamics of c scfv in the absence or presence of antigen, experiments involving diffracted x-ray tracking (dxt) were performed [ ] . c scfv with an n-terminal his-tag was immobilized on substrate surfaces using tag chemistry, and au-nanocrystals were labeled on the surface of scfv as tracers. the motions of c scfv were analyzed in two rotational directions representing tilting (u) and twisting (v) mean square displacement (msd) analysis from more than trajectories showed that the slope for c scfv without antigen, especially in the u direction, was greater than that for c scfv with antigen, suggesting that the motion of scfv was suppressed on antigen binding. the antibiotic resistance enzyme aph( '')-ia confers antimicrobial resistance to aminoglycoside antibiotics in staphylococci and enterococci. this kinase phosphorylates aminoglycosides such as gentamicin and kanamycin, chemically inactivating the compounds. we have determined multiple structures of the enzyme in complex with nucleoside and aminoglycoside substrates and cofactor magnesium. introduction of aminoglycoside to crystals of aph( '')-ia induce gross conformational changes in crystallo, illustrating several important stages of the catalytic cycle of the enzyme. an interaction between nucleoside triphosphate and an amino acid residue on a conserved loop has also been identified that appears to govern a conformational selectivity and modulates the enzyme activity when no substrate is present. comparisons between multiple protein molecules both within and between crystal structures allow us to infer functional states of the enzyme as it carries out catalysis. these structures collectively highlight an enzymatic flexibility that not only allows the binding of diverse aminoglycosides, but also appears to transition from a stabilized, inactive enzymatic state to a catalytically active enzyme with an active site geometry identical to distantly-related eukaryotic protein kinases. mechanistic insight gained from these studies begin to demystify a widespread staphylococcal resistance factor, and provide a starting point for the development of anti-infectives toward this important antimicrobial resistance machine. ryan godwin , william gmeiner , freddie salsbury wake forest university -department of physics, wake forest university health sciences -department of cancer biology the zinc-finger of the nf-jb essential modulator (nemo) is a ubiquitin binding domain, and an important regulator of various physiological processes including immune/inflammatory responses, apoptosis, and oncogenesis. the nominally functioning residue monomer ( jvx) is represented by a bba motif, with a cchc active site coordinating the zinc ion. here, we investigate the effects of a single point mutation that has been linked to the disease states associated with ectodermal dysplasia. the single mutation of the last binding cysteine (residue ) to a phenylalanine ( jvy) distorts the available conformation and dynamics of the protein, as shown via microsecond, gpuaccelerated molecular dynamics simulations. we examine these two proteins in various states of zinc-binding and coordinating cysteine protonation. in addition to destabilization of the alphahelix induced by the cysteine to phenylalanine mutation, prominent conformations show the bsheets turned perpendicular to the alpha-helix, providing a possible mechanism for the induced disease state. , catalytic ( - aa) and c-terminal ( - aa)) were expressed in e. coli. several truncated in variants containing amino acids - , - , - and - were also prepared. a full-size ku with a gst-tag on its n-terminus was purified from e. coli. all the experiments performed showed that neither n-terminal nor c-terminal domains of hiv- in are essential for its binding with ku despite a weak binding capacity retaining to the c-terminal domain. the catalytic core ( - aa) as well as the mutant lacking c-terminal domain ( - ) both demonstrated affinity to ku comparable to the affinity of the full-size in, whereas its truncated variant ( - aa) bound to ku protein only weakly. we also expressed a c-terminal ha-tagged full-length in and its - variant in hek t cells together with a wt ku - flag and showed that both in variants are stabilized by co-expression with ku by approx. twofold. we hypothesize that the binding surface within in lies in the region from to a.a. that is a long a-helix. we have shown that a homologous integrase from prototype foamy virus that lacks this structural element does not bind to ku . it is worth noting that ku does not affect the interaction of in with its major cellular partner -ledgf/p as well as its interaction with the dna substrate. this work was supported by an rfbr grant - - and by an rscf grant - - . the nadph-dependent cytochrome p oxidoreductase (cypor) is large amino-acid long microsomal multidomain enzyme responsible for electron donation to its redox partner cytochrome p (cyp) involved in drug metabolism. electron transfer (et) chain is mediated by two riboflavin-based cofactors -flavin mononucleotide (fmn) and flavin adenine dinucleotide (fad) within their respective domains and nicotinamide adenine dinucleotide phosphate (nadph). during this electron transfer cypor undergoes several structural changes in open and closed state of both domains in different degree of contact. in spite of the fact that cyp-cypor complexes play a key role in drug metabolism, the atomistic mechanism of structural rearrangements during complex electron transfers is still lacking. here, we present the results of our study on structural changes during cypor multidomain complex movement between individual electron transfers using classical molecular dynamics (md) and metadynamics (mtd) simulations with cofactors of nadph, fad and fmn in resting state. homology model of human cypor in both forms (opened and closed) were embedded into pure dioleoylphosphatidylcholine (dopc) bilayer. after system equilibration (figure ), structural changes of protein, anchor and cofactor movement were studied. we were able to select possible cypor-membrane orientation which would allow interaction with cytochrome p . in addition, spontaneous closing of open cypor was observed. however structural changes between crystal structures and structures obtain from md simulations lead us to the use of metadynamics in order to speed up the process. fmn and fad cofactor remained in close van der waals contact during the -ns long simulation stabilized by p stack interaction of fad with trp , whereas continual movement of nadph continually weakens its p stack interaction with fad. after ns of classical md additional metadynamics simulations were performed in order to investigate internal motion of cofactors during electron transfer. atoms c n (nadph) and n (fad) which are responsible for et were able to move closer to the distance of Å after adding biasing potential. this distance is more than sufficient for electron transfer to occur. after switching back to classical md cofactors got into resting positions ( Å) again. our results show that cypor undergo several structural changes and internal motions of cofactors in order to transfer electrons to its redox partner -cyp. research & utilization div., jasri/spring- , grad. school frontier sci., univ. tokyo, grad. sch. sci., univ. hyogo, japan, national institute of advanced industrial science and technology, japan, pentameric ligand-gated ion channels (plgics) are a major family of membrane receptors that open to allow ions to pass through the membrane upon binding of specific ligands. plgics are made up of five identical (homopentamers) or homologous (heteropentamers) subunits surrounding a central pore. structural information about their multiple allosteric states, carrying either an open or a closed channel, has become available by recent studies by x-ray crystallography. however, dynamic information are needed to understand their mechanism of gating, notably the long-range allosteric coupling between the agonist binding site and the ion channel gate. here we used the diffracted x-ray tracking (dxt) method ( ) to detect the motion of the extracellular and transmembrane domain two plgics: the nicotinic acetylcholine receptor (nachr) and a proton-gated bacterial ion channel from gloeobacter called glic. dxt is a powerful technique in biological science for detecting atomic-scale dynamic motion of allosteric proteins at the single molecular level and at tens of micro seconds timescale resolution. the dynamics of a single protein can be monitored through trajectory of a laue spot from a nanocrystal which is attached to the target protein immobilized on the substrate surface ( , ). dxt detects two kinds of rotational motions of nanocrystal, tilting and twisting, based on x-ray incident beam axis. dxt analysis with . ms/f time resolution showed that tilting motion of the transmembrane domain of glic and both tilting and twisting motions of the extracellular domain of glic and nachr were enhanced upon application of agonists (lowering the ph for glic, and binding of acetylcholine for nachr). the detailed dynamic information, including size effect of gold nanocrystal to the motion of them, is discussed. [ proteins possess unique structure-encoded dynamics that underlie their biological functions. here, we provide experimental evidence for an evolutionary mechanism driven solely by long-range dynamic motions without significant backbone adjustments, catalytic group rearrangements, or changes in subunit assembly. crystallographic structures were determined for several ancestral gfp-like proteins that were reconstructed based on posterior sequence predictions, using members of the stony coral suborder faviina as a model system. the ancestral proteins belong to the kaede-type class of gfps, a group of proteins that undergoes irreversible green-to-red photoconversion and is therefore frequently employed in superresolution microscopy. surprisingly, we find that the structures of reconstructed common green ancestors and evolved green-to-red photoconvertible proteins are very similar. therefore, we analyzed their chain flexibility using molecular dynamics and perturbation response scanning. we find that the minimal number of residue replacements both necessary and sufficient to support lightinduced color conversion provide for increased fold stiffness at a region remote from the active site. at the same time, the allosterically coupled mutational sites appear to increase active site conformational mobility via epistasis. these data suggest that during evolution, the locations of fold-anchoring and breathing regions have been reversed by allosteric means. therefore, we conclude that the green-tored photoconvertible phenotype has arisen from a common green ancestor by migration of a knob-like anchoring region away from the active site diagonally across the beta-barrel fold. based on titration experiments, we estimate that at ph , . % of the protein population harbors neutral side chains for his and glu , residues that form an internal salt bridge near the chromophore. we propose that this reverse-protonated subpopulation constitutes the catalytically competent state. in the electronically excited state, light-induced chromophore twisting may be enhanced, activating internal acid-base chemistry that facilitates backbone cleavage to enlarge the chromophore. in this way, a softer active site appears to be coupled to a mechanism involving concerted carbon acid deprotonation and betaelimination. dynamics-driven hinge migration may represent a more general platform for the evolution of novel enzyme activities by tuning motions in the active site. the binding of an agonist to a gpcr causes a conformational change in the receptor that leads to its activated functional state. rhodopsin, the membrane receptor responsible for photoreception in the vertebrate retina, is a prototypical gpcr and has been extensively used in structural, biochemical and biophysical studies of this class of receptors. different small molecules have been described to be capable of binding to rhodopsin. in addition, mutations in rhodopsin have been associated with retinal diseases and efforts have been carried out in order to find potential ligands that can offset the effect of these mutations. cyanidins, a group of flavonoids within the larger family of polyphenols, have been reported to stimulate chromophore regeneration of rhodopsin by means of the formation of regeneration intermediates. the aim of the current study was to evaluate the effect of the flavonoid quercetin on the conformational properties of both native bovine rhodopsin and heterologously expressed recombinant rhodopsin. rhodopsin was purified from bovine retinas by immunoaffinity chromatography, and photobleaching, thermal stability, metarhodopsin ii decay and chromophore regeneration assays were carried out in the absence or in the presence of mm quercetin. for recombinant rhodopsin, a plasmid encoding wild-type opsin was transfected into mammalian cos- cells, in the absence or in the presence of mm quercetin, harvested, regenerated with -cis-retinal, or -cis-retinal, and subsequently purified in dodecyl maltoside solution. no differences in photobleaching behavior, upon illumination, could be detected in the purified quercetin-containing samples compared to those in the absence of this flavonoid. in the case of rhodopsin, and the recombinant wild-type protein regenerated with -cis-retinal, quercetin did not significantly alter the thermal stability and rate of regeneration of the purified proteins under our experimental conditions. however, a two-fold increase in the thermal stability and a % increase in chromophore regeneration were observed for the recombinant wild-type protein regenerated with -cis-retinal in the presence of quercetin. in contrast, the presence of quercetin did not alter the electrophoretic and basic spectroscopic properties of rhodopsin, or those of the recombinant wild-type protein, suggesting no important structural alterations as a result of quercetin binding to the receptor. the positive effect of quercetin on the stability, and chromophore regeneration of rhodopsin, could be potentially used to counteract the effect of naturally-occurring misfolding mutations in rhodopsin. thus, quercetin could help stabilizing rhodopsin mutants associated with retinal diseases such as retinitis pigmentosa. furthermore, docking of the ligand, carried out on the crystallographic structure of rhodopsin (entry gzm), reveals several favorable sites for quercetin binding. one of this would be compatible with -cis-retinal suggesting a complementary binding to the receptor of this isomer which would not be compatible with -cis-retinal binding. identification of prospective allosteric sites of p by computational methods protein function is intrinsically associated with structural flexibility, so that understanding the functional properties of proteins requires going beyond the static picture produced by x-ray diffraction studies. structural flexibility can also be interpreted as a dynamic exchange between different conformational states with low energy barriers at room temperature. allosterism is a mechanism to regulate protein function associated with the plasticity exhibited by proteins. allosteric sites can be considered transient cavities that can be occupied by a small molecule with the subsequent modulation of the protein plasticity. occupation of these sites may modify the affinity of the protein for its native substrate that can be positive when the affinity increases or negative when the affinity decreases. allosterism can be used for the design of non-competitive ligands as new therapeutic agents. this mechanism of activity modulation is particularly interesting for those targets that use a common substrate for activation, like in the case of kinases to search for selective compounds. proteins can be viewed in solution as an ensemble of diverse energy accessible conformations. binding of an allosteric ligand produces a redistribution of the population of the diverse conformational states, which at the end modulate the affinity of the native substrate. allosteric sites can be characterized using computational methods by ensemble docking. it consist of characterize a set of structures that represent the accessible conformations of a protein that can then be used to perform virtual screening. in the present work we have studied prospective allosteric sites of p using computational methods. the protein is a member of the mitogen-activated protein kinases (mapks), a highly regulated group of enzymes that control a variety of physiological processes, including mitosis, gene expression, apoptosis and metabolism movement among others. the conformational profile of p was assessed using a us trajectory of accelerated molecular dynamics as sampling technique in explicit solvent. we used as starting structure the apoform of p in its inactive conformation (entry p ). the conformational features of the protein were assessed through the analysis of the variance of the most flexible regions of the protein using principal component analysis. the snapshots of the trajectory were projected onto the two principal components. subsequent cluster analysis permitted us to select a few structures for further studies. specifically, prospective biding sites were identified using a hydrophobic probe as implemented in the sitemap program. the results show previously described regulatory sites and some new prospective ones. hydrogen/deuterium exchange-mass spectrometry provides clues on the mechanism of action of min e maria t. villar , kyung-tae park , joe lutkenhaus , antonio artigues cell division in most bacteria is initiated by the formation of the z ring, an essential cytoskeletal element that serves as a scaffold for the cytokinesis machinery, at the mid body of the cell. in e coli the spatial location of the z ring is regulated by the min protein system, comprised by three major proteins: minc, mind and mine. the dynamic interaction between these proteins results in the formation of an oscillating protein gradient between the poles of the cell. this oscillation determines the position of the formation of the z ring. many aspects of this simple mechanism are beginning to be understood. in particular, the conformational changes associated with the interaction of the three min proteins between them and with the cell membrane, are of especial interest. hydrogen/deuterium exchange mass spectrometry (hdx ms) is a sensitive technique for the detection of changes in protein conformation and dynamics. the main advantages of this methodology are the ability to study native proteins in solution, the requirement for low protein concentrations, the potential to discriminate multiple coexisting conformations, and the lack of an upper limit to the size of protein to be analyzed. here we use hdx ms to analyze the dynamics of the wild type mine and of its inactive double mutant d a d a. our results show significant differences in the rates of exchange and in the total amount of deuterium exchanged at the end of the reaction between these two forms of mine. the wild type protein exchanges most of the amide hydrogen during the first few seconds of initiation of the exchange reaction. on the other hand, the mutant protein exchanges only % of the total amide hydrogen atoms during the first seconds of initiation of the exchange, and the remaining % amide hydrogen atoms are exchanged more slowly during the next few minutes of the reaction. our data are consistent with the existence of a highly flexible structure for the wild type protein and the coexistence of at least two rigid conformations for the double mutant that are undergoing a cooperative transition. interestingly, the central b-sheet forming the interface between the two subunits is protected against exchange on both proteins. these results provide insights into the conformational changes that mine undergoes during its interaction with mind. biased signalling and heteromization of the dopamine d receptor in schizophrenia and parkinson's disease pablo herrera nieto , james dalton _ , jes us giraldo _ universidad aut onoma de barcelona biased signalling and heteromization of the dopamine d receptor in schizophrenia and parkinson's disease as a significant component of dopamine signalling in the brain, the dopamine d receptor (d r), a member of the class a gpcr family, is an important target in the treatment of neurological conditions such as schizophrenia and parkinson's disease. d r shows a variety of signalling pathways through g proteins, including adenylyl cyclase inhibition, gbgpotentiation of adenylyl cyclase , and erk kinase activation, in addition to b-arrestin recruitment,. these pathways are differentially activated by some agonists and it has been suggested that d r ligands with gai/o antagonist and b-arrestin agonist activity may have anti-psychotic behavioural activity with reduced extra-pyramidal side effects. d r has also been found to form homodimers or higher-order hetero-oligomers with other gpcrs, which may modulate d r conformation and activity, thus constituting an additional form of allosteric receptor regulation. based on these findings, we have computationally modelled the full-length structure of d r, including its long intracellular loop (icl ) that is residues in length and absent in all homologous gpcr crystal structures. using state-of-the-art tools, such as rosetta for ab initio protein folding and acemd for micro-second molecular dynamics (md) simulations we have successfully de novo folded icl , which primarily consists of extensions to transmembrane helices (tmh) and and an intervening disordered histidine/proline-rich region, which is highly flexible. the latter is observed to interact with other receptor intracellular loops (icl and icl ) and appears to restrict access to the g-protein binding-site. in addition, we have docked a structurally diverse collection of ligands (biased agonists, antagonists and allosteric modulators) into our d r model and observed characteristic binding patterns suggestive of different biased signalling mechanisms. finally, through protein-protein docking with rosettadock, we have generated a complete heterodimer model of d r with the adenosine a a receptor (aa ar), where a mutual interface is formed between their respective tmhs and , as well as an association between the c-terminus of aa ar and icl of d r. this may be a particularly relevant biological complex in the treatment of parkinson's disease where antagonists of aa ar have been shown to ameliorate disease effects, potentially through direct interaction with d r. bis-ans as a tool to monitor conformational changes upon assembly of binary and ternary complexes of eif e, e-bp inhibitory protein, and the mrna 'cap specific recognition of the mrna ' terminal cap structure by the eukaryotic initiation factor eif e is the first and rate-limiting step in the cap-dependent translation. small e-binding proteins, e-bp , e-bp , and e-bp , inhibit the translation initiation by competing with eif g initiation factor for the same binding site, and by blocking the assembly of the translation machinery [ ] . our recent studies revealed intricate cooperativity between the cap and e-bp binding sites of eif e [ ] . here, we applied a fluorescent dye, , '-dianilino- , '-binaphthyl- , '-disulfonate (bis-ans) to investigate conformational changes upon assembly of binary and ternary complexes composed of human eif e, e-bp , and the mrna 'cap analogue, m gtp. the fluorescence quantum yield of bis-ans increases significantly upon binding to hydrophobic sites of proteins, making the probe a convenient tool to determine the accessibility to hydrophobic surfaces, and to monitor structural reorganisation of macromolecules [ ] . we characterised the interaction of bis-ans with eif e and e-bp by fluorescence titration. the association processes takes up to several hours until the saturation of the fluorescence signal is achieved, reflecting high flexibility of the protein structures. the association constants kas of eif e/bis-ans complexes are very high for the non-specific interaction. the kas values for eif e/bis-ans and eif e/ e-bp /bis-ans are similar ( m ), whereas the presence of m gtp results in ca. -fold weaker binding of the probe to eif e. the affinity of bis-ans for e-bp is -fold lower than that for eif e. we found no effect of either m gtp or e-bp on the fluorescence of bis-ans in complex with eif e, thus indicating lack of conformational changes around the probe on eif e/m gtp or eif e/ e-bp complex formation. it also testifies that bis-ans does not bind to the cap-binding site, despite the hydrophobic nature of this eif e region. on the contrary, addition of m gtp to the eif e/ e-bp /bis-ans complex causes an increase of the probe fluorescence, which indicates differences in the structural reorganisation in the binary, m gtp/eif e, compared with the ternary, m gtp/eif e/ e-bp , complexes, and confirms the spatial cooperation between the cap and e-bp binding sites. we also observed an increase of fluorescence for bis-ans bound to e-bp in the presence of eif e, pointing out that e-bp partially folds upon association with eif e. in summary, our results provide a deeper insight into the structural aspects of the molecular interaction at early stages of the cap-dependent translation. acknowledgements: this work was supported by the bst /bf project from university of warsaw background: beta -glycoprotein (b gpi) is a protein abundantly present in human plasma and highly conserved in all mammals. b gpi has been identified as the major antigen in the antiphospholipid syndrome (aps), a severe thrombotic autoimmune disease. despite its importance in the pathogenesis of aps, the physiological role of b gpi is still elusive. in a previous work we have demonstrated that b gpi significantly prolongs the clotting time in fibrin generation assays, and inhibits aggregation of gel-filtered platelets (ic . um), either isolated or in whole blood, by inhibiting cleavage of par on intact platelets (ic . um) and in solution. importantly, b gpi does not alter the ability of thrombin (fiia) to generate the anticoagulant protein c, with or without thrombomodulin added. hence, we concluded that b gpi inhibits the key procoagulant properties of fiia, without affecting its unique anticoagulant function. we also proposed that b gpi, together with other more efficient anticoagulant pathways such as thrombomodulin-fiia -protein c and antithrombin iii-fiia, may function as a mild anticoagulant in vivo especially in those compartments were the efficacy of thrombomodulin is limited, as in the large vessels, or is even absent, as in the brain vasculature. aims: lacking the threedimensional structure of b gpi-thrombin complex, the aim of this work is to identify the peptide regions either on thrombin and b gpi involved in complex formation. results: data obtained by fluorescence and surface plasmon resonance (spr) indicated that b gpi interacts whit fiia whit physiological affinity (kd nm). kd values calculated by reverting the interacting systems are very similar to each other (kd nm), suggesting that b gpi in the mobile phase has a conformation which is competent for the binding to immobilized fiia. the affinity of fiia for immobilized b gpi is markedly decreased by increased ionic strength (i.e. kd increases by -fold going from . m to . m), suggesting the electrostatic interactions play a key role in fiia -b gpi recognition. filling/inactivation or perturbation of fiia active site does not alter the affinity of fiia for immobilized b gpi, confirming that the active site is not involved in the interaction. mapping of thrombin binding sites with specific exosite-directed ligands (i.e. hirugen, gpibalpha, hd aptamer) and thrombin analogues having the exosites variably compromised (i.e. prothrombin, prethrombin- , alpha-thrombin), reveals that the positively charged exosite-ii of fiia plays a key role in b gpi binding. from the docking model of the bb gpi-thrombin complex, we identified a highly negatively charged segment - in domain v of b gpi interacting with positively charged pathes in thrombin exosite ii. the synthetic peptide b gpi( - ) was able to bind to fiia with an affinity (kd nm) comparable to that of full-length b gpi, deduced from fluorescence or spr measurements and to compete in spr measueremnts with the binding of full-length b gpi to thrombin. hence, combining experimental and theoretical data, we obtained a reliable model of the b gpi-thrombin complex. metalloproteases are one of the most diverse types of proteases, presenting a wide range of folds and catalytic metal ions. in the case of the merops ma clan, where most of the known metalloproteases are grouped based on the consensus hexxh sequence motif, a single catalytic zinc ion and common fold architecture [ ] . despite these common features, members from distinct families present distinct domain composition and topology. given our interest in developing new tailor-made metalloproteases for bioengineering applications, an in-depth understanding of the factors governing their function is required. protein internal dynamics includes the space of functionally-relevant structural changes occurring during an enzymatic reaction, and there is an increasing understanding on how it relates with protein sequence and structure evolution. therefore, we have recently assessed how the structural heterogeneity of metalloproteases relates with the similarity of their dynamical profiles [ ] . first, the dynamical profile of the clan ma type protein thermolysin, derived from the anisotropic network model, was evaluated and compared with those obtained from principal component (pc) analysis of a set of crystallographic structures and essential dynamics (ed) analysis of a ns molecular dynamics simulation trajectory [ ] . a close correspondence was obtained between normal modes (nm) derived from the coarse-grained model and experimentally-observed conformational changes (rmsip between nm -nm and pc of . ), corresponding to functionally-relevant hinge bending motions that were shown to be encoded in the internal dynamics of the protein (cumulative overlap of ed -ed and pc of . ). next, dynamics-based comparison methods that employ a related coarse-grained model (b-gaussian elastic network model) was made for a representative set of ma clan members [ ] , allowing for a quantitative description of its structural and dynamical variability. although members are structurally similar ( % pairs with dalilite z-score > . ), they nonetheless present distinct dynamical profiles ( % of pairs with aladyn p-value > . ), with no identified correlation between structural and dynamical similarity. for cases where high dynamical similarity was observed, the respective modes corresponded to hinge-bending motions encompassing regions close to the active site. further inspection of the produced alignments indicates that for ma clan metalloproteases, conservation of internal dynamics has a functional basis, namely the need for maintaining proper intermolecular interactions between the protein and respective substrate. previously unnoticed dynamical similarity between clan members botulinum neurotoxin type a, leishmanolysin and carboxypeptidase pfu was also found. together, these results suggest that distinct selective pressure mechanisms acted on metalloprotease structure and dynamics through the course of evolution. this work shows how new insights on metalloprotease function and evolution can be assessed with comparison schemes that incorporate additional information of protein dynamics. glucokinase from antarctic psychrotroph pseudoalteromonas sp. as- (psgk) has a higher specific activity at low temperatures and a higher thermal stability than its mesophilic counterpart from e. coli (ecgk). in order to elucidate the structural basis for cold-adaptation and thermal stabilization of psgk, we have determined the crystal structure of psgk at . Å and compared it with the ecgk structure. psgk is a homodimer of the subunit of amino acid residues. each subunit consists of two domains, a small a/b domain (residues - and - ) and a large a b domain (residues - ). the active site is located in a cleft formed between the two domains. the identity of amino acid sequence between psgk and ecgk was %, but three dimensional structures of them are very similar to each other, having the conserved catalytic residues and substrate-binding residues. the analysis of the mainchain temperature factors revealed that the regions of small domain and the hinge region connecting two domains of psgk showed higher temperature factors with a lower number of intramolecular hydrogen bonds and ionic interactions than the corresponding regions of ecgk. however, the large domain regions of psgk showed lower temperature factors with a higher number of intramolecular hydrogen bonds than ecgk. furthermore, the atomic temperature factors of catalytic asp on the small domain were higher, but those of glucose-binding glu , his , and glu on the large domain were lower than ecgk. these results suggest that highly flexible hinge region and the catalytic residue on the small domain of psgk may contribute to its cold-adaptation, namely higher activity at low temperatures, whereas a more rigid structure of the large domain of psgk stabilizes its overall structure more strongly than ecgk. nowadays non-waste technologies in synthetic chemistry become more and more popular. such processes are often carried out using different enzymes. dehydrogenases represent the large group of enzymes, which are widely used in synthesis of chiral compounds and other useful molecules. such enzymes need nadh or nadph as a cofactor and due to high cost of reduced coenzymes a cofactor regeneration system is an obligate part in such kind of processes. it was shown that formate dehydrogenase (fdh, ec . . . .) is one of the best enzymes for nad(p)h regeneration. fdh catalyses the reaction of formate oxidation to carbon dioxide coupled with reduction of nad(p) to nad(p)h. the main advantages of fdh are the irreversibility of catalyzed reaction, low price of formate ion and wide ph optimum of activity. our laboratory has the largest collection of formate dehydrogenases from different sources. many fdh genes from bacteria, yeasts and plants were cloned and enzymes were expressed in active and soluble forms. mutant formate dehydrogenases from bacterium pseudomonas sp. show the highest thermal stability as well as activity in comparison with other reported formate dehydrogenases. now we have focused on eukaryotic genes. the recombinant enzymes from soya glycine max (soyfdh), arabidopsis thaliana (athfdh), moss physcomitrella patens (ppafdh) and yeast ogataea parapolymorpha (opafdh) were obtained by genetic engineering methods. it was revealed, that soyfdh has the best michaelis constants among all known fdhs, but it's less thermally stable compared to other fdhs. new mutant forms of soyfdh with excellent catalytic characteristics and high thermal stability were obtained by protein engineering. other enzymes (athfdh, ppafdh and opafdh) are comparable in their stability with majority of bacterial enzymes (but not with psefdh), so all the new obtained fdhs can be successfully used for cofactor regeneration. marmara university, wellesley college, antibiotics are essential therapeutic drugs widely used in the treatment of bacterial infections. unfortunately, misuse of these drugs resulted in the development of bacterial defense mechanisms. blactamase synthesis is among these mechanisms that renders b-lactam antibiotics ineffective. understanding the dynamic behavior of this enzyme is an important step in controlling its activity. in a former study, the importance of highly conserved w in modulating the hinge type h motion was reported. in the light of this information, mutant tem- b-lactamase enzymes with w a, w f and w y substitutions were constructed. wild-type and mutant tem- b-lactamases purified with ni affinity chromatography were subjected to enzyme assay using centa as the substrate. with w f and w y mutations, the remaining activity was approximately % of the initial activity. however with the w a mutation, activity was totally lost. structural studies of the w a mutant with cd and florescence spectroscopy indicated that there was no major change in the overall structure. however this mutation disrupted the interactions of w which resulted in an increase in the flexibility of this region of the protein. this project was supported by t € ub _ itak project no m . light-switchable zn binding proteins to study the role of intracellular zn signaling stijn aper , maarten merkx zn plays an important catalytic and structural role in many fundamental cellular processes and its homeostasis is tightly controlled. recently, free zn has also been suggested to act as an intracellular signaling molecule. to get increased understanding of the signaling role of zn we are developing light-switchable zn binding proteins to perturb the intracellular zn concentration using light. these protein switches consist of two light-responsive vivid domains and the zn binding domains atox and wd , linked together with flexible peptide linkers. in the dark, zn is tightly bound in between the two zn binding proteins. light-induced dimerization of the vivid proteins disrupts this interaction and thus results in zn release. the fluorescent proteins cerulean and citrine were attached to the vivid domains to allow the different conformational states of the protein switch to be monitored using fret. zn titrations revealed a -fold decrease in zn affinity going from dark-to light-state for the initial design, which was further improved to -fold by optimizing the linkers between the protein domains. in addition, the zn affinities of both states were tuned to be optimal for intracellular applications. switching between the high affinity dark-state and the low affinity lightstate was found to be reversible for at least two light-dark cycles. following the in vitro characterization, we are currently assessing the performance of this genetically encoded 'caged' zn in mammalian cells. proteins as supramolecular building blocks: engineering nanoscale structures school of biological sciences, university of auckland, school of biological sciences, victoria university proteins hold great promise in forming complex nanoscale structures which could be used in the development of new nanomaterials, devices, biosensors, electronics and pharmaceuticals. the potential to produce nanomaterials from proteins is well supported by the numerous examples of self-assembling proteins found in nature. we are exploring self-assembling proteins for use as supramolecular building blocks, or tectons, specifically the n-terminal domain of a dna binding protein (nterm-lsr ) and a typical -cys peroxiredoxin (hsprx ). non-native forms of these proteins have been designed undergo selfassembly into supramolecular structures in a controllable manner. self-assembly of nterm-lsr is initiated via proteolytic cleavage, thereby allowing us to generate supramolecular assemblies in response to a specific trigger. we will show that the degree of oligomerisation can be controlled by variations in environmental conditions such as ph and protein concentration. furthermore, via protein engineering, we have introduced a new "switch" for oligomerisation via enteropeptidase cleavage. the new construct of nterm-lsr can be activated and assembled in a controlled fashion and provides some ability to alter the ratio of higher ordered structures formed. hsprx has been shown to oligomerise into dimers, toroids, stacks and tubes in response to specific triggers such as ph and redox state. in this work we have utilised the histidine tag to further control the assembly of this versatile protein tecton. we will show that minute variations in ph can induce oligomersation of hsprx toroids into stacks and tubes. furthermore, by utilising the histidine tag as a ligand we can bind divalent metals to these supramolecular structures. this not only drives the formation of higher ordered oligomers but also provides a facile route which may facilitate the functionalisation of these protein nanoscale structures after they have been assembled. danielle basore , , rajesh naz , scott michael , sharon isern , benjamin wright , katie saporita , donna crone , christopher bystroff , , biological sciences, rensselaer polytechnic institute, cbis, rensselaer polytechnic institute, chemical and biological engineering, rensselaer polytechnic institute, computer science, rensselaer polytechnic institute, obstetrics and gynecology, west virginia university, unintended pregnancy is a worldwide public health concern, with million pregnancies being classed as unintended in . the magnitude of this number clearly indicates an unmet need in terms of contraception. methods that are currently available are effective, but exhibit many problems. side effects, ease of use, cost, and availability are all concerns. we propose a contraceptive vaccine that would be safe, effective, long-lasting, cheap, and reversible. our vaccine would prevent pregnancy by targeting sperm with antibodies raised in the woman's body. several approaches have been taken to developing a contraceptive vaccine in recent years. the most successful so far has been using human chorionic gonadotropin (hcg), a hormone produced during pregnancy, as an antigen . the hcg vaccine progressed to phase clinical trials, but only displayed an % efficacy, which is insufficient for a contraceptive. our lab uses a structure based approach to the design of an anti-sperm antigenic protein. we believe this will raise a more vigorous immune response that will produce a longer lasting titer. the catsper complex is a heterotetrameric calcium channel found in the tail region of sperm . each subunit of the complex contains an exposed loop known as the p-loop. the p-loop is unique on the surface of sperm because it is not glycosylated, allowing antibodies to potentially recognize and bind it. ylp is a twelve residue peptide that mimics the glycans in the glycocalyx of sperm . ylp is a member of the flitrx library, and in mice, produced protective titers that were reversible both voluntarily and involuntarily. our designs will introduce these two potential antigens into a loop of the l protein of human papilloma virus. l spontaneously assembles into virus like particles, and will aid in the production of a robust immune response. protein carriers for passage of the blood-brain barrier sinisa bjelic medical solutions that help protein therapeutics accumulate into the brain are crucial for future treatment of neurological disorders. biodrugs have a tremendous potential to treat disorders of the nervous system, but their efficiency has been severely restricted. to reach the brain all drugs must traverse the blood-brain barrier (bbb) -a permeable wall that separates blood from the brain -whose main function is to protect the nervous system from environmental influences of bacteria and toxins. unfortunately the bbb is also the culprit that effectively blocks access to therapeutics required for treatment of neurological diseases. a way to boost exposure of therapeutics across the bbb is to piggyback onto the transferrin receptor, a multidomain protein anchored in the membrane, which is involved in the physiological facilitation of iron uptake. here i present research that aims at successfully developing potent protein carriers for transferrin receptor-mediated passage of the bbb by using computational protein design in combination with yeast display methodology for hit validation and optimization. the longterm goal is to couple therapeutics -as for example drugs against alzheimer's -to the designed carriers to increase the brain uptake and cure neurological disorders. medium-throughput multistep purification of coagulation factor viia jais r. bjelke , gorm andersen , henrik Østergaard , laust b. johnsen , anette a. pedersen , tina h. glue there is a need of medium-to-high throughput purification of low-titre recombinant protein variants for screening to identify the final biopharmaceutical lead. such proteins include coagulation factors to be used for treatment of haemophilia and other bleeding disorders. at novo nordisk we have established a platform for production of recombinant coagulation factor viia variants, which include a spectrum of single-point mutations to large domain insertions. the variants were produced using transiently transfected hek f, hkb or choebnalt (qmcf technology) suspension cells. harvest cultivations were typical in the range of . -to l. a -step continuous, multistep purification method was implemented on € aktaxpress systems (ge healthcare). the interlinked process steps include capture using an immunoaffinity column, polish, concentration and buffer exchange using an anion-exchange column and proteolytic activation of the zymogen variant forms using a coagulation factor xaimmobilized column. buffers were designed such that elution from the capture column was aligned with binding conditions on the polish column to avoid a desalting step in-between. the following and final enzymatic activation was optimized with regards to flow rate to ensure full conversion while minimizing unwanted secondary cleavages in factor viia. the final products were fractionated in sharp chromatographic peaks ready for characterization. hplc and sds-page analyses showed a solid quality of the produced variants and more than variants have been produced in sub mg scale using the outlined method. biomimetic sequestration of co : reprogramming the b domain of protein g through a combined computational and experimental approach esra bozkurt , ruud hovius , thereza a. soares , ursula rothlisberger ecole polytechnique f ed erale de lausanne, federal university of pernambuco protein engineering is a powerful tool to generate highly specific enzymes for biomimetic production of chemicals. among many applications, the development of enzymes to accelerate carbon dioxide fixation is a possible route to limit co emission. in this project, we are inspired by the ancient enzyme carbonic anhydrase which efficiently catalyzes the reversible hydration of carbon dioxide in the presence of a zinc ion active site. to create an efficient biocatalyst, the engineered gb domain containing a his cys zn (ii) binding site was used as a starting point. in subsequent work, b domains comprising of his wat zn (ii) binding sites have been rationally designed to produce carbonic anhydrase mimics. the re-engineering was accomplished through a series of mutations to orient the zinc bound reactive species to form a hydrogen bond network in the active site while retaining the native secondary structure. we performed classical molecular dynamics (md), quantum mechanics/molecular mechanics (qm/ mm) simulations and metadynamics, with the aim to explore potential catalytic roles of the reengineered b domains and to elaborate the reaction mechanism. briefly, we introduced novel zn (ii) binding sites into thermostable b domain. in parallel, experiments are underway. wild-type protein was expressed and purified. structural and mutagenesis studies are ongoing. the results emphasize the power of theoretical work to enable the mimicking of nature's enzymes for desired catalytic functions. the roles of entropy and packing efficiency in determining protein-peptide interaction affinities diego caballero , , corey o'hern , , , , lynne regan , , physics, yale university, integrated graduate program in physical and engineering biology, yale university, mechanical engineering and materials science, yale university, applied physics, yale university, molecular biophysics and biochemistry, yale university, chemistry, yale university despite many recent improvements in computational methods for protein design, we still lack a quantitative and predictive understanding of the driving forces that control protein stability, for example, we do not know the relative magnitudes of the side-chain entropy, van der waals contact interactions, and other enthalpic contributions to the free energy of folded proteins. in addition, we cannot reliably predict the effects of point mutations on enzyme specificity or sequence tolerance in ligand binding sites. the tetratricopeptide repeat (tpr) motif is a common and versatile protein system that has been used as a model to study protein-protein interactions. for example, recent studies have experimentally measured the binding affinity and specificity for different tpr binding pockets and peptide ligands and generated a ranking of the protein-peptide pairs with the highest affinity. to gain a fundamental understanding of the interplay between atomic close packing and fluctuations of side-chain conformations in protein-peptide binding pairs, we performed all-atom langevin dynamics simulations of key residues near the binding interface of tpr proteins and their cognate peptides. the langevin dynamics simulations enabled us to calculate the entropy and potential energy of side chain conformations in the presence of backbone fluctuations for each protein-peptide pair. we compile rankings of the stability and affinity of mutant tpr-peptide structures to those obtained from experimental studies. this research has enhanced our ability to rationally manipulate protein-peptide interfaces. advances from this research will enable the design of tpr modules that specifically recognize biologically important proteins. monitoring protein-protein interactions using tripartite split-gfp complementation assays protein-fragment complementation assay (or pca) is a powerful strategy for visualizing protein-protein interactions in living cells. previously described split-gfp based sensors suffer from the poor solubility of individual pca fragments in addition to background signal originating from their spontaneous selfassembly ( ). we developed a new encoded genetic reporter called "tripartite split-gfp" for visualizing protein-protein interactions in vitro and in living cells. the assay is based on tripartite association between two twenty amino-acids long split-gfp tags, gfp and gfp , fused to interacting protein partners, and the complementary gfp - detector. when proteins interact, gfp and gfp selfassociate with gfp - to reconstitute a functional gfp ( ). using coiled-coils and frb/fkbp model systems we characterize the sensor in vitro and in escherichia coli. we extended our studies to mammalian cells and examine the fk- inhibition of the rapamycin-induced association of frb/fkbp . the small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence and for screening modulators of complex formation in cell-based assays. aldehyde dehydrogenases (aldhs) catalyze the oxidation of aldehydes to their corresponding acids using nad(p) as coenzyme. these enzymes are responsible for the detoxification of lipid peroxidation products, which have been involved in the etiology and pathogenesis of different diseases involving increments in oxidative stress. recent data from our group, showed that aldh a is resistant to inactivation by lipid peroxidation products, even at concentrations - times higher than those required to inactivate aldh a and aldh . the amino acids sequence of the aldehyde-binding site of the three enzymes was analyzed, and it was found that the enzymes susceptible to the effect of lipid peroxidation products (aldh a and aldh ), have cys residues flanking the reactive cys (position ), based on this criteria and considering that these aldehydes react preferentially with cysteine, a mutant of aldh was generated changing the cys residues adjacent to cys . the mutant aldh -cys thr-cys val, was resistant to the inactivation by acrolein and -hne, even at concentrations -fold higher than those required to inactivate aldh . however, the mutant presented values of km , and -fold higher for acrolein, propionaldehyde and acetaldehyde, respectively, compared to the wild type enzyme, but showed a catalytic efficiency similar to the parent enzyme. these data revealed that cys residues near to the reactive cys in aldh are important in the inactivation process induced by lipid aldehydes, but also participate in determining the specificity for the substrates in this enzyme. small molecule-assisted shutoff: a widely applicable method for tunable and reversible control of protein production h. kay chung , conor jacobs , yunwen huo , jin yang , stefanie krumm , richard plemper , , roger tsien , michael lin department of biology, stanford university, department of pediatrics, stanford university, department of pharmacology, university of california san diego, department of pediatrics, emory university, institute for biomedical sciences, georgia state university, department of chemistry and biochemistry, university of california san diego, howard hughes medical institute, university of california san diego, the ability to quickly control the production of specific proteins would be useful in biomedical research and biotechnology. we describe small molecule-assisted shutoff (smash), a technique in which proteins are fused to a self-excising degron and thereby expressed in a minimally modified form by default. degron removal is performed by a cis-encoded hepatitis c virus (hcv) protease, so that applying clinically available hcv protease inhibitors causes degron retention on subsequently synthesized protein copies and suppresses further protein production. we find that smash allows reversible and dosedependent shutoff of various proteins with high dynamic range in multiple cell types, including yeast. we also successfully use smash to confer drug responsiveness onto a rna virus for which no licensed drug inhibitors exist. as smash does not require permanent fusion of a large domain, it should be useful when control over protein production with minimal structural modification is desired. furthermore, as smash only uses a single tag and does not rely on modulating protein-protein interactions, it should be easy to generalize to multiple biological contexts. top, a protein of interest is fused to the smash tag via a hcv ns protease recognition site. after protein folding, the smash tag is removed by its internal ns protease activity, and is degraded due to an internal degron activity. bottom, addition of protease inhibitor induces the rapid degradation of subsequently synthesized copies of the tagged protein, effectively shutting off further protein production. vaccine development has emerged, epitope-focused immunogens, but in the past these have failed to deliver the expected outcome. here, we employed a new computational design methodology (rosetta fold from loops or ffl) to design epitope-focused immunogens. ffl was devised to insert structurally defined functional sites into protein scaffolds. throughout the ffl stages the structure of the scaffold is folded and its sequence designed to stabilize the desired functional conformation of the inserted site. we used ffl to design epitope-focused immunogens for the respiratory syncytial virus (rsv), for which despite the intense research we are still lacking an approved vaccine. we designed three-helix bundles harboring an rsv epitope, that was previously co-crystallized with the neutralizing antibody motavizumab. the designs were thermodynamically stable (tm > ˚c) and showed extremely high affinities to motavizumab (kd pm). structural characterization through x-ray crystallography of antibodybound and unbound scaffolds showed good agreement to the computational models in the overall structure (rmsd - . Å) and exquisite mimicry of the epitope region (rmsd - . Å), when compared to the peptide-epitope in complex with motavizumab. the designed immunogens were used to immunize non-human primates (nhp), and approximately % of the cohort developed rsv neutralizing activity, in some instances with high potency. to evaluate the therapeutic relevance of the elicited neutralization activity, we compared the nhp neutralization titers to those of human sera after natural rsv infection, which generally yields protective levels of antibodies. the neutralization potency of the best nhp responders was comparable to that of the human sera. to better understand the features of the antibodies elicited, we isolated several rhesus monoclonal antibodies (rhmabs) from the animal that exhibited the most potent neutralization. two of the rhmabs bound to the immunogen with very high affinity (kd pm) and were potent rsv neutralizers. interestingly, these rhmabs were approximately fold more potent than the fda-approved prophylactic antibody palivizumab. our results provide the first proof-of-principle for epitope-focused vaccine design, and demonstrate the power of the ffl figure . schematic of nucleotide binding, exchange and hydrolysis in tubulin, and its coupling to mt assembly. exchange of gdp (orange) for gtp (magenta) at the e-site in b-tubulin (blue) happens in the unpolymerized dimer (left). the active, gtpbound tubulin dimer adds to a growing mt (right). interaction of the incoming a-tubulin (green) with the e-site nucleotide at the plus end of a mt (with b-tubulin exposed) results in gtp hydrolysis. the mt cartoon (bottom right) shows an oversimplified representation of a gtp cap as it first grows by tubulin addition and then shrinks by polymerization-coupled gtp hydrolysis (here b-tubulin that is bound to gtp is shown in red and that bound to gdp is shown in blue). cryo-em density map (emdb- ) and atomic model (pdb: jak) for an eb -decorated mt bound to gtpgs. a-tubulin, b-tubulin and eb are colored green, blue, and orange, respectively. computational methodology. we anticipate that ffl will be useful for a variety of other challenges in the computational design of functional proteins. designed repeat proteins as templates for photoactive molecules and fluorescent nanoclusters sara h. mejias , , antonio aires , , javier l opez-andarias , pierre couleaud , , begoña sot , , carmen atienza , nazario mart ın , , aitziber l. cortajarena , imdea nanoscience, c/faraday, , ciudad universitaria de cantoblanco , cnb-csic-imdea nanociencia associated unit "unidad de nanobiotecnolog ıa", departamento de qu ımica org anica i, facultad de qu ımica, universidad complutense self-assembly of biological molecules into defined functional structures has a tremendous potential in nanopatterning, and the design of novel bionanomaterials and functional devices. molecular selfassembly is a process by which complex three-dimensional structures with specified functions are constructed from simple molecular building blocks. we present first the study and characterization of the assembly properties of modular repeat proteins, in particular designed consensus tetratricopeptide repeats (ctprs), and their application as building blocks in order to generate functional nanostructures and biomaterials. ctpr proteins can be assembled into self-standing thin films, and thin nanometer fibers in solution. in this work, we show the use of the designed consensus repeat proteins as scaffolds to template: ( ) photoactive organic molecules, and ( ) fluorescent nanoclusters. .we explore the potential of ctpr proteins to arrange donor-acceptor pairs for electro-active materials. in particular, porphyrin rings arranged by ctprs in a defined distance and orientation for favoring face-to-face orientation which should lead to an improvement in the optoelectronic properties. our results confirm the successful ability of ctpr proteins to be used as scaffold for ordering organic chromophores, while preserving their structure. the unique self assembly properties of ctpr scaffolds have been exploited to generate ordered conductive films of the protein-porphyrin conjugates. these results open the door to fabricate hybrid protein-based solid devices. .we show results on the ability of ctpr to encapsulate and stabilize fluorescent gold nanoclusters. we investigated the influence of the protein sequence in the final properties of the nanoclusters. the structural and functional integrity of the protein template is critical for future applications of the protein-cluster complexes. therefore synthetic protocols that retain the protein structure and function have been developed. as a proof of concept, a ctpr module with specific binding capabilities has been successfully used to stabilize nano clusters. biohybrid photoelectrochemical cells have been developed by functionalizing the hematite photoanode with the light-harvesting cyanobacterial protein c-phycocyanin (pc) yielding a substantial enhancement of the photocurrent density. photoelectrochemical cells combining light-harvesting proteins and inorganic semiconductors have potential for the use in artificial photosynthesis. in this work we present processing routes for the functionalization of hematite photoanodes with pc, including in situ co-polymerization of pc with enzymatically-produced melanin and using a recombinantly produced pc . moreover, recombinant forms of the light-harvesting protein c-phycocyanin from synechocystis sp. pcc were engineered to carry a peptide with affinity for hematite. similarly, a bacterial laccase was engineered to acquire affinity for hematite. results obtained from the different approaches to hematite functionalization and the advantages offered by protein engineering will be presented. minimizing a suitable free energy expression is arguably the most common approach in (ab initio) protein structure prediction. the achieved accuracy depends crucially on the quality of the free energy expression in use. here, we present corrections to existing free energy expressions which arise from the thermal motion of the protein. we (i) devise a term accounting for the vibrational entropy of the protein, and (ii) correct existing potentials for 'thermal smoothing'. (i) vibrational entropy is almost always neglected in free energy expressions as its consideration is difficult. this practice, however, may lead to incorrect output because distinct conformations of a protein can contain very different amount of vibrational entropy, as we show for the chicken villin headpiece explicitly [ ] . for considering vibrational entropy, we suggest a knowledge based approach where typical fluctuation and correlation patterns are extracted from known proteins and then applied to new targets. (ii) at ambient conditions, timeaveraged potentials of proteins are considerably smoothened due to thermal motion where the strength of this effect varies strongly between atoms. distinguishing these inhomogeneities by introducing new atom species regarding their locale environment can therefore increase the precision of time-averaged potentials [ ] . extraction of general principles from the continually growing protein data bank (pdb) has been a significant driving force in our understanding of protein structure. atomistic or residue-level statistical potentials, secondary-structural propensities, and geometric preferences for hydrogen bonding are among the classical insights that arose from observations in the pdb. given the magnitude of structural data available today, it is likely that many quantitative generalizations remain to be made. here we hypothesize that the pdb contains valuable quantitative information on the level of local tertiary structural motifs (terms), with term statistics reflecting fundamental relationships between sequence and structure. we define a term to be the structural fragment that captures the local secondary and tertiary environments of a given residue, and put our hypothesis through a series of rigorous tests. first, we show that by breaking a protein structure into its constituent terms, and querying the pdb to characterize the natural ensemble around each, we can estimate the compatibility of the structure with a given amino-acid sequence through a metric we term "structure score." considering submissions from recent critical assessment of structure prediction (casp) experiments, we find a strong correlation (r . ) between structure score and model accuracy, with poorly predicted regions readily identifiable. this performance exceeds that of leading atomistic statistical energy functions. next, we show that by considering the terms of a structure that are affected by a given mutation, and mining the pdb to characterize sequence statistics associated with each, we are able to predict mutational free energies on par with or better than far more sophisticated atomistic energy functions. finally, we ask whether term statistics are sufficient to enable the design of proteins de-novo. we demonstrate that given a native backbone conformation, term considerations alone with no input from molecular mechanics correctly predict roughly the same fraction of amino acids from the corresponding native sequence as state-ofthe-art computational protein design methods. knowledge-based energy functions have already put pdb statistics to good use by parsing structural environments into geometric descriptors, generally assuming their conditional independence. our results suggest that it may now be possible to instead consider local structural environments in their entirety, asking questions about them directly. if this is the case, then the pdb is an even larger treasure trove of information than it has been generally known to be, and methods of mining it for term-based statistics should present opportunities for advances in structure prediction and protein design. comprehensive understanding of a protein fold is intertwined with successful design. recent advances in designing de novo structures have shown that proteins can be designed for a few globular and helical folds. however, designing all-b structures and barrels remains challenging because loops and intricate long range interactions that are important in these topologies are difficult to control. for designing novel catalysts, the (a/b) -barrel (or tim-barrel) fold is one of the most important examples, for it is the most common topology for enzymes. for almost year, attempts in designing de novo tim barrel structures have all resulted in poorly folded proteins. here we describe the successful design of a -fold symmetrical (a/b) barrel directly from geometrical and chemical principles. designed variants with a wide range of stabilities from being molten globules to cooperatively folded proteins were experimentally characterized, and the results revealed the importance of sidechain-backbone hydrogen bonding for defining the characteristic a/b-barrel. the residue tim barrel structure is among the smallest tim-barrels and has a fully-reversible melting temperature of c. the x-ray crystal structure shows atomic-level agreement with the design model. despite this structural similarity, psi-blast searches do not identify sequence similarities to known tim-barrel proteins. more sensitive profile-profile searches suggest that the design is sufficiently distant from other native tim-barrel superfamilies to be in a superfamily of its own, further implying that nature has only sampled a subset of the sequence space available to the tim-barrel fold. the ability to de novo design tim-barrels opens new possibilities for custom-made enzymes. university of texas southwestern medical center, biofrontiers institute, university of colorado creation of new molecular sensors and actuators based on fluorescent proteins relies on methods for identifying complex photophysical phenotypes and subsequently performing separations on cell populations. we developed a microfluidic flow cytometry approach tailored to interrogating the performance of genetically-encoded fluorophores and present the results of studies employing this technology. the system screens cell-based libraries on the basis of multiple photophysical parameters relevant to imaging, including brightness, photostability, and excited-state lifetime (i.e. a proxy for fluorescence quantum yield) at a rate of up to cells/sec. in a first generation of experiments, molecular dynamics-guided design was used to create a library of mcherry mutants that was screened with this system, resulting in the identification of a variant with a higher stability b-barrel and improved photostability but with a decreased brightness due to reduction in the fluorescence quantum yield. to avoid inadvertent decreases in this important performance criterion, subsequent rounds of selection were performed on the basis of both photostability and excited-state lifetime as sorting criteria. in these second generation selections, mutations were designed to target pathways of oxygen access through the bottom of the bbarrel in addition to a position that directly interacts with the chromophore. furthermore, subsequent rounds of screening were used to improve folding and maturation. the multiparameter sort identified multiple clones with up to -fold improved photostability and up to double the excited-state lifetime of the parent mcherry fluorescent protein. the best mutant we identified produces one order of magnitude more photons before photobleaching compared to mcherry, at excitation conditions characteristic of confocal fluorescence microscopy. our results demonstrate the utility of combining moleculardynamics-guided library design with technology for photophysics-based selections. we anticipate that the new fluorescent proteins obtained in this work will find use in low-copy-number and long-duration imaging live cell imaging applications in cell-lines created by genomic editing techniques. targeted protein degradation achieved through a combination of degrons from yeast and mammalian ornithine decarboxylase rushikesh joshi , ratna prabha c. the maharaja sayajirao university of baroda targeted protein degradation achieved through a combination of degrons from yeast and mammalian ornithine decarboxylase targeting the over accumulated protein in the cell for degradation using specific degrons is an emerging research area. the degradation of the vast majority of cellular proteins is targeted by the ubiquitin-proteasome pathway. but in the case of ubiquitin independent protein degradation, odc/az system is more effective in achieving targeted protein degradation than other types of degradation . ornithine decarboxylase (odc) is key regulatory enzyme in the biosynthesis of polyamines. the protein has two domains namely, n terminal a/b barrel domain and c-terminal b-sheet domain. degradation of odc is mediated by polyamine inducible protein, antizyme (az). antizyme interacts with odc on n-terminal region, which results in degradation of odc by proteasomes. in mammalian odc the c-terminal has an unstructured tail of residues, which pulls odc into proteasome for degradation. it was reported earlier by coffino's group that the unstructured tail acts as a degron in chimeric fusion with gfp . in yeast, same function is achieved by n-terminal residues . present study focuses on accomplishing targeted protein degradation in saccharomyces cerevisiae by adding these two degradation signals or degrons of yeast odc and mammalian odc as tags to a reporter protein. we have selected two degrons namely, n terminal a/b barrel domain of yeast odc and c-terminal residues of mouse odc and grafted them to n and c-terminus of the reporter protein yegfp. degradation of yegfp and yegfp fusion with degrons of odc (degron-yegfp) were monitored by western blot using anti-gfp antibody and fluorescence spectroscopy. initially, the amount of degron-yegfp fusion protein was very low compared to control yegfp. it means that the chimeric protein underwent rapid degradation in the cells. after inhibition of proteasome, increase in the level of degron-yegfp was observed, confirming that the degrons cause rapid degradation of reporter protein through proteasome. earlier, we have also tagged ubiquitin from yeast with last residues of modc and observed enhanced degradation of ubiquitin in saccharomyces cerevisiae. therefore, both the degrons of odc alone and in combination are capable of decreasing stability of reporter protein in the cells. however, the combination of degrons is more effective than either of them in isolation. enzymes fold into unique three-dimensional structures, which underlie their remarkable catalytic properties. the requirement that they be stably folded is a likely factor that contributes to their relatively large size (> , dalton). however, much shorter peptides can achieve well-defined conformations through the formation of amyloid fibrils. to test whether short amyloid-forming peptides might in fact be capable of enzyme-like catalysis, we designed a series of -residue peptides that act as zn dependent esterases. zn helps stabilize the fibril formation, while also acting as a cofactor to catalyze acyl ester hydrolysis. the fibril activity is on par with the most active to date zinc-protein complex. such remarkable efficiency is due to the small size of the active unit (likely a dimer of -residue peptides), while the protein is at least -fold larger in molecular weight. the observed catalytic activity is not limited to ester hydrolysis. we have designed copper binding peptides that are capable oxygen activation. these results indicate that prion-like fibrils are able to not only catalyze their own formation -they also can catalyze chemical reactions. thus, they might have served as intermediates in the evolution of modern-day metalloenzymes. these results also have implications for the design of self-assembling nanostructured catalysts including ones containing a variety of biological and nonbiological metal ions. rational design of the cold active subtilisin-like serine protease vpr with improved catalytic properties and thermal stability abstract proteinase vpr, from a psychrophilic vibrio species and its thermophilic structural homologue, aqualysin i (aqui) from thermus aquaticus, we set out to design a mutant of vpr which would be more thermostable, but would retain the high catalytic activity of the wild type enzyme. our starting protein template was a previously stabilized mutant containing two inserted proline residues close to the nterminus of vpr (n p/i p). this vpr_n p/i p mutant was shown to have a significantly increased thermal stability but displayed a concomitant tenfold loss of catalytic efficiency. from our previous studies we selected two mutations, one which increased catalytic activity (q k) of the enzyme significantly and another which stabilized the protein against thermal denaturation (n d). the n d mutation had been shown to introduce a salt bridge into the structure of the cold adapted proteinase, yielding higher stability but without negative effects on activity. the q k exchange had been shown to double the turnover number (kcat) to that of the wild type enzyme. insertions of these selected mutations into the vpr_n p/i p mutant were according to predictions; the q k increased the kcat tenfold, and the n d mutation increased the thermal stability. in the combination mutant, vpr_n p/i p/n d/q k, thermal stability was increased by c and c, in terms of tm and t %, respectively. furthermore, the catalytic activity of the mutant was somewhat higher than that of the wild type enzyme. critical peptide stretches may not serve as faithful experimental mimics for protein amyloidogenesis bishwajit kundu , dushyant garg certain amino acid stretches are considered critical to trigger the amyloidogenesis in a protein. these peptide stretches are often synthetically produced to serve as experimental mimics for studying amyloidogenesis of the parent protein. here we provide evidence that such simple extrapolation may be misleading. we studied the amyloidogenesis of full length bovine carbonic anhydrase ii (bcaii) and compared it with those formed by its critical amyloidogenic peptide stretch - (pepb). under similar solution conditions and initial monomeric concentrations, we found that while amyloid formation by bcaii followed aggregation kinetics dominated by surface-catalyzed secondary nucleation, pepb followed classical nucleation-dependent pathway. the afm images showed that bcaii forms short, thick and branched fibrils, whereas pepb formed thin, long and unbranched fibrils. atr-ftir revealed parallel arrangement of cross b sheet in bcaii amyloids, while pepb arranged into antiparallel b sheets. amyloids formed by bcaii were unable to seed the fibrillation of pepb and vice versa. even the intermediates formed during lag phase revealed contrasting ftir, far uv cd signature, hydrophobicity and morphology. we propose that for any polypeptide, the sequences flanking a critical region are equally effective in modulating the initial nucleation events, generating prefibrillar and finally fibrillar species with contrasting characteristic. the results have been discussed in light of amyloid polymorphism and its importance in the design of therapeutic strategies targeting such toxic regions. aksana labokha , ralph minter all approved biological drugs target extracellular proteins and not the majority of the expressed human genome, which resides within intracellular compartments. included in the latter category are many important, disease-relevant targets which cannot be easily addressed by small molecule approaches, such as the oncology targets c-myc and k-ras. although bacteria and viruses have evolved strategies to deliver biological material to the cell cytoplasm and nucleus, our ability to engineer recombinant proteins to replicate this is somewhat limited by (i) our nascent understanding of protein uptake and trafficking pathways and (ii) the ability to easily quantify cell delivery to the cytoplasm and cellular organelles. the aim of my project is to address these challenges by developing an effective assay for cytoplasmic uptake and then using it to measure the delivery efficiency of recombinant proteins which mimic natural delivery strategies e.g. cell penetrating peptides fusion, exotoxin mimics, and supercharged proteins (proteins with high surface charge which can enter cells). i also intend to explore the influence of the rab superfamily, which are the master regulators of protein trafficking, to influence and control both the kinetics and final subcellular destination of exogenous proteins. protein engineering: what's next? with the growing industrial need for engineering enzymes for the deconstruction and transformation of plant biomass in biorefineries, there is a want for the development of new approaches for designing special purpose biocatalysts. techniques, such as directed evolution, which mimic the natural selection process by evolving proteins towards the improvement of a given property, have unquestionably demonstrated their value and are routinely used in large industrial companies. nevertheless, the brute force employed in these methods, could significantly gain from an all-atom description of the underlying catalytic mechanisms, to center the efforts on more limited areas of the protein. in the last years, we have developed computational tools, which combine the electronic structure description of qm/mm methods with the potential to model long time scale processes of pele, to study the details of a variety of reactions. examples, which will be discussed, include rationalizing the selective oxyfunctionalization of steroids using fungal enzymes and the study of the effect of point mutations on the oxidation efficiency of laccases. these methods have shown their potential not only at the descriptive level but, more importantly, through their high predictive capability that opens many opportunities for their use in biotechnology. in this talk, we will show how recent advances in in silico approaches are setting new grounds for future computer guided directed evolution. several orthogonal bioreactions take place simultaneously within membrane bound organelles in eukaryotes and proteinaceous microcompartments in bacteria. these subcellular structures contain sets of enzymes co-involved in metabolic pathways. towards the goal of creating artificial protein microreactors, we seek to develop an artificial organelle that emulates the metabolic activity of the carbon fixating organelle of autotrophic bacteria, the carboxysome. here, we show that the two key carboxysomal enzymes, ribulose- , -bisphosphate carboxylase/oxygenase (rubisco) and carbonic anhydrase (ca), can be efficiently co-encapsulated using our previously reported encapsulation system which is based on a bacterial capsid formed from the protein lumazine synthase (aals- ). our preliminary results suggest that the enzymes can act in tandem and that the co-encapsulation of ca with rubisco in the capsid is necessary for enhanced rubisco activity in vitro. we attribute this observation to the high local concentrations of the rubisco substrate, co , produced by ca within the capsid. we are developing a theoretical model of a minimal carboxysome using the kinetic rate constants of our rubisco and ca variants and aals- as the shell to complement these experiments. next, we will incorporate our minimal carboxysome within an expression host such as e.coli, opening up the possibility of further optimization through directed evolution. in the past targeting and engineering of chemokines has led to several interesting drug candidates. [ ] amongst them, met-rantes, a met-ccl with high g protein-coupled receptor (gpcr) affinity but no subsequent signal transduction, as well as mutants addressing the interaction with the so-called glycosaminoglycans (gags) seem to be the most promising candidates. both, gag knockout as well as gag affinity matured chemokine isoforms have been considered as anti-inflammatory drug candidates, out of which an il- mutant with modifications reached clinical phase where it was profiled for acute neutrophil-related exacerbation in copd. [ ] cxcl (ip- ) is a proinflammatory chemokine released by various cells following stimulation by interferon g (ifn-g) . it is therefore considered as a late chemokine being responsible for the attraction of different lymphocytes. [ ] any therapeutic indication is consequently related to chronic and multiple applications. we have therefore engineered cxcl very conservatively at positions to ultimately generate dominant-negative mutants with a mildly improved gagbinding affinity and an entire knock off gpcr activity. the first steps of our engineering approach were in silico modelling of the mutants and the establishment of a suitable upstream-and downstreamprocessing protocol. next we generated a fluorescently engineered cxcl variant for our fluorescence-based affinity studies which was subjected to biocomparability investigations relative to the native, non-fluorescent protein. compared to the wild type, the fluorescently engineered mutant exhibited similar biological, chemotactic and gag-binding properties. next we started to produce sufficient amounts of the members of our nascent mutant library which were tested with respect to their biophysically behavior as well as to their knocked out chemotactic potency on cells. these experiments included gel electrophoresis and western blot analysis to determine identity and purity; circular dichroism (cd) and chaotrope-induced unfolding to approximate structure; isothermal fluorescence titration (ift); surface plasmon resonance (spr) and isothermal titration calorimetry (itc) to quantify gagbinding affinity and boyden chamber experiments to determine the chemotactic activity. our results show that we are able to tune the gag binding strength along with the gpcr activity of human cxcl which could lead to therapeutic applications in the future. nanodiscs are composed of a nanometer-sized phospholipid bilayer encircled by two a helical, amphipathic membrane scaffold proteins (msps). these particles provide a unique detergent free lipid bilayer model enabling biochemical and biophysical characterization of membrane proteins in a physiologically relevant medium. previously, the largest diameter reported of a nanodisc assembled using msps was about - nm. here we present a method to create large nanodiscs (up to nm in diameter) assembled with covalently circularized msps (cmsp). we can observe the homogeneity in nanodiscs diameter as a narrow distribution using negative-stain em. using our method, we have created nm nanodiscs and used them to study poliovirus ( nm diameter) entry and rna translocation. a nm nanodisc is sufficiently large to accommodate multiple copies of the cd receptor (also known as the poliovirus receptor), and has enough surface area to act as a surrogate membrane for the rna translocation complex during viral uncoating. the nm nanodiscs functionalized with the his-tagged ectodomain of poliovirus receptor, cd , were generated by adding lipids derivatized with a nta nickel- chelating head group to the lipid mixture during nanodisc assembly. cd receptor was added to the already assembled nanodiscs and incubated for minutes at room temperature. the receptordecorated nanodisc complex was purified by size exclusion chromatography. the purified complex was then incubated with poliovirus for minute at c, and then heated to c for minutes to initiate receptor-mediated viral uncoating. virus binding to nanodisc-cd complex and subsequent insertion of viral components into and across the membrane were confirmed by negative-stain electron microscopy (figure c) . to obtain a high-resolution structure for the rna translocation complex we conducted single-particle cryo-em studies using a polara f microscope. unlike liposomes, generating a reconstruction of samples containing nanodiscs is less complicated since the nanodiscs are more homogenous in size, and allow for thinner ice. also, the viral rna can be visualized more easily. the method for making large nanodiscs as well as the negative stain and cryo-em data will be will be presented and discussed. parametric design of alpha-helical barrels and pore-like assemblies with very high thermodynamic stabilities computational design of novel protein structures and enzymes with new functions is a promising tool to create superior biological materials with tailor-made properties, new pharmaceuticals, complex fine chemicals or renewable fuels. it also challenges our understanding of protein folding, protein evolution, molecular recognition and catalysis. here we present a procedure for designing proteins with backbones produced by varying the parameters in the crick coiled-coil generating equations [ ] . combinatorial design calculations using the software suite rosetta identify low energy sequences for alternative helix supercoil arrangements. after that, loop modeling is applied to connect the designs with lowest energy. the extent to which the designed sequences encode the designed structures is evaluated using large-scale structure prediction calculations, as well as symmetric and asymmetric protein-protein docking calculations. subsequently, synthetic genes are generated for sequences that converge strongly on the designed structure for experimental characterization. we applied this approach to monomeric three and four helical bundle structures as well as a pentameric five-helix bundle structure using idealized coiled-coil geometries [ ] . recently we expanded this approach to higher complexity backbones, which resulted in the de-novo design of monomeric, antiparallel six-helix bundles with untwisted, left-and right-handed geometries. circular dichroism (cd), size-exclusion coupled multi-angle light scattering measurements (sec-mals), negative stain electron micrographs (em) and small angle x-ray scattering (saxs) of these designs suggest that they indeed form the designed structures. in addition, we used rosetta protein-protein interface design functionality to computationally design oligomers out of our previously published three and four helix bundle structures to generate self-assembling pore-like structures with the potential use as channels or transporters. again, experimental validation of these designs by cd, sec-mals, em and saxs show that the designs are correct. we are currently undertaking further structural investigation of all these designs by x-ray crystallography. the designs described above can act as templates for protein or small molecule binding, holding a catalytic machinery or for scaffolding enzymes in reaction cascades. some of these applications are currently under investigation, including a self-sufficient redox system employing two copper-centers, binding of heme-moieties as a prosthetic group and tailoring the pore-like geometries to be used in nanopore sequencing. university of washington, university of california, san francisco, repeat proteins are an example of how evolution proceeds by building on existing structures and functions, but also a source of modular protein scaffolds for molecular recognition and biomaterials. however, it is unclear whether the limited number of folds and families that we know today is the result of the intrinsic limitations of polypeptide chains or the consequence of the path followed by evolution. we explored this hypothesis by computational design of repeat proteins based on modular units formed by two alpha helices and two loops of variable lengths, without relying on information from available repeat protein families. the automated sampling of the conformational space resulted in a large number of architectures from which de novo designs were selected for experimental characterization. % of the proteins were stable up to c and monodisperse and designs were structurally validated by small angle x-ray scattering. crystal structures were solved for of them, with root mean square deviation from the models between . Å and . Å. the designs differ from known proteins both at the sequence and structure levels and cover a broader range of geometries than observed in naturally occurring repeat protein families, indicating that existing architectures represent only a small fraction of what can be achieved. our results show that it is possible to expand the range of repeat protein architectures beyond the naturally occurring families, and that computational design can provide new scaffolds and enable the design of proteins tailored for specific applications. the serpin family of proteins consists of over members, all with a highly conserved native structure that is metastable ( ). serpins use this metastability to control the activity of proteases, via a specific inhibitory process. the serpin binds to its target protease through specific residues within the reactive centre loop, the protease cleaves the loop and results in a large conformational change causing the protease to become distorted and catalytically inactive whilst the serpin becomes much more stable ( , , ) . the metastable nature of aat is therefore required to facilitate the rapid and gross conformational changes required for its inhibitory function ( , ) . several disease-causing mutants of aat have been identified, the most common of them being the z-variant ( ). the z-variant has an increased propensity to polymerize in the endoplasmic reticulum of hepatocytes leading to cell death and liver damage ( ) . during the past fifteen years, many groups have unsuccessfully screened a number of serpins and a vast range of solution conditions to identify a combination of serpin and conditions that will enable the folding reaction of a serpin to be characterized. we have now taken an alternative approach and designed a synthetic "model" serpin that folds reversibly to its native state. in order to do this, we used a consensus design approach, analysing a sequence alignment of serpin sequences and determining the prevalent amino acid residue at each position, we termed this serpin conserpin (consensus serpin). here we present the structural, biophysical and functional characterisation of conserpin. combined crystallographic and folding studies reveal the characteristics of conserpin that likely dictate its unique stability and folding behaviour, whilst retaining activity as a serine protease inhibitor. the development of enhanced protein binding scaffolds is a key for engineering protein inhibitors and biosensors with advanced characteristics. utilizing the structural variability and designability of repeat proteins offers a means for designing protein binders where the overall shape is customized to optimally match a target molecule. we developed a computational protocol for the design of repeat proteins with a predefined geometry. by combining sequence optimization of existing repeats and de novo design of capping structures, we designed leucine-rich repeat (lrr) proteins where the building blocks assemble into a novel structure. the suggested design procedure was validated by engineering an artificial donut-like ring structure, which is constructed from ten self-compatible repeats. characterization of several designed constructs further suggests that buried cysteines play a central role for stability and folding cooperativity in certain lrr proteins. this effect could provide a means for selectively stabilizing or destabilizing specific parts of an lrr-based protein binder. the computational procedure may now be employed to develop repeat proteins with various geometrical shapes for applications where greater control of the interface geometry is desired. engineering apobec g enzymes for altered specificity and processivity louis scott , muhammad razif , aleksandra filipovska , , oliver rackham , harry perkins institute of medical research, school of chemistry and biochemistry, the university of western australia apobec g (a g) is a host-encoded protein involved in the defense against hiv- and other retroviral infections. a g is a cytidine deaminase with a ' to ' processive nature, causing targeted c to t mutations along a dna strand. the catalytic and processive activity of a g leads to the hypermutation of nascent retroviral cdna, resulting in premature termination codons and dysfunctional proteins. ultimately, the action of a g inhibits viral replication. the ability of a g to jump and slide along a dna strand, deaminating at targeted sequences, makes it an interesting candidate for protein engineering. engineered a g enzymes for increased activity, altered specificity, and altered processivity are attractive options for expanding the dna modifying enzyme toolbox. mutation of catalytic residues, residues thought to affect its processive nature and those thought to be involved in target recognition, can create novel a g enzymes. using structure guided selection, residues in key functional sites that are amiable to mutation will be chosen. individuals from the resulting libraries of mutants will be selected by directed evolution for desired characteristics. the resulting a g enzymes will be examined for the relationship between their structure and function. such engineered a g enzymes could be targeted to catalyse the reversion of deleterious genetic mutations. furthermore, engineered a g enzymes could be used in mutational studies that call for targeted deamination along a dna strand, or mutational studies that call for unspecific and high throughput dna deamination. engineering porous protein crystals as scaffolds for programmed assembly thaddaus huber , luke hartje , christopher snow a key motivation for nano-biotechnology efforts is the creation of designer materials in which the assembly acts to organize functional domains in three dimensions. crystalline materials are ideal from the validation perspective because x-ray diffraction can elucidate the atomic structure. relatively little work has focused on engineering protein crystals as scaffolds for nanotechnology, due to the technical challenges of coaxing typical proteins into crystallizing, and the likelihood of disrupting the crystallization process if changes are made to the monomers. we have circumvented these limitations by installing guest protein domains within engineered porous crystals ( nm pore diameter) that have been rendered robust using covalent crosslinks. the retention of the scaffold structure despite changes to the solution conditions and macromolecule uptake can be validated through x-ray diffraction. we have engineered scaffold crystals for the non-covalent and covalent capture of guest macromolecules. by controlling the reversible loading and release, we can prepare "integrated" crystals with spatially segregated guest loading patterns. as assessed using confocal microscopy, such host-guest crystals are highly stable. ultimately, the resulting crystals may serve as a robust alternative to dna assemblies for the programmed placement of macromolecules within materials. engineering ultrasensitive protein probes of voltage dynamics for imaging neural activity in vivo francois st-pierre , , michael pan , , helen yang , xiaozhe ding , , ying yang , , thomas clandinin , michael lin , department of bioengineering, stanford university, department of pediatrics, stanford university, nervous systems encode information as spatiotemporal patterns of membrane voltage transients, so accurate measurement of electrical activity has been of long-standing interest. recent engineering efforts have improved our ability to monitor membrane voltage dynamics using genetically encoded voltage indicators. in comparison with electrophysiological approaches, such protein-based indicators can monitor many genetically defined neurons simultaneously; they can also more easily measure voltage changes from subcellular compartments such as axons and dendrites. compared with genetically encoded calcium indicators, voltage sensors enable a more direct, accurate, and rapid readout of membrane potential changes. however, several challenges remain for in vivo voltage imaging with genetically encoded indicators. in particular, current voltage sensors are characterized by insufficient sensitivity, kinetics, and/or brightness to be true optical replacements for electrodes in vivo. as a first step towards addressing these challenges, we sought to develop new voltage indicators that further improve upon the performance of the fast voltage sensor accelerated sensor of action potentials (asap ). in asap , voltage-induced conformational changes in a natural voltage-sensing domain perturb the fluorescence emission of a covalently linked green fluorescent protein (gfp). using a structurebased approach to guide mutagenesis, we discovered several amino acids that tune the kinetics and voltage sensitivity of asap . these residues are not only located in the voltage-sensing domain, but also in the fluorescent protein and in the linkers bridging sensing domain and gfp. our most improved variant, asap , exhibits improved sensitivity to voltage transients such as neuronal action potentials and subthreshold depolarizations. we sought to characterize the ability of these new voltage sensors to monitor neural activity in vivo using laser-scanning two-photon microscopy, a technique that allows imaging with lower autofluorescence and deeper tissue penetration. we report that asap sensors were able report stimulus-evoked voltage responses in axonal termini of the fly visual interneuron l . asap sensors enabled voltage imaging with dramatically improved temporal resolution compared to three recently reported calcium and voltage sensors. overall, our study reports novel voltage indicators with improved performance and highlights how specific amino acids can tune the performance of a proteinbased fluorescent sensor. we anticipate that these results will pave the way for further engineering of voltage sensing proteins, and that our new sensor asap will facilitate current and future efforts to understand how neural circuits represent and transform information. assembly of armadillo repeat proteins from complementary fragments erich michel , randall watson , martin christen , fabian bumback , andreas pl€ uckthun , oliver zerbe demonstrated that complementary fragments of a designed consensus armadillo repeat protein (armrp) recognize each other [ ] . the two fragments ym : ma, in which y, m and a denote the n-cap, internal repeats and the c-cap, respectively, form a : complex with a nanomolar dissociation constant, which is essentially identical to the crystal structure of the continuous ym a protein. we further demonstrate that structurally intact armadillo repeat protein complexes can be reconstituted from fragments obtained at various split sites -essentially after every repeat but also within repeats. the fragments display variable affinities towards each other, depending on the split site. the low affinity of some complementary pairs can be dramatically increased upon addition of peptide ligands. while a number of proteins are known that can be reconstituted from fragments we believe that the fact that armadillo repeat proteins can be reconstituted from various complementary fragments is novel and opens new interesting perspectives and applications in biochemistry. a reliable method for generating optically controllable proteins would enable researchers to interrogate protein functions with high spatiotemporal specificity. we recently engineered a tetrameric fluorescent protein, dronpa n, that undergoes light-induced monomerization, then developed a general architecture for lightinducible proteins based on this light-induced transition. we created proteins whose active sites were blocked by fused dronpa n domains in the dark, but would become unblocked by light. here we present further two extensions to this concept that together enabled the generalization of this method to additional classes of proteins. first, we engineered a photodissociable dimeric dronpa (pddronpa) with tunable affinity, faster photoswitching speed, and decreased level of protein aggregation, enabling better performance of fusion proteins. second, we introduce the concept of caging a protein active site by insertion of dronpa domains into loops rather than strictly at the protein termini. we use the pddronpa system to impose optical control on kinases and the cas endonuclease. the resulting light-inducible mek kinase, raf kinase, and cas endonuclease showed high caging efficiency of protein activities in the dark, and robust protein activation upon light illumination. we believe that our efforts on further improving and generalizing this method would bring the power and benefits of light control to a broad community of biologists. exploring the evolution of folds and its application for the design of functional hybrid proteins saacnicteh toledo patiño , birte h€ ocker the structural diversity of proteins may appear endless, nevertheless even large protein complexes can be decomposed into protein domains and smaller sub-domain sized fragments. only recently, we could identify such fragments employing sequence-based comparisons of different folds, as the tim-barrel and the flavodoxin-like fold (farias-rico et al., ) . as an extension of this work, we compared all a/b proteins and identified several fragments shared by different folds illustrating how nature may have achieved structural and functional diversity from a reduced set of building blocks. inspired by this combinatorial concept, we searched for homologous fragments bearing active sites to engineer a functional fold-chimera. we extracted the vitamin-b binding part from methylmalonyl coa mutase, which belongs to the flavodoxin-like fold (fl) and used it to replace the corresponding fragment in uroporphyrinogen iii synthase, which belongs to the hemd-like fold (hdl). the new hybrid resulted in a stable and well-folded protein whose structure was determined by x-ray crystallography. moreover, cobalamin-binding function was successfully transferred to the new protein from the fl parent, which shows the advantage of using this approach for the design of new functional proteins. in addition, profile alignments revealed sequence and structural evidence that suggested an evolutionary path for hdl from fl by gene duplication. to test this hypothesis, we expressed a modified c-terminal half of uroporphyrinogen iii synthase and solved its structure by nmr spectroscopy, thereby confirming the predicted fl architecture. altogether, our approach facilitates the detection of common ancestry among different folds contributing to our understanding of protein development. furthermore, our results show how new complex proteins can be designed using fragments of existing proteins that serve as building blocks in a lego-like manner. we believe that combining fragments containing existing properties will provide a successful method for the design of novel functionalities in the future. [ ] . the active site cysteine plays a key role in the reaction mechanism and we investigated this residue in more detail by exchanging this moiety with selenocysteine (sec) and homocysteine (hcy). the sortase mutants were generated by semisynthesis using expressed protein ligation (epl). the resulting cys-, sec-and hcy-sortase enzymes were characterized and showed a moderate - -fold reduction of activity for sec-sortase. the activity of hcysortase was barely detectable with less than % of wildtype activity. the alkylation efficiency of the active site nucleophiles correlated with the expected pka values of sec, cys and hcy. analysis of the ph dependency of the transpeptidation reactions showed that the activity optimum of sec-sortase was shifted towards more acidic conditions. these investigations provide further insights into the reaction mechanism of sortase a and the semisynthetic enzymes may provide new tool for further biochemical studies. propanediol oxidoreductase from escherichia coli (fuco) uses nadh/nad as cofactors to catalyze the conversion of s-lactaldehyde to s- , -propanediol and vice versa. fuco is an attractive enzyme in the search for possible biocatalysts producing a-hydroxy aldehydes, which are important for the synthesis of natural products and synthetic drugs. enzymes catalyzing these types of reactions are unique in catalytic power and stereoselectivity. the usage of fuco in synthetic industry is limited by the restricted substrate scope, which makes fuco inactive with larger phenyl-substituted alcohols. we used reengineering and directed evolution to enable fuco to catalyze the regio-and enantioselective oxidation of arylsubstituted vicinal diols, such as phenylpropanediols, into a-hydroxy aldehyde products. we mutated amino acids considered to restrict the entry into the active site, and modeled the mutants that were most active with the substrates phenylacetaldehyde and s- -phenyl- , -propanediol and performed docking studies with them. as expected, our experimental and in silico results show that the mutations enlarge the active site cavity and enable the mutant enzymes to accommodate the new substrates. we also found specific amino acids in the active site, which need to be conserved to allow the substrates to make stabilizing interactions. interestingly, an asparagine residue makes the mutant enzymes able to discriminate between phenylacetaldehyde and s- -phenyl- , -propanediol. in conclusion, we successfully re-engineered the specialist enzyme fuco to accept also bulkier molecules as substrates, thereby making it more useful for industrial purposes. one way to gain insight into the sequence-structure-function relationship in proteins is to de novo design artificial proteins. despite impressive successes in de novo protein design, designing a folded protein of more than amino acids still remains a challenge. using this approach, an idealized (beta/ alpha) fold protein was designed leading to the production of a protein of amino acids (octarellin v). this protein showed a low solubility and stability. through directed evolution we produced a soluble variant, octarellin v. . the biophysical characterization of octarellin v. shows a well folded monomeric and thermostable protein with a tm over c. however, after several screenings, we could not find crystallization conditions for this protein. as an alternative, we decided to co-crystallize octarellin v. with a protein partner that helps the crystallization process. we used protein partners: alpha-reps and nanobodies. the first one is characterized to interact through a large surface contact, whereas the second is characterized to recognize an specific small epitope. crystallization of both complexes was performed successfully by vapor diffusion and the structures were solved. the experimental structures correspond to the first for an artificial protein of this size and it will allow to criticize the computational design of the octarellin v. generation of synthetic antibodies against membrane proteins in nanodiscs for use in structural biology methods. here, we describe a robust strategy for generating a class of high performance antibodybased affinity reagents that have proven useful in determining the structures of relevant functional states of membrane proteins. these reagents are fab fragments that are generated by phage display from fully synthetic libraries and are called synthetic antibody fragments, or sabs. we have developed phage display sorting strategies that can trap a desired conformational state, making it accessible to structural analysis, or target a particular epitope on the protein surface. however, to maximize this technology for membrane proteins, several limitations of phage display sorting in detergent formats had to be overcome, the greatest being that using detergents can produce non-native conformational biases. we sought to address these limitations by embedding membrane proteins into nanodiscs, soluble lipidfilled discoidal particles, to better mimic the native membrane environment. nanodiscs stabilize the membrane protein and allow it to respond to conformation-inducing stimuli such as ligands, ions and ph during phage display selections. we have established and validated an improved protocol using two membrane protein systems: ) mj , an archaeal membrane protein of unknown function, and ) cora, a pentameric magnesium ion channel. using mj , we compared the nanodisc protocol with the standard method performed in detergent, and as an important byproduct, we characterized the influence of the membrane protein environment on the apparent affinity of sabs to their cognate antigen. using cora, we developed a more sophisticated sorting strategy resulting in a variety of sabs specific to either the open or closed conformation of the channel. finally, using sabs as crystallization chaperones we obtained the structure of mj at . Å resolution, and crystallized cora in several new conditions. lipocalin-type prostaglandin d synthase (l-pgds) is a member of the lipocalin superfamily, and binds a large variety of small hydrophobic molecules. using this function of l-pgds, we have already reported the feasibility of l-pgds as a novel drug delivery vehicle for the poorly water-soluble drugs [ ] . sn- , -ethyl- -hydroxy-camptothecin, is a semi-synthetic analogue of anti-cancer alkaloid camptothecin that targets dna topoisomerase i. despite of the potent anti-tumor activity, however, sn- was not used directly in a clinical practice due to its poor water solubility. thus, irinotecan hydrochloride (cpt- ), which is the water-soluble prodrug of sn- , is used for the cancer treatment. however, cpt- shows approximately . % cytotoxic activity of sn- against the various cancer cell lines in vitro, and its metabolic conversion rate is % of the original volume of cpt- . here, we show the development of the drug delivery system utilizing l-pgds, which enables a direct clinical usage of sn- . first, we investigated the effect of l-pgds on the solubility of sn- . in the presence of mm l-pgds, the concentration of sn- was . mm, which was , -fold as compared with that in pbs. then, we carried out isothermal titration calorimetry measurements to investigate the detailed binding mode of sn- to l-pgds. as a result, it was revealed that l-pgds binds three molecules of sn- , and the dissocia- control over the sensitivity with which artificial biomolecular receptors respond to small changes in the concentration of their target ligand is critical for the proper function of many cellular processes. such control could likewise be highly useful in artificial biotechnologies in which highly responsive behavior is of value, such as biosensors, genetic logic gates, and "smart" materials and delivery devices. in nature, the control of molecular responsiveness is often achieved using "hill-type" cooperativity, a mechanism in which sequential binding events on a multivalent receptor are coupled such that the first enhances the affinity of the next, producing a steep, higher-order dependence on target concentration. here we use an intrinsic-disorder-based mechanism that can be implemented without requiring detailed structural knowledge to rationally introduce this potentially useful property into several normally noncooperative biomolecules. to do so we fabricate a tandem repeat of the receptor that is destabilized (unfolded) via the introduction of a long, unstructured loop. the loop spatially separates the two sets of the two halves of the binding sites, preventing a complete binding site that enables target molecule binding without prior closure of the loop. thus, the first binding event requires the energetically unfavorable closing of this loop, reducing its affinity relative to that of the second binding event, which, in contrast occurs at a pre-formed site. using this approach we have rationally introduced cooperativity into three unrelated aptamers, achieving in the best of these a hill coefficient experimentally indistinguishable from the theoretically expected maximum. the extent of cooperativity, and thus the steepness of the binding transition, are, moreover, well modeled as simple functions of the energetic cost of binding-induced folding, speaking to the quantitative nature of this design strategy. essential and non-essential amino acid species for an ancestral protein satoshi akanuma the translation system is an essential element for life because it links genetic information embedded in genes to functional molecules, proteins. the modern genetic code, which encodes the standard amino acids (and three terminations) using triplet codons, is shared by most of the extant organisms on the earth. a number of theories have been proposed for the origin and evolution of the genetic code, and these theories suggest that only a fewer amino acids were used in primitive proteins and later the amino acid repertoire gradually increased up to through the course of evolution. if so, one would wonder how many number of and which types of amino acids were involved in the primitive proteins. i have begun to address this issue experimentally. i first resurrected several ancestral proteins and then restricted the amino acid usage of one of the resurrected proteins. i targeted nucleoside diphosphate kinase (ndk) that catalyzes the transfer of a phosphate from a nucleoside triphosphate to a nucleoside diphosphate. ndk may have arisen early because at least one gene that encodes ndk is present in most extant organisms. the first step in the reconstruction of ancestral ndk sequences is to prepare multiple amino acid sequence alignments using homologous sequences of ndk from extant species. then, phylogenetic trees were built. ancestral sequences of ndk that represent the last common ancestors of archaea and of bacteria were reconstructed using the information contained in the predictive phylogenetic trees. the reconstructed ancestral kinases are extremely thermally stable [akanuma et al., ] . then, using the most thermally stable ancestral ndk, arc , as the starting molecule, i restricted its amino acid usage. arc does not contain any cysteine residue and therefore consists of amino acid species. i completely replaced one of the amino acid species by other amino acid species and thus created proteins each of which consisted of amino acid species. then, i evaluated the stabilities and activities of the resulting arc variants to assess the individual contributions of the amino acid species. as the result, i found that the amino acid species do not equally contribute to the stability and activity of arc and that some amino acid species can be easily lacked but others are important or essential for its stability and function. the result clearly shows that the full amino acid species are not necessarily essential and supports the hypothesis that proteins in the early stage of evolution were made from a reduced amino acid set. the protein surface recognition for protein-protein interactions (ppi) is involved in signal transduction, immune reaction, and creation of the nanostructures in living cells. the methods for rational designing of ppi that could provide non-antibody scaffolds and nanostructured materials are required for the therapeutic and nanotechnological applications. although there have been some successful rational designs with computational methods, it is still difficult to design freely the ppi onto arbitrary proteins. the reason for this limitation is decreased solubility in the designed protein due to the additional hydrophobic residues in order to drive ppi. another reason is a limited set of design modes by which proteins can interact, because the target proteins have individual surface structures. therefore, many methods of constructing an interface for numerous target scaffold proteins without loss of their solubility are necessary. surface exposed a-helices are often observed in natural globular proteins. moreover, there are many examples for naturally occurring oligomeric proteins where an a-helix from each subunit interacts to form an intermolecule coiled coil. further, the works related to designing of artificial helical bundle reported by the several other groups have provided information about how to generate and tune the interaction between a-helices. therefore, a surface exposed a-helix would be a good target for designing a de novo interface onto the scaffold protein. here we engineered two different proteins, sulerythrin and cys-larfh, to form the cys-larfh-sulerythrin dimer-cys-larfh heterotetramer via an intermolecular helix-helix interaction. wild-type sulerythrin forms a dimeric eight-helix bundle. cys-larfh is a designed monomeric protein that forms four-helix bundle containing interhelical s-s bonds. both sulerythrin and cys-larfh are extremely thermostable. to design protein-protein interfaces onto the individual proteins, we first introduced six leucines to the two a-helices of sulerythrin and three leucines to a a-helix of cys-larfh. as expected, the introduction of the hydrophobic amino acids reduced their solubilities. to recover the solubility, we then introduced six aspartates or glutamates around the hydrophobic surface of the sulerythrin (hereafter referred to as l d or l e). similarly, three arginines were introduced around the artificial hydrophobic surface of the cys-larfh (hereafter referred as iv- l r). the solubilities of the mutants with the hydrophobic interface and additional charged residues were recovered their solubility. in addition, the sulerythrin mutants l d and l e exist mainly as dimer. the cys-larfh mutants iv- l r, also exists as monomer. we then examined the interaction between l e or l d and iv- l r. a pull-down experiment, in which co beads bound to either his-tagged cys-larfh and iv- l r were used to pull down wild-type sulerythrin, l d, or l e, demonstrates that l d or l e specifically interacts to iv- l r. furthermore, when analysed by size exclusion chromatography, the dominant peaks of the mixture of l d and iv- l r appeared at the volume expected for the heterotetrameric complex. thus we successfully created the de novo ppi by using a very simple concept involving hydrophobic interaction in combination with charge interactions. in vitro selection of liposome anchoring peptide by cdna display naoto nemoto , ryoya okawa , yuki yoshikawa , toshiki miyajima , shota kobayashi a liposome-anchoring peptide (la peptide) was selected against liposomes composed of dioleoyl-snglycero- -phosphocholine (dopc) by in vitro selection using cdna display method. the selected peptide la peptide consists of the n-terminal region (hydrophobic) and the c-terminal region (basic) in a characteristic manner. thus, la peptide was synthesized chemically and the interactions between la peptide and particular types of liposomes were investigated and confirmed by confocal laser scanning microscopy. designing of a novel platinum-binding amino acid sequence on a protein surface asumi kaji , hiroya niiro , satoshi akanuma , tetsuya uchida , akihiko yamagishi designing of a novel interaction between a metal and a protein is a key to create hybrid materials between organic and inorganic materials. for example, in a glucose biosensor, which is widely used for measuring glucose concentration in blood, glucose oxidoreductase molecules are immobilized on a platinum electrode by polyacrylamide gel. a metal-binding tags that is added to the n-or cterminus of a protein is also used for fix the protein to a metal. however, a technique to create a metal binding site on a desired position of a protein has not been invent. if such a technique would be established, the technique would contribute to developing and improving biosensors and to producing new bionanoelectronic materials. in this study, we created a platinum-binding site on a loop located at a protein surface. we used an artificial protein, larfh, that had been synthesized by connecting four identical alpha helices originated from the c-terminal segment of the escherichia coli lac repressor with three identical loops. we randomized the ser, gly, gln, gly, gly, ser sequence within one of the inter-helical loops and then selected for binding to platinum by a t phage display system. most of the selected larfh variants contained the tyr, lys, arg, gly, tyr, lys (ykrgyk) sequence in the randomized segment. we then evaluated the affinity of the larfh variant to platinum by means of quartz crystal microbalance analysis. we found that the variant binds to platinum more strongly than does the original larfh. in the annual symposium, we will also report about the affinity of the isolated ykrgyk sequence to platinum and about the crucial role of the first tyrosine in binding to platinum. engineering of an isolated p a subunit of pi ka permits crystallization and provides a platform for structure-based drug design pi ka remains an attractive target for development of anticancer targeted therapy. a number of p a crystal structures in complex with the nsh -ish fragment of p regulatory subunit have been reported, including a few small molecule co-crystal structures, but the utilization of this crystal form is limited by low diffraction resolution and a crystal packing artifact that partially blocks the atp binding site. taking advantage of recent data on the functional characterization of the lipid binding properties of p a, we designed a set of novel constructs allowing production of isolated stable p a subunit missing the adapter binding domain (abd) and lacking or featuring a modified c-terminal lipid binding motif. while this protein is not catalytically competent to phosphorylate its substrate pip , it retains ligand binding properties as indicated by direct binding studies with a pan-pi ka inhibitor. additionally, we determined apo and pf- bound crystal structures of the p a ( - ) subunit at . Å and . Å respectively. comparison of isolated p a ( - ) with the p a/p complex reveals a high degree of structural similarity, which validates suitability of this catalytically inactive p a for iterative sbdd. importantly, this crystal form of p a readily accommodates the binding of non-covalent inhibitor by means of a fully accessible atp site. the strategy presented here can be also applied to structural studies of other members of pi kia family. identification of structural determinants involved in the differential conformational changes of ef-hand modules emma liliana arevalo salina , joel osuna quintero , humberto flores soto , gloria saab rinc on instituto de biotecnolog ıa, universidad nacional aut onoma de m exico identification of structural determinants involved in the differential conformational changes of ef-hand modules calcium signals are regulated by several proteins, most of which belong to the ef-hand superfamily. the ef-hand motif is formed by a helix-loop-helix that binds calcium through its loop . these motifs occur in adjacent pairs, forming a single globular domain which is the basic structural and functional ca binding unit. the proteins in this family can be classified as calcium sensors or modulators, according with their function. the first group undergoes a major conformational change upon calcium binding, while the second one remains practically unchanged , . to explain the biophysics behind the different behavior of these proteins upon ca binding, we have sought to identify structural determinants that could account for these features, especially for the difference in the conformational change. we examined the primary structure from two ef-hand motifs: a sensor ef-hand from chicken troponin c (sciii) and a modulator ef-hand from bovine calbindin d k (clbn). the main differences were in the binding ca loop and a group of charged residues in the h helix of the modulator ef-hand. then, we constructed chimeric clbn motifs containing the loop or the loop and h from sciii motif (h clbnsciii and h h clbnsciii). these constructs were analyzed using a reporter system that discriminates ef-hand-sensor motifs from signal-modulators at the single-motif level. this reporter is based on the fusion of genes codifying for the ef-hand and the prephenate dehydrogenase from e. coli (tyra), a protein which is active only as a dimer. isolated ef-hand motifs have the ability to homo-dimerize and in the fusion can stabilize and activate tyra. the sensor motif exhibits a conformational change by binding calcium and in doing so, destabilizes the dimeric conformation of tyra and virtually eliminates its activity. in the modulators, on the other hand, the rather small conformational change only gives rise to a decreased tyra activity. both constructed chimeric ef-hand fusions showed a loss of activity upon ca binding, indicating that the residues connector of the sensor ef-hand from sciii is sufficient to confer the conformational change. in addition we used cd and extrinsic fluorescence spectroscopies to analyze any conformational change in the h h clbnsciii and h clbnsciii isolated modules, not finding any difference between the ca free and ca bound chimeras, suggesting that the change in activity of the reporter protein is due to a change in the orientation of the helices in the ef-hands induced by calcium. the effect of ca binding of the chimeras in the context of the entire calbindin d k protein is under investigation. mapping side chain interactions at the n-and c-termini of protein helices nicholas e newell, independent researcher interactions involving one or more amino acid side chains near the ends of protein helices stabilize helix termini and shape the geometry of the adjacent loops, contributing to supersecondary structure. side chain structures that have been identified at the helical n-terminus include the asx/st n-caps, the capping box, and hydrophobic and electrostatic interactions. at the cterminus, capping is often achieved with main-chain polar groups, (e.g. the schellman loop), but here also particular side chain motifs clearly favor specific loop geometries. key questions that remain concerning side chain interactions at helix termini include: ) to what extent are helix-terminal motifs that include multiple amino acids likely to represent genuine cooperative interactions between side chains, rather than chance alignments? ) which particular helix-terminal loop geometries are favored by each side chain interaction? ) can an exhaustive statistical scan of a large, recent dataset identify new side chain interactions at helix termini? in this work, three analytical tools are applied to answer the above questions for both n-and c-termini. first, a new perturbative least-squares d clustering algorithm is applied to partition the helix terminal structures in a large ( , example), low-redundancy pdb dataset by loop backbone geometry. the clustering algorithm also generates a set of structural exemplars, one for each cluster, that is used to represent the most important loop geometries at each terminus. next, cascade detection (newell, bioinformatics, ), an algorithm that detects multi-amino acid cooperativities by identifying overrepresented sequence motifs, is applied to each cluster separately to determine which motifs are most important in each loop geometry. finally, the results for each motif are displayed in a capmap, a d conformational heatmap that depicts the distribution of motif abundance and overrepresentation across all loop geometries by projecting these quantities onto the structural exemplars generated by clustering. the capmap reveals the loop conformations most favored by a motif. actual structures from the clusters corresponding to these favored conformations are then examined in a structure browser to characterize the side chain interaction associated with the motif. this work identifies a 'toolkit' of side chain motifs which are good candidates for use in the design of synthetic helix-terminal loops with specific desired geometries, because they are used in nature to support these geometries. highlights of the analysis include determinations of the favored loop geometries for the asx/st motifs, capping boxes, big boxes, and other previously known and unknown hydrophobic, electrostatic, h-bond, and pi-stacking interactions. a goal of future work is to make these results available in a structurally-addressable database that would enable researchers to immediately retrieve the side chain interactions most compatible with a desired loop geometry. generation of fluorescent protein-tagged gp mutants to analyze the intracellular distribution of hiv- envelope protein shuhei nakane , zene matsuda green earth research center, green earth institute co., ltd., res ctr for asian infect dis, inst of med sci, the univ of tokyo, lab of struct virol and immunol, institute of biophysics, cas hiv- is a causative enveloped virus of aids. its envelope protein (env) has two non-covalently associated subunits, gp and gp , which are proteolytically processed from a gp precursor. the gp subunit is a surface protein and gp is a transmembrane protein. the gp and gp subunits are responsible for the receptor recognition and membrane fusion, respectively. the cytoplasmic tail (ct) of gp is about amino acids long and is believed to play a critical role in intracellular trafficking of env. to visualize dynamic trafficking, the c-terminus of gp has been tagged with fluorescent proteins such as gfp. however, tagging of ct may cause a concern to affect the interactions between the ct and cellular proteins that are involved in intracellular trafficking. to avoid this problem, here we tried to insert gfpopt, a gfp variant, into five variable regions of gp . we have analyzed the phenotypes of env mutants, such as the cell surface expression, processing of gp , membrane fusion activity, and virion incorporation. among variable regions of gp , the v region was most sensitive to insertion. v /v region was less sensitive than v . consistent with the recently revealed structure, exteriorly located v and v were highly tolerant to insertion. we used the mutant with the gfp insertion in the v region to analyze the intracellular distribution of env with and without ct. we found that deletion of ct increased the presence of vesicles colocalized with late endosome markers. this is consistent with the hypothesis that the ct region contains a motif regulating intracellular trafficking. our results showed that env with gfpopt insertion in its gp subunit is a useful tool for the study of intracellular dynamics of hiv- env. these mutants would also be useful to trace the fate of virus particles during infection. pi- ngs-guided phage panning: comparison to conventional panning strategy buyung santoso , dorain thompson , john nuss , john dwyer phage display is a powerful tool for generating binders to a target protein. multiple rounds of panning with conventional phage display strategies typically result in a number of hits, which are then individually screened using in vitro assays. clones screened at this stage are a combination of specific binders, sequences that are selected due to amplification bias, and non-specific binders. if the number of specific clones is low relative to the non-specific sequences, a larger number of clones have to be screened to ensure sufficient diversity of early leads. with the advent of next generation sequencing (ngs) technology, we aim to test whether we can increase the diversity of specific hits and decrease the number of non-specific sequences. in our experiment, four rounds of conventional panning produced ten peptide binders to target protein. ngs analysis after two rounds of panning was done in parallel, yielding more than ten thousand sequences, ranked by abundance. all ten binders from conventional panning were found in the top most abundant ngs hits. more importantly, additional hits were found in ngs analysis but not in conventional panning, highlighting this strategy as a promising alternative for hit discovery with the significant upside of more diverse and higher affinity leads. numerous processes in pharmaceutical development, including construct screening, structural genomics, protein engineering and expression optimization among others, require the use of higher throughput plasmid dna purification. the majority of issues encountered in mini, midi, and maxiprep purification kits involve flocculate removal following alkaline lysis, and there is currently no easy way to produce large amounts of plasmid dna without the addition of complicated and time consuming clarification steps. the existence of a hassle-free automated system that is not restricted by sample size would significantly help in cutting time and costs during the initial processing steps of plasmid purification. the autoplasmid mea instrument provides a fully automated solution to traditional problems faced in plasmid purifications, allowing mini, midi, and maxiprep plasmid purifications to be performed on a single instrument. the data presented here on plasmid yield, purity, and suitability for sequencing and transfection/transformation illustrate a new strategy for automated plasmid preps. by eliminating traditional clarification methods, cell culture volumes between - ml can be processed leading to yields ranging from - lg. this flexible system was developed in order to satisfy a wide variety of concentration and yield requirement, while eliminating the time consuming steps previously needed to obtain similar results. the ability to perform fully automated mini, midi, and maxi plasmid preps on one instrument allows for a customized all-in-one purification system that is not restricted by traditional clarification methods, eliminating manual intervention, and streamlining the purification process. the modular nature of protein architectures suggests that proteins have evolved through duplication and fusion to give rise to modular, often symmetric forms, which later diversified under the influence of evolutionary pressure. we have developed a computational protein design method termed reverse engineer evolution (re volution) to create symmetrically self-assembling protein building blocks. we have used this method to design a perfectly symmetric b-propeller protein called pizza. subsequently, we have engineered a metal binding site into this pizza protein. this new pizza variant carries two nearly identical domains per polypeptide chain, and forms a trimer with three-fold symmetry. the designed single metal ion binding site lies on the symmetry axis, bonding the trimer together. two copies of the trimer associate in the presence of cadmium chloride in solution, and high resolution x-ray crystallographic analysis reveals a nano-crystal of cadmium chloride, sandwiched between two trimers of the protein. this nano-crystal, containing seven cadmium ions lying in a plane and twelve interspersed chloride ions, is the smallest reported to date. our results indicate the feasibility of using rationally-designed symmetrical proteins to biomineralize nano-crystals with applications in bionanotechnology. bacillus licheniformis trehalose- -phosphate hydrolase structures suggest keys to substrate specificity chwan-deng hsiao , min-guan lin , long-liu lin , yuh-ju sun institute of molecular biology, academia sinica, department of applied chemistry, national chiayi university, depaertment of life science, national tsing hua university trehalose- -phosphate hydrolase (trea) of the glycoside hydrolase family (gh ) catalyzes the hydrolysis of trehalose- -phosphate (t p) to yield glucose and glucose- -phosphate. products of this reaction can be further metabolized by the energy-generating glycolytic pathway. here we present the crystal structures of bacillus licheniformis trea (bltrea) and its r q mutant complexed with p-nitrophenyl-a-d-glucopyranoside (r q/ ppng) at . Å and . Å resolution, respectively. the overall structure of bltrea is similar to other gh family enzymes. however, detailed structural comparisons revealed that the catalytic groove of bltrea contains a long loop adopting a different conformation from those of gh family members. unlike the homologous regions of bacillus cereus oligo- , -glucosidase (bcogl) and erwinia rhapontici isomaltulose synthase (nx- ), the active site surface potential of bltrea exhibits a largely positive charge, contributed by the four basic residues his , his , lys and lys . mutations at these residues resulted in significant decreases of bltrea enzymatic activity. strikingly, a hhlk motif and the lys residue played critical roles in bltrea substrate discrimination. crystal structure of engineered lrrtm synaptic adhesion molecule and a model for neurexin binding anja paatero , katja rosti , alexander shkumatov , cecilia brunello , kai kysenius , prosanta singha , henri huttunen , tommi kajander institute of biotechnology, university of helsinki, helsinki, finland, dept of pharmaceutical and pharmacological sciences, ku leuven, leuven, belgium, neuroscience center, university of helsinki synaptic adhesion molecules are key components in the development of the brain, and in the formation of neuronal circuits, as they are central in the assembly and maturation of the chemical synapses. several families of neuronal adhesion molecules have been identified such as ncams, neurexins and neuroligins, and in particular recently several leucine rich repeat protein families, e.g. netrin g-ligands, slitrks and lrrtms. the lrrtms form a family of four proteins. they have been implicated in excitatory glutamatergic synapse function, and were specifically characterized as ligands for neurexins in excitatory synapse formation and maintenance. in addition, lrrtm and lrrtm have been found to be ligands for heparan sulphate proteoglycans. we report here the crystal structure of a stability-engineered mouse lrrtm , with a tm c higher than the wild type protein, while retaining its function. we localized the neurexin binding site to the concave surface based on protein engineering, sequence conservation and prior information on the ligand interaction with neurexins, allowing us to propose a tentative model for lrrtm:neurexin interaction compex. cell culture studies and binding experiments show that the engineered protein is functional and capable of forming synapse-like contacts. small angle x-ray scattering data suggests that the wild type protein forms transient dimers, which may have importance for the function. the structural and functional data presented here provide the first structure of an lrrtm protein, and a model for molecular mechanism of lrrtm function in adhesion. computational design of phenylalanine binder olga khersonsky , gil benezer , sarel fleishman recently, abdesign algorithm was developed in our lab for de novo design of antibodies ( ). it is guided by natural conformations and sequences, and exploits the modular nature of antibodies to abstract generate an immense space of conformations, which can be used as scaffolds for design of stable highaffinity binders. we have used abdesign to design a binder of phenylalanine. , antibody scaffolds were obtained by splicing h and l fragments into a template (pdb id brr), and subsequent optimization of vh and vl orientation. phenylalanine binding site, based on native phenylalanine binders, was introduced into the scaffolds with rosettamatch ( ), and the sequences were subsequently optimized by rosetta enzyme design protocol ( ) . designs were experimentally tested by yeast display for binding of biotinylated phenylalanine ligand. several designs were found to bind the ligand, and we plan to further characterize this affinity and improve it using directed evolution techniques. in collaboration with the group of prof. johnsonn, the resulting phenylalanine binder will be incorporated in a bio-luminescent (lucid) sensor for phenylalanine ( ) . phenylalanine monitoring device would be of primary importance for patients with phenylketonuria, a genetic disease with phenylalanine metabolism problem. cold-adapted enzymes are interesting because of their higher catalytic activity compared to mesophilic and thermophilic homologues. alkaline phosphatase (ap) from a psychrophilic vibrio marine bacteria (vap) has an unusual large surface loop that extends from each of its monomers to stabilize a homodimeric structure ( ). in many cold-adapted enzymes, the loop regions are longer compared to proteins of mesophilic organisms and our aim was to study the functional and structural role of this loop. three substitutions (r l, y f and f y) were introduced within the large surface loop as directed by microsecond molecular dynamics (md) simulations. with the r l mutation, two hydrogen bonds were broken that connect the loop to residues on the adjacent subunit, and further two hydrogen bonds broken with the adjacent q . as a consequence, r l displayed a % higher kcat compared with wild-type and a slight decrease in the km value. overall, the catalytic efficient improved by %. the global heat stability (tm) and the active site sensitivity to heat (t %) were reduced by c and c, respectively. md simulations showed that hydrogen bonds to arg are important for longrange communication to the active site. certain rotamers of two important residues in the catalytic site, ser and arg , were favored, presumably toward states more competent for catalysis upon the replacement of arg with leu. in the y f variant, removal of one hydrogen bond between the loop and the other subunit caused a small drop in stability parameters, whereas both kcat and km were reduced by about half, giving similar kinetic efficiency (kcat/km) to that of wild-type. finally, we changed a residue at the root of the large loop (f y) such that one new intersubunit hydrogen bond could form. this variant maintained the wild-type characteristics. in conclusion, removing hydrogen bonds connecting the major loop of one subunit to the protein surface of the other subunit in vap produced higher catalytic activity and this shows functional connections between loop mobility and the active site. our study also demonstrates that interactions between residues in the large disordered loop and the opposite subunit in the dimeric vap are determinants of its stability. thus, we managed to show that loosening of interface contacts between the two vap subunits by replacement of crucial residues provides a way to orchestrate structural and kinetic dynamics in a productive way. the de novo design of artificial proteins arises as a stringent test of our understanding of the relationship between sequence, structure, and function. examples include the design of a four a-helix bundle, a new protein topology called top , and a series of artificial (ba) -barrels called octarellins. however, de novo design has proven difficult for larger proteins with more than amino acids. here we present two methods to generate the backbone and to perform the de novo design of (ba) -barrel proteins through the use of the software rosetta; both have different advantages and limitations. the first method for generating the backbone is knowledge-based, with a first analysis of a non-redundant database of natural (ba) -barrel proteins in order to obtain statistical analysis on preferred secondary structure element length and amino acidic propensities. with this information we use the rosetta cm software to create more than models which are then ranked in term of rosetta energy. the second method is performed with the parametricdesign package of rosetta, in which only geometrical information are requested (number of strands and helices, radius of the b-and a-barrels, degree of inclination, orientation of the side chains, among others). both methods contain a step of loop refinement and multiple steps of sequence design with the package rosetta design, in order to find low scoring amino acid sequences for each of the starting backbone conformations. thousands of models will be generated by both methods and then analyzed in term of sequence similarity, secondary and tertiary structure prediction, and stability by molecular dynamics simulations. the best candidate sequences will be selected for the experimental verification. in order to identify a putative successfully design, we added a metal binding site during the design step. all the proteins will be expressed in e. coli. the solubility of the designed proteins inside bacteria will be determined thanks to the fusion to green fluorescence protein (gfp). solubility, stability, secondary structure, and cooperativity of folding will be assessed for each protein before determination of their three-dimensional structure. construction of protein capsule possessing drugs controlled release ability shota shimizu , masatoshi nakatsuji , keisuke yamaguchi , yuya sano , yuya miyamoto , takashi inui most compounds that exhibit anti-tumor activities are water-insoluble, thus limiting their clinical use. chemical modification of these compounds and the use of solubilizing agents such as organic solvents, surfactants and ph modifiers improve their solubility. however, chemical modification of compounds decreases their potency, and the use of solubilizing agents causes toxicity in many cases. thus, drug delivery systems (dds) for poorly water-soluble anti-tumor drugs which exploit liposomes, cyclodextrins, and lipid nanoparticles have been studied intensely. in these dds, the controlled release of drugs from the delivery vehicle is one of the most important functions. selective release in target cells leads to adequate therapeutic efficacy with few side effects. in our laboratory, we have already demonstrated that lipocalin-type prostaglandin d synthase (l-pgds), an intravital transporter protein, is a novel and valid drug delivery vehicle for sn- , a poorly water-soluble anti-tumor drug. in this study, we generated l-pgds-based protein capsules with a controlled-release function by introducing a disulfide bond into the upper part of the drug-binding cavity of l-pgds. the intracellular concentration of glutathione ( . mm) is known to be substantially higher than the extracellular concentration ( mm). therefore, it is expected that in the extracellular oxidative environment the disulfide bonds in the protein capsule remain stable, avoiding premature release of the internal drugs during circulation of blood, after reaching the target cells, the disulfide bonds are cleaved in the intracellular redox-environment, and then the internal drugs are released. we generated three kinds of protein capsules which have disulfide bonds in different positions, w c/w c, k c/h c, k c/w c, based on tertiary structure information of human l-pgds (pdb id: o y). firstly, we performed circular dichroism (cd) measurements to confirm the structure of each capsule. the cd spectra of three protein capsules were similar to that of wild-type l-pgds in the far-uv region. therefore, the secondary structures of three protein capsules were not changed from wild-type l-pgds by introducing the mutations. quantitative analysis of the free thiol group in the protein capsule by dtnb assay revealed that the intermolecular disulfide bond was formed by h o -induced oxidation and cleaved by dithiothreitol-induced reduction. in addition, to investigate the solubility of sn- in the presence of protein capsules, we mixed the protein capsule of reduced-form with sn- suspension, and stirred at c for hours. the resulting concentrations of sn- in pbs with mm w c/w c, k c/h c, and k c/w c were mm, mm, and mm, respectively. these values were approximately -fold higher than without protein capsules. sds-page analysis showed that the bond formation decreased in a time-dependent manner, and that new intermolecular disulfide bond was not formed in the protein capsules after hours' incubation. from the above, we succeeded in generating drug delivery vehicles possessing openable and closable lids that are responsive in an oxidation-reduction environment. takaaki miyamoto , mai kuribayashi , satoshi nagao , yasuhito shomura , yoshiki higuchi , , shun hirota graduate school of materials science, nara institutte of science and technology, graduate school of science and engineering, ibaraki university, department of life science, graduate school of life science, university of hyogo, domain swapping has been of interest as a mechanism of protein oligomerization, where a secondary structural region or a domain of one protein molecule is replaced with the corresponding region or domain of another protein molecule. we have previously shown that c-type cytochromes and myoglobin form oligomers by domain swapping. , in this study, we show that a four-helix bundle protein cyt cb , in which the heme of cyt b is attached to the protein moiety by insertion of two cys residues, forms a domain-swapped dimer. dimeric cyt cb was more stable than dimeric cyt b at c, showing that attachment of the heme to the protein moiety stabilizes the domain-swapped structure. absorption and cd spectra of dimeric cyt cb were similar to the corresponding spectra of the monomer, showing that the active site and secondary structures were similar between the dimer and monomer. the redox potential of dimeric cyt cb was also similar to that of its monomer. the dissociation temperature of dimeric cyt cb was c, and its dh on dissociation to monomers was . kcal/mol (per dimer). according to x-ray crystallographic analysis, dimeric cyt cb exhibited a domain-swapped structure, where the two helices in the n-terminal region (helices and ) in a protomer and the other two helices in the c-terminal region (helices and ) of the other protomer interacted between each other. the heme coordination structure of the dimer was similar to that of the monomer. we have previously shown that domain-swapped oligomers of horse cyt c form through intermolecular hydrophobic interaction between the n-and c-terminal a-helices at the early stage of folding. it has been suggested that helices and form first at the initial stage of folding in wild-type apo cyt b . therefore, we propose that cyt cb forms a domain-swapped dimer when helices and interact intermolecularly at the initial stage of folding, whereas the intramolecular interaction of helices and results in formation of a monomer. a highly buried and conserved tryptophan residue close to the dimer interface in a cold-adapted phosphatase is phosphorescent and important for activity. jens g. hj€ orleifsson and bjarni asgeirsson. department of biochemistry, science institute, university of iceland, dunhagi , reykjavik, iceland. alkaline phosphatase (ap) from vibrio g - is a cold-adapted dimeric enzyme with one of the highest catalytic efficiency reported for known aps. it contains five intrinsic tryptophan (trp) residues and one additional trp located on the c-terminal streptag used for expression and purification. in this study, we made several single trp-substitutions to determine the role of each of the trp in the fluorescence emission spectrum. we also determined their solvent exposure by acrylamide fluorescence quenching. the results indicate that trp , trp and trp are mostly responsible for the fluorescence emission. quenching experiments with acrylamide indicated that all the trp residues were about equally accessible for quenching, except trp which was shown to be highly buried in the core of the protein. interestingly, the enzyme was found to be highly phosphorescent at c, having two phosphorescence lifetimes. the longer lifetime is due to trp . trp is located close to the dimer interface and points towards a helix in the active site where his binds an active-site zinc ion. in other aps, an aromatic amino acid is conserved in the location occupied by the trp residue. in most cases for cold-adapted aps it is indeed a trp. interestingly, the mutation of the trp to a phenylalanine affected both stability and activity of the enzyme. kcat/km was -fold lower than for wild-type. overall, this study reveals that trp can be used as a phosphorescent probe of local dynamics and could possibly also serve to study the dimer-monomer equilibrium due to proximity to the dimer interface, an area clearly crucial for enzyme activity and stability. modulating protein-protein interaction with a molecular tether helen farrants , oliver hantschel , kai johnsson ecole polytechnique f ed erale de lausanne (epfl) high-affinity scaffolds for protein-protein interactions, such as monobodies and darpins can be engineered in vitro to bind to protein targets. we speculate that the affinity for the target protein can be modulated by incorporating these evolved scaffolds and a synthetic intramolecular tether into protein switches, in a protein construct of composed of snap-tag, a monobody and a circular permutated dihydrofolate reductase. the tether, attached to the construct via snap-tag, was composed of a linker and trimethoprim, which interacts reversibly with the circular permutated dihydrofolate reductase. we have investigated the affinity between the n-sh domain of the phosphatase shp and an evolved monobody in such a protein construct using a fret assay. when the intramolecular tether was bound the circular permutated dihydrofolate reductase ("closed" conformation), there was an increase in the affinity of the construct to the target n-sh . in the presence of a small molecule competitor ("open" conformation) the affinity of the monobody construct to its target was reverted to the value reported in the literature. the intramolecular tether in these protein constructs combined with engineered scaffolds for protein-protein interactions may be a general approach towards protein switches. because most proteins are long polymers of amino acids with twenty or more chemically-distinct sidechains, there are an enormous number of potential protein sequences. here, we report the construction of biologically active proteins with minimal chemical diversity. transmembrane domains of proteins can specifically interact with other transmembrane domains to modulate the folding, oligomerization, and function of transmembrane proteins. for example, the bovine papillomavirus e protein is a -amino acid transmembrane protein that transforms fibroblasts to tumorigenicity by binding directly to the transmembrane domain of the platelet-derived growth factor b receptor (pdgfbr), resulting in ligandindependent receptor activation and cell transformation. these studies showed that a free-standing transmembrane domain could fold properly in cells and act in trans to modulate the activity of a larger transmembrane protein target. because of the relative chemical simplicity of transmembrane domains and this ability to act even when not linked to more complex soluble protein domains, we reasoned that short transmembrane proteins could be used to define the minimal chemical diversity sufficient to construct biologically active proteins. to accomplish this, we infected cultured mouse cells with a retroviral library expressing -amino acid proteins consisting of an initiating methionine followed by a randomized sequence of leucines and isoleucines, two hydrophobic amino acids that differ only by the position of a single methyl group, and selected rare proteins with transforming activity. we isolated numerous proteins consisting of diverse sequences of leucine and isoleucine that cause morphologic transformation, escape from contact inhibition and focus formation, and growth factor independence. genetic and biochemical analysis of these proteins indicate that like e they interact with the transmembrane domain of the pdgfbr to specifically activate the receptor and transform cells. mutational analysis of individual proteins identified specific leucines and isoleucines required for transforming activity, and insertion of a single isoleucine at a particular position in a stretch of leucines is sufficient for activity. these proteins identify the minimal chemical diversity required to generate a biologically active protein and have important implications for biochemistry, protein evolution, protein engineering and synthetic biology. yusuke azuma , donald hilvert virus-like particles that are precisely loaded with functional cargo are an important tool to study the effect of spatial confinement and create novel entities with application in biotechnology and medicine. by genetic fusion to a positively supercharged green fluorescent protein (gfp( )), an enzyme retroaldolase (ra) was efficiently targeted to the negatively charged lumen of an engineered protein cage, aquifex aeolicus lumazine synthase variant (aals- ). the encapsulation is quantitative under mild aqueous condition up to a mixing ratio of guest enzymes per host cages. the chromophoric tag is used for precisely quantifying the enzyme concentration, which allows detailed characterization of the effect of encapsulation on the enzyme activity. the generality of the encapsulation system was examined with structurally different enzymes. introduction and purpose: in the immune system, high affinity antibodies are generated by selection of b cells activated by antigen-stimulation followed by additional optimization through somatic hyper mutation of antibody genes. in the artificial antibody libraries, such as phage libraries, selection of specific antibody clones from the library is performed by in vitro selection process called biopanning and the subsequent binding screening. however, in spite of high efficiency of enrichment in biopanning, there is a possibility that we overlook the minor antigen-specific clones in the screening because of the limitation of the number of clones employed for screening. in recent years, high-throughput analysis of dna sequences by the next-generation sequencer (ngs) has become available not only for genomic analysis of organisms but also repertoire analysis of antibodies. in this presentation, we report the successful isolation of a variety of antigen specific antibodies from patients-derived antibody phage library by a combination method of high throughput sequence analysis on ngs and biopanning. method: we constructed two kinds of human single chain fv (scfv) antibody libraries from pooled mrna of five cancer patients and of a wheat allergy patient, respectively. after biopanning against a cancer antigen or wheat allergy antigen "gluten", the phagemid vector dna prepared from the pooled phages before or after biopanning was used for pcr amplifications of vh genes, adding the index and adapter sequences for ngs analysis. the high throughput sequencing was performed on miseq (illumina) using miseq reagent kits v . after discarding the short sequences and low quality data, '-and '-reading sequences were unified by a merge program. the frequencies (%) of all vh sequences were evaluated using a program based on usearch . clustering software and the changes of the frequency (%) of each sequence between before and after panning were assigned as amplification rate. results and discussion: vh sequences at each round of pooled phages after biopanning against cancer antigen were analyzed on ngs. after three rounds of biopanning, three clusters of antibody sequences were specifically enriched suggesting these are specific binders. to check this, scfv gene were regenerated by pcr using h-cdr specific primers and scfv-displaying phages reconstructed were subjected to binding analysis. all three phages showed a clear specific binding to cancer antigen in elisa. subsequently, to test the usefulness of this method, we applied it to identify allergen-specific scfv from allergy patient-derived antibody phage library. the phylogenetic tree analysis of vh sequences which showed the amplification rate higher than . by a single round of biopanning elucidated total eleven clusters of vh sequences. the vh sequences in the two clusters with the highest amplification factor were selected and the regenerated scfv-displayed phages were tested for binding analysis. the prepared scfvdisplayed phages and also scfv proteins showed a clear binding ability to allergen. thus, it is suggested that the analytical method of vh sequences on ngs before and after biopanning is very useful to isolate a variety of disease related antigen-specific novel antibodies quickly with high degree of certainty. biochemical analysis of the recognition helix of z-dna binding proteins: roles in conformational specificity yang-gyun kim , xu zheng , so-young park conversion of right-handed b-dna into left-handed z-dna is one of the dramatic structural transitions in biological processes including gene regulation and chromatin remodeling. z-dna binding motif, zalpha (za), was first discovered from human adar . subsequently, with sequence and structure similarity to the hzaadar , families of proteins including viral e l, interferon-induced protein dai (zbp ) and pkz has been identified to have za domain(s). interestingly, the za domain of the e l protein from vaccinia virus (vvzae l) was confirmed to have the ability of z-dna-binding, but it does not have the b-to-z conversion activity. here, we showed that the replacement of the a -helix of vvzae l (vvzae l-a ) with that of hzaadar results in acquiring the ability to converting b-dna to z-dna. the detailed biochemical analysis of the a -helix mutants of vvzae l further suggested that the contribution of positively charged residues in the c-terminal part of the a -helix is crucial during the b-to-z transition. in addition, hydrophobic residues of the n-terminal part of the vvzae l-a also influence on the b-to-z conversion activity, possibly through forming a tightly-packed structure. in conclusion, our results revealed the previously-unknown contribution of amino acid residues existed in the a -helix of the za domains to the b-to-z conversion. moreover, it strongly implies that such residues may play important roles in initiating conformational changes of dna structure during the b-to-z conversion event. the ability of switching the activity of proteins at will is of great interest from an application point of view. one promising approach utilizes a protein modification with an organic photochromic molecule. linking two protein side chains with the photochrome that undergoes a light induced conformational change, protein secondary and tertiary structure can be stabilized or destabilized and thus the structure dependent activity can be switched "on" and "off" by light irradiation. for this the photochrome must fulfil several requirements. foremost, it must possess two states of comparable stability that differ significantly in their geometry. it must further be water soluble and non-toxic, and should not experience fatigue phenomena upon multiple irradiations. there are two classes of molecules that fulfil those requirements: azobenzenes and spiropyrans. we are pursuing two different strategies for the design of photoswitchable proteins. in the first approach we attach an azobenzene compounds to side chains of the alpha-helical antifreeze protein type i. the end to end distance of the photochromic molecule is sterically compatible with the folded helix only in one form, photoisomerization therefore switches the folding state between an active helical state and an inactive unfolded form. in a second, more general approach we use the trp-cage domain as a switching unit. the trp-cage is the smallest known folded protein ( amino acids). its folding is induced by hydrophobic interactions of a tryptophan side chain in a short helical segment. after modification with a photochromic molecule in appropriate positions, its structure is rendered sensitive to the state of the chromophore. by creating protein chimera of such a trp-cage and biologically active peptides with helical propensity, we aim at conferring the light-dependent fold of the cage to the attached peptide moiety. salt-bridges are electrostatic interactions between groups of opposite charges. net interaction energy (ddgnet) of a salt-bridge is partitioned into bridge (ddgbrd), desolvation (ddgdsolv) and protein (ddgprot) energy-terms of which estimation of ddgdsolv and ddgprot are only possible by computational means. thus, general purpose poisson-boltzmann equation solver: "delphi" (in commercial package of insight-ii) and "apbs" (open-source) are popularly used to determine these energy-terms. nevertheless, the computation-method is highly involved one than other uses of these solvers. moreover, protein-specific saltbridges, grid-points, center, hydrophobic-isosteres-mediated mutation-files of original charge-radius file and others are to be worked out prior to the computation. this might answer as to why only limited numbers of structure files ( % of crystal-structure-database) are worked out till date. at this juncture, an efficient fully automated all-in-one-procedure that could analyze large dataset in a single run would be useful. to the best of our knowledge, such procedure is truly lacking in public domain. at this end, our fully automated all-in-one procedure: adsetmeas (available freely at http://sourceforge.net/projects/adsetmeas/along with detailed documentation) uses "apbs" method to compute component as well as net energy-terms of salt-bridges and redirect compact output in excelformat. further, micro-environments of salt-bridges are also been reported based on the presence of polar, dipolar, acidic, basic and hydrophobic side-chains in their proximity. the procedure provides versatility to users in choosing a] model for computation of energy-terms to-date available in the literature and b] method (default or advanced) for parametric optimization in "apbs" calculations. it works in unix like environment including cygwin. it processes all proteins present in the working directory with any number of salt-bridges in them. a pre-released version of the procedure was successfully applied for energy-terms on salt-bridges from halophilic proteins. overall, our adsetmeas provides intricate details on salt-bridge energetic from crystal structures and find application in the field of computational structural biology. these and other results will be discussed in the conference. next generation analgesics -targeting ion channels with antibody-drug conjugates (adcs) anna wojciechowska-bason , clare jones , chris lloyd postdoctoral fellow, adpe, medimmune, cambridge, ria, medimmune, cambridge, adpe ion channels are common targets for chronic pain therapies. small molecule analgesics are widely used therapeutically, but due to poor specificity they often cause a wide range of side effects. as a result, efficacy of existing treatments is very limited. we believe that to achieve the required specificity and efficacy, a novel and innovative approach is required that would combine the potency of the small molecule with the selectivity of an antibody. therefore, we propose to apply antibody-drug conjugates (adcs) to deliver small molecules or peptides to ion channels in order to specifically modulate pain signalling pathways. voltage-gated sodium channel nav . has a well characterised role in the perception of pain. here we present the activity of the peptide huwentoxin-iv (hwtx-iv) and small molecule inhibitors ptc-a, ptc-b and ptc-c on voltage gated sodium channels nav . and nav . . in novel findings, we report that these inhibitors show little selectivity between the voltage-gated sodium channel family members, nav . and nav . , and that the ic values and the impact on channel biophysics (voltage-dependence of activation and fast inactivation) of the inhibitors are largely similar for both channel types. therefore, the use of hwtx-iv and other small molecule inhibitors of nav . for pain therapy could be dose-limited due to side effects mediated by the inhibition of channel nav . . in conclusion, we propose that hwtx-iv and the investigated small molecule inhibitors could be used for the treatment of pain as part of a nav . antibody-drug conjugate (nav . -adc), establishing nav . specificity and minimising side effects. maria antonietta carillo , daniel varon silva malaria is one of the most infectious diseases caused by plasmodium species parasites. the merozoite surface protein (msp ) is the most abundant protein on the surface of the plasmodium species merozoite stage, which plays an important role during the erythrocytes invasion process [ ] . msp is synthesized as a -kda glycosylphosphatidylinositol (gpi) anchored protein precursor which is processed at the end of the schizogony into four different fragments. the primary processing step produces a complex of four fragments that are present on the merozoite surface. the secondary processing step at erythrocytes invasion results in the detaching of the complex from the surface, except for the cterminal -kda domain (msp ), which remains anchored to the parasite surface by the gpi moiety. in human malarial infections, the gpi is considered to be a toxin that causes the expression of various host genes and induces a pro-inflammatory immune response, making it a valuable candidate for the development of anti-malarial drugs. in order to study the function of the gpi and evaluate the effects, msp fragment has been expressed, purified and anchored to the synthetic gpi molecule using protein trans-splicing strategy based on the split intein method [ ] . the role of the gpi moiety will be studied through protein folding experiments and the effect of the anchored protein will be evaluated in vitro in order to understand the function of the gpis. assessment of uch-l substrate selectivity using engineered ubiquitin fusions with varying linker lengths peter suon, mario navarro, john love san diego state university, san diego state university, assessment of uch-l substrate selectivity using engineered ubiquitin fusions with varying linker lengths peter suon, mario navarro, and john j. love san diego state university the ubiquitin proteasome system (ups) is a complex system composed of multiple structural and functional elements that play key roles in cellular processes such as signal transduction, cell cycle regulation, apoptosis, and protein degradation. proteins destined for degradation are first tagged with the protein, ubiquitin, which is covalently attached to internal lysine residues. once the target has be degraded by the proteasome; the enzyme ubiquitin carboxy hydrolase l (uch-l ) is believed to prepare ubiquitin for additional rounds of ubiquitination by cleaving small peptides and chemical adducts from the ubiquitin c-terminus. previously in our laboratory, protein substrates of uch-l were engineered and used to characterize uch-l substrate selectivity. the engineered substrates consisted of n-terminal monoubiquitinated test variants derived from streptococcal protein g (protein gb ) and staphylococcal protein a (spab). the thermal denaturation temperatures (tm) of the fusion proteins were measured using circular dichroism and span a range of over c. more importantly, the rate of hydrolysis for the fusion proteins is demonstrated to be directly correlated to the tm of the test variant fused to the c-terminus of ubiquitin. previously, the engineered substrates were designed to emulate natural ubiquitin fusions and thus did not contain any 'linker' residues between the c-terminus of ubiquitin and the n-terminus of the test protein. to explore the effects of linker length on uch-l hydrolysis we are engineering new uch-l substrates that contain an unstructured amino acid linker between ubiquitin and the test protein. to further explore the catalytic efficiency of uch-l we will revisit diubiquitin (ub-ub), which is not hydrolyzed by uch-l , and will make mutations in the hopes of generating a hydrolysable substrate. using rational design, the new variants will be engineered to destabilize the c-terminal ubiquitin to determine if this results in hydrolysis of the new ub-ub construct. the thermal stability of these new fusion protein substrates will be measured using circular dichroism spectroscopy (cd) and uch-l hydrolysis rates will be characterized using existing assays. our goal is to continue the use of engineered substrates to further explore the catalytic properties of uch-l activity and the potential role in protein trafficking and degradation within living cells. we present a biophysical study of a suite of helical proteins that have been modified to contain and -amino acid additions on their termini that impart increased resistance to degradation in e. coli abstract recombinant expression systems. the b domain of staphylococcal protein a (ab) and the homeobox dna-binding domain from d. melanogaster engrailed (en) are small -helix bundles. these domains do not appreciably accumulate in the e. coli bl (de ) cytoplasm when expression in a pet vector is chemically induced. this is likely due to host protein degradation/recycling factors that function to efficiently degrade these two proteins. addition of sequences encoding either of two amino-terminal beta-hairpins to either the n-or c-terminus of ab and en results in the accumulation of large amounts of these new chimeric proteins. additionally, destabilization of the ab or en sequence does not abolish the expression enhancement effect of the beta-hairpin addition. we have investigated the biophysical origins and effects of the beta-hairpin additions using circular dichroism (cd) spectroscopy, and have determined that the added sequence does not significantly perturb the secondary structure of ab or en, nor does it significantly influence the unfolding temperature (tm). while investigation into the origin of the accumulation effect is ongoing, we hypothesize that the addition of the sequence is disruptive to recognition events in the native protein degradation machinery in e. coli. thus, this approach represents both a biotechnological tool for expressing helical peptides recalcitrant to expression, as well as a system well-suited to probing mechanisms of protein recycling and homeostasis. a special class of these proteins are lipidated proteins containing a glycosylphosphatidylinositol (gpi) glycolipid moiety at the c-terminus. the lipid chains of the gpi anchor molecule are responsible for the membrane association of the attached protein. a unique feature of gpi-anchored proteins is that after isolation they can be reinserted into the membrane of recipient cells with the retention of the biological function. accordingly, the exogenous introduction of fluorescent gpi-anchored protein analogues into cell membranes is a useful method for visualizing the cellular traffic of membrane associated proteins and for engineering cell surfaces. we have recently shown that cholesterol can be applied for anchoring proteins to the plasma membrane of live cells without perturbing the membrane. in order to introduce proteins containing covalent modifications that are not genetically encoded, an enzymatic method was considered and fused with the c-terminal cholesterylation method. the usefulness of the method is demonstrated via the preparation of multimeric model proteins of kda monomers, that is an appropriate representation of the ligation of domain size proteins. transmembrane domain dimerization drives p ntr partitioning to lipid rafts irmina garc ıa carpio , marc¸al vilar sociedad de biof ısica de españa. sbe p neurotrophin receptor (p ntr), is best known for its role in mediating neuron cell death during development or after injury but it also regulates cell proliferation, axon guidance or survival. the key to understand its signaling could rely in its structure and conformational states. it has been described that p forms disulfide-linked dimmers through the cys in the transmembrane domain which are essential for its ngf mediated signaling. previous studies have shown that p is present in lipid rafts, where it interacts with intracellular adaptors to activate different signaling pathways. we design several p mutants in the tm domain that impairs dimerization and study the role of tm domain dimerization in lipid rafts recruitment. our analysis suggests that p tm domain dimerization influences lipid raft partitioning. these results could be a key role to understand its signaling and processing pi- bioluminescent sensor proteins for therapeutic drug monitoring of the monoclonal antibody cetuximab martijn van rosmalen , remco arts , brian janssen , natalie hendrikse , dave wanders , maarten merkx therapeutic drug monitoring (tdm) -adapting the drug dosage scheme to the individual patient's pharmacokinetic and pharmacodynamic characteristics -is still uncommon for therapeutic monoclonal antibodies, despite preliminary studies showing its potential benefits. one of the factors impairing tdm implementation is the lack of equipment and trained personnel to regularly measure drug concentrations in patients receiving treatment. point-of-care diagnostic devices which could be used by patients themselves or by their general practitioners would greatly advance the feasibility of tdm. here we present a biosensor for the therapeutic monoclonal antibody cetuximab. we developed a series of cyclic peptides that specifically recognize cetuximab, covering a fourfold range of affinities, and incorporated these cyclic peptide sequences into a set of luminescent sensor proteins. the sensors translate cetuximab concentrations into a change in emission color that can be read out using a mobile phone camera. together, these sensors can quantify cetuximab levels within the relevant therapeutic concentration range and we propose that they can be used for therapeutic drug monitoring applications. genetically encoded biosensor for cell permeability of inhibitors of the p -hdm interaction silvia scarabelli , thomas vorherr , kai johnsson ecole polytechnique f ed erale de lausanne, the evaluation of the permeability across the cellular membrane is a key step in the development of therapeutics, since it affects the distribution and the efficacy of the latters. reliable and versatile techniques for the determination of structural permeability determinants of molecules and information about the entry kinetics are still missing. we introduced in the past a class of semi-synthetic ratiometric sensor proteins (snifits) that has been shown to be suitable for the measurement of intracellular metabolites concentrations. here we describe a totally genetically encoded sensor based on the snifits modular design for the assessment of the cell permeability of small molecules and peptides inhibitors of the protein-protein interaction between p and hdm . we show that our sensor detects the presence of hdm -binding stapled peptides in vitro, and, when expressed in mammalian cells, it responds to the perfusion of the known small molecule hdm inhibitor nutlin- a. moreover, experiments made with an automated microscope show that the sensor is suitable for measuring and comparing the kinetics of entry of different kinds of inhibitors in the cytosol of living cells. in parallel, we are developing an hcaii-based sensor protein for the sensing of sulfonamides and eventually their peptide derivatives. we show that the sensor responds to the presence of different kinds of hca-inhibitors in vitro and in perfusion experiments. this second sensor would broaden the range of molecules and peptides whose permeability can be studied with our tools beyond the family of the hdm -binders. our sensors overcome the limitations of the already existing techniques for measurements of permeability while offering a simultaneous measurement of the cell permeability and of the binding efficiency of small molecules and peptides of interest. archer: predicting protein function using local structural features. a helpful tool for protein redesign. jaume bonet , javier garcia-garcia , joan planas-iglesias , narcis fernandez-fuentes , baldo oliva structural bioinformatics lab, grib, upf, division of metabolic and vascular health, university of warwick, the advance of high-throughput sequencing methodologies has led to an exponential increase of new protein sequences, a large proportion of which remain unannotated. the gap between the number of known proteins and those with assigned function is increasing. in light of this situation, computational methods to predict the function of proteins have become a valid and necessary strategy. here we present archer, a server that exploits archdb's hierarchy of super-secondary structures to map go and enzyme functions upon protein regions and, thus, infer the function of a protein. the server relies on either the sequence or structure of the protein of interest and returns the mapping of functional subclasses extracted from archdb. moreover, it computes the functional enrichment and significance of each subclass, combines the functional descriptors and predicts the function of the query-protein. combining the functional enrichment analysis of the super-secondary structures with the structural classification of archdb, users can select variants of the target sequence that swap the region of a supersecondary structure by another that putatively fits in the same scaffold minimizing the effect on the global tertiary structure. only variants that modify the predicted function are offered for selection, thus providing a rational, knowledge-based, approach for protein design and functionalization. the archer server is accessible at http://sbi.imim.es/archer. phytochromes are natural photoreceptors known to regulate photosynthesis in plants, fungi and bacteria. phytochromes found in bacteria share common architecture and consist of a pas-gaf-phy photosensory core and a c-terminal output module, responsible for biological function. a bacterial phytochrome, bphp , from rhodopseudomonas palustris undergoes reversible conversion from the farred absorbing state (pfr) to the red-absorbing state (pr) followed by the conformational change upon nm light irradiation. as most of bacterial phytochromes, bphp forms a dimer. it was shown that nm light causes a protomer swapping between the bphp dimers; and likely, the output module is involved in this process. however, the mechanism of the light-induced swapping is poorly studied. we tested an ability of the protomer swapping between bphp dimers using pull-down biochemical assay. for this, strep-tagged bphp was immobilized on strep-tactin sepharose beads in the presence of untagged bphp fused to mruby at different concentrations. after incubation, the proteins were eluted and visualized in sds-gel using a zinc-induced fluorescence assay. an amount of the bound to beads protein was estimated by densitometry. it was found that more than % of heterodimers (streptagged-bphp and bphp -mruby ) form within . h of incubation under nm light at -fold excess of one of the interacting partners. in darkness, the swapping was much slower. in the similar setup we checked the amount of heterodimers after , and min of incubation. no difference was observed for different time points, suggesting that the protomer swapping is relatively fast process. next, a role of the c-terminal effector domain of bphp in the light-induced interaction was studied. for this, kinetics of the pfr-to-pr transition was analysed by measuring of absorbance at nm and nm for full-length bphp and a bphp mutant with the deleted c-terminal domain. while full-length bphp showed the normal pfr-to-pr transition, absorbance of the mutated bphp at nm did not raise. however, nm absorbance changes were similar for both proteins; and surprisingly, the similar dark relaxation kinetics was observed. we propose that the impaired pfr-to-pr transition is caused by restricted pr conformation in the mutant rather than by fast pr-to-pfr relaxation. understanding the mechanisms of the bphp light-induced structural changes and the protomer interaction should advance engineering of bacterial phytochromes into fluorescent probes and optogenetic tools. antibody detection is an integral part of many diagnostic strategies, most crucially so when infectious diseases are involved. currently used assays, such as elisa or spr, enable detection of antibodies in the laboratory with high sensitivity, yet a translation of these technologies to an application outside of the laboratory setting is far from trivial. problematically, the burden of disease for many infectious diseases is carried precisely by those countries where access to laboratory facilities is severely limited. we therefore developed a novel, one-step assay that allows the detection of antibodies directly in solution using a luminescent sensor protein. our strategy is based on the use of a bright luciferase, nanoluc, tethered to a green fluorescent protein (mneongreen) via a semi-flexible linker containing two epitope sequences. crucially, two small helper domains were fused to the protein termini. these domains keep nano-luc and mneongreen in close proximity in the absence of antibody, enabling efficient bioluminescence resonance energy transfer (bret). binding of antibody to the epitopes in the sensor proteins linker domain pulls the bret partners apart, effectively changing the color of emission from green to blue. the assay allowed the detection of picomolar amounts of anti hiv -p antibodies directly in solution, both under optimized buffer conditions and in blood plasma. in principle. the modular sensor architecture should allow detection of any antibody with a well-defined epitope of sufficient affinity. to demonstrate this, the hiv-epitopes were substituted for two ha-tag epitopes, yielding a sensor that enabled the detection of picomolar amounts of anti-ha antibodies. the simple optical readout provided by the sensor system allowed us to record the emitted signal with a conventional mobile phone camera. a simple software application that analyzes the image based on rgb values sufficed to interpret the recorded image vis-a-vis the presence of antibody. bearing in mind the eventually envisioned application in a point-of-care diagnostic setting, this combination of sensor recording and interpretation using nothing more than a mobile phone and a software application holds considerable diagnostic potential. beyond point-of-care diagnosis of infectious diseases, a simple assay to detect and quantify antibodies directly in solution could also have a substantial impact in other fields. antibodies are ubiquitous in biotechnology, and this is reflected by the plethora of potential sensor applications, which range from a role in microfluidic circuits or monitoring the biotechnological production of antibodies, including validation of bispecificity, to veterinary applications, diagnosis of autoimmune diseases and monitoring the success of vaccination campaigns. the continually growing protein data bank (pdb) has been a key resource for general principles of protein structure. for example, parsing structural observations in the pdb into simple geometric descriptors has given rise to statistical energy functions. here we present a novel strategy for mining the pdb on the basis of local tertiary structural motifs (term). we define a term to be the structural fragment that captures all local secondary and tertiary structural environments of a given residue, and query the pdb to obtain quantitative information for each terms. first, we show that by breaking a protein structure into its constituent terms, we can describe its sequence-structure relationship via a new metric we call "structure score." using submissions in recent critical assessment of structure prediction (casp) experiments, we find a strong correlation (r . ) between structure score and model accuracy -a performance that exceeds leading atomistic statistical energy functions. next, we show that querying terms affected by point mutations enables the quantitative prediction of mutational free energies. our simple approach performs on par with state-of-the-art methods fold-x and popmusic on mutations, and provides superior predictions in certain cases where other methods tend to fail. in all, our results suggest that the data available in the pdb are now sufficient to enable the quantification of much more sophisticated structural observations, such as those associated with entire terms, which should present opportunities for advances in computational structural biology techniques, including structure prediction and design. exploiting natural sequence diversity for protein crystallization sergio mart ınez-rodr ıguez , valeria risso , jos e m sanchez-ruiz , jos e a. gavira , departamento de qu ımica-f ısica, universidad de granada, laboratorio de estudios cristalogr aficos, iact-csic-ugr granada during the last decade, different rational and high-throughput approaches have been successfully applied in the protein crystallography field to widen thejjso-called "protein crystallization bottleneck" [ , ] . despite the enormous efforts carried out by our community, the statistics presented by structural biology consortiums [ ] suggest that so far only the easy-to-pick fruit has been attained; thus, new approaches are necessary to further expand the crystallization limiting step to relevant targets. on the basis of previous hypothesis suggesting that the difficulties found in protein crystallization might be a result of evolutionary negative design [ ] , we have used two different protein engineering approaches exploiting natural sequence diversity using beta-lactamase as toolbox: i) ancestral reconstruction and ii) consensus approach [ ] . both approaches resulted in hyperstable and promiscuous ancestral derivatives. furthermore, our initial crystallization results also suggest that both approaches increased the crystallizability of the resulting enzymes when compared to the extant tem- beta-lactamase. the adipocyte-derived hormone adiponectin has become a key player for the understanding of overweight related diseases like obesity, diabetes, atherosclerosis or the metabolic syndrome. one of its abstract major functions are the insulin sensitizing effects, which are mediated by the activation of ampk, p -mapk and ppara ( ). furthermore adiponectin is involved into glucose regulation and fatty acid oxidation. recently, three adiponectin receptors adipor , adipor and t-cadherin have been described while an unknown fourth receptor is hypothesized ( ) . for only two of them (adipor and adipor ) the signaling transduction via adiponectin has been confirmed ( ). in order to find new binding partners or co-receptors, we cloned and expressed full length adiponectin as a fusion protein with a c-terminal intein and a chitin binding domain (cbd) as well as an n-terminal his -tag. by using the impactsystem, the fusion protein was cleaved to form the corresponding thioester. to separate the starting materials as well as the cleaved intein chitin binding domain, the purification was performed with chitin beads. furthermore, the product was concentrated by ni-nta-affinity chromatography. accordingly, the obtained adiponectin thioester was reacted with a tamra-or a biotin labeled peptide, respectively, to receive the corresponding ligation product. finally the functionalized adiponectin was purified by size exclusion chromatography. further studies will allow screening for interacting molecules in cell and tissue derived samples. departamento de quimica fisica, facultad de ciencias university of granada, dpto. de quimica fisica biologica. instituto de quimica fisica rocasolano, departamento de quimica organica, facultad de ciencias university of granada, rational design of non-natural enzyme activities has proved challenging. here, we report the introduction of catalysis of the kemp elimination (a model of proton abstraction from carbon) in scaffolds corresponding to precambrian nodes in the evolution of the antibiotic resistance protein b-lactamase. we used a single-mutation, minimalist approach based on chemical intuition, and obtained catalysis levels similar to those reported in the literature for computational kemp-eliminase designs involving multiple mutations. remarkably, the approach was unsuccessful when performed on modern b-lactamases. we provide experimental evidence that enhanced conformational flexibility contributes to the success of the minimalist design in the ancestral scaffolds. this work has implications for the understanding of function emergence in protein evolution and demonstrates the potential of ancestral protein resurrection in enzyme engineering and design. exploring the importance of dimerization for dj- function through engineered domain fusions sierra hansen , jiusheng lin , mark wilson parkinson's disease is a progressive neurodegenerative disease that affects approximately . million people worldwide and is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. dj- (park ) is one of several genes that are mutated in rare forms of familial parkinsonism. dj- is a dimeric cytoprotective protein that defends against oxidative stress and preserves mitochondrial function. dimerization of dj- is thought to be essential for this function, as some diseaseassociated mutations cause poor folding and disrupt the dj- dimer. however, recent reports suggest that dj- may be functional as a monomer. to test this, we have engineered a non-dissociable dj- dimer that is a fusion of two human dj- domains. this construct cannot dissociate into monomers and thus will provide a stringent test of the importance of monomeric dj- . our engineered construct is modeled on plant dj- homologs, which feature naturally occurring duplicate dj- domains separated by a small ( amino acid) linker region. using x-ray crystallography, we confirmed that this engineered non-dissociable human dj- dimer has identical structure to the naturally occurring dimeric protein. we have investigated the influence of enforced dimerization of the pathogenic effects of the parkinsonian l p and l p mutations. cd spectroscopic analysis reveals that single and double l p mutations in the non-dissociable dj- dimer maintain a higher degree of structure than l p mutations in the native protein. additional characterization of the protective capacity and subcellular trafficking of this non-dissociable dj- dimer is underway. the purification, crystallization and preliminary characterization of sdre from s. aureus the purification, crystallization and preliminary characterization of sdre from s. aureus staphylococcus aureus (s.aureus) is an important human opportunistic pathogen which colonizes about % of the human population persistently [ ] . surface proteins of s.aureus can excretion a kind of sortase, which represents a surface organelle responsible during the pathogenesis of bacterial infection the host circulation [ ] . sdr proteins were a component of cell wall anchored family proteins, including sdrc, sdrd and sdre [ ] . sdre could combine with the complement regulatory protein factor h to escape the alternative pathway of complement [ ] . to further investigate the functions of sdre, we have expressed and purified the adhesive domain (residues -' : ), and crystallized the recombinant protein. in addition, we also constructed the mutant s.aureus, and the cell experiments confirmed that sdre gene participate in the bacteria invasion. bacterial microcompartments (bmcs) are proteinaceous organelles that sequester key metabolic reactions to increase enzymatic efficiency or to prevent the loss of volatile or toxic intermediates. there is an increasing desire to engineer bmcs for non-native enzymatic processes. it is thought this will increase multi-enzyme pathway efficiency and allow the expression pathways that may produce toxic or volatile intermediates in bacteria. the mechanisms of small molecule transport and retention of toxic intermediates by bmcs remain poorly understood. better understanding of the bmcs pores critical to engineer bmcs for these non-native pathways. in order to better understand the bmc pore we have undertaken structure-guided modifications of the the hexameric pdua shell protein of the , -propanediol utilization microcompartment (pdu mcp). these modifications include pore mutations in an attempt to alter substrate specificity and permutations of pdua to allow more drastic alterations to the structure of the protein. crystal structures of pdua pore mutants, solved to atomic resolution ( - . Å) provide evidence of the pore residues that confer specificity. further, a pdua permutation (pduap) has resulted in a closed icosahedral cage. this novel pduap cage shows a ph and salt dependent assembly and may serve as a reaction vessel or be utilized for cargo delivery. ( , ) . anm-mc is used to identify targeted transition pathways and intermediates between open and closed states of proteins. at each step of this iterative technique, the protein is deformed along the collective anm mode showing the best overlap with the target direction and its energy is minimized via short mc run. in this work, optimization of simulation parameters (number of mc moves and their perturbation strength, anm deformation factor in each cycle and force constant for backbone bonds) was performed in order to increase the efficiency of this technique. as a result, this technique can now be applied to much larger systems and conformational changes. the transition pathway between apo and dna-bound conformations of the yeast rna polymerase, which is a hetero- -mer with more than residues, will be presented here. moreover, the pathway intermediates for more than diverse proteins were analyzed in terms of changes in local strain energy and backbone torsional angles during apo-to-complex transitions. certain residues interacting with the ligand are detected to exhibit large changes with respect to any of these two parameters for more than half of the proteins in our dataset. department of chemistry and chemical biology, harvard university, howard hughes medical institute, harvard university, transgenic crops have radically reshaped the agricultural landscape. since their introduction in the late s, transgenic crops have affected economic gains greater than us$ billion globally due to reduced production costs and increased yield gains. crops modified to produce biological insecticides derived from the soil bacterium bacillus thuringiensis (bt) are among the most robust methods of pest control. bt toxins offer many advantages over traditional insecticides, chiefly their inability to affect human biology and exquisite selectivity for defined pest species. however, the evolution of resistance to bacillus thuringiensis oendotoxins (bt toxins) in insects has been widely observed in the field, and greatly threatens the use of this mechanism of pest control in the future. we developed a phage-assisted continuous evolution (pace) platform for the rapid generation of high-affinity protein-protein interactions and validated the system by evolving known high affinity antibody mimetics in < days of pace. we applied this system to the evolution of the bt toxin protein cry ac to recognize a non-cognate cadherin-like receptor from trichoplusia ni, a pest for which bt toxin resistance has been observed in both the laboratory and the field. the resulting evolved cry ac variants exhibits high affinity for the target receptor, and kill insect cells more potently than wild-type cry ac. our findings establish that the directed evolution of novel receptor recognition in bt toxins can be used to target resistant pests, and has far-reaching implications for biological reagents and therapeutics. optimization of a designed protein-protein interface brian maniaci , collin lipper , john j. love san diego state university, university of california protein-protein interactions play key roles in practically every biological process. protein-protein interactions vary with composition, affinity, and lifetime of the complex. studying designed protein-protein interactions will provide insight into the underlying principles of complex assembly and formation. computational protein docking and amino acid sequence design were used previously to generate protein dimers from monomeric proteins. the normally monomeric b domain of streptococcal protein-g (gb ) was computational docked to itself, followed by optimization of the interfacial side chains. two variants, monomera and monomerb, were computationally derived as a result of a designed protein-protein interface. these designed proteins were characterized using analytical ultracentrifugation and heteronuclear nmr techniques. this design resulted in a pair of protein monomers that formed a heterodimer of modest binding affinity. a tetrahedral metal-templated interface design strategy was implemented in an attempt to strengthen the monomera-monomerb complex by introducing cross-monomer metal coordination. another advantage of using the metal-templated interface is the ability to control the protein-protein interaction both temporarily and spatially. a number of newly engineered variants of monomer a and monomer b with metal coordination sites were designed, produced, and tested for increased affinity of the protein-protein complex. while the generation of a metal-templated monomera-monomerb complex was unsuccessful, we were able to obtain monomera variants that form a homodimer assembly only in the presence of zinc (ii) ions. the crystal structures of metal-templated monomera variants in the presence of zinc provide an explanation for the observed dimer formation. the crystal structure indicates that the protein-protein interaction is not driven by the designed protein interface, but rather non-specific association via edge-strand interactions. new variants were designed with the goal of engineering a high affinity homodimer in a helix-to-helix orientation as the originally designed protein-protein interface. current evaluation of monomera variants for self-association via metal coordination are being evaluated using size exclusion chromatography with a multi-angle light scattering detector for oligomerization state quantification. the results of this protein design project should lead to a greater understanding of the biophysical parameters that drive natural protein-protein interactions. continuous evolution of site-specific recombinases with highly reprogrammed dna specificities the ability to precisely modify the genome of human cells has enormous potential as a novel therapy and a powerful research tool. in contrast to reprogrammable nucleases, such as talens or a cas / sgrna pair -which specifically cleave dna but then rely on stochastic host cells processes to effect gene insertion -site specific recombinases directly catalyze genomic integration with high efficiency. a major limitation of this approach is that recombinases, such as cre, natively bind with high specificity to long dna target sequences (loxp in the case of cre) that do not exist in the human genome. previous attempts at evolving cre resulted in modest changes to its specificity, or required hundreds of rounds of manual protein evolution. we developed and validated a phage assisted continuous evoluiton (pace) selection for rapidly altering the dna specificity of cre recombinase towards a site present in a human genomic safe harbor locus. the pace experiments resulted in cre variants capable of recombining a substrate with nearly % of the nucleotides altered compared to loxp. we successfully used one of these variants to integrate exogenous dna into the genome of unmodified human cells. we are currently using sequencing methods to determine the specificity of the new recombinase clones. aleardo morelli , burckhard seelig generation of comprehensive deletion libraries mediated by in vitro transposition analysis of protein enzymes and ribozymes from nature, and from in vitro evolution, revealed that deletions of up to dozens of amino acids (or nucleotides) can be structurally tolerated. furthermore, shortened variants can exhibit better stability and increased catalytic activity. in order to investigate the effects of deletions, we developed a new procedure based on in vitro transposition to build libraries of more than , deletion mutants in three to four days. we tested our procedure on dna sequences coding for an artificial rna ligase called ligase c. we used the generated library for an mrna display selection, and isolated two active mutants containing and amino acids n-terminal deletions. structural characterization of ppsc, a multi-domain polyketide synthase from mycobacterium tuberculosis using a fragment-based approach alexandre faille , nawel slama , anna grabowska , david ricard , annaik qu emard , lionel mourey , jean-denis pedelacq polyketide synthases are of great interest in numerous scientific fields. they are composed by multiple domains, each having a different role to play in the catalysis of sequential reactions including condensation, reduction and esterification. their reaction products, named polyketides, represent a large variety of chemical compounds, from antibiotics to immunosuppressors or even anticancer drugs. ppsc is a kda polyketide synthase, organised into six catalytic domains (ks-at-dh-er-kr-acp) with singular functions. along with other type i polyketide synthases, ppsc is responsible for the biosynthesis of an essential polyketide for the virulence of mycobacterium tuberculosis (mtb) and thus is a target of choice for the design of inhibitors. to date, no structural information of any type i polyketide synthase in its entire form has been described. main reasons are the length of these large size enzymes and the flexibility imposed by the linkers between domains, thus making them very difficult to crystallize. numerous questions about domain-domain interactions, spatial arrangement of this complex machinery, substrate specificity and stereochemistry are still unanswered. addressing the structural and functional characterization of ppsc would then help answering these questions and provide valuable information for drug design. to overcome the length-and flexible-dependent problem originating from the presence of multiple domains and linkers, we decided to study domains expressed alone. for this purpose, we used our domain trapping strategy to identify soluble fragments representing a single domain from ppsc [ ] . it has the advantage of not relying on the bioinformatically designed domain boundaries and can even sometimes include parts of linkers to obtain more soluble fragments. using this strategy, we were able to identify relatively small and highly soluble fragments representing each domain of ppsc, thus facilitating the downstream structural and functional characterization. more than fragments have been submitted to crystallization trials. among these, gave crystals and allowed us to determine the x-ray structure of ppsc at, er, in addition to the dh domain in complex with a substrate analog for which activity was confirmed in vitro. the computational design of proteins that bind small molecules remains a difficult challenge in protein engineering. the ability to computationally design native-like interactions with high accuracy and efficiency would be an asset towards therapeutic development, enzyme design, and engineering functional proteins. we have developed a systematic approach to designing interfaces. we first identify ligands with naive binding affinity to our protein scaffold, then use rosettaligand to computationally dock the ligand while designing the interface for a tighter interaction. this way, we are taking a 'shot in dim light' for design as opposed to a 'shot in the dark', allowing us to more thoroughly investigate the successful and not-so-successful designs, and improve the computational methods. of ligands screened, we identified weakly-binding hits in the range of - mm. thus far, rosettaligand has successfully designed one tighter protein-ligand interface, from mm to mm. in progress experiments include designing and experimentally validating more designed interfaces. structural studies of human acidic fibroblast-growth factor (fgf ) mutants with a probable anticancer activity lectins are carbohydrate-binding proteins ubiquitously present in nature. they play a role in biological recognition phenomena involving cells and proteins. the interaction lectin-carbohydrate is highly specific, and can be exploited for the development of nanoparticles containing on their surface lectins specifically directed to carbohydrate residues present only on malignant cells and absent on healthy ones ( ) . lectins have been found to possess anticancer properties and they are proposed as therapeutic agents, binding to cancer cell membranes or their receptors, causing cytotoxicity, apoptosis and inhibition of tumor growth. some lectins are able to prevent the proliferation of malignant tumor cells because they recognize the t-antigen (gal b - galnac) found specifically on the surface of tumor cells ( ) . the main problem is that their use as a detection agent for the t-antigen in clinical studies is not possible because the immune system can recognize them as foreign molecules and develop an immune response. previous studies with x-ray crystallography made in our laboratory have characterized a lectin found in mushrooms called bel b-trefoil which has antiproliferative activity on tumor cell lines, because it contains three binding sites for the t-antigen. unlike other lectins with this property, bel b-trefoil shows structural homology with a human protein, acidic fibroblast growth factor (fgf ) ( ). superposition of their structures suggests that the human protein could be mutated to contain at least one of the binding sites for the t-antigen. such mutations should create in fgf the potential capacity of recognizing tumor cells with less immunogenicity than the fungal protein. fgf is mitogenic and chemotactic, and mediates cellular functions by binding to transmembrane receptors, which are activated by ligand-induced dimerization requiring heparin as co-receptor. to reach our purpose, the fgf cdna was cloned into a bacterial plasmid and then mutated in five different positions to eliminate its mitogenic activity and to engineer in the protein the t-antigen binding capacity. attempts to crystalize the mutants of fgf were made using the hanging drop technique with the final aim to carry out their structural characterization by x-ray diffraction analysis of the crystals. the de novo synthesis of proteins in response to the activation of cellular signaling pathways is a crucial element of many high-level biological processes, including the synaptic plasticity underpinning memory formation in the brain. while of fundamental biological importance, there has been a shortage of tools with which to specifically target pools of newly synthesized proteins of interest for study. thus, we have developed timestamp and smash, methods for drug-dependent tagging, or destruction, respectively, of newly synthesized copies of proteins of interest. both methods rely on protein tags that remove themselves by default via an internal hepatitis c virus (hcv) ns protease, but which are retained in the presence of cell-permeable small molecule protease inhibitors. the timestamp tag contains split yfp halves and epitope tags which are reconstituted and preserved, respectively, on proteins of interest following drug application, whereas the smash tag contains a strong degron which remains attached to proteins of interest following drug application, resulting in their clearance. one limitation of time-stamp and smash is that they can only be used to independently manipulate one protein of interest at a time. furthermore, the application of timestamp and smash to study endogenous protein pools in mammals has not yet been explored. here, we report on efforts to extend these techniques by reengineering ns proteases which can be inhibited by two different drugs orthogonally to one another. by incorporating different drug resistance mutations into two ns protease variants, we engineered ns protease domains that are inhibitable either by asunaprevir only, or by telaprevir only. we found that these tags permit simultaneous and independent control over the newly synthesized pools of two proteins of interest within the same population of cells. we also report the development of transgenic knock-in mouse strains incorporating timestamp and smash tags, which allow the interrogation of newly synthesized pools of specific endogenous synaptic proteins in the context of their endogenous regulatory elements, and without relying on overexpression. infectious diseases are often diagnosed by the presence of specific antibodies that are produced in response to the invading pathogen. one example are antibodies that are present in patient blood after infection with the dengue virus serotype and that are directed against an epitope on the virus' nonstructural protein (ns- ). traditional antibody diagnosis relies on time-consuming multi-step assays that require sophisticated equipment in a laboratory environment. a promising alternative are protein switches that are based on bioluminescence resonance energy transfer (bret). these switches comprise a luciferase (nanoluc) and a green fluorescent protein (mneongreen), which are connected via a semiflexible linker. the linker contains two epitope sequences of ns- to which the antibodies bind specifically. if no antibodies are present nanoluc and mneongreen are held in close proximity via two helper domains and bret can occur; thus green light originating from mneongreen is visible. if antibodies are present, they bind to the specific epitopes in the linker of the switch and cause stretching of the linker and therewith break the interaction of the helper domains. as a result, nanoluc and mneongreen are separated in such a way that bret cannot occur anymore; thus only blue light originating from nano-luc remains visible. using this principle, monoclonal anti-ns- antibodies were detectable in a controlled buffer system and in spiked plasma samples. furthermore, the developed antibody switch was applied to plasma samples of macaques after a primary infection with dengue virus serotype . signal readout was possible using a laboratory-based plate reader as well as the camera of a standard smartphone. we demonstrate that this bret-based protein switch can quickly detect antibodies in solution in a single-step assay format using simple equipment for signal readout, such as a standard smartphone. this simplified antibody detection platform has the potential to be carried out outside of a laboratory, thus in areas with limited laboratory infrastructure and a high number of diverse infectious diseases. proteins expressed from more than two-thirds of the human genome reside within intracellular compartments. of these proteins many are important disease-related targets such as kras and c-myc which cannot be easily addressed by conventional small molecule approaches. some of the weaknesses of small molecules can be addressed by biologic drugs, for example high target specificity and inhibition of protein-protein interactions. the challenge for biologics is how to engineer recombinant proteins to access the intracellular space. one strategy is to use systems evolved by bacteria and viruses to deliver material inside the cells. an example of such pathway is used by pseudomonas exotoxin a (pe). the modularity of pe allows the catalytic domain to be replaced with a biologic payload against desired intracellular target. an additional benefit of pe-based delivery is a possibility of targeting the drugs only to relevant cells in the body by modifying the cell-targeting domain of the pe. the aim of this project is to deliver functional payloads against k-ras and c-myc into the cell using a pseudomonas exotoxin a translocation domain. we used phage and ribosome display to select antibody mimetics that bind k-ras and c-myc. here, we present their activity in biochemical assays and the initial results on generation of pe-based constructs. ( ) . hemagglutinin is synthesized as ha molecule assembled as noncovalently bound homotrimers on the viral surface. this precursor protein is cleaved by trypsin-like proteases to yield two subunits ha and ha linked by a single disulphide bond ( ) . ha is also post-translationally modified by n-glycosylation ( ). it is well established that the virus hemagglutinin is the main antigen, inducing the neutralizing antibodies. in the attempt towards developing influenza vaccine production (the egg-based manufacturing lasts several months) that would be faster and safer the utilization of recombinant antigen alone is currently being observed. recently we demonstrated that yeast produced influenza h protein although cleaved into two subunits induced strong immunological response in mice ( ) . in this report, we describe the biochemical and immunological characterization of the h antigen, based on hydrolytic domain of the h n gene, with deletion of multibasic cleavage site and expressed in yeast system. the ha encoding gene from h n virus with deletion of nucleotides was cloned into ppiczac vector. rha fusion protein with his -tag was secreted into the culture medium and was purified to homogeneity in one step using ni-nta agarose. the efficiency of the antigen purification was mg/l. glycosylation sites of rha were determined using lc-ms-ms/ms. analysis of the n-linked glycans revealed that the rha is glycosylated at the same sites as the native ha in the vaccine strain. next we investigated if the hemagglutinin with deletion of the cleavage site oligomerize into higher molecular forms. to determine the oligomeric forms of the recombinant antigen various approaches were applied e.g. native-page, size exclusion chromatography or dynamic light scattering. as a final experiment to measure the size of oligomers in a protein sample a combined technology sec-mals was conducted, using multi angle light scattering (mals) as a detector. the immunological activity of rha was tested in chicken and mice, where antigen elicited high immune response. the data presented here demonstrate that new influenza antigen produced in p. pastoris is highly immunogenic and might be consider as a candidate for subunit vaccine. structural motifs capture redundant patterns that frequently occur in proteins. motifs associated with contiguous fragments of structure (i.e., secondary structural motifs) are well studied and have been successfully used to capture "rules" describing sequence-structure relationships in protein design and structure prediction. we have extended this concept to motifs that capture tertiary information-(i.e., tertiary structural motifs or terms. we have discovered that a relatively small alphabet of terms describes the known structural universe (all secondary, tertiary and quaternary information in the pdb) at sub-angstrom resolution. this alphabet of universal motifs reveals the remarkable degeneracy of the protein structure space, with just a few hundred terms sufficient to accurately capture half of the known structural universe. we have begun to demonstrate the considerable promise this structural alphabet has for applications such as protein design, structure prediction, and docking. we have developed a novel protein design framework that selects amino acid sequences, given a desired structure, using solely information from the universal terms. we show that given a native backbone, this framework recovers the native sequences to a level on par with state-of-the-art atomistic protein design methods, indicating that the motifs capture the salient structural rules governing native proteins. further, predicted sequence distributions agree closely with observed evolutionary variation. given the apparently high degeneracy among even complex features of protein structure, methods based on mining the pdb for tertiary information should provide ample opportunities for advancement in problems of computational structural biology. sortase-mediated synthesis of protein-dna conjugates for sensitive biosensing bedabrata saha , marieke op de beeck , remco arts , maarten merkx in recent years, semisynthetic protein-dna conjugates have emerged as attractive biomacromolecules for different applications in bio-nanotechnology, biosensing, diagnostics and therapeutics. in protein-dna conjugates, synthetic oligonucleotides allow the construction of desired molecular architecture with high specificity, while maintaining the original functionality of the protein molecules for desired application. however, the synthesis of site-specific and stoichiometric protein-dna conjugates can be challenging. due to the diversity in composition and physico-chemical properties of the proteins, few generic strategies are available for conjugation of protein molecules to a dna scaffold. a common approach is to use thiol-based covalent conjugation, but the introduction of additional cysteines can lead to the formation of intermolecular disulfides or interfere with the formation of native disulfide bonds. as an alternative, here we have developed a site-directed protein-dna conjugation strategy based on sortase mediated trans-peptidation reaction. the sortase recognizes a 'sorting motif' (i.e. lpxtg, x any amino acid), which is recombinantly introduced by site-directed mutagenesis at the cterminal end of the protein molecule. the sortase cleaves the t-g peptide bond and catalyzed the formation of a new amide bond between the lpxt peptide and the n-terminal amine of any molecule bearing an n-terminal oligoglycine motif. for this purpose, a triglycine motif was introduced at the 'end of single-stranded dna (ssdna). on-column synthesis of triglycine modified ssdna, protected on a controlled pore glass beads, simplified the purification process and enhanced the yield of triglycinemodified ssdna (> %). we used this conjugation strategy in several biosensing applications. for example, we used the method to conjugate ssdna linkers at the c-termini of a range of single-chain antibody fragments (scfv) and applied these constructs to allow oriented display of capture molecules on biosensor surfaces. ssdna-scfv were using an excess of triglycine modified ssdna, we achieved % conversion scfv-ssdna conjugate, which can be further purified by in two step purification process consisting of ni-nta affinity column and ion-exchange chromatography. we also extended this sortase-based conjugation strategy to develop a bioluminescence based assay for sensitive target oligonucleotide detection. in this regard, the ' and ' end triglycine-modified ssdna molecules were successfully conjugated with a bret protein pairs, nanoluc luciferase and mneongreen fluorescent protein. the introduction of a c-terminal sortase-his tag and and n-terminal strep-tag allowed efficient purification of theseprotein-ssdna conjugates from excess oligonucleotides and unreacted protein. mass spectrometry based proteomics to identify the protein differences in human breast milk from breast cancer patients and controls devika channaveerappa , roshanak aslebagh , kathleen f. arcaro , costel c. darie breast cancer is the second leading cause of cancer death in women. about % women in the us develop breast cancer. death rates due to breast cancer have been declined over the years due to advancements in mammography and treatment. although, mammography helps in the early detection of breast cancer, it has few limitations. dense breast tissue makes mammogram less accurate. breast milk can be assessed to evaluate the risk of one getting breast cancer by comparing the proteomes of breast milk from healthy and breast cancer suffering individual. this study makes use of mass spectrometry based proteomics to identify the differences between the control and cancerous samples which would further help in identifying potential biomarkers for breast cancer. firstly, sds-page was used to separate the proteins from the whole milk sample. the gel bands for each sample was then excised and cut into small pieces. the gel pieces were washed and trypsin digested in order to extract the peptides. peptide mixtures in the solution were cleaned using c zip-tipp and then analyzed by liquid chromatography-tandem mass spectrometry (lc-ms/ms). minutes and minutes gradient were used for lc-ms/ms analysis. raw data obtained were converted to pkl files using proteinlynx global served (plgs version . ). raw data were then submitted to mascot database search for protein identification. the mascot results were then exported as .dat files and further analyzed using scaffold version . software. three breast cancer milk samples were investigated against healthy control milk samples. in the sds-page gel, after coomassie staining, the protein patterns did show minor differences. after lc-ms/ms analysis, the proteins identified by mascot database search were imported into the scaffold software and compared for the relative ratio between the proteins from the milk sampled from control donors and the donors with breast cancer. there were significant differences identified in the proteomes of the two sets of samples. some of the proteins were upregulated in the breast cancer samples and some were down regulated when compared with the controls. additional investigation of more breast milk samples is ongoing. this study focuses on identifying biomarkers directly in the milk of donors with breast cancer. leukolike vectors: leukocyte-inspired nanoparticles claudia corbo , , alessandro parodi , , roberto palomba , , roberto molinaro , michael evangelopoulos , francesco salvatore , , ennio tasciotti the houston methodist research institute, fondazione irccs sdn, nanomedicine aims to improve drug efficiency by enhancing targeting and biocompatibility, and reducing side effects. multiple surface modifications have been proposed to provide nanocarriers with these features, based on complex synthesis processes and very often inefficient in contemporary providing biological tolerance and targeting properties [ ] . bio-inspired approaches based on surface coatings developed from the purified cell membrane of immune cells represents a new paradigm shift for the development of carrier enable of prolong circulation and proper tumoritropic capabilities. we showed that nanoporous silicon (nps) particles coated with leukocyte cellular membranes -leukolike vectors (llvs) -possess cell-like properties [ ] . llvs can escape macrophage uptake, delay sequestration by the reticulo-endothelial system, target tumor inflamed vasculature and accumulate within the cancer parenchyma [ ] . llvs were fully characterized for their shape, size, surface charge and coating through dynamic light scattering and scanning electron microscopy. in addition we characterized the content and function of the leukocyte's proteins transferred onto the llvs coating through high-throughput proteomic analysis and the results revealed the presence and the correct orientation of several important markers of leukocytes: cd , cd and mhc-i were identified as key players in determining llvs biocompatibility, while leukocyte associated function- (lfa- ) and mac- contributed to the llvs targeting ability and bioactivity towards inflamed endothelium [ ] . recent investigation showed that the coating induced the formation of a singular protein corona (i.e. the protein adsorption layer) on the surface of the nanoparticles compared to negative control following in vivo injection. in addition, the proteolipid coating favored active extravasation of the llvs in the tumor vasculature by molecular mechanisms similar to those used by tumor infiltrating leukocytes. this work shows that is possible to transfer biologically active leukocyte membrane proteins onto synthetic nanoparticles, thus creating biomimetic carriers retaining cell-like functions that are not affected by the protein corona effect that occurs in vivo. the targeting of the inflamed endothelium can be applied to a broad range of diseases and the approach used to formulate the system could open new avenues for the fabrication of the next generation of personalized treatments by using as cell membrane source the immune cells of patients. references: [ ] alessandro parodi, claudia corbo, armando cevenini, roberto molinaro, roberto palomba, laura pandolfi, marco agostini, francesco salvatore, ennio tasciotti. enabling cytoplasmic delivery and organelle targeting by surface modification of nanocarriers. nanomedicine uk. accepted. steroid hormone receptors are intracellular receptors that initiate signal transduction in response to steroid hormones, including oestrogen and androgens. generally, the binding of the steroid to the nuclear receptor induces the protein to form a dimer and relocate onto the chromatin, although the order of these events may vary. the location of receptor binding on the chromatin is defined by specific hormone response elements (hre). once located, the receptor promotes gene activation by the recruitment of other co-factors. it is this process that makes the complex of receptor protein and co-factors play a pivotal role in the regulation and activation of genes. the failure to regulate this process correctly is a key step in the development of several endocrine-driven cancers. for example: oestrogen receptor positive (er ) breast cancer is one of the most common forms of cancer and accounts for % of all breast cancer cases. in er tumours, the oestrogen receptor (er) drives the tumour growth and cell proliferation. understanding the interactions of the er with other proteins, either directly or indirectly, can provide vital insight to the regulation of the system that drives this cancer. the progesterone receptor (pr) has also been implicated in breast cancer, and the androgen receptor (ar) is a known driver in the majority of prostate cancers. to meet the challenges of elucidating these systems, we have developed methods to purify and analyse cross-linked regulatory complexes bound to dna by mass spectrometry (chip-ms). this allows for the enrichment of proteins involved in gene regulation. chip-ms, combined with tandem mass tags (tmt), makes it possible to realise a quantitative method to investigate the dynamic network of interactions between proteins within complexes that undertake the regulation of biological systems. chip-seq is a well-established method for identifying where these protein complexes are bound to the genome. this work focuses on how to combine these technologies with my previous development of cross-linking coupled mass spectrometry techniques (xcms) to provide a strategy for visualising the dynamic organisation of the proteins on the chromatin. global kinetic analysis of caspase protein substrates in cell lysate reveals selective roles and target specificity olivier julien , min zhuang , arun wiita , james wells caspases are cysteine proteases that play important roles in development, cell differentiation and cell death. however, the limited number of known caspase substrates hinders our understanding of caspase function. here we performed a non-biased identification and kinetic analysis of caspase- and caspase- proteolytic substrates in cell lysate, using an enzymatic n-termini enrichment approach followed by mass spectrometry. we identified and potential substrates for the initiator caspase- and putative executioner caspase- , respectively. our results not only confirm known substrates but also identify many more new substrates with the precise location of proteolysis. given the emerging roles of caspases- and in inflammation and neurodegeneration, these new substrates may provide molecular insight into the progression of related diseases. the sequence consensus logo of caspase- targets was very similar to a classical executioner caspase motif (devd), while caspase- revealed a vevd motif. using selected reaction monitoring (srm), we quantified the kinetics of proteolysis of a large subset of these substrates by measuring the appearance of the caspase cleavage product over time. in the end, we measured and kcat/km values for individual substrates cut by caspase- and caspase- , respectively. by comparing these data with our previous analysis of caspase- , , and , we found that substrates that are shared between caspases are often cleaved at rates that differ by orders of magnitude. thus, despite having nearly identical primary sequence motifs, the caspases exhibit remarkable substrate specificity that may reflect their specialized roles within the cell. the rockefeller university, new york university school of medicine, johns hopkins university school of medicine line- (l ) retrotransposons are catalysts of evolution and disease whose sequences comprise a significant proportion of the human genome. despite tremendous influence on genome composition, l rnas only encode two proteins. consequently, l particles include a combination of permissive host factors that are essential to their lifecycle as well as repressive factors that constitute defenses against l 's mutagenic activity. we previously characterized host proteins associated with synthetic and natural human l retrotransposons, as expressed in cell culture, using a combination of techniques including metabolic labeling and affinity proteomics. to build on these analyses, we have implemented a series of d separations and post-purification treatments to produce a multi-dimensional interactomic characterization of affinity isolated l s. these studies have revealed the presence of at least two populations of putative transposition intermediates that may exhibit distinctive intracellular localizations. we report a comprehensive, quantitative survey of the proteins partitioning within these distinct l populations and their associated in vitro activity. our observations provide a basis for the classification of l interactors with respect to their physical and functional links, facilitating hypotheses to direct in vivo experimentation. polyubiquitin recognition by continuous ubiquitin binding domains of rad probed by modeling, small-angle x-ray scattering and mutagenesis sangho lee , trung thanh thach , namsoo lee , donghyuk shin , seungsu han , gyuhee kim , hongtae kim rad is a key protein in double-strand break dna damage response (ddr) pathways by recognizing k -linked polyubiquitylated chromatin proteins through its bipartite ubiquitin binding domains ubz and lrm with extra residues in between. rad binds k -linked polyubiquitin chains as well as k linked ones and mono-ubiquitin. however, the detailed molecular basis of polyubiquitin recognition by ubz and lrm remains unclear. here, we examined the interaction of rad ( - ), including ubz and lrm, with linear polyubiquitin chains that are structurally similar to the k -linked ones. rad ( - ) binds linear polyubiquitin chains (ub , ub , ub ) with similar affinity to a k -linked one for diubiquitin. ab initio modeling suggests that lrm and the extra residues at the c-terminus of ubz (residues - ) likely form a continuous helix, termed 'extended lr motif' (elrm). we obtained a molecular envelope for rad ubz-elrm:linear ub by small-angle x-ray scattering and derived a structural model for the complex. the rad :linear ub model indicates that elrm enhances the binding of rad with linear polyubiquitin by contacting the proximal ubiquitin moiety. consistent with the structural analysis, mutational studies showed that residues in elrm affect binding with linear ub , not monoubiquitin. in cell data support that elrm is crucial in rad localization to dna damage sites. specifically e seems to be the most critical in polyubiquitin binding and localization to nuclear foci. finally, we reveal that the ubiquitin-binding domains of rad bind linear ub more tightly than those of rap , providing a quantitative basis for blockage of rap at dsb sites. taken together, our data demonstrate that rad ( - ) forms continuous ubiquitin binding domains, comprising ubz and elrm, and provides a structural framework for polyubiquitin recognition by rad in the ddr pathway at a molecular level. optimization of a protein extraction method for the proteomic study of pozol cynthia teresa leyva-arguelles , carmen wacher , rosario vera , romina rodr ıguez-sanoja instituto de investigaciones biom edicas, unam., facultad de qu ımica, unam., instituto de biotecnolog ıa, unam key words: proteomics, fermentation, pozol pozol is a mexican traditional no alcoholic beverage elaborated by various ethnic groups in the southeastern of mexico. pozol is obtained from the natural fermentation of nixtamal (heat-and alkali-treated maize) dough. the main carbohydrate in maize dough is starch ( - %), because others such as sucrose, glucose and fructose are mostly lost during nixtamalization; so, the starch remains as the major carbohydrate available for fermentation [ ] . a wide variety of microorganisms have already been isolated from the fermentation of pozol; these microorganisms include fungi, yeasts, lactic acid bacteria, and non-lactic acid bacteria [ ] . however, only few bacteria are amylolytic in this fermentation and all of them are weakly amylolytic [ ] . in an attempt to explain how a very low content of soluble sugars can support a diverse and abundant microbiota, a proteomic approach was designed to understand the fermentation of pozol [ ] . nevertheless, the extraction of proteins from pozol remains a limiting step in proteomic analysis mainly due to the complexity of the sample. on the basis of the aforementioned reasons, the aim of this work was to obtain a suitable extraction method of proteins for proteomic analysis. therefore, the fermentation of pozol was continued for h and samples were taken at , , and h. for each sample, the total sugar content was determined by the dubois et al. method [ ] and protein extraction was performed by two methods: a) direct extraction from the dough [ ] and b) initial extraction of microorganisms and soluble proteins (this work). comparison between the two protein methods was performed on two-dimensional gels with silver stain. then, gels underwent to image analysis by the image master d platinum software. comparing the d-gels, more proteins spots were obtained with method b than that with method a, indicating a more efficient protein extraction with method b. although, using method a higher concentration of total proteins was observed, they were mostly maize proteins, that in turn overlap and reduce the efficiently extraction of the microbial low abundant proteins. then, method b allows a better extraction of those low abundant proteins and removes sample components that may interfere with the determination. these results could help us to find the proteins involved in carbohydrate metabolism of the microbiota and finally elucidate the dynamics of pozol fermentation. proteomics has been applied to the enology field for numerous purposes including fermentation control, improvement of fermentation processes, ensuring wine quality, etc. according to rodriguez et al., ( ), the information provided by wine proteomics is not only useful for these intentions, but also offers excellent prospects for innovation and diversification of winemaking processes in the near future. in this context, our group has focused research on the identification of proteins that might be important for yeast survival under typical wine elaboration conditions (standard fermentation, sherry wine biological aging and sparkling wine second fermentation) as well as proteins that configure the content of metabolites which are ultimately responsible for wine quality. by using novel proteomic (offgel fractionator and ltq orbitrap xl ms) and metabolomic techniques (sbse-td-gc-ms) we have identified a high amount of up-regulated proteins involved in processes like oxidative stress response (in biological aging) or protein biosynthesis (in second fermentation) as well as thirty-three proteins directly involved in the metabolism of glycerol, ethanol and seventeen aroma compounds excreted by the yeast under biological aging conditions. further, in order to validate proteome data; null mutants of genes codifying proteins up-regulated in the biological aging condition were constructed. analyses of correlated phenotypes are in progress. this technique and its combination with metabolomics within the enology context will provide enough knowledge to design or choose yeasts or conditions that satisfy wine production and/or wine characteristics such as color/aroma/texture/flavour profile demands of winemakers and consumers. additional binding sites for cytochrome c on its redox membrane partners facilitate its turnover and sliding mechanisms within respiratory supercomplexes blas moreno-beltr an , antonio d ıaz-quintana , katiuska gonz alez-arzola , alejandra guerra-castellano , adri an vel azquez-campoy , miguel a. de la rosa , irene d ıaz-moreno ibvf, ciccartuja, universidad de sevilla -csic, bifi -iqfr (csic), universidad de zaragoza, departamento de bioqu ımica y biolog ıa molecular celular, universidad de zaragoza, gliding mechanisms of cytochrome c (cc) molecules have been proposed to shuttle electrons between respiratory complexes iii and iv within plant and mammalian mitochondrial supercomplexes, instead of carrying electrons by random diffusion across the intermembrane bulk phase [ ] [ ] . in this work, the binding molecular mechanisms of the plant and human cc with mitochondrial complexes iii and iv have been analyzed by nuclear magnetic resonance and isothermal titration calorimetry. our data reveal that both cc-involving adducts possess a : stoichiometry -that is, two cc molecules per adduct -. the presence of extra binding sites for cc at the surfaces of complexes iii and iv opens new perspectives on the mitochondrial electron transport chain, where membrane respiratory complexes can be either in independent, free diffusional motion or forming macromolecular assemblies. in the latter context, such new binding sites for cc facilitate the turnover and sliding mechanisms of cc molecules within supercomplexes. indeed, the accommodation of several cc molecules between complexes iii and iv in supercomplexes provide a path for cc diffusion from complex iii to iv. such path could have physiological significance in the electron flow, which is controlled in supercomplexes to optimize the use of available substrates [ ] [ ] [ ] . can bio-functionalities be deciphered from protein sequence information using computational approaches? background: the processes of uncovering bio-functionalities such as pharmacological activities, disease processes, physiological and structural properties by means of clinical approaches are irrational. this is because they are resource and time consuming. sometimes, they involve sophisticated and expensive equipments, reagents and animal tissues. contrarily, sequence information-based computerized approaches are rational and have become relevant in assessing bio-functionalities. they include geno pheno [coreceptor] [ ] , position-specific scoring matrix (pssmsi/nsi and pssmcxcr /ccr ) [ ] , and informational spectrum method (ism)-based phylogenetic analysis (istree) [ ] . aim: this presentation demonstrates how bio-functionalities could be deciphered from sequence information using computational approaches. method: ism procedure and peptides, vipmfsals and capagfail are engaged. results: protein sequences of the peptides are converted into bio-functionality (affinity). affinity between the two peptides is demonstrated as significant amplitudes at the point of common interaction also referred to as consensus frequency, signifying remarkable affinity. discussions: bio-functionalities of bio-molecules are known to be expressed in one or two genes, which have been found to provide as much biological information as the bio-molecules. this indicates that biological characteristics, represented in these genes and proteins can now be extracted from their sequence information. for example, multi-drug resistances arising from a variety anti-microbial agent from several classes including alkaloids, flavonoids, etc can be retrieved from the sequence information of their encoding genes (mdr and mdr ). similarly, translation of hiv infection to aids disease can be extracted from the protein sequence alterations in the hiv gp . similarly, effectiveness of anti-retroviral agent, maraviroc on the hiv isolate h bx and ndk can be deciphered from the sequence information of their v observed at the predicted sequences. these positions are important as they surround the cleavage site in the three-dimensional structure, and are probably less tolerant to change. moreover in previous studies, cys at p position has been shown to be the dominant determinant for cleavage efficiency, while cys, pro and glu at p position have also been shown to be correlated with increased cleavage efficiency of ns / a protease. for adv cysteine protease, on the other hand, bsst produces similar significant results for both type (xgx-g) and type (xgg-x) consensus cleavage sites, where p and p ' positions have gly with highest percentage in type (xgx-g) while p and p positions have gly in type (xgg-x). these indicate that the bsst seems to provide a powerful methodology for predicting the substrate specificity for the hcv ns / a serine protease and adv cysteine protease, which are targets in drug discovery studies. protein plasticity improves protein-protein binding description chiara pallara , juan fern andez-recio an accurate description of protein-protein interactions at atomic level is fundamental to understand cellular processes. however the current structural coverage of protein-protein interactions (i.e. available experimental structures plus potential models based on homologous complex structures) is below % of the estimated number of possible complexes formed between human proteins. , for these reasons, computational docking methods aim to become a complementary approach not only to solve the structural interactome but also to elucidate the basis of the protein-protein association mechanism. in spite of the advances in protein-protein binding description by docking, dealing with molecular flexibility is a major bottle-neck, as shown by the recent outcomes of the capri (critical assessment of prediction of interactions) experiment. this data clearly confirms that the protein dynamics plays a key role in protein-protein association. the use of conformational ensembles generated from unbound protein structures in combination with computational docking simulations might represent a more realistic description of protein-protein association. here, we present the first systematic study about the use of precomputed unbound ensembles in docking, as performed on a set of cases of the protein-protein docking benchmark . . the primary aim of our work is to understand the role of the protein conformational heterogeneity in protein-protein recognition. to do this, small conformational ensembles were automatically generated starting from the unbound docking partners, and then an extensive analysis of their binding properties was performed in the context of pydock docking scheme. the results show that considering conformational heterogeneity of interacting proteins can improve docking description in cases that involve intermediate conformational changes in the unbound-to-bound transition. more interestingly, we found that protein plasticity increases chances of finding conformations with better binding energy, not necessarily related to bound geometries. the relevance for future docking methodology development and for understanding protein association mechanism will be discussed. purpose of the research: there is increasing interest in the development of protein scaffolds that can be used to develop affinity reagents that are alternatives to antibodies. the affimer scaffold is based on the cystatin protein fold. the affimer scaffold is biologically inert, biophysically stable and capable of presenting a range of designed or random binding surfaces defined by peptides inserted at different loops. the result is highly specific, high affinity interactions with a wide range of targets including ones that are inaccessible to antibodies. affimers are designed to work in the same way as the very best antibodies, but with a number of key advantages. affimers are quick to develop (typically weeks) without using animals. they contain no disulphide bonds, are expressed easily in e. coli and have no batch to batch variability. affimers are small molecules ( aa, kda), robust and stable (resistant to ph range, thermally stable and not sensitive to edta). affimers can be a direct replacement for antibodies -no process or workflow change required -and perform identically to antibodies in assays such as elisa, facs, ihc, western blots, affinity purification, microarray and potentially therapeutics. we describe some applications of the technology in regards of affimer development for custom targets on one hand and for the biomarker discovery workflow using affimer microarrays on the other. main results: by screening of our very large ( x ) library against yeast sumo protein we identified affimers with high affinity allowing their use for elisa. moreover, no cross-reactivity was observed when affimers were used on western blots leading to a unique band specific to yeast sumo when compared to human proteins. a library of , random affimers, expressed in e. coli, was printed on glass microscope slides and challenged with plasma from children (n ) with sepsis and from healthy children (n ). unsupervised hierarchical clustering based on the , affimers allowed differentiation between the control and patient samples. affimers were found to differentially bind proteins between the groups with a > fold change. the affimer arrays identified a strong signature of sepsis and roc curve analysis allowed confident prediction of disease (auroc of . ). affinity purification and preliminary mass spectrometry analysis identified known biomarkers of sepsis and also potentially novel biomarkers not previously associated with this disease. major conclusions: this work demonstrates the scope of affimer affinity reagents to develop alternative binders to antibodies, where affimers perform identically in most assays without the disadvantages associated with antibodies. moreover, affimers enable a new protein microarray-based biomarker-discovery workflow and we predict that array-based validation of signatures identified using discovery arrays prior to affinity purification and mass spectrometry will offer a cost-and time-effective methodology compared to purely mass spec-driven workflows. tau pathologies, called 'tauopathies', are related to several neurodegenerative diseases including alzheimer disease (ad). in ad, tau protein is observed hyper-phosphorylated and aggregated as paired helical filament (phf). the neuronal tau protein is an intrinsically disordered proteins (idps). nuclear magnetic resonance spectroscopy (nmr) is here used to study the tau protein phosphorylations and protein-protein interactions (ppis). in in vitro assays, tau phosphorylation by rat brain extract is considered as an hyperphosphorylation model that was furthermore pointed out to enable tau aggregation [ ] . in a first step, we have identified all the phosphorylation sites of rat brain extract phosphorylated-tau, using the analytical capacity of nmr. we showed that the protein is modified at ser/thr sites. among the kinases that we have characterized so far using tau as substrate, only the extracellular signal-regulated kinase (erk ) shows an ability to modify in vitro tau protein on so many sites. we have indeed identified phosphorylated ser/thr-pro motifs out of potential phosphorylation sites in the sequence of full length -residue tau. in addition, we showed using transmission electron microscope (tem) a similar in vitro aggregation capacity of erk-phosphorylated tau protein compared to that of rat brain extract phosphorylated-tau. this shows that phosphorylation by the erk kinase generates an hyperphosphorylated tau. given the high efficiency of erk towards tau, we have next looked into the mechanism of tau recognition. erk kinase possesses two well-characterized docking domains: d recruitment sites (drs) and f recruitment sites (frs), which recruit complementary docking sites and increase the specificity and efficiency of the interaction with both its upstream regulators and downstream substrates [ ] . as the interaction between tau protein and erk kinase is analyzed by nmr spectroscopy, multiple sites of interaction are observed along the tau sequence, similar to drs docking sites, all located in the so-called microtubule binding domain of tau. these sites are short sequences loosely matching the reported consensus for d sites w - uxu (w, u, and x refer to positively charged, hydrophobic, or any intervening residues, respectively) [ ] , and also the reverse sequence uxuw - .to confirm the mapping of the interaction, two tau recognition sites were produced as recombinant peptides of about amino-acid in fusion with an n-terminal his-tag sumo. interaction assays using d [ h, n] hsqc spectra of the peptides confirm their binding to erk kinase. the potential of these peptides to inhibit erk activity with tau as substrate is now being investigated. while rigid-body docking has become quite successful for predicting the correct conformations of binary protein complexes, determining whether two given proteins interact remains a difficult problem. successful docking procedures often give equally good scores for pairs of proteins for which there is no evidence of interaction. studies investigating what we define as the 'pre-docking' problem via in silico approaches have only recently become feasible with the help of supercomputers and gridcomputing systems. in a previous work, on a restricted set of protein complexes, we showed how predictions of interacting partners could be greatly improved if the location of the correct binding interface on each protein was known. experimentally identified complexes are found to be much more likely to bring these two interfaces into contact, at the same time as yielding good interaction energies. we present data from a complete cross-docking (cc-d) study of a database of proteins, including the treatment of more than , potential binary interactions. the performance of the interaction index we developed to predict binding probability compares well with other methods. by studying the interaction of all potential protein pairs within a dataset, cc-d calculations can also help to identify correct protein interaction interfaces. the present large-scale study also reveals the influence of various protein families (enzyme-inhibitor, antibody-antigen, antigen-bound antibody, etc.) on binding specificity, showing, in particular, the distinctive behavior of antigenic interfaces compared to enzymes, inhibitors or antibodies. the performance of our approach is encouraging. although identifying interaction interfaces significantly helps in the identification of interacting proteins, further refinements will be necessary to make in silico cross-docking a viable alternative to high-throughput experimental methods. whole-protein mass spectrometry reveals global changes to histone modification patterns in hypoxia sarah wilkins , kuo-feng hsu , christopher schofield chemistry research laboratory, oxford university cells respond to limiting oxygen availability (hypoxia) by altering the gene expression profile. this primarily involves changes at the level of transcription via the activity of hypoxia-responsive transcription factors, although increasing evidence suggests that changes in chromatin structure (i.e. from a condensed 'silent' state to a more open or 'active' state) are required in order for transcription to take place. in particular, post-translational modifications (ptms) to histones have an important regulatory function in gene expression under hypoxic conditions. the n-terminal tails of histone proteins are accessible to a set of enzymes capable of 'writing' and 'erasing' ptms including acetylation, methylation, ubiquitylation, sumoylation and phosphorylation. to date, studies in hypoxia have employed antibody-based methods to investigate changes in histone modifications, and so have focused on individual marks in isolation. the interplay between coexisting ptms is thought to be much more important than the effect of any single mark. therefore, a global view of the histone modification profile is essential to gain a complete understanding of the function of histone ptms and their roles in gene regulation. in this study, we apply whole protein mass spectrometry to investigate hypoxia-induced changes in histone marks. this 'top-down' approach provides insight into combinational modification patterns that are difficult to establish by antibody-based methods or peptide ms analysis. we investigated changes in the global ptm profiles of histones from a range of human cell-lines and tissues under severe hypoxia (< . % o ). we find that hypoxia causes a shift in the overall profile towards a more highly modified state, with significant changes in methylation and phosphorylation. marked changes in histone ptms were also observed following treatment of cells with epigenetic inhibitors and commonly used hypoxia mimetics, including several iron chelators currently in clinical trials for the treatment of anaemia. finally, we show that this method can be used to identify the histone variant h ax, whose phosphorylation at serine is an indicator of double-stranded dna breaks in cancer. overall, these data provide important insights into the epigenetic changes associated with hypoxia in normal and disease contexts. we hope to further develop this method in combination with different labelling strategies to enable quantitative analysis of histonemodifications in cells. mass spectrometry-based protein biomarker discovery in neurodevelopmental disorders interactions. there is currently no biological diagnosis or known cause of asd. slos is characterized by a cholesterol deficiency due to a mutation on the dhcr gene. approximately / , babies are born with slos. diagnosis is achieved by measuring cholesterol and -dehydrocholesterol ( dhc) levels in the blood, however, there is currently no proven treatment for slos. because of this, research is increasing to determine biomarkers for these disorders. here, samples from people with asd (sera and saliva) and slos (saliva), and matched controls were analyzed using a combination of gel electrophoresis (tricine-page, sds-page and blue native page), in gel digestion or insolution digestion and nanoliquid chromatography-tandem mass spectrometry (nanolc-ms/ms) to investigate differences between the proteomes of people with these neurodevelopmental disorders and matched controls. several alterations in protein expression were identified. these differences may lead to potential biomarkers for diagnosis, possible therapeutic targets and an altogether better understanding of the disorders. understanding protein recognition using structural features protein-protein interactions (ppis) play a crucial role in virtually all cell processes. thus, understanding the molecular mechanism of protein recognition is a critical challenge in molecular biology. previous works in this field show that not only the binding region but also the rest of the protein is involved in the interaction, suggesting a funnel-like recognition model as responsible of facilitating the interacting process. further more, we have previously shown that three-dimensional local structural features (groups of protein loops) define characteristic patterns (interaction signatures) that can be used to predict whether two proteins will interact or not. a notable trait of this prediction system is that interaction signatures can be denoted as favouring or disfavouring depending on their role on the promotion of the molecular binding. here, we use such features in order to determine differences between the binding interface and the rest of the protein surface in known ppis. particularly, we study computationally three different groups of protein-protein interfaces: i) native interfaces (the actual binding patches of the interacting pairs), ii) partial interfaces (the docking between a binding patch and a non-interacting patch), and iii) back-to-back interfaces (the docking between non-interacting patches for both of the interacting proteins). our results show that the interaction signatures in partial interfaces are much less favoured than the ones observed in native and back-to-back interfaces. we hypothesise that this phenomenon is related to the dynamics of the molecular association process. back-to-back interfaces preserve the exposure of the real interacting patches (thus, allowing the formation of a native interface), while in a partial interface one interacting patch is sequestered and becomes unavailable to form a native interaction. structural characterization of the cytoplasmic mrna export platform laboratory of cellular and structural biology, the rockefeller university., laboratory of mass spectrometry and gaseous ion chemistry, the rockefeller univ., university of california, san francisco, the new york structural biology center, department of biochemistry, faculty of medicine, university of montreal mrna biogenesis is an intricate process that begins within the nucleus and culminates with the remodeling and nuclear export of the mrnp particles through the nuclear pore complex (npc). defects in this conserved mechanism have been shown to cause serious human diseases. the protein assembly that performs the last steps in mrnp biogenesis and export is located at the cytoplasmic face of the npc and is formed by different proteins, organized into several subcomplexes whose arrangement and molecular architecture are poorly understood. in this study we applied an integrative approach, combining cross-linking and mass spectrometry (cx-ms), electron microscopy and available high-resolution structures, to describe the molecular architecture of the endogenous npc cytoplasmic mrnp export machinery. we generate a hybrid, close-to-atomic structure of the yeast native nup complex, the core of the assembly. our map also reveals how the nup complex organizes the entire cytoplasmic mrnp export machinery, and how this in turn docks into the architectural core of the npc. mapping of phenotypic profiles into our structures allows us to generate a first functional map of the ensemble. we expect that our map will serve as a framework to understand the molecular mechanisms underlying this key step of mrnp biogenesis. study of candidate proteins to pore associated with p x receptor in different cell types carla oliveira , anael alberto , mônica freitas , luiz alves laborat orio de comunicac¸ão celular -fiocruz, centro nacional de ressonância magn etica nuclear -ufrj aim: the p x r is a purinergic receptor, which differs from others subtypes due to its structural and pharmacological characteristics. when exposed for extended time or to high concentrations of its agonist (atp), promotes an increase in membrane permeability, allowing the passage of molecules up to da. there is a controversy among several authors that leave in doubt if this receptor needs a second protein for the pore formation and which protein could be. we select five pore-forming proteins: trpv , trpa , connexins- (cx- ), pannexin- (panx- ) and vdac. we believe that different mechanisms and proteins could be associated with p x r, depending on the cell type and their microenvironment stimuli. in this context, our main goal is identify possible proteins that could be associated with the p x r pore in different cells and species. methods and results: we started with rt-pcr technique of cell lines: j .g , n a, u , u , hek- and primary cells from wistar mouse and swiss mice. we used different primers and pcr cycle for each target at different species. we observed that the p x , panx- and cx- are the most abundant and are present in all cell types except the absence of p x in u cells and panx- in mice macrophages and u cells (n> ). however, trpv was seen at n a and u cells and trpa in and primary cells from mouse and mice and in j .g cells (n> ). regarding to the vdac, it is present in mouse macrophages, j .g and hek- cells (n> ). the further steps, we verified if those proteins could be physically associated with the p x r. we coimmunoprecipitated the p x r of j .g (with or without atp), mice macrophages, hek- and u cells. the samples were applied in two separated . % bis/acrylamide gels: one destined to mass spectrometry (ms) and the other to western blot. at this point, we confirmed the presence of p x r, and observed several others proteins associated to p x r at different cell conditions, mainly when we exposed, j .g cell, to mm atp (n ). at this condition, we found by ms, hsp , , and ; alpha and b tubulin; myosin va; alpha, b and g actin; malate and lactate dehydrogenase (n ). although u and hek- had not received atp treatment, we found several proteins associated to p x . the next step was to immunoprecipitated those proteins in j .g (treated or not with atp) and use it to verify if p x are physically associated to them. as result we saw the p x associated to panx- in j .g cells. conclusion: we conclude that the p x r activated by extracellular atp triggers the recruitment of variety different proteins. at this condition, we can suggest that maybe there is a conformational change, regardless of the numerous recruitment structural proteins. in addition, apparently, the pore-forming protein pannexin- is associated with p x r, and the others pore forming proteins (vdac, cx- , trpv , trpa ) seems not be linked to p x r at j . recently, we developed a series of molecular modeling tools for structure-based studies of protein functions and interactions. these tools are publicly available as web servers that are easily operated even by non-specialists: cabs-fold server for protein structure prediction [ ] ; cabs-flex server for modeling of protein structure flexibility [ ] ; aggrescan d server for prediction of protein aggregation propensities and rational design of protein solubility [ ] ; and cabs-dock server for prediction of peptide binding sites and peptide docking [ ] . the web servers are freely available from the laboratory website: http://biocomp.chem.uw.edu.pl/tools sandy on , pinghui feng university of southern california, keck school of medicine, developing a technique to detect deamidated proteins and peptides using rig-i sandy on, pinghui feng university of southern california, norris comprehensive cancer center, department of microbiology, and molecular biology, los angeles ca perhaps the most notable type of post-translational modification of proteins and peptides into a higher order structure is deamidation of asparagine and glutamine. deamidation occurs when an amine group is removed, degrading the molecule for purpose of regulating intracellular levels. previous studies have demonstrated that this notable post translational modification has been uncovered over time for use in dna recombinant technology as well as use as a biological clock to facilitate the rapid turnover of biologically important components of the cell. while the effects of this non-enzymatic chemical reaction have been widely studied, the method to uncover modification sites over a large quantity of proteins remains an issue. one of the most common types of deamidation is of asparagine and glutamine residues. at this time, most researchers will depend on mass spectrometric based proteomic techniques for identification of these post-translational sites. the issue is that mass spectral analysis of deamidated proteins and peptides is complication and can lead to misassigned identification attributed by an overlapping of c peak of the amidated form with the deamidated monoisotopic peak; these two peaks are only separated by . mda. while these issues can be mediated by using a mass spectrometer with a high mass measurement accuracy, and high resolving power, it is essential to establish simpler methods for identifying substrates that have undergone deamidation. if deamidation is present, different protein bands will be exhibited in the western blot, which will be compared to a triple mutant rig-i, which resists deamidation, to observe the location of this modification on the protein. with enough testing, i will determine specific sites of digestion and use this information to make conclusions of unknown proteins. i will make results regarding whether the protein has been modified based on the digestion sites. i will use mass spectrophotometry analysis to compare the proteins on a wider scale and double check my results. i have narrowed it down to a couple of different digestion sites that indicate deamidation. though the analysis work can be tedious, it is crucial to ensure the sites we isolate are accurate in order to establish this technique. from my research, we can apply this method for wider scale use such as in clinical settings. in areas of inflammation of parkinson's' patients, we can review specifically the infected cells versus uninfected and isolate the proteins, usually deamidated, responsible often smaller in size and more specific. in addition, research articles have already shown that suppressing modification of certain cells such as bcl-xl playing a major in leading the regulation of cancer cell death by apoptosis. by leading the discovery of a simpler methods to uncovering deamidation in cells, researchers will more easily and quickly be able to scan through various proteins, some of which discovered eventually may play pivotal roles in cancer research. influenza virus (iv) hemagglutinin (ha) is a homotrimeric integral membrane glycoprotein that mediates receptor-binding and membrane fusion. it constitutes the prominent viral surface antigen and a main target for neutralizing antibodies. bacterial, recombinant ha-based vaccines indicate high potential to confer protection against highly pathogenic (hp) avian iv (aiv) h n and arise as alternative for the traditional egg-or cell culture-based manufacturing. relatively short time of bacterial has production can be of great importance in case of a pandemic. escherichia coli produced protein, based on the ha sequence of a/swan/poland/ - v / (h n ) hpaiv*, has been successfully expressed in the form of inclusion bodies at institute of biotechnology and antibiotics. refolded and purified antigen was obtained in a soluble form, isolated by reversed phase hplc and identified with peptide mass fingerprinting using matrix-assisted laser desorption/ionization time of flight mass spectrometry (maldi tof/tof ms). the performed research in a great extent allowed to confirm the amino acid sequence of the recombinant ha (rha) assumed based on the cdna and allowed to establish the location of a total of six disulfide bridges. however, during purification and storage of the rha, apart from desired higher order rosette-like structures of the protein, other non-native species resulting from posttranslational modifications, misfolding, aggregation and degradation may occur what results in reduced vaccine potency. here, besides the properly folded monomers, we indicate non-native aggregates induced by disulfide crosslinking. moreover, several free cysteine residues and unexpected intrachain s-s were identified in rha tryptic peptide maps. cys was found most susceptible to formation of disulfide bridges between the distinct chains of rha. the above findings allow to assume that not all rha particles fold to form the native structure. reduced cys residues exhibit tendency to undergo oxidation and uncon- new strategies and approaches to understand how antibodies recognize and neutralize snake toxins represent a challenge to improve the antivenoms. the neurotoxic activity of micrurus venom is carried majority by two distinct proteins families, ftx and pla . the conserved structural folding of these toxins can be appreciated as model to generate inhibitors against them. in this regard, monoclonal antibodies (mabs) can be used as tool to find hot spots for inhibit the toxins and represent the first step in order to develop recombinant neutralizing molecules. in this work our goals were analyse a set of monoclonal antibodies against the most toxic components of m. altirostris venom by proteomics approaches. the venom was fractionated; its major toxic proteins identified by in vivo tests based on murine lethal toxicity analyses (approved by the ethical committee for animal experimentation from center of health and science of the federal university of rio de janeiro -no. . / - ). the toxic components were used to generate a panel of five monoclonal antibodies. elisa and antivenomics results allowed us identify the specificity of all mab and their neutralizing efficacy was measured by in vitro tests. three mabs showed reactivity towards ftx and two against pla . all monoclonal antibodies against ftx lack a broad recognition. however, we identified a pair of monoclonal antibodies able to recognize all pla molecules of m. altirostris venom and showed a synergism to inhibit the catalytic activity of them. moreover, we challenge monoclonal antibodies against to micrurus venom for inhibit the pla activity of naja naja, specie taxonomically out of micrurus cluster. our results showed that pla of m. altirostris venom share a pair of conserved antigenic regions and draw attention to use these epitopes to miming antigen to generate antibodies for antivenom production. moreover, face to the cross reactivity and the pla activity inhibition capability by mabs towards the naja naja venom, our results highlight the conservation of neutralizing epitopes across the elapidae family. protein-protein interactions are known to play key roles in the most important cellular and biological processes such as signaling, metabolism, and trafficking. one major goal of structural biology is the structural characterization of all protein complexes in human and other organisms. these efforts can be complemented by computational approaches. in this context, computational docking attempts to predict the structure of complexes from their monomeric constituents. the docking problem presents two main challenges: the generation of structural poses or sampling, and the identification of the correct structures with a scoring function (sf). docking methods can be successful if the interacting partners undergo small conformational changes. however, in a general situation, these algorithms generate a large number of incorrect predictions, and therefore the predictive success strongly depends on the accuracy of the sf used to evaluate the docked conformations. a variety of strategies have been developed to score putative protein-protein docked complexes. they are usually based on atomic level potentials, residue level potentials, or a combination of both. in current work, we have evaluated different sf, taken from cchappi server, on the results of different rigid body docking methods, ftdock, zdock, and sdock, using the docking benchmark . and a docking set built from capri scorers experiment. our results show sf that showed better or similar success rate than the in-built sf. some of these sf increase the docking success rates especially for flexible or weak-binding cases, which are the most challenging for docking. of them are residue level sf robust enough to detected solutions in cases with large conformation change. in particular we found two sf that shows outstanding robustness, one designed for protein modeling and shared among docking methods, and the other is for protein docking which is also the best success rate in the top ranking in the capri scorers set. the other atomic level sf display high success rate to find a solution within weak binding proteins. the most successful sf are shared between the docking methods and display high success rate in the hard cases of the benchmark . and in the capri scorers set. the difference between them in the resolution level at which they work, one being atomistic the other residue-based. we found that they success rate vary according to the docking method chosen, allowing them to explode different properties of the sampling used. this way to characterize a protein complex can help to develop new combined scoring functions in protein docking or a new ranking strategy to enhance the success rate. multi-ptk antibody: a powerful tool to detect a wide variety of protein tyrosine kinases (ptks) isamu kameshita , noriyuki sueyoshi , yasunori sugiyama the eukaryotic protein kinases consist of large families of homologous proteins and play pivotal roles in various cellular functions. these enzymes are classified into two major groups; protein serine/threonine kinases and protein tyrosine kinases (ptks). ptks are believed to be involved in various cellular events such as cell cycle, proliferation, differentiation, apoptosis, and cell adhesion in multicellular eukaryotes. as many as ptk genes have been identified in the human genome and many of these ptks are known to be closely correlated with various diseases such as cancer. therefore, it is important to elucidate the expression profiles of the entire ptk family in cells and tissues. to investigate the expression profiles of the cellular ptks, we produced an antibody that detects a wide variety of ptks. for production of the antibody, antigenic peptides corresponding to amino acid sequences of a highly conserved region (subdomain vib) of ptks were synthesized and immunized to balb/c mice. among various antigens, a peptide with amino acids, cyvhrdlraan, efficiently produced a polyclonal antibody with a broad reactivity to ptks. we established a hybridoma cell line producing a monoclonal antibody, yk , which appeared to cross-react with various ptks. at least ptks could be detected by yk antibody, as evidenced by its reactivity with the recombinant src tyrosine kinases whose subdomain vib had been replaced by those of the other ptks. when differentiated hl- cells were analyzed by western blotting after two-dimensional electrophoresis with yk antibody, we observed significant changes in the immunoreactive spots in hl- cell extracts along with the changes in the morphology of the cells. these results suggest that the multi-ptk antibody, yk , will be a powerful tool for the analysis of a variety of cellular ptks. analysis of the siglec- and hvap- interactions leonor carvalho , vimal parkash , heli elovaara , sirpa jalkanen , xiang-guo li , tiina salminen structural bionformatics laboratory, department of biosciences, medicity research laboratory, department of pharmacology, drug development and therapeutics, sialic acid-binding immunoglobulin (ig)-like lectins (siglec) are type i transmembrane proteins. siglec- has an n-terminal v-set domain followed by two c -set domains in the extracellular region. it contains an immunoreceptor tyrosinebased inhibitory motif (itims) in its cytoplasmic tail and can function as an inhibitory receptor by dampening the tyrosine kinase-driven signaling pathways. these proteins are expressed primarily on leukocyte subsets and, thus, are thought to be involved in regulation of leukocyte functions during inflammatory and immune responses. recently, phage display screening experiments identified siglec- as leukocyte surface ligand for human vascular adhesion protein (hvap- ; aoc gene product) and their interaction was confirmed by cell adhesion and enzymatic assays (kivi et al., ; aalto et al., ) . based on our preliminary data, hvap- sugar units with sialic acid (sa) might mediate interactions with the v-set domain in siglec- . furthermore, it is known that the siglec peptides binding to hvap- are located in the ce loop of the second c -set of domain (siglec- _c ). based on current hypothesis an arginine in siglec- _c interacts with the tpq residue in the active site of hvap- . the ce loop of siglec- _c has two arginines (r and r ) and, therefore, the interacting arginine is unclear. we will now study the interaction mode of hvap- and siglec- in silico to predict the role of the arginines in the c domain and the role of sa-binding using the d model of the full-length ectodomain of siglec- and the hvap- crystal structure. the in silico analysis will be conducted in parallel with experimental site-specific mutational studies and the result will be combined to elucidate the mechanism of hvap- -siglec- interaction. adam middleton , catherine day attachment of ubiquitin to substrate proteins regulates almost all cellular processes, including protein degradation and cell division. ubiquitylation involves a cascade of three families of proteins: ubiquitin activating (e ), ubiquitin conjugating (e ) and ubiquitin ligase (e ) enzymes. the . kda protein can be attached as a monomeric moiety or as a polyubiquitin chain, and the type of modification spells out the 'ubiquitin code' that directs the fate of the substrate. polyubiquitin chains can be formed via eight different linkage types, and the arrangement of chain formation is typically directed by the e enzymes. forming a polyubiquitin chain involves binding of two molecules to the e : the donor (ubd) and acceptor (uba) ubiquitin. ubd is linked to the e via a thioester bond between its c-terminal gly and the active site cys of the e , and when primed for catalysis it interacts with a particular face of the e . in contrast, coordination of uba by e s is transient and cannot be easily measured; however, uba binding defines the linkage type of polyubiquitin chains. the e , ube k, directs lys chain synthesis, which results in modified proteins being degraded by the proteasome. we generated a stable form of the ube kub conjugate and crystallized it, and showed that both ube k and its ubiquitin conjugate are monomeric. using molecular docking, we modelled the position of both ubd and uba and investigated the interfaces with site-directed mutagenesis. these experiments led to a molecular model that revealed how ube k can synthesise lys -linked ubiquitin chains. this molecular explanation provides a foundation for understanding how other e s generate lys -linked polyubiquitin chains. the two chromophorylated linkers of r-phycoerythrin in gracilaria chilensis marta bunster, francisco lobos-gonz alez, jos e aleikar v asquez, carola bruna, jos e mart ınez-oyanedl fac de cs biol., universidad de concepci on the two chromophorylated linkers of r-phycoerythrin in gracilaria chilensis. francisco lobos-gonz alez, jos e aleikar v asquez, carola bruna, jos e mart ınez-oyanedel, marta bunster. departamento de bioqu ımica y biolog ıa molecular, facultad de ciencias biol ogicas, universidad de concepci on. phycoerythrin is a phycobiliprotein present in phycobilisomes in gracilaria chilensis as a complex with chromophorylated linker proteins. our interest is to discover the role of these linkers in the function of phycobilisomes. phycobilisomes(pbp) are auxiliary light harvesting protein complexes in charge of channeling energy towards photosystem ii in alga, cyanobacteria and cryptophyta. this is possible thanks to fluorescent proteins called phycobiliproteins (pbp) and the chromophores (phycobilins, open-chain tetrapyrrols) attached to specific cysteines. phycobiliproteins share a common general structure; they are organized as (alfab) heterodimers which themselves assemble as trimers(alfab) or hexamers (alfab) ; this complexes are organized in high order structures to form the core and the rods. besides pbps, pbs have linker proteins in charge of the assembly and stabilization of the complex, and also it has been proposed that they collaborate in the fine tuning of the energy transfer steps between chromophores. these linkers are located within the rods, the rod-core interface, the core and the core-membrane interface. although most linker proteins are colorless, chromophore bearing linkers have been described, which suggest its participation in the energy transfer process. two of them, g and g are associated to r-phycoerythrin in gracilaria chilensis, nevertheless the information available on these linkers in eukaryots is still limited. to understand how these linkers collaborate with the function of the phycobilisome, we need structural information, especially the coordinates of all the chromophores present in the complex; we have sequenced both linkers from the genomic dna, performed sequence analysis and also we have purified the linkers by anion exchange, molecular sieve and hpl chromatography. the characterization was performed by denaturant electrophoresis, absorption and emission spectroscopy and by mass spectrometry. the results show that they have molecular masses as predicted, with a peptide signal for chloroplasts, an internal sequence repeat; residues - with residues - for g and residues - with residues - for g , and the presence of conserved cysteine residues putative sites of chromophorylation. the spectroscopy shows that they have different composition of phycobilins and a very short t / . a preliminary model for both linkers shows that they belong to aa structural class and that they share a common fold (heat like motifs) frequently involved in protein-protein interactions. dept. of phys., chuo univ., grad. sch. of inform. sci. and eng., tokyo tech, rigid-body docking algorithms are useful for predicting tertiary structures of near-native protein complexes. however, this algorithms generate many protein complex poses including false positives. then, near-native poses are searched in a post-docking process. there are many computational softwares with rigid-body docking algorithms, for example, zdock. we developed a high-performance protein-protein interaction prediction software, megadock, which is basically used on supercomputing environments for a large scale and network level in this work, we then tried to use these docking softwares and the profile method for understanding mechanisms of protein-protein interactions. we focused on some physicochemical properties, electrostatic and hydrophobicity, of a set of protein complex poses generated by a rigid-body docking process. from these poses, we obtained sets of possible interacting amino acid pairs. a set of interaction profiles has some information of docking spaces. from the view of a network prediction, the docking spaces of a set of protein complex poses are one of the properties for discriminating native protein-protein pairs from non-native pairs. in this work, ensemble docking process is performed by megadock ver. . and zdock ver. . . . cluster analysis is used with profiles of physicochemical properties. we used a dataset composed of typical monomer-monomer protein pairs and will discuss mainly differences between native and non-native protein pairs. the structural studies of the two thermostable laccases from the white-rot fungi pycnoporus sanguineus marta orlikowska , grzegorz bujacz institute of technical biochemistry, lodz university of technology, poland laccases (ec . . . , benzenodiol oxygen oxidoreductases) are enzymes that have the ability to catalyze the oxidation a wide spectrum of phenolic compounds with the four-electron reduction of molecular oxygen to water [ ] . it has been found that the active site is well conserved in between laccases from different organisms. it contains four copper atoms: one paramagnetic type cooper (t ) that is responsible for their characteristic blue color and where the oxidation of the reducing substrate occurs, one type cooper (t ) and two type coopers (t ) that conform a trinuclear cluster in which molecular oxygen is reduced to two molecules of water [ ] . laccases are present in many different species and they have been isolated from plants, fungi, prokaryotes, and arthropods in most cases laccases are monomeric glycoproteins of around amino acids with molecular weights in the range of - kda. the various functions carried out by those enzymes include the antagonistic ones such as their involvement in lignin biosynthesis (in plants), lignin degradation, pigment production, fruiting body formation, pathogenesis (in fungi) and spore protection against uv light (in bacteria) [ , ] . the diversified functions of laccases make them an interesting enzyme for study from the point of view of their structure, function and application. laccases of white-rot fungi (wrf) are of special interest because one of its role is to degrade lignin and most of them are extracellular enzymes helping purification procedures [ ] . during the last two decades, there has been an increasing interest in the genus pycnoporus for its ability to overproduce high redox potential laccases as the ligninolytic enzymes. we present the crystal structures of two thermostable lacasses produced by strain pycnoporus sanguineus cs (laci and lacii). the molecular weights of laci and lacii, determined by sds-electrophoresis, is and kda, respectively [ ] . both isoforms shows high amino acids sequence similarity ( %) between them and high thermal stability, at c and c. they remained active at high concentration of organic solvent (acetonitrile, ethanol or acetone). the unique properties make them promising candidates for industrial applications in wasterwater treatment. laci exerted a higher thermal and ph stability, tolerance against inhibitors and was a more efficient catalyst for abts and dmp (laccases substrate) then lacii [ ] . based on the structures we would like to understand the isoforms differences that confers laci a markedly better performance than lacii in ph and thermal stability as well as better resistance to inhibitors. analysis of liver proteome in cystathionine ß-synthase deficient mice using d ief/sds-page gel electrophoresis, maldi-tof mass spectrometry, and label-free based relative quantitative proteomics izabela bieli nska , Łukasz marczak , hieronim jakubowski , institute of bioorganic chemistry, polish academy of sciences, rutgers university, new jersey medical school homocysteine (hcy) arises from the metabolism of the essential dietary protein amino acid methionine. levels of hcy are regulated by remethylation to met and transsulfuration to cys. cystathionine bsynthase (cbs) catalyzes the conversion of homocysteine to cystathionine (first step of transsulfuration reaction). human cbs deficiency is a recessive inborn error of homocysteine metabolism that casues severe hyperhomocysteinemia (hhcy) and diverse clinical manifestations, including fatty liver disease [ ] . although the causes of fatty liver disease in cbs deficiency have been studied the underlying mechanism is not understood. we hypothesize that cbs deficiency induces changes in gene expression that could impair liver homeostasis. to test this hypothesis and gain insight into hepatic functions of cbs we analyzed the liver proteome of cbs -/-and cbs / mice [ , ] using d ief/sds-page gel electrophoresis and maldi-tof mass spectrometry (n ) we identified twelve liver proteins whose expression was significantly altered as a result of the cbs gene inactivation. expression of three proteins was upregulated and of nine down-regulated by the cbs-/-genotype. two up-regulated liver proteins are involved in iron metabolism (ftl and fth). those proteins are associated with oxidation stress and inflammation. third up-regulated liver protein (cbr ) is related to oxidation-reduction process. the downregulated protein are involved in the hydrolysis of n-acylated or n-acetylated amino acids (acy ), regulation of endopeptidase activity (a at ), cholesterol biosynthetic process (fpps), amino acid degradation (huth), cellular calcium ion homeostasis and l-ascorbic acid biosynthetic process (rgn). using label-free based relative quantitative proteomics (n ) we identified fourteen liver proteins whose expression was significantly altered as a result of the cbs gene inactivation. expression of four proteins was up-regulated and of ten proteins was down-regulated. the down-regulated liver proteins are linked with regulation of bone mineralization and inflammatory response (ahsg) or regulation of mrna splicing (roa ). the up-regulated liver proteins are involved in tricarboxylic acid cycle (suca), oxidation-reduction process (cy ), cholesterol metabolic process, iron ion homeostasis (fech), fatty acid metabolic process (ssdh; eci ) and response to oxidative stress (lonm). our findings suggests that cbs interacts with diverse cellular processes, including lipid metabolism, that are essential for normal liver homeostasis. deregulation of genes involved in lipid metabolism provides a possible explanation for fatty liver disease associated with cbs deficiency. transcription factors play central roles in coordinating developmental processes, as evidenced by the increasing number of transcription factor-related developmental disorders being uncovered by nextgeneration sequencing and genome-wide studies of copy number variation. the action of a transcription factor in regulating gene expression depends on interactions with other transcription factors, coactivators/co-repressors and chromatin modifying and remodeling complexes. transcription factors are commonly regulated by post-translational modifications. however the study of protein-protein interactions and post-translational modifications of transcription factors by common techniques such as coimmunoprecipitation and mass spectrometry is hampered by the difficulty in preserving interactions and modifications through cell lysis. to circumvent this issue, we developed a bioluminescence resonance energy transfer (bret) assay, which allows protein-protein interactions to be observed in live cells. in this assay, a protein of interest is expressed as a fusion with luciferase from renilla reniformis, and its putative interaction partner as a fusion with yellow fluorescent protein (yfp). upon addition of a cell-permeable substrate, the distance-dependent non-radiative transfer of energy from luciferase to yfp is quantified by measurement of light emission at two wavelengths to assess the interaction between the two fusion proteins. to validate the utility of this assay for investigating transcription factor interactions, we confirmed homodimerization of the foxp transcription factor, haploinsufficiency of which causes a rare and severe speech and language disorder, as well as interaction of foxp with other members of the foxp family. we also confirmed the interaction between foxp and multiple candidate interactors identified through yeast two-hybrid assays, including the autism-related transcription factor tbr , the co-repressors ctbp and ctbp , and post-translational modification enzymes of the pias family. the role of pias enzymes in sumoylation -the covalent modification of proteins with small ubiquitin-like modifier (sumo) proteins -led us to further explore this process, which is notably difficult to investigate because of the dynamic and labile nature of the modification, which is also typically present on only a minor fraction of molecules of a given protein. combining the bret assay with gel-shift techniques we demonstrated that foxp is sumoylated. finally, we used the bret assay to examine the effects of etiological foxp variants in speech and language disorder on protein-protein interactions and post-translational modification. in summary, the bret assay is a sensitive, reliable and potentially high-throughput technique for exploring protein biology in the context of live cells. we have demonstrated applications of the assay in validating putative protein-protein interactions, assessing posttranslational modifications, and investigating functional effects of protein variants identified in patient cohorts. these investigations have provided novel insights into the function of the foxp transcription factor in neurodevelopment and into the etiology of foxp -related speech and language disorder. the directly interaction between pres of human virus b and human heat shock protein (hsp ) deqiang wang , chen ke , jun zhang key laboratory of molecular biology on infectious disease, the department of cell biology and genetics the directly interaction between pres of human virus b and human heat shock protein (hsp ). hepatitis b virus (hbv) has infected billion people worldwide, and million of them are chronically infected. the chronic virus infection, a major public health problem worldwide, leads to bout two-thirds of hepatocellular carcinoma (hcc). the hbv envelope consists of the large (l), middle (m) and small (s) envelope proteins, which contain pres -pres -s, pres -s, and s domain alone, respectively [ ] . the pres domain is believed to mediate virus attachment to the high-affinity receptor. yan et al employed a novel technique to propose sodium taurocholate co-transporting polypeptide (ntcp) as the candidate hbv receptor, and consequently, ntcp is a target for a new family of anti-hbv agents [ ] . whereas, it remains a query to clarify that ntcp is the only or major hbv receptor in vivo. to illuminate if other host proteins cooperatively participate the hbv infection, we detect the interaction between pres and many candidate host proteins. fortunately, we have found that the human heat shocking protein (hsp ) could directly interact with the pres domain of the hbv virus protein. both the pull down and the size exclusion chromatography experiments verify that the grp have the ability binding to pres . whereas, whether the interaction between hsp and pres relates to the hbv infection need further experiments to clarify. the member sponsorship in the vast world of naturally occurring peptides, where more than peptides are known and approximately peptide therapeutics are currently being evaluated in clinical trials (fosgerau & hoffmann, ), the rapid and accurate determination of their physicochemical properties is key in peptide drug discovery. among these properties, hydrophobicity is crucial for understanding molecular recognition and biomolecular aggregation. hence, there is a great interest in determining hydrophobicity scales for amino acid structures. in this work, octanol/water partition (log p) and octanol/water distribution (log dph, fig. ) of n-acetyl-l-amino-acid methyl amides were determined by means of quantum mechanical ief-mst solvation calculations taking into account the intrinsic conformational preferences of each amino acid according to dunbrack's libraries (dunbrack & karplus, ; ) . the results reveal log d . differences for a-helical and b-sheet conformations in arg, lys, hid, asn, gln, met, cys, leu and ile. furthermore, by decomposing the octanol/water transfer free energy into electrostatic and non-electrostatic components, we estimated that the non-electrostatic cost of transferring the amino acid side chain amounts to . . cal/mol.Å , in agreement with previous estimates reported in the literature. comparison of our scale with other theoretical and experimental hydrophobicity scales yields satisfactory results, leading to correlation coefficients ranging from . to . . additionally, the mstderived hydrophobicity scale led to significant correlations with the rp-hplc retention factors measured for eight decapeptides (r . ) and for influenza virus hemagglutinin -mer (ac-ypydvp-dyaslrs-amide) peptides (r . ). finally, the hydrophobicity scale was able to reproduce the experimental log p for random neutral peptides (r . ) and log d . for ' : random charged peptides (r . ), fig. . future studies will address the application of this methodology to nonproteogenic amino acids, the prediction of peptide hydrophobicity at global and atomic level in peptides, and the scoring of peptide-protein interactions. docking-based tools for discovery of protein-protein modulators docking-based tools for discovery of protein-protein modulators. protein-protein interactions (ppis) play an essential role in many biological processes, including disease conditions. strategies to modulate ppis with small molecules have therefore attracted increasing interest over the last few years. although protein-protein interfaces (ppifs) are considered difficult to target with small molecules given its lack of well defined cavities. successful ppi inhibitors have been reported into transient cavities from previously flat ppifs. recent studies emphasize on hotspots (those residues contribute for most of the energy of binding) as promising targets for the modulation of ppi. pydock algorithm is one of the few computational methods that use energy of solvation to predict protein-protein interfaces and hotspots residues. we present an approach aimed at identifying hotspots and transient pockets from predicted proteinprotein interfaces in order to find potential small molecules capable of modulating ppis. the method uses pydock to identify ppifs and hotspots and molecular dynamics (md) techniques to propose putative transient cavities. we benchmarked the protocol in a small set of protein-protein complexes for which both structural data and ppi inhibitors are known. the method applies to the unbound proteins of the complexes the fast fourier transform algorithm, followed by the energy-based scoring from pydock to calculate the normalized interface propensity (nip) values derived from rigid-body protein docking simulations to predict the ppifs and hotspots residues without any prior structural knowledge of the complex. then we used md to describe the possible fluctuations of the interacting proteins in order to suggest transient pockets that could be useful as targets of small molecules for the modulation of ppis. finally, we evaluated by ligand docking, the validity of predicted hotspots and pockets for in silico drug design. we found that the nip-based method from pydock protein-protein docking identifies hotspots residues that are located within the binding site of known inhibitors of ppis. predicting ppifs from a three dimensional structure is a key task for the modulation of ppis. the use of the nip-based hotspots prediction method improve the identification of transient cavities from md simulation when compared to known binding cavities. this approach can be extremely useful in a realistic scenario of drug discovery targeting ppifs, when there is no information at all about the protein-protein complex structure. protein complexes are the fundamental molecular organizations that assemble multiple proteins to achieve various biological processes. identification of protein complex membership should provide a genotype-phenotype map to elucidate human gene-disease associations. it has been routinely assumed that network clusters with dense connections inside and sparse connections outside would form functional protein complexes. therefore, searching highly modular subgraphs in protein-protein interaction networks was explicitly or implicitly implemented in the algorithms to find protein complexes. however, to our surprise, we found a large portion of complexes with a medium-to-low modularity from the analysis of experimentally confirmed protein complexes. we also discovered that these complexes have cellular functions enriched in highly time-and space-dependent expression, such as signal transduction or subcellular localization. we further developed an algorithm to find such complexes by weighing network connections to capture transient interactions with intrinsically disordered regions. we confirmed that our method improved the identification of biologically relevant members of protein complexes and covered more complexes with a medium-to-low modularity. furthermore, newly discovered subunits in protein complexes could explain more disease-gene associations, indicating its utility to expand current genotype-phenotype map of human diseases. expanding template-based protein-protein complex prediction using ab-initio docking sergio mares-s amano , luis angel rodr ıguez-lumbreras , juan fern andez-recio structural characterization of protein-protein interaction (ppi) networks is crucial for understanding the underlying molecular mechanisms whereby life processes and disease arise. however, due to inherent limitations of experimental techniques, such characterization only covers an extremely reduced fraction of the human ppi network (interactome). recent studies have shown that although available structural templates may suffice to model a significant proportion of the interactome, model accuracy and binding specificity remain unsolved problems. consequently, improving the ability to predict ppis structurally will help to provide a better d profile of the known interactome, which may ultimately lead to the development of new therapeutic applications. here we show a novel approach that combines templatebased modeling with protein-protein computational docking to the structure-based prediction of ppis. our approach samples different protein-protein structural models derived from docking simulations. models are subsequently ranked using a function that incorporates an energy-based scoring term and a structural template similarity score. the energy-based scoring function includes electrostatics, van de waals and desolvation calculations, whilst the template similarity score accounts for the degree of structural similarity of models against a high-resolution and diverse dataset of structural templates. our approach highly improved the predictive success rate over individual ab-initio docking and templatebased techniques across a large benchmark dataset, including protein-protein complexes. when compared to the performance of the ab-initio docking algorithm, we found that the approach increased consistently the success rate, by approximately %, for the top , top and top solutions. the success rate improvement was even more notorious when the comparison was performed against the predictions from the traditional template-based docking. though incorporating ab-initio docking expands considerably the scope of the template-based docking method, challenges remain for interacting proteins in which high conformational changes occur upon binding and also the size and diversity of the repertoire of structural templates needs to be increased. is essential for the development of multicellular organisms. in mammalian cells, early events in pcd involve the release of cytochrome c (cc) from mitochondria to the cytoplasm, so letting cc play a key role in assembling the apoptosome and triggering apoptosis. in plants, pcd is part of a general process -the so-called hypersensitive response -in which mitochondrial cc is likewise released into the cytosol but its further role and cytoplasmic partners remain veiled. such a coincidence in cc release made us think of a common link for pcd in such evolutionarily distant species along evolution. to go deeper in understanding the pcd-dependent role of cc, a proteomic approach based on affinity chromatography with cc as bait was run using human and plant cell extracts. upon combining this approach with bimolecular fluorescence complementation (bifc), a total of eight and nine unknown proteins interacting with cc under pcd conditions were identified in human and plant cells, respectively [ , ] . such novel cc-partners -which are located in the cytoplasm and even in the nucleus -are involved in protein folding, translational regulation, oxidative stress, dna damage, energetic and mrna metabolism [ ] . strikingly, some of the novel human cc-partners are closely related to those for plant cc, so indicating that the evolutionarily well-conserved event of cc release from mitochondria could involve a common signalosome consisting of a wide range of common targets [ ] . to also understand such a promiscuity of cc from a structural point of view, the cc surface residues involved in complex formation with each one of its counterparts were mapped by using nmr spectroscopy. the resulting data shows that the heme crevice of cc is at the cc-partner interface in most of the complexes, which is in agreement with the vast majority of known redox adducts of cc. in contrast, however, to the high turnover number of the redox cc adducts inside the mitochondria, the complexes formed by cc under pcd conditions lead to the formation of rather stable nucleo-cytoplasmic ensembles. altogether, these findings suggest that extra-mitochondrial cc interacts with nuclear and/or cytoplasmic pro-survival, anti-apoptotic proteins in both humans and plants so as to lead living cells to dye. keywords: cytochrome c, programmed cell death, signalosome. post-translational phosphorylation often modulates the function of proteins. in particular, they affect the role that cytochrome c (cc) plays in cell life and death [ ] . cc is phosphorylated in vivo in tyr and tyr residues [ , ] , but recently, two new phosphorylation sites have been described at positions and [ ] . hence, we aim at understanding the structural and functional changes induced by thr and ser phosphorylation cc. for this purpose, we designed two phosphomimetic mutants of cc by replacing either thr or ser by the canonical amino acid aspartic acid (t d and s d). as control, two other mutants at the same two positions (t a and s a) were analyzed so as to differentiate the effects due to the presence of a negatively charged residue. remarkably, the s a mutant is significantly less stable than the wild-type species. we found that phosphorylation at position thr diminishes the redox potential and oxygen consumption. in addition, t d mutation affects the ability of cc to bind the distal site pcc , thereby suggesting that phosphorylation at this position affects the electron carrier capacity of cc. mass spectrometry (ms) is widely used techniques to gain knowledge about biomolecules [ , ] . it produces a high amount of data which is often presented as a list containing thousands of proteins. that list usually contains few hits interesting for our research. the pocess to select those proteins may include integrating experimental with annotation data. it requires spending some time in both, performing calculus and searching in databases. in this poster we present msbiodata analysis tool, a web service thought to deal with this tedious work. with this tool, researchers can set rules to select the most interesting hits in his lists using both, experimental data and gene ontology [ ] annotation. the data can be upload to the web using an excel spreadsheet or a flat files in a mztab format, and rules are easily constructed by means logical sentences. those sentences are composed by one or more terms linked by logic operators (and and or). each term in the logical sentence indicates to our program the conditions that selected hits must meet. once the alysis is finished, the results are delivered by email. msbiodat analysis tool do not requires any programming knowledge to be used and is freely available at: http://msbiodata.innomol.eu keywords bioinformatics/data analysis/proteomics/data mining/ mass spectrometry. beside the rate of protein synthesis, the regulation of protein degradation plays a crucial role in the white muscle protein accumulation and overall fish growth. intracellular proteolysis in salmonid species, such as atlantic salmon, salmo salar l. and rainbow trout, oncorhynchus mykiss walb., was studied to evaluate the basic mechanisms of protein degradation that could possess a potential target to regulate the body mass accumulation in farmed fish. a number of white muscle proteases such as cathepsins b, l, and d, proteasomes, and calcium-dependent proteases (m-and m-calpains), was studied in the juvenile specimens of different size-and age-groups both wild and farmed salmonids. the correlations between the protease activity and expression levels and morphometric characteristics of fish were found. the size-and age-related differences in intracellular protease activity revealed in fish muscles indicate both general role of proteolysis regulation in salmonid growth and the specific role of the individual proteolytic enzymes as well. the data on negative correlation of cathepsin d and calpain activity in muscles and the rate of weight increase in juvenile salmonids were obtained. a revealed positive correlation of cathepsin b activity and morphometric parameters in fish young presumably indicates its primary contribution to non-myofibrillar protein turnover. ubiquitin-proteasome system seems to contribute to background protein turnover as the proteasome activity was not corresponded with growth rate. summarizing the data obtained the autophagy-lysosomal and calpain-related protein degradation pathways were recognized to be directly involved in body growth and muscle protein retention in salmonid fish. the work was carried out using technical facilities of ib karrc ras equipment centre and financially supported by the russian science foundation, grant no. - - "salmonids of the north-west russia: ecological and biochemical mechanisms of early development". solving the proteomic organization of fitness-related genes in uropathogenic escherichia coli in life threatening sepsis. nowadays, complete genomes for almost all major bacterial pathogens are available, helping researchers to identify virulence factors. however we still ignore how these genes are organized at the proteome level and how this association influences bacteria pathogenicity. we integrated available databases on upec e. coli (strain cft ) to investigate the genomic and proteomic organization of genes related to upec fitness in the host. intriguingly, we found that most fitnessrelated genes have orthologs not only in other pathogenic strains but also in non-pathogenic bacteria such as e. coli k- . these genes are organized in clusters and operons with similar structure. by integrating protein-protein interaction data we observed that genes with high impact on fitness also display a highly clustered organization when compared to other genes. overall, our results show that proteinprotein interaction clusters associated to upec fitness in the host represent a promising target for the design of new antibiotics. elucidating the molecular mechanisms by which the hnh endonuclease gp activates the terminases in bacteriophage hk ( ) . hnh endonucleases are characterized by two highly conserved his residues and an asn residue( ). gp is essential for phage head morphogenesis, likely because gp enhances the activity of the hk terminase enzymes toward the cos site ( ) . notably, enhancement of the terminase-mediated cleavage of the phage cos site requires the presence of an intact hnh motif in gp . mutation of the canonical metal binding his in the hnh motif abrogates gp mediated-terminase activity. although phages are widely studied, there is no definitive structural or mechanistic evidence as to how the hnh endonuclease within gp functionally interacts with the adjacent terminase enzymes to facilitate phage morphogenesis. previous work on hnhcontaining bacteriophage proteins does not address explicitly how the requirement for divalent metal binding at the hnh endonuclease site induces interaction with the terminase enzymes that are so crucial for phage dna packaging during morphogenesis ( , ) . in addition, gp possesses no sequence similarity to hnh proteins for which the structure has been determined ( ), making structural studies of gp necessary. toward these ends, we use nuclear magnetic resonance (nmr) spectroscopy to probe metal and terminase binding of gp in the wild type state and bearing metal binding mutations. we also report backbone resonance assignment of gp . our nmr studies have elucidated residues within gp required for metal binding and terminase activity. these data are being used to assess the role of specific gp residues in phage morphogenesis. together, this work will identify the enigmatic role describing how metal binding in hnh endonucleases is crucial in the replication and morphogenesis of phages. meat production from pigs for human consumption is a resource heavy process, indeed every part of the animal that is not used constitutes a protein food-chain loss, which is neither economically nor environmentally viable. the goal of this project is to better harness slaughterhouse waste such as the keratin rich pig bristles and nails through microbial conversion. instead of using identified single microorganisms, it is the goal to define microbial consortia where microorganisms synergistically show the ability of efficient keratin degradation/conversion. candidate consortia have been obtained by selecting for microorganisms growing on enriched media that contains milled pig bristles as sole carbon and nitrogen source. by using mass spectrometry and various biochemical analyses to investigate keratinolytic enzymes, methods will be established for identifying and characterizing suitable consortia. protein families likely to be involved are keratinases, which are specialized proteases including serine, cysteine and metallo proteases, as well as systems capable of reducing or otherwise breaking disulfide bonds which are highly abundant in hair and nails. furthermore, interactions and symbiosis of microorganisms in a consortium will be investigated at the meta-proteomics level. the project will lead to development of biotechnological degradation of keratin rich fibers, and provide new insights into functional dynamics and efficacy of microbial consortia. a comprehensive protein domain analysis to map cancer-type-specific somatic mutations interpretation of the genome-wide association studies (gwas) of cancer patients to find cancer-typespecific biomarker is challenging due to the mutational heterogeneity of cancer types. network approaches to find cancer-type-specific variants and biological pathways are increasing since genes tend to act together to display phenotypic or disease outcomes. phenotype similarity has proven to reflect the relationship of functionally related genes. we applied phenotype similarities between various diseases for expanding molecular connections of cancer-type-specific variants to discover cancer-type-specific modules. specifically, cancer-type-specific variants of cancer types from the cancer genome atlas (tcga) were analyzed to find phenotype-inferred relationships among the variants. we find that cancer variants that cause the similar disease phenotypes tend to be linked as a cluster of biological pathways or functions. moreover, cancer-type-specific modules could explain the underlying pathogenicity of specific symptoms which manifest in particular cancer types. cancer-type-specific modules and pathways found from phenotype similarity/dissimilarity based on cancer symptoms improved the discrimination performance to sort cancer-type-specific variants to accurately predict patient groups. our method will be further developed to find genetic biomarkers for the diagnosis or prognosis of specific cancer types pk- engineering a stable, symmetric membrane protein scaffold amanda duran , jens meiler computational protein engineering has the potential to contribute to various fields including drug design, protein therapeutics, and materials science. protein-ligand interface design and the construction of large, stable proteins rely on stable scaffolds. symmetry is a great tool for protein stability both in protein engineering and nature. several membrane protein structures exhibit pseudo-symmetry and are proposed to be the result of gene duplication, fusion and diversification events originating from a monomeric gene. aquaporins (aqp) are a class of membrane proteins that exhibits a two-fold inverted pseudo-symmetry. the escherichia coli aqp glycerol facilitator protein (glpf) was originally computationally engineered to be perfectly symmetric in sequence and presumably in structure. the symmetric gene was assembled, cloned, and expressed. however, after facing many challenges experimentally, the computational study has been expanded to aqps of known structure for a more extensive symmetric backbone search. mammoth structural alignment was used to align the structures to their inverted counterparts. cutpoints were calculated based on a-carbon distance. finally, the rosetta protein modeling software suite was used to refine and energetically minimize the symmetric backbones. from over generated symmetric backbones, candidates were chosen for experimental verification. these studies are ongoing.currently, the symmetric backbone models have scored to be more stable than the wild-type proteins. experimental verification of these symmetric backbones will provide valuable information for the current state of membrane protein modeling and design using computational methods. intrinsically disordered proteins drive heritable transformations of biological traits daniel jarosz , james byers , sohini chakrabortee , sandra jones , amelia chang , david garcia stanford university, whitehead institute for biomedical research, rockefeller university the transmission of information from one generation to the next generally occurs via nucleic acids. the only known protein-based molecular memories are prions, which drive heritable biological traits based upon self-templating changes in protein conformation. these protein-based genetic elements have previously been identified systematically, but at least three do not share the sequence biases or structural characteristics that have informed such studies. here we employed a comprehensive library of yeast proteins to examine the breadth of protein-based inheritance. transient overexpression of more than forty proteins created new traits that were heritable and beneficial. some shared properties of known prions, but most employed distinct genetic and biochemical mechanisms to act as elements of inheritance. traits with these characteristics were common in wild yeast strains and could also be elicited using orthologous mammalian proteins. the inducing proteins were strikingly enriched in intrinsically disordered sequences that have been widely conserved across evolution. intrinsically disordered proteins are associated with human disease and with dosage sensitivity in yeast, flies and worms. our results suggest another widespread role for such intrinsically disordered sequences: induction of heritable epigenetic switches that transform phenotypic landscapes and drive adaptation to stressful environments. prediction of binding affinity in protein complexes: contacts do matters almost all critical functions in cells rely on specific protein-protein interactions. understanding these is therefore crucial in the investigation of biological systems. despite all past efforts, we still lack a thorough understanding of the energetics of association of proteins. here, we introduce a new and simple approach to predict binding affinity based on functional and structural features of the biological system, namely the network of interfacial contacts. we assess its performance against a protein-protein binding affinity benchmark and show that both experimental methods used for affinity measurements and conformational changes have a strong impact on prediction accuracy. using a subset of complexes with reliable experimental binding affinities and combining our contacts-and contact types-based model with recent observations on the role of the non-interacting surface in protein-protein interactions, we reach a high prediction accuracy for such a diverse dataset outperforming all other tested methods. free radical oxidation -a new method for obtaining stable protein coatings on magnetic nanoparticles magnetically targeted nanosystems (mtnss) are now considered to be applicable in different areas of biology and medicine such as hyperthermia, magnetic resonance imaging, immunoassay, cell and molecular separation, a smart delivery of drugs to target cells. proteins are promising materials for creation of coatings on magnetic nanoparticles (mnps) due to their biocompatibility, an ability to protect magnetic cores from influence of biological liquids and prevent agglomeration of mtnss in dispersion, their possible functional activity as therapeutic products and biovectors. the creation of stable protein coatings with retention of native properties of molecules is still an important biomedical problem because of disadvantages of the commonly used methods such as formation of a polydisperse ensemble of particles, nonselective linking of proteins leading to cross-linking of macromolecules in solution, and desorption of coatings. a novel method in obtaining stable single-layer coatings assembled from protein molecules on the surface of magnetite nanoparticles has been developed. it is based on protein liability to free radical modification, leading to the formation of intermolecular covalent cross links. free radicals are locally generated on the surface of nanoparticles via the fenton reaction thereby proteins adsorbed on the surface are subjected to the cross-linking. o-phenylenediamine was used for detection of free radical generation initiated by nanoparticles. the proteins drastically differing in their structure and properties, namely, serum albumin, thrombin and immunoglobulin g were selected for creating the protein coatings. the properties of the obtained coatings and their stability have been studied with the help of dynamic light scattering (dls), uv/vis spectrophotometry, antibody-antigen test and the method of spectral-fluorescent probes. albumin molecules in mnps coatings have been shown to retain their capability of binding with a dye and be conformationally stable. the dye , '-di-(g-sulfopropyl) , 'diphenyl- -ethiloxacarbocyanine-betaine interacting with albumin with a growth of fluorescence and with partial cis-trans conversion of the dye has been used. it has been proven that coatings composed of protein macromolecules are ) stable, ) formed around individual nanoparticles and ) have several nanometers in thickness. the free radical linking of thrombin and immunoglobulin g on the surface of nanoparticles has been shown to almost completely keep native properties of the protein molecules. the free radical linking method reveals new possibilities for design of single-layer multiprotein polyfunctional coatings on the surfaces of all the nano-, micro-and macroobjects containing metals of variable valence (for example, fe, cu, cr). the spectral-fluorescent investigation was supported by the russian foundation for basic research, project nos. - - and - - mol_a. regulation of neuronal snares by accessory proteins shrutee jakhanwal , reinhard jahn regulation of neuronal snares by accessory proteins shrutee jakhanwal and reinhard jahn department of neurobiology, max planck institute of biophysical chemistry, fassberg, goettingen, germany- . synaptic vesicle exocytosis lies at the heart of the process of neurotransmitter release. and, the family of proteins that is central to the process of synaptic vesicle exocytosis is the family of snare proteins. there are three kind of neuronal snare proteins namely syntaxin, snap and synaptobrevin. these three snare proteins interact through their snare-motifs to form a highly stable four-helix bundle, which in turn, pulls two membranes together to mediate fusion. years of work in this field have established that the four-helix bundle is critical for the membrane fusion to occur. however, the process of regulation of snare-mediated fusion remains very poorly understood. the major regulatory proteins involved in the process are munc , munc , synaptotagmin and complexin. the major aim of my project is to obtain a closer look at the regulation process of snare-mediated fusion by focusing on the interaction between the snare proteins and the regulatory proteins. to achieve this objective, i express and purify the different proteins involved in the process of snare-mediated fusion and thereafter subject them to appropriate biochemical characterization. in order to assess the role of the purified proteins in the process of fusion, i reconstitute them into liposomes and perform in-vitro lipidmixing assays. these assays are based on f orster resonance energy transfer (fret). based on the discretion of assessing the protein-protein or protein-lipid interactions, either the proteins or the lipids can be fluorescently labeled. also, the lipid compositions can be varied in order to assess the effect of lipid on the function of the respective protein. fluorescence-based anisotropy measurements can also provide information about the degree of freedom of a protein, indirectly providing information about the kinetics of a reaction. employing these techniques, i observe that munc - leads to displacement of syntaxin from a complex of syntaxin and snap . also, a complex of syntaxin and munc is resistant to the action of the aaa-atpase, nsf and its co-factor asnap, implicating this complex as a strong candidate for acting as the starting point for the process of neurotransmitter release. munc also appears to enhance lipid-mixing by interacting with the snare-complex. further investigations on the same lines can provide very useful insights into the process and can help us unravel the secrets that underlie the beauty of the exquisitely regulated process of neurotransmitter release. binding of thymidine nucleotides to a viral thymidine monophosphate kinase aldo a. centro de investigaci on en alimentaci on y desarrollo theme: biochemistry there is great interest in the evolution and activities of fish trypsins, since they appear to have evolved into different families. the cdna for trypsin iii from the monterey sardine (sardinops sagax caerula) was obtained and its deduced amino acid sequence matched its identity with a purified protease from the fish by mass spectrometry analysis. molecular modeling of sardine trypsin iii compared to other homologs showed a typical trypsin fold with all the cognate components for catalysis, and specific amino acid distribution that are possible factors that explain the cold adaptation. from phylogenetic analysis, sardine trypsin iii belongs to the novel y family, which is proposed to have evolved for cold adaptation. the obtained recombinant trypsin iii showed a low catalytic efficiency, but it remained active at cold temperatures, similar to other cold-adapted trypsins. the cold-adaptation of sardine trypsin iii opens a wide range of biotechnological applications for this protease and is also interesting from the serine protease structure-function relationship point of view. fungicidal mechanism of scolopendin , a cationic antimicrobial peptide from centipede heejeong lee , dong gun lee drastically (from . x - to x - colonies) upon deletion of this residues domain from the full length trai. we are investigating the structure and function of this very c-terminal end of trai using nmr spectroscopy. for the backbone assignment we used slice-selectively homonuclear broadband decoupled spectra along with standard experiments. three-bond scalar coupling constants were obtained through real-time j-upscaling experiments. with the backbone assignments, we have the first hand evidence which shows that his domain is for the most part intrinsically disordered, but contains short a-helical regions. structural development, interaction studies to find the binding partner and transition of disorder to order orientation of this domain will be further investigated in this project. here we investigated a model system where mab aggregation is induced by increasing the ionic strength (nacl) at low ph. the aggregation depends both on protein and sodium chloride concentration. with nanoparticle tracking analysis (nta) and micro flow imaging (mfi) the aggregation formation was further characterized. aggregation can be partially reverted by lowering the ionic strength as determined by soluble monomer concentration measurement using se-hplc: parts of insoluble aggregates could be solubilized as soluble aggregates, dimers or even monomers. a quasi equilibrium is formed in between the subtypes. the whole aggregation process was examined by ftir and cd-spectroscopy to identify structural changes of the mab. screen of protective additives: the effect of osmolyte additives on aggregation kinetics and final aggregate concentration is investigated, revealing protective effects in both cases. in a screen with more than compounds not only the aggregation propensity was studied but also structural changes. the aggregation index (quantity for colloidal stability) and the melting point (quantity for conformational stability) measured by differential scanning fluorimetry were determined. the used mtp format screen has potential for buffer optimization and formulation development. structural biology and protein dynamics tetraspanin cd has a broad range of cellular functions, such as integrin association forming tetraspanin-enriched domains, synapse formation between b and t cells, cell adhesion, motility, invasion and signalling. furthermore, cd is one of the four receptors involved in the cell entry of hepatitis c virus (hcv) and therefore infection onset, one of the major causes for chronic liver disease resulting in cirrhosis and hepatocarcinoma. human cd large-extracellular-loop (hcd lel) is composed of a "stalk" and a "head" subdomain; with the latter interacting with hcv-e glycoprotein. we present four novel hcd lel crystal forms. analysis of the fourteen independent observed hcd lel high-resolution x-ray structures suggests that the dynamism of the hcd lel head-subdomain is an inherent molecular property, an observation supported also by molecular dynamics (md) studies. we classify the conformations in three distinct clusters (closed, intermediate and open) , which are seen both in the crystal structures and in the molecular dynamics simulations. the md simulations also show that conformational variability is modulated by ph changes, with distinct probability for each cluster at acidic and neutral ph. furthermore, in silico docking of the recent e core structure with three of the major types of hcd lel head-subdomain clusters highlights hydrophobic interactions as the major forces in the e core: hcd lel recognition mechanism. we propose that the flexibility of the hcd lel is exploited by hcv at different stages of cell entry from virus attachment to internalization and fusion with the endosomal membrane. our results provide important insights on the basic mechanism governing hcv binding to hcd , and can help structure-based drug design of entryinhibitors of hcv. allophycocyanin of gracilaria chilensis: from gene to function jorge dagnino-leone , jos e martinez-oyanedel , marta bunster-balocchi universidad de concepci on theme: structure-function relationship of proteins the phycobilisomes (pbs) are auxiliary photosynthetic complexes that allow cyanobacteria and red algae to enhance the energy uptake in the range of - nm. in gracilaria chilensis, an eukaryotic red algae, pbs is composed of phycoerythrin (pe), phycocyanin (pc) and allophycocyanin (apc); these proteins possess chromophores which capture energy and then transfers it to photosytems. pbps are oligomers of a ab heterodimer; it oligomerizes into a trimer (ab) , this trimer has discoidal shape and it is associated in hexamers (ab) , several of this hexamers forms cylinder-like structures. pbs has components: antennas and core. the antennas are composed of pe and pc, whose function is to capture energy between - and - nm respectively and transfer it to the core. the core is formed by apc, which can absorb energy in the - nm range. apc emission allows transferring energy to the photosystems with high efficiency. pbs is also composed by linker proteins which allow the correct assembly of pbs and possibly regulate the energy transfer. the main goal in our group is to build an atomic model of the gracilaria chilensis phycobilisome. we have solved the crystal structure of pe and pc and created an antenna model. at present we are working in apc and the chromophorilated linker proteins. the objective of the present work is to create a model of the core of gracilaria chilensis; to achieve these we have used molecular biology, biochemistry and bioinformatics techniques. we designed oligonucleotides primers for the four allophycocyanin subunits genes and for the globular domain of the apce linker. these primers were used in pcr experiments to obtain the genes sequences. the sequences were translated to a aminoacid sequences and used to build a d model for apc subunits and trimers using the software modeller. on the other hand we purified and analyzed the spectroscopic properties of apc from gracilaria chilensis using absorption and fluorescence spectroscopy. we also determined apc oligomerization state using gel filtration. molecular docking using the cluspro server was performed to obtain a hexamer and apc cylinder models. based on electron micrographs obtained by our lab a tri-cylindric core model was built. all the models were submitted to a molecular dynamics using gromacs software. finally we determine possible energy transfer pathways in the core model applying the extended forster equation, spectroscopic data from literature and the transition dipole moments of each of the chromophores present in the core. as conclusion of this work we built the first atomic model of gracilaria chilensis phycobilisome core and propose energy transfers pathways inside the core in the context of a phycobilisome. novel practical strategies to access artificial metalloenzymes marco filice , jose miguel palomo departamento de biocat alisis, instituto de cat alisis, csic protein chemistry and engineering since the first report, the design of artificial metalloenzymes has rapidly been converted into an important topic in biological and inorganic chemistry due to their potential applications in synthetic chemistry, nanoscience and biotechnology. the combination of a catalytically active organometallic moiety with a macromolecular host has permitted the creation of biohybrids, a new kind of heterogeneous catalytic entities combining the attractive features of both homogeneous and enzymatic systems. presenting our most recent achievements in this research area, here we describe two novel powerful and promising approaches focusing the practical synthesis and large scale production of heterogeneous artificial metalloenzymes showing chimeric activity. the first strategy is based on the in situ synthesis of noble metal nanoparticles and their supramolecular assembly with a microbial lipase from candida antarctica (fraction b) finally creating an ultra-active organometallic-enzyme heterogeneous nanobiohybrid. in the second approach, combining different protein engineering protocols (molecular biology, orienting immobilization, solid-phase bioorganic modification and bioinformatic tools), an orthogonal solid-phase strategy creating novel unnatural catalytic sites was designed and optimized. the application of such a strategy onto the structure of the lipase from geobacillus thermocatelunatus permitted the generation of a heterogeneous artificial metallolipase with chimeric activity. as proof-of-concept, the combinatorial library of generated artificial metalloenzymes obtained by both strategies was successfully assessed in a set of different synthetic reactions (selective c-c bond formation as suzuki, heck or diels-alder reactions) and also combining both activities (metallic and enzymatic) in cascade processes such as dynamic kinetic resolution of amines or production of arylamines. the obtained results were excellent in all cases. extending this strategy to other enzymes, proteins and catalytic metals, we envisage the creation of a combinatorial library of programmable artificial enzymes useful for a wide set of applications (i.e. fine organic and medicinal chemistry, bioremediation or biomedicine). proteomic examination of the yeast nuclear pore complex dynamics protein turnover and exchange nuclear pore complexes (npcs) are proteinaceous assemblies situated in nuclear envelopes of eukaryotic cells. the main function of the npc is the selective transport of macromolecules. npcs also partake in other functions, such as nuclear organization and gene regulation. the core scaffold of the npc is thought to be a stable structure, while the peripheral components exchange at various rates. however, these phenomena have not been elucidated in detail. the recent findings that yeast daughter cells get a higher proportion of the old npcs and the core scaffold hardly turns over raise the possibility that the exchange of the peripheral nucleoporins can be a repair mechanism. yeast provides a useful organism for the interrogation of nucleoporin exchange, as it performs closed mitosis; hence the only mixing of npc constituents is due to exchange. we have developed a panel of genetic tools providing for conditional induction and repression of nucleoporins. by combining these switches with stable isotope metabolic labeling and affinity capture, cross linking coupled to mass spectrometry, we are able to distinguish between pre-existing and newly synthesized proteins and quantify their relative amounts in the npc. our preliminary findings are in agreement with results obtained in other organisms: the core scaffold of the npc (inner ring, outer ring) appears to be stable, however does exchange slowly over time, while peripheral components exchange faster. by looking at the exchange rates of yeast nucleoporins we hope to gain insight into the npc biology of actively dividing eukaryotic cells. active site clustering identifies functional families of the peroxiredoxin superfamily angela harper , janelle leuthaeuser , patricia babbitt , jacquelyn fetrow department of physics, wake forest university, department of molecular genetics and genomics,-wake forest university, departments of physics and computer science, wake forest university bioinformatics understanding the relationships between proteins is vital to increasing our knowledge of the protein universe. while there are large databases of sequence information, the massive data influx over the past decade has prevented adequate classification of proteins at the molecular function level. however, it has been previously suggested that a protein's active site information may correlate with these known molecular functional differences; thus, active site profiling was developed to use residues around the active site of a protein to relate proteins. subsequently the deacon active site profiler (dasp) was developed to create these active site profiles and search them in a database, such as gen-bank, in order to find proteins with similar active site environments. by using dasp to computationally cluster proteins based on the similarity of their active site profiles, the peroxiredoxin (prx) superfamily was analyzed through active site similarity methods. the residues from the active site of each prx structure were extracted and clustered, and these profiles were iteratively searched in genbank through a multi-level iterative sequence searching technique (misst). the prx superfamily has been studied by experts, allowing the results of these searches to be compared to a well-annotated group of proteins. while previous sequence based evolutionary methods have been unable to identify functional differences between some subgroups of the prxs, notably the ahpc-prx and prx subgroups, misst discretely separates these subgroups. classifying prx proteins into functionally relevant groups using computational active site similarity methods lays the foundation for an automated process for identifying protein functional groups beyond the prx superfamily. synthesis and conformational studies of glycoprotein n homolog of bovine herpesvirus (bhv- ) by using cd, nmr and molecular modelling it serves as a chaperone for viral glycoprotein m and, in its gm-unbound form, acts as an inhibitor constraining the transporter associated with antigen processing (tap). the ul . /gm complex formation is required for the maturation and proper trafficking of both viral proteins. in the absence of gm, ul . blocks transport of antigenic peptides by tap and their mhc i-restricted presentation. the molecular mechanism of ul . activity still remains elusive. in order to investigate the structural requirements for biological function ul . study was conducted using cd, nmr and molecular dynamics methods. the data obtained with the use of high purity synthetic peptides encompassing ul . confirmed the presence of an alpha-helix structure, formed preferentially in the presence of dodecylphosphocholine (dpc) micelles as a membrane-like environment. in order to determine the three-dimensional structure of ul . protein in the present work its nmr solution structure in the presence of membrane-like environment was performed. the nmr data were used as a set of restraints for a simulated annealing protocol that generated dstructures of the colin johnson , sara codding membrane proteins resealing of tears in the sarcolemma of myofibers is a necessary step in the repair of muscle tissue. defects in this repair process are responsible for muscular dystrophy and cardiomyopathy. the repair pathway is triggered by the influx of calcium through lesions in the membrane, which result in membrane fusion and patching of the wound. recently dysferlin has been identified as a calcium binding protein essential for sarcolemma repair, as well as other snare mediated exocytotic events including cytokine and acid sphingomyelinase secretion. in this presentation we demonstrate a direct interaction between dysferlin and the snare proteins syntaxin and snap- . in addition, fret and in vitro reconstituted lipid mixing assays indicate that dysferlin accelerates snare heterodimer formation and snare mediated lipid mixing in a calcium sensitive manner. our results suggest a model whereby dysferlin acts as a calcium sensing snare effector for exocytosis and membrane fusion. exploring the therapeutic potential of a peptide derived from a poxviral immune evasion protein: nmr determination of the solution structure of viper and its inactive mutant toll-like receptors (tlrs) have a role in viral detection leading to cytokine and ifn induction, and as such they are targeted by viruses for immune evasion. the poxviral protein a has been identified to inhibit tlr signaling by interacting with tir domain-containing proteins of the receptor complex to collectively inhibit all tlr adaptor proteins that positively regulate transcription-factor activation ( ). one aa peptide (kysf-klilaey) termed viper (viral inhibitory peptide of tlr ) was reported to retain the inhibitory properties of full length a against tlr signaling. a r homopolymer delivery sequence at the c-terminus provided delivery of the peptide into cells. structural comparisons are presented between r-viper, which is active in preventing tlr -dependent cytokine induction in cell culture, and a mutant that exhibited loss of function ( r-viper l a,e a), through solution nmr spectroscopy. we find that despite a relatively minor sequence difference, the loss of hydrophobicity as well as negative electrostatic interactions result in subtle but potentially significant differences in the region of the peptide proposed to interface with tlr . reference: wake forest university, wake forest university, university of california san francisco protein function prediction the elucidation of protein molecular function lags far behind the rate of highthroughput sequencing technology; thus, it is essential to develop accurate and efficient computational methods to define functional relationships. protein clustering based on sequence similarity has emerged as a simple, high-throughput method for defining protein relationships, but sequence-based techniques often inaccurately define molecular function details. active site profiling (asp) was previously developed to identify and compare molecular details of protein functional sites. protein similarity networks were created using both active site similarity and sequence similarity for four manually curated superfamilies, and results demonstrate that asp-based clustering identifies detailed functional relationships more accurately than sequence-based clustering. building on this, two iterative pipelines were developed using active site profiling and profile-based searches to cluster protein superfamilies into functional groups. first, the two level iterative clustering process (tulip) utilizes active site profiling and iterative pdb searches to divisively cluster protein structures into groups that share functional site features. across eight superfamilies, tulip clusters exhibit high correlation with expert functional annotations. subsequently, the multi-level iterative sequence searching technique (misst) utilizes iterative profile-based genbank searches to identify protein sequences that belong in each tulip group. the results indicate that these asp-based methods accurately and efficiently identify functionally relevant groups through a process that can be applied systematically and on a large-scale. moreover, the approach can be applied more quickly than detailed manual curation, suggesting its value in guiding annotation efforts. dept. biochemistry and molecular biology. university of valencia, lab of peptide and protein chemistry. centro de investigaci on pr ıncipe felipe membrane proteins changes in the equilibrium between pro-survival and pro-apoptotic members of the b-cell lymphoma- (bcl- ) protein family at the mitochondrial outer membrane (mom) induce structural changes that committed cells to apoptosis. bcl- homology- (bh )-only proteins participate in this process activating pro-apoptotic effectors and promoting permeabilization of the mom. the membrane association of bh -only proteins is a controversial issue due to the lack of a canonical carboxyl-terminal (c-terminal) transmembrane (tm) domain. we used an in vitro transcription/translation system to study the insertion capacity of these hydrophobic c-terminal regions of the bh -members bik, bim, noxa, puma and bmf into microsomal membranes, and an escherichia coli complementation assay to validate our results in bacterial cells. furthermore, we have fused these hydrophobic regions to gfp to investigate the subcellular sorting. these results will allow further refinement in the elaboration of the bcl- protein-protein and protein-membrane interactome network. alexis peña , flaviyan jerome irudayanathan , shikha nangia syracuse university, dept. of biomedical and chemical engineering computational modeling, biostatistics, biomedical and chemical engineering tight junctions (tj) are vital intracellular barriers that are responsible for regulating paracellular transport. claudins, a family of abstract small transmembrane proteins with approximately members, are an integral part of the tj strands. tight junctions provide molecular-level protection and prevent infection and toxins from entering the body; in the same sense tjs allow nutrients and vital solutes to pass through. claudins are associated with various diseases including metastatic cancer as well as an entry point for many viruses. despite their importance and abundance in all cell membranes and their ubiquitous nature, the exact -d structure of claudins has remained elusive to traditional x-ray crystallographic and nmr studies. in this investigation, a computational approach was used to determine the claudin structure of claudin - . homology modeling, molecular dynamic simulations, and reverse mapping were employed to predict the protein structures with relative accuracy. understanding structure of claudin proteins and its interaction at the molecular level can lead to effective drug delivery technology. determination of optimal conditions for an isothermal titration calorimetry essay to obtain kinetic parameters of trypsin i from pyloric caeca of monterey sardine (sardinops sagax caerulea) idania emedith quintero reyes , francisco javier castillo y añez , enrique fernando vel azquez contreras , roc ıo sugich miranda , david octavio corona mart ınez , aldo alejandro arvizu flores , ivet cervantes dom ınguez protein kinetics determination of optimal conditions for an isothermal titration calorimetry essay to obtain kinetic parameters of trypsin i from pyloric caeca of monterey sardine (sardinops sagax caerulea) trypsin is the most studied alkaline protease and it s very common to found isoforms from this protein as the case for monterey sardine (sardinops sagax caerulea); as it shows an expression of trypsin i and trypsin iii according to the cdna characterization. trypsin i was determine to be a cold adapted enzyme as it shows a higher catalytic efficiency (kcat/km) than the mesophilic counterparts. the kinetic parameters were obtained by spectrophotometric essays, which are not fallible for all the enzymes because native, recombinant or mutant enzyme activity could be below the detection limit of the assay, opaque or turbid solutions interfere with spectrophotometric detection, etc. alternative tools as the isothermal titration calorimetry (itc) can measure enzyme kinetics using thermal power generated by the enzymatic conversion of substrate to product; were the rate of reaction is directly proportional to thermal power. the objective of this study was to stablish the optimum conditions to obtain kinetic parameters of trypsin i from pyloric caeca of monterey sardine using itc. to reach the objective trypsin i was purified from viscera of monterey sardine using molecular exclusion and affinity chromatography obtaining a yield of . mg/ml. at c kcat and km of tryipsin i form monterey sardine were . s- and . mm respectively. at c were . s- and mm (kcat and km) and at c kcat was . s- and km . mm. the kinetic parameters obtained by spectrophotometric assay at c were kcat and km s- and . mm respectively. at c the kcat was . s- and km . mm and at c kcat s- and km mm. comparing the values obtained for kcat with the spectrophotometric essay were higher fold than those obtained by itc and the values in km were similar by both methods. even though the differences in kcat, we can reassert the psychrophilic behavior of trypsin i as the catalytic efficiency is higher by both methodologies. in the understanding that the kinetic behavior of enzymes is important to not only understanding biochemical pathways and catalytic mechanisms but is again a fruitful area for drug discovery and development; so the itc provides a universal approach to determining the kinetic behavior of enzymes and can yield in a single experiment a complete set of kinetic parameters for an enzyme-catalyzed reaction that can be applied for the different alkaline proteases from pyloric caeca of monterey sardine (sardinops sagax caerulea). mysterious world of stress-responding sigma factors in bacillus subtilis olga ramaniuk protein-dna interaction bacterial transcription is mediated by the rna polymerase holoenzyme containing sigma factors -essential proteins for the initial step of transcription that recognize and bind to promoter dna. the primary sigma factor is essential in exponential phase of growth while alternative sigma factors are active during transcription under stress conditions. this project has three main aims. the first aim is to explore the binding properties of b. subtilis alternative sigma factors; specifically, whether sigma factors lacking the autoinhibitory domain . can bind to promoter dna in the absence of rnap. the second aim explores whether rnap associated with alternative sigma factors is regulated by the concentration of the initiation nucleoside triphosphate. the third aim is to define the regulon of sigma i. in order to achieve our aims, out of alternative sigma factors were successfully purified using affinity chromatography and ion exchange chromatography. we set up in vitro transcription system with selected sigma factors and initiated experiments with sigma i regulon determination. results named above and our future findings will help to better understand gene expression regulation on the level of transcription initiation. this work was supported by grant no. p - -g from the czech science foundation. assessing the costs and benefits of protein aggregation protein aggregation and cell fitness protein aggregation has been associated with numerous diseases but also with important cellular functions such as epigenetic inheritance. here we present a population genetics approach to infer the costs and benefits of protein aggregation on cell fitness. this information is crucial to understand how cellular systems tolerate the formation of protein deposits and which factors modulate this event. using our experimental system, we measured different protein aggregation effects (deleterious, neutral or beneficial) within the same genomic background. single cell analyses, within the same population, showed stochastic variability in the aggregate's size and in its effect on cell fitness. our data indicates that, in certain conditions, protein aggregation can enhance population variability and survival expectancy. overall, these results suggest that the presence and formation of protein aggregates could be almost harmless whereas the associated gain and loss of function are critical for the cell. revealing the key role of negatively charged residues of heme sensor proteins involved in geobacter sulfurreducens' signal transduction pathways marta a. silva , telma c. santos , teresa catarino , carlos a. salgueiro ucibio-requimte, departamento de qu ımica, fct-unl., instituto de tecnologia qu ımica e biol ogica, unl signal transduction proteins bacterial chemotaxis systems sense and regulate the microbe mobility in response to environmental conditions. such mechanisms constitute a striking example of cell motility to gain advantages for cell survival and permit the bacteria to fill important niches in a diversity of anaerobic environments [ ] . geobacter sulfurreducens (gs) is an anaerobic bacterium with a considerable respiratory versatility whose genome encodes for an unusual family of methyl-accepting chemotaxis proteins (mcp), each containing at least one heme c-binding motif [ ] . these sensor proteins, gsu and gsu , are involved in signal transduction pathways mediated by chemotaxis-like systems [ ] . the thermodynamic and kinetic characterization of the sensors gsu and gsu by visible spectroscopy and stopped-flow techniques, at several ph and ionic strength values revealed that sensor gsu midpoint reduction potentials are lower than those of gsu at all ph and ionic strength values and the same were observed for the reduction rate constants [ ] . the origin of the different functional properties of these closely related sensor domains are rationalized in the structural terms showing that gsu has two extra negatively charged residues in the vicinity of the heme group, which have no counterpart in gsu : glu and asp . residue asp is less exposed compared to glu and it was suggested that its carboxylic group might have a role in the modulation of the heme reduction potential of gsu . to investigate this, both residues were replaced by a positively charged amino acid (lysine) and by a neutral one (asparagine or glutamine). for the mutants with enough expression, a functional characterization was carry out, using several spectroscopic techniques, including uv-visible and cd, together with kinetics and potentiometric measurements. significant changes on the reduction potential values are observed when a negative charge is replaced by a positive one at position or . therefore, the decrease of the reduction potential in asp and glu mutants reinforces the hypothesis that the higher reduction potential observed for heme sensor domain gsu is related with the less negative electrostatic surface around the heme. this work provides, for the first time, evidence for the co-existence of two similar methyl-accepting chemotaxis proteins functioning in different working potential ranges. these proteins are responsible to allow geobacter sulfurreducens triggering an adequate cellular response in different anoxic subsurface environments. national autonomous university of mexico, faculty of medicine, national autonomous university of mexico, faculty of chemistry, national autonomous university of mexico, institute of chemistry molecular evolution the glycolytic enzyme triosephosphate isomerase (tim) is an oligomeric (b/alpha) barrel that catalyses the interconversion of d-glyceraldehyde -phosphate and dihydroxyacetone phosphate in a diffusion-limited reaction. although each subunit has its own active site, naturally occurring monomeric tims have not been reported; in fact, monomer association is very tight. tim topology is well conserved among the three domains of life. nevertheless, their folding mechanism and inhibition properties vary across species. comparative studies of proteins have proved to be very useful in understanding the relationship between sequence and physicochemical properties, however, they lack the capacity to give a more integrative and evolutive correlation. in order to elucidate how the catalytic properties, the oligomerization state and the stability of extant tims arose, in this work we examined the molecular history of eukaryotic tim through ancestral protein reconstruction methods (maximum likelihood) and the subsequent physicochemical characterization of the resurrected enzymes. we first characterized in detail the protein corresponding to the last common ancestor of animals and fungi (tim ). the cd and fluorescence spectra of tim are similar to those of extant tims. secondary structure is lost in a cooperative transition with tm . c. the enzyme loses activity upon dilution suggesting that only the dimer is active. dilution experiments followed by isothermal titration calorimetry indicate that dissociation enthalpy is small; moreover the heat capacity change observed is three times higher than the one predicted for a rigid body dissociation process, suggesting partial unfolding of the monomers. when compared with extant tims, the catalytic efficiency of tim is reduced -fold, whereas binding of pgh, a transition-state analogue, shows a similar thermodynamic signature. these data indicate that although monomer association may have been less tight in ancestral tims, catalysis has been always linked to oligomerization. analysis of the crystal structure of tim , obtained at . Å resolution, suggests that the lack of four salt bridges observed in the interface of extant tims is responsible for the low dimer stability. in order to test this hypothesis we also studied the stability of four younger reconstructed ancestors that acquired the salt bridges in two different phylogenetic lineages. we found a correlation between the appearance of stabilizing interactions in the interface, dimer stability and catalysis; suggesting that these salt bridges are partially responsible for extant dimer stability and shed light on the dimeric nature of extant tims. receptor protein-tyrosine phosphatases: dimerization, receptor kinase interaction and allosteric modulation elizabeth dembicer , damien thevenin department of chemistry, lehigh university theme: receptor tyrosine kinase and receptor protein phosphatase signaling many cell-signaling events are regulated through reversible tyrosine phosphorylation of proteins, which is controlled by the counterbalanced actions of two key enzyme families: protein tyrosine kinases and protein tyrosine phosphatases. interestingly, both families include transmembrane receptor-like enzymes, namely the receptor tyrosine kinases (rtks) and the receptor-like ptps (rptps). while the regulation and actions of many rtks are well characterized, the mechanisms controlling the enzymatic activity of rptps and how they interact with their substrates remain to be fully explained. thus, understanding how these receptors function and interact will give fundamental insights into how tyrosine phosphorylation is finely tuned in cells, and how it can be modulated. increasing evidence indicates that rptps, like rtks, are regulated by homodimerization. however, it appears that homodimerization inhibits the activity of most rptps. even though the transmembrane (tm) and the juxtamembrane domains have been proposed to be involved in this process, there is no clear structure-based proposal for the role of these regions. moreover, several rptps have been identified as candidate regulators of rtks. in particular, the receptor-type tyrosine-protein phosphatase eta (ptprj; also known as dep or cd ) is capable of attenuating egfr tyrosine phosphorylation. physical interactions of egfr with ptprj at the cell surface have been documented, but the basis for these interactions is unknown. here, using a dominant-negative transcriptional activator-based assay (dn-aratm), and mutagenesis analysis, we show that: ( ) ptprj has a strong tendency to homodimerize, ( ) ptprj heterodimerizes with egfr through tm-tm interactions, ( ) these interactions are mediated by specific residues, and can be modulated by the delivery of peptide binders. this work represents the first structure-function study of rptp-rtk interaction, and may not only result in significant progress towards a better understanding of the basic biology of rptps in cancer cells, but also offer new possibilities for targeting protein tyrosine phosphatases for therapeutic modulation of egfr in oncology. inhibiting egfr dimerization and signaling through targeted delivery of juxtamembrane domain peptide mimics using phlip anastasia thevenin , kelly burns , janessa guerre-chaley , damien thevenin regulating receptor tyrosine kinase signaling the elevated phosphorylation of key regulatory tyrosines on oncogenic signaling proteins that result from aberrant protein tyrosine kinases activity plays well-abstract established roles in promoting tumorigenesis and in the high frequency with which resistance arises to existing therapeutic treatment. for instance, this is the case for the epidermal growth factor receptor (egfr). thus, there is a clear need for novel specific targeting methods to inhibit the activity of receptor protein tyrosine kinases, such as egfr, in cancer. egfr becomes activated upon ligand binding to the extracellular domain, leading to receptor dimerization. the juxtamembrane (jm) domain of egfr is critical for intrinsic tyrosine kinase activity and receptor dimerization by stabilizing the active conformation of egrr through the formation of a antiparallel helical dimer. therefore, peptides mimicking the jm domain -if specifically delivered to cancer cells -have the potential to prevent egfr dimerization, receptor activation, downstream signaling, and thus to attenuate aberrant egfr activity in cancer cells. here, phlip (ph low insertion peptide), a peptide that can selectively target cancer cells and tumors based solely on their extracellular acidity, is used to selectively translocate the jm domain of egfr in cancer cells to prevent egfr dimerization. at ph above , phlip is soluble and unstructured, however, when exposed to lower ph such as observed in tumors, phlip inserts as a transmembrane (tm) alphahelix, allowing the direct translocation of cargo molecules into the cytoplasm. using the dominant negative arac-based transcriptional reported assay (dn-aratm), which assesses jm and tm domain interactions in cells membranes of e. coli, we show that phlip-jm is able to disrupt egfr dimer by %. current work is focused on testing the ability of such phlip-jm peptide conjugate to perturb egfr homodimerization and decrease downstream signaling through soluble kinases, such as akt and erk, in cancer cells. the thumb subdomain of yeast mitochondrial rna polymerase is involved in processivity, transcript fidelity and mitochondrial transcription factor binding gilberto velazquez , luis brieba , rui sousa universidad de guadalajara, langebio cinvestav, university of texas healthsscience center at san antonio dna protein interaction abstract single subunit rna polymerases have evolved two mechanisms to synthesize long transcripts without falling off a dna template: binding of nascent rna and interactions with an rna:dna hybrid. mitochondrial rna polymerases share a common ancestor with t-odd bacteriophage single subunit rna polymerases. herein we characterized the role of the thumb subdomain of the yeast mtrna polymerase gene (rpo ) in complex stability, processivity, and fidelity. we found that deletion and point mutants of the thumb subdomain of yeast mtrna polymerase increase the synthesis of abortive transcripts and the probability that the polymerase will disengage from the template during the formation of the late initial transcription and elongation complexes. mutations in the thumb subdomain increase the amount of slippage products from a homopolymeric template and, unexpectedly, thumb subdomain deletions decrease the binding affinity for mitochondrial transcription factor (mtf ). the latter suggests that the thumb subdomain is part of an extended bindingsurface area involved in binding mtf . design principles of membrane protein structures vladimir yarov-yarovoy , diane nguyen membrane protein structure membrane proteins play key role in cellular signaling and ion transport. statistical analysis of expanding database of high-resolution membrane protein structures in protein data bank (pdb) provides useful information about membrane protein structure and function. we used rosettamembrane software (yarov-yarovoy v et al ( ) proteins) to analyze unique alpha helical membrane protein structures in pdb and derive knowledge based energy function for membrane protein structure prediction, membrane protein-protein docking, and membrane protein design. the rosettamembrane residue environment energy term is based on amino acid propensities in hydrophobic, interface, and water layers of the membrane and depends on the residue burial state -from being completely buried within a protein environment to being completely exposed either to the lipid or water environments. residue buried state is determined from the number of residue neighbors within and Å spheres. the rosettamembrane residue-residue interaction term is based on the propensities of amino acid pairs to be in close proximity to each other within hydrophobic, interface, and water layers. results of our statistical analysis reveal fine details of favorable and unfavorable environments for all amino acids types in all membrane layers and residue burial states. we find that large hydrophobic amino acids are favorable facing the hydrophobic core of the lipid bilayer. small amino acids are favorable facing the protein core within the hydrophobic layer of the membrane. aromatic or positively charged amino acids and favorable facing the lipid head groups. residue-residue interactions are often favored between polar and charged amino acids and also between some of small and large hydrophobic amino acids inside of the protein core within the hydrophobic layer of the membrane. these data will be useful for rational design of novel membrane protein structures and functions. coordinated gripping of substrate by subunits of a aaa proteolytic machine ohad yosefson , andrew nager , tania baker , robert sauer protein quality control' or 'protein degradation' hexameric aaa protein-remodeling machines use conserved loops that line the axial pore to apply force to substrates during the mechanical processes of protein unfolding and translocation. an open question in the aaa field is whether pore loops from different subunits of the hexameric ring grip the substrate coordinately (all six subunits involved), independently (one subunit at a time involved), or partially coordinated (two or three subunits at a time). to answer this question, we studied covalently linked hexamers of the e. coli clpx unfoldase bearing different numbers and configurations of wild-type and mutant pore loops and challenged these variants with protein substrates with a broad range of stabilities. we find that successful unfolding of increasingly resistant substrates requires the coordinated action of a greater number of wild-type pore loops. our results support a mechanism in which a power stroke initiated in one subunit of the clpx hexamer results in the simultaneous movement of all six pore loops, which coordinately grip and apply force to the substrate. structure and function of the toc m-domain, and its role in targeting the preprotein receptor to the chloroplast outer envelope membrane matthew smith , shiu-cheung lung , prem nichani , nicholas grimberg , j. kyle weston , shane szalai , simon chuong deartment of biology, wilfrid laurier university, department of biology, university of waterloo chloroplast biogenesis and function rely on the import of thousands of nucleus-encoded preproteins from the cytosol. preprotein import is supported by the toc and tic (translocon at the outer and inner envelope membranes of chloroplasts) complexes, which work cooperatively to translocate preproteins across the double-membrane envelope to the chloroplast interior. toc is one of the preprotein receptors of the toc complex, is also encoded in the nucleus and post-translationally targeted to the chloroplast, and is comprised of distinct domains: ) the intrinsically disordered n-terminal acidic (a-) domain; ) the central gtpase (g-) domain; and ) the c-terminal membrane (m-) domain that anchors the protein to the chloroplast outer membrane (com) through an unknown mechanism. the m-domain has no known homologues and does not contain a predicted trans-membrane domain, but does contain intrinsic chloroplast targeting information at the extreme c-terminus. the m-domain also contains a predicted b-helix motif, which may be important for anchoring the protein to the com. we are interested in characterizing the structure of the m-domain and determining the nature of its association with the com, as part of our larger goal of understanding the role toc plays in protein import into chloroplasts. we are also interested in defining the precise nature of the targeting information contained within the extreme c-terminus of toc , elucidating the targeting pathway that is used, and whether other com proteins use this pathway. we will present our most recent data on the structure, function and targeting of the toc m-domain. structural investigation of nlpc/p protein acquired by trichomonas vaginalis through a lateral gene transfer event jully pinheiro , , augusto simoes-barbosa , david goldstone microbiology, school of biological sciences, university of auckland, structural biology, school of biological sciences, university of auckland trichomonas vaginalis is an extracellular flagellated protozoan parasite that causes the most common non-viral sexually transmitted disease, with approximately million cases worldwide annually. nevertheless, the biochemical processes behind t. vaginalis infection and its interaction with the vaginal microbiota are still not well defined. in the draft genome sequence of trichomonas vaginalis strain g was described, identifying , protein-coding genes. of these, nine genes encode nlpc/p -like members. this superfamily is widely represented in the different kingdoms of life and has diverse enzymatic functions, such as amidases, endopeptidases and acetyltransferases. previous studies have shown that members of this superfamily hydrolyze specific peptide linkages in bacterial cell walls affecting germination, vegetative growth, sporulation and division or cell lysis/invasion. as a typical eukaryote, the protozoan parasite t. vaginalis does not have a cell wall itself. previous studies suggest that the t. vaginalis nlpc/p genes were acquired via lateral gene transfer from bacteria and must have an important function, possibly controlling the vaginal microbiota and aiding parasite invasion and infection. to investigate the function of the nlpc/p family of proteins in t. vaginalis we have expressed, purified and crystallized a member tvag_ and report its three-dimensional structure, determined at . Å resolution, by x-ray diffraction. the structure of the protein reveals a typical papain-like fold resembling peptidoglycan hydrolases from the nlpc/p family with a conserved cysteine and histidine; forming the catalytic residues. the protein contains two bacterial sh domains at the n-terminus. this domain acts as a general binding domain and is likely to aid the interaction of the nlpc/p domain with substrate components. combined with biochemical and enzymatic characterization, the structure of this nlpc/p protein will help to elucidate the molecular origin of its hydrolase activity and to decipher their putative role in the parasite infection. novel dna polymerases from red sea brine-pools: new potential polymerases for pcr application masateru takahashi , etsuko kimura , mohamed salem , ulrich stingl , samir hamdan protein biotechnology the polymerase chain reaction (pcr) is a key tool in medical and biological research. the most common pcr reaction relies on the thermal cycling method that consists of repeated cycles of heating and cooling steps for dna melting and extension by the dna polymerase, respectively. the introduction of new dna polymerases to the market is a major area of development that tremendously helped in improving the performance and quality of pcr. nonetheless, pcr still requires optimization of salt and metal ion concentrations leaving a room in the market for introducing new dna polymerases that are robuster in their salt and metal ion concentration dependence. in this study, we will present the characterization of a novel archaeal dna polymerase from the red sea brine-pool (termed br ) and demonstrate how its enzymatic activity reflects on every aspects of the environment of the brine-pool -high tolerance to concentrations and types of salts and metal ions including utilization of zn ions in its active site. these results suggest that the brine-pool microorganisms are likely to contain novel chemical pathways to deal with its exterior harsh conditions. we will further show the mechanism of br polymerase how it was adjusted to be active in harsh condition. structural basis for the identification of the n-terminal domain of coronavirus nucleocapsid protein as an antiviral target ming-hon hou , shing-yen lin , chia-ling liu , yu-ming chang , jincun zhao , stanley perlman institute of genomics and bioinformatics, national chung hsing university., institute of biological chemistry, academia sinica., department of microbiology, the university of iowa drug discovery coronaviruses (covs) cause numerous diseases, including middle east respiratory syndrome and severe acute respiratory syndrome, generating significant health-related and economic consequences. covs encode the nucleocapsid (n) protein, a major structural protein that plays multiple roles in the virus replication cycle and forms a ribonucleoprotein complex with the viral rna through the n protein's nterminal domain (n-ntd). using human cov-oc (hcov-oc ) as a model for cov, we present the d structure of hcov-oc n-ntd complexed with ribonucleoside '-monophosphates to identify a distinct ribonucleotide-binding pocket. by targeting this pocket, we identified and developed a new coronavirus n protein inhibitor, n-( -oxo- , -dihydrophenanthridin- -yl)(n,n-dimethylamino)acetamide hydrochloride (pj ), using virtual screening; this inhibitor reduced the n protein's rna-binding affinity and hindered viral replication. we also determined the crystal structure of the n-ntd-pj complex. on the basis of these findings, we propose guidelines for developing new n protein-based antiviral agents that target covs. thermal and structural stability of ß-glucosidases gh maira artischeff frutuoso departamento de bioqu ımica do instituto de qu ımica da universidade de são paulo enzymology we compared the stability of thermophilic b-glucosidases gh to mesophilic ones in the presence of denaturants as urea and high temperature by following the transitions between the native and unfolded states by tryptophan fluorescence, enzymatic activity and differential scanning fluorimetry (dsf). the bacterial b-glucosidases (bgla) and (bglb) of the mesophile paenibacillus polimyxa and bglucosidase (bglthm) of the thermophile thermotoga maritima were expressed as recombinant proteins in novablue (de ) and purified by affinity chromatography (ni-nta resin). these recombinant enzymes have very similar folding type structure (b/a) barrel, as shown in crystal structures and exhibited a characteristic peak between and nm in the tryptophan fluorescence spectra, indicating that those proteins are folded. circular dichroism analysis in the far-uv region ( nm to nm) also showed typical spectra of folded proteins with secondary structure composition of % of a-helix and % of b-sheets for bgla, % of a-helix and . % of b-sheets for bglb and % of a-helix and % of b-sheets for bglthm. the average degree of accessibility to the exposed tryptophan residues in the native enzyme to increasing concentrations of the acrylamide suppressor (stern-volmer constant -ksv) is greater to bgla ( . ), but similar to bglb ( . ) and bglthm ( . ). the thermal stability determined by dsf was higher for bglb (tm . c) than for bgla (tm . c) . the bglthm was stable at c and remained stable for up to h at c. in addition the thermal inactivation kinetics at c evaluated by the relative remaining activity showed that bgla denaturation (kinactivation of . s- ) is faster than bglb (kinactivation of . s- ). on the other site, bglthm inactivation at c was a two-step process, which exhibited an initial fast step (kinactivation of . s ) followed by a slow step (kinactivation of . s- ). the chemical denaturation by urea followed using tryptophan fluorescence showed a transition pl- covalent structure of single-stranded fibrinogen and fibrin oligomers cross-linked by fxiiia. the influence of free radical oxidation anna bychkova , vera leonova , alexander shchegolikhin , marina biryukova , elizaveta kostanova , mark rosenfeld n. m. emanuel institute of biochemical physics, russian academy of sciences protein structure and function native fibrinogen is a key blood plasma protein whose main function is to maintain hemostasis by virtue of producing the cross-linked fibrin clots under the effect of thrombin and fibrin-stabilizing factor (fxiiia). fxiiia-mediated isopeptide g-g bonds are known to be produced between g polypeptide chains of adjacent fibrinogen or fibrin molecules. but there are apparently conflicting ideas regarding the orientation of g-g bonds. in this study several peculiarities of self-assembly of fibrin(ogen) and induced oxidation of the proteins have been studied with the aid of elastic and dynamic light scattering, uv-, ftir-and raman spectroscopy methods. in the presence of fxiiia both the non-oxidized and oxidized fibrinogen molecules has been shown to bind to each other in the "endto-end" fashion to form the flexible covalently cross-linked fibrinogen homopolymers. to identify the orientation of g-g bonds in fibrin protofibrils a novel approach based on self-assembly of soluble cross-linked fibrin protofibrils and their dissociation in the urea solution of moderate concentrations has been applied. the results of elastic and dynamic light scattering coupled with analytical ultracentrifugation indicated the protofibrils to exhibit an ability to dissociate under increasing urea concentration to yield single-stranded structures entirely brought about by g-g bonds. the results of this study provide an evidence to support the model of the longitudinal g-g bonds that form between the g chains end-to-end within the same strand of a protofibril. since fibrinogen is known to be sensitive to ros the mechanisms of fibrinogen and fibrin self-assembly under induced oxidation have been investigated. in both cases the polypeptide chains of the oxidized fibrin(ogen) proved to be involved in the enzymatic cross-linking more readily than those of unaffected molecules. the enhancing role of the d:d interaction under oxidation could be considered as an compensatory mechanism in the assembly of fibrin when the d:e interaction is impaired. the experimental data on fibrinogen and fibrin oxidation acquired in the present study, being combined with our earlier findings, make it reasonable to suppose that the spatial structure of fibrinogen could be evolutionarily adapted to some ros actions detrimental to the protein function. the study was supported by the rfbr, research projects - - mol_a and - - a. structural and thermodynamic analysis of co-stimulation receptor cd phosphopeptide interactions with grb , gads, and pi -kinese sh domains in addition to the signaling produced by the binding of antigen-major histocompatibility complex to tcell receptors, co-stimulatory signals from other receptor-ligand interactions are required for full activation of t-cells. the cd receptor on the t-cell surface has been well characterized, and the binding of ligand to cd is critical for producing co-stimulatory signals. cd has no enzymatic activity and its cytoplasmic region consists of amino acids that contain the sequence ymnm, in which the tyrosine residue is phosphorylated by kinase. the phosphorylated sequence, pymnm, is recognized by src homology (sh ) adaptor proteins, such as growth factor receptor binding protein (grb ), grb related adaptor downstream (gads), and the phosphatidylinositol -kinase (pi -kinase) regulatory subunit, p . the consensus sequence for the binding of grb sh and gads sh is pyxnx, and that of p n-terminus sh (nsh ) and c-terminus sh (csh ) is pyxxm. we reported the high-resolution crystal structure of grb sh in complex with the cd phosphopeptide [higo et al., plos one , e , ] , and recently determined those of gads sh , p nsh , and p csh . these data along with the results of binding thermodynamics analyzed using isothermal titration calorimetry, helped to elucidate the molecular recognition mechanisms of cd by adaptor proteins. the sh proteins were overexpressed in escherichia coli, and were purified using affinity and gel-filtration chromatography. the cd phosphopeptides, -residue (octp) and -residue (ddcp ), were synthesized using the solidphase supported technique, and were purified using reversed-phase chromatography. the crystals were obtained by the hanging-drop vapor diffusion method. x-ray diffraction data were collected at synchrotron radiation facilities, and the structures were determined by the molecular replacement method. the models of grb sh , gads sh , p nsh , and p csh in complex with octp were refined at . , . , . , and . Å resolutions, respectively. the crystal structures showed that the phosphotyrosine phosphate moiety directly interacted with the side-chain of arginine in sh , which is common in all complex structures. in the grb sh and gads sh complexes, the side-chain of asparagine at the py position forms a pair of hydrogen bonds with the main-chain amide and carbonyl groups of lysine in sh . alternatively, in the p nsh and csh complexes, the side-chain of methionine at the py position is located in hydrophobic pockets of nsh and csh , in which the hydrophobic interactions of csh would be stronger than those of nsh . this idea is supported by the observed binding thermodynamics. the binding affinity of csh to ddcp , because of a favorable enthalpy change, is about -fold higher than that of nsh . the binding affinity of grb sh to ddcp is similar to that of gads sh to ddcp , and is about -fold lower than that of nsh to ddcp . these results indicate that the contribution of hydrophobic interactions of nsh and csh at the py position are stronger than those of hydrogen bonds of grb sh and gads sh at the py position. novel kinetochore protein complex from silkworm holocentric chromosomes takahiro kusakabe , hiroaki mon , jaeman lee the kinetochore, which consists of centromere dna and a multilayered protein complex, plays important roles in chromosome organization and segregation. interactions between chromosomes and spindle microtubules allow chromosomes to congress to the middle of the cell, and to segregate the sister chromatids into daughter cells in mitosis, which is followed cytokinesis. in contrast to monocentric chromosomes, in which the centromere is normally present at a single region on each chromosome, the holocentric chromosomes have centromeric activity along the entire length of the chromosome. it has been known that the silkworm, bombyx mori, has holocentric chromosomes since s, none of silkworm kinetochore proteins, however, have been identified so far. here we report the identification of a novel set of genes for outer kinetochore proteins in silkworm by using bioinformatics and rna interference-based screening. under the hypothesis that depletion of essential kinetochore genes causes cell cycle arrest in mitosis, we performed rnai in the silkworm cell line, bmn -sid , targeting a set of candidate genes. knockdown of five genes caused significant cell cycle arrest at the g /m phase. we also found that these five proteins make a complex, and that all of them are localized along the chromosome arms, indicating that the silkworm kinetochore extends along the chromosome. inactivation of bine aldehyde dehydrogenase from spinach by its physiological substrate bine aldehyde to contend with osmotic stress caused by drought, salinity, or low temperatures some plants synthesize the osmoprotectant glycine bine (gb) from bine aldehyde (bal). the last step-the irreversible nad dependent oxidation of bal-is catalyzed by aldh enzymes that exhibit bine aldehyde dehydrogenase (badh) activity. we here report that the spinacia oleracea badh (sobadh) is reversibly inactivated by bal in the absence of nad in a time-and concentration-dependent mode to approximately % of the original activity. inactivation kinetics are consistent with a partial reversible, two-steps mechanism that involves the formation of an active non-covalent enzyme•bal complex before formation the inactive enzyme-bal complex. crystallographic evidence indicates that in the enzyme previously inactivated by bal the aldehyde forms a thiohemiacetal with the nonessential cys (sobadh numbering) located at the aldehyde-entrance tunnel, thus totally blocking the access to the catalytic cysteine. accordingly, bal does not inactivate the c s sobadh mutant. two crystal structures of the inactivating enzyme-bal complex showed that the trimethylammonium group of bal is inside the active-site aromatic box, as in the productive way of binding. this explains why the inactivation of the a i mutant-where the binding of the trimethylammonium group is hindered-requires non-physiologically high bal concentrations, while the a c mutant-where the binding is allowed-is inactivated similarly to the wildtype enzyme. cys- is conserved in most plant aldh enzymes of known sequence, and in all of them with proven or predicted badh activity. inactivation by bal appears therefore to be a common feature of plants badhs. this short-term regulation may be of great physiological importance since the irreversibility of the badh-catalyzed reaction would unbalance the nad /nadh ratio if the aldehyde concentrations are high, the nad concentrations low and the reaction is not slowed down. plants badhs are prone to this situation since they work under osmotic stress conditions, when high bal concentrations are required for the synthesis of high levels of the osmoprotectant gb. the partial nature of the decamer possesses a donut shaped structure with calcium ions on the surface available for interactions with carbohydrate molecules. binding specificity was evaluated for carbohydrates using differential scanning fluorimetry (dsf) that showed bjcul interacts with galactose and lactose but less with glucose and sacarose. surprisingly, high levels of thermostabilization of bjcul was achieved with the antibiotic aminoglycosides geneticin (g ) and gentamicin in a calcium concentration dependent manner, but not kanamycin. intriguingly, while lactose and galactose inhibited erythrocyte agglutination by bjcul, g and gentamicin did not affect hemagglutination implying a second site of binding. dsf analysis also suggested the presence of a second binding site for the antibiotics and crystallization of the complexes are in progress in order to understand fully this new binding mechanism of c-type lectin with antibiotics. ab initio modelling of structurally uncharacterised antimicrobial peptides mara kozic institute of integrative biology, university of liverpool ab initio modelling of structurally uncharacterised antimicrobial peptides mara kozic * institute of integrative biology, biosciences building, university of liverpool, crown street, liverpool l zb, united kingdom * mara.kozic@liverpool.ac.uk antimicrobial resistance within a wide range of infectious agents is a severe and growing public health threat. antimicrobial peptides (amps) are among the leading alternatives to current antibiotics, exhibiting broad spectrum activity. an understanding of the structure of a protein can lead us to a much improved picture of its molecular function. furthermore, an improved understanding of structure-function relationships facilitates protein design efforts to enhance their activity. currently, the d structures of many known amps are unknown. to improve our understanding of the amp structural universe we have carried out large scale ab initio d modelling of structurally uncharacterised amps. such ab initio modelling is facilitated by the typical small size of amps as well as their tendency to contain disulphide bonds, these providing valuable additional information to simulations. preliminary results reveal unexpected similarities between the predicted folds of the modelled sequences and structures of well-characterised amps. for example, lacticin q was revealed to contain a helical bundle fold that bears a striking resemblance to enterocin a. we also found a remarkable similarity between the predicted structure of silkworm peptide and b-hairpin amps such as tachyplesin i. our results improve the understanding of the structure-function relationship of amps. surface aggregation-propensity as a constraint on globular proteins evolution susanna navarro , marta diaz , pablo gallego , david reverter , salvador ventura institut de biotecnologia i biomedicina and departament de bioquimica i biologia, institut de biotecnologia i biomedicina, universitat aut onoma de barcelona in living cells, functional protein-protein interactions compete with a much larger number of nonfunctional interactions. theoretical studies suggest that the three-dimensional structures of present proteins have evolved under selective pressure to avoid the presence of aggregation-prone patches at the surface that may drive the establishment of anomalous protein contacts. however, no experimental evidence for this hypothesis exists so far. the a-spectrin sh domain (spc-sh ) has been used as a protein model to decipher the sequential aggregation determinants of proteins. here we use it to address the structural determinants of protein aggregation and their link to protein evolution. to this aim we exploit aggrescan d (a d), a novel algorithm developed by our group, which takes into account both protein structure and experimental data to project aggregation propensities on protein surfaces. we used a d to design a series of spc-sh variants with progressively stronger aggregationprone surfaces and characterized their thermodynamic, structural and functional properties. our data support evolution acting to constraint the aggregation propensities of globular protein surfaces in order to decrease their potential cytotoxicity and the protein quality control machinery acting to buffer this negative selective pressure. utilizing d structure for the annotation of structural motifs in the conserved domain database narmada thanki-cunningham , noreen gonzales , gabriele marchler , myra derbyshire , james song , roxanne yamashita , christina zheng , stephen bryant , aron marchler-bauer , farideh chitsaz conserved domain database, structure group cbb/ncbi/nlm/nih the conserved domain database (cdd) is a protein classification and annotation resource comprised of multiple sequence alignments representing ancient conserved domains. cdd protein domain models are curated by ncbi and use d protein structure explicitly to define domain extent and the location of conserved core structures, and to provide accurate alignments between diverse family members via structure superposition. cdd also imports external collections such as pfam and tigrfam. recently, a novel class of annotation labeled as "structural motifs" has been introduced to supplement current capabilities. these annotations define compositionally-biased and/or short repetitive regions in proteins, which are difficult to model as functional domains conserved in molecular evolution. structural motifs include transmembrane regions, coiled coils, and short repeats with variable copy numbers. for many types of short tandem repeats, a few position-specific score matrices (pssms) suffice to annotate more than % of the known instances of that structural motif. unfortunately, a lack of sequence similarity within coiled-coil regions prohibits the development of only a few generic models; therefore, models for coiled-coil regions in the context of specific families have been developed using the spiricoil database as a reference. increased coverage of coiled-coil regions in cdd, specific site annotations of these structural motifs as well as their representation on the webpages will be discussed. specific in vivo ultrasound imaging of e-selectin expression in tumors using a microbubble contrast agent covalently attached to the peptide ligand iellqar, known to bind to e-selectin [ ] . however, it was observed that this probe has a limitation in the imaging of cardiovascular diseases where higher shear stresses prevent microbubbles from remaining attached to the target. therefore, peptides with higher eselectin affinity are needed to design probes capable of imaging these diseases. in this context, automated docking and molecular dynamics methodologies were combined and applied to different e-selectin binding peptides. these studies predicted the energetically more favorable binding mode as well as the key interactions between the peptide ligands and the e-selectin receptor. some of these peptides were prepared by solid-phase peptide synthesis and their interactions with e-selectin analyzed by surface plasmon resonance technique. the results showed that these peptides have different affinities for e-selectin. these data were correlated with the computational studies and evaluated to obtain crucial information of the key recognition elements needed for higher e-selectin affinity. these recent results will be presented. burkholderia pseudomallei is the causative agent of melioidosis, a serious invasive disease of animals and humans in tropical and subtropical areas. sedoheptulose- -phosphate isomerase from b. pseudomallei (bpgmha) is the antibiotics adjuvant target for melioidosis. in general, bpgmha converts dsedoheptulose- -phosphate to d-glycero-a-d-manno-heptopyranose- -phosphate (m p). this is the first step of the biosynthesis pathway of ndp-heptose responsible for a pleiotropic phenotype. therefore, this biosynthesis pathway is the target for searching novel antibiotics increasing the membrane permeability of gram-negative pathogens or adjuvants synergistically working with known antibiotics. the crystal of this enzyme has been solved at . Å resolution. there is an active site pocket where a putative metal binding site is located. to find out inhibitors of bpgmha, in-silico virtual screening with zinc, a free database of commercially-available compounds, has been performed. tens of thousands of chemical compounds were docked into the active site of bpgmha. a number of putative bpgmha binding compounds better than m p were found using surflex-dock included in the sybyl software package. characteristics of these compounds were surveyed and classified to identify common binding properties with bpgmha. mapping the structure of laminin using cross-linking and mass spectrometry gad armony , toot moran , yishai levin , deborah fass weizmann institute of science, department of structural biology, weizmann institute of science, israel center for personalized medicine laminin, a kda heterotrimer, is a major element in the extracellular matrix (ecm). within the ecm, laminin contributes to the adhesion and migration of cells, both in health and disease. the laminin trimer was observed by rotary shadowing electron microscopy to be cross shaped: the three short arms of the cross are formed by the amino-terminal halves of the three subunits, whereas the long arm of the cross holds the three chains together in a long coiled coil. the narrow and flexible arms of the laminin cross complicate studying its structure to high resolution by crystallography or electron microscopy single particle reconstruction. to advance our understanding of this remarkable quaternary structural assembly, we have used cross-linking and mass spectrometry to analyze the organization of the laminin trimer. this technique was validated by known crystal structures of isolated laminin domains. in all cases the crystal structure distances agree with the cross-linker length. the identified cross-links were particularly helpful in assigning the register and the subunit order of the long coiled coil due to the high content of cross-linkable residues in this region. using known x-ray crystal structures, homology modeling, and distance restraints provided by two cross-linker chemistries, a clearer picture of the laminin quaternary structure is obtained. non-sequential protein structure alignment program mican and its applications shintaro minami , george chikenji , motonori ota dept. of info. sci., nagoya univ., dept. of comp. schi. & eng., nagoya univ. in some proteins, secondary structure elements are arranged spatially in the same manner, but they are connected in the alternative ways. analysis on such non-sequential structural similarity in proteins is important because it provides a deeper understanding of the structural geometry of protein. this can be also observed even in the homologous proteins, indicating the non-sequential structural similarity is significant in the protein evolution. however, the non-sequential structural similarity in proteins is less investigated. we developed a novel non-sequential structural alignment program mican, which can handle multiple chains, inverse direction of chains, c$lpha$models, alternative alignments, and non-sequential alignments. we performed comprehensive non-sequential structural comparison among homologous proteins in the same scop superfamily by using the mican program. based on the result, we found that approximately % of superfamilies include at least one protein pairs showing non-sequential structural similarity. % nonsequential structurally similar pairs are aligned in a simple way, e.g. circular permutation, $$strand flip/ swap, but % are complicated. interestingly, most of such complicated non-sequential similarities can be explicable by combination of - simple non-sequential relationships. this result indicates that accumulation of simple structural changes in the course of protein evolution produces completely different fold homologs. as early as , ritter surmised that the cell's molecules cooperate to form a "special apparatus and an organised laboratory". despite supporting evidence from srere, mcconkey and others, efforts to understand molecular organisation in vivo are still in their infancy. however, important aspects of the cell interior have already been revealed. for example, weak molecular interactions structure the cytoplasm into time-evolving, functional zones. weak interactions are difficult to capture and can preclude protein detection in cells by many biophysical techniques, including nmr spectroscopy. , we explored the effects of cell-like milieus on the cytochrome c (cyt c)-flavodoxin (fld) interaction. these oppositely charged proteins interact weakly with a number of cognate partners. neither cyt c nor fld is detectable by nmr in escherichia coli confirming their "sticky" nature ( figure a) . the cyt c-fld interaction was assessed in buffer, % polyacrylamide gels and in solutions containing g/l of macromolecular crowders ( figure b) . h, n hsqc nmr revealed that the interaction was transient in buffer, proceeding via the known binding site for both proteins. substantial line broadening was effected in crowded and confined solutions suggesting that the cyt c-fld complex is stabilised under native-like conditions. the stabilising effect of macromolecular crowders was also observed by native gel electrophoresis and crystallization. these findings coincide with spitzer and poolman's model for cytoplasmic structuring, emphasising the role of charge-charge interactions and crowding in the formation of macromolecular "clusters". the implications for cytoplasmic structuring will be discussed alongside related investigations of cationic protein interactions in e. coli extracts. , detergent:protein ratio. the transmembrane b-barrel of bama is folded in either micelles, bicelles or nanodiscs, however an n-terminally attached single potra domain is flexibly unfolded, due to the absence of stabilizing contacts with other protein domains. measurements of backbone dynamics show distinct time scales of dynamic behavior for bama b-barrel and parts of its extracellular loop l , revealing high local flexibility within the the lid loop. this work presents the first high-resolution d solution nmr spectra of the bama barrel and establishes improved biochemical preparation schemes, which will serve as a platform for structural and functional studies of bama and its role within the bam complex. protein arginine methylation is a widespread and important posttranslational modification in eukaryotic cells, shown to be involved in the activation or repression of transcription, modification of the splicing machinery, signaling, and dna repair. mammalian protein arginine methyltransferases include a family of nine sequence-related enzymes that transfer one or two methyl groups onto the terminal guanidino groups on arginine residues, producing monomethylarginine only (mma, type iii), symmetric dimethylarginine (sdma) and mma (type ii), or asymmetric dimethylarginine (adma) and mma (type i). while prmt , , , , , and have been characterized as type i enzymes, and prmt as a type ii enzyme, the role and activity types of the two final members of this family of enzymes, prmt and prmt , had been unclear due to conflicting results in the literature, and the substrates for these enzymes had been elusive. both prmt and prmt are distinct members of the family with two methyltransferase or methyltransferase-like domains and containing acidic residues in otherwise well-conserved substrate double e binding motif, features not seen in the other prmt enzymes. recent work in our laboratory confirmed prmt as the only type iii mma-forming enzyme in the group, with a unusual low temperature optimum for activity, and a heretofore not seen preference for a basic stretch of residues in an r-x-r sequence for methylation. mutations of the acidic residues in the substrate-binding motif results in a loss of the specific r-x-r activity and the appearance of a g-r-g specificity typical of many of the other prmts. the physiological substrate of prmt has yet to be confirmed, although histone h b is an effective in vitro substrate. prmt , on the other hand, had no reported activity, until immunoprecipitation from hela cells showed it pulled down two splicing factors, sf b and sf b , in a complex. amino acid analysis showed that prmt methylates sf b to produce both mma and sdma, thus making it the second type ii enzyme in mammals. prmt knockdown results in modulation of alternative splicing events. this enzyme appears to be relatively specific for the sf b protein; a peptide containing the methylatable arginine residue was not found to be a substrate, and typical substrates of other prmts are not recognized by prmt . we found that the position of the methylated arginine residue in sf b is important, and the acidic residues in the substrate-binding motif also play an important role in substrate recognition. thus, prmt and prmt represent unique members of the mammalian prmt family. hydrogen peroxide levels, endogenous hormones (cytokinins, salycilic acid, as well as jasmonic acid and its conjugates), polyphenolics and terpenoids in a model system of a. alba in vitro with inhibition of rootng and stimulation of callusogenesis by means of individual and combined cytokinin and cytokinin/ auxin treatments. results: it was established that inhibition of rooting and stimulation of callusogenesis caused by benzyl adenine (ba) or combinations of ba and indole- -butiric acid (iba) in vitro were related to elevation of sesquiterpenoids in the essential oils, as well as polyphenolics content, accompanied by a drop of stress hormones, bioactive cytokinins and preservation of oxidative stress and lipid peroxidation levels, as compared with non-treated control. individual treatments with either iba or ba, also increased the sesquiterpenoid content in the essential oil of the plant, in a concentration related manner, this effect being more profound after ba treatment. in addition, ba treated plants exhibited a drop of protein levels of the aerial samples, as well as profound differences of enzymatic activity in the callus tissues, as compared with callus of plants treated with different combinations of ba and iba. conclusion: the results of the present work indicate that alterations of endogenous phytohormonal levels, caused by exogenous plant growth regulators treatment, might be the mediator between primary and secondary metabolism by means of affecting protein levels and activity of key enzymes in vitro. three different additives (( . % (v/v); formic acid, acetic acid, ammonium format with formic acid) have been investigated in response to ion intensity of esi-ms for individual hnp - in saliva. kinetex v r column separation efficiency was evaluated using two different column dimensions ( x . mm and x mm.) and two different stationary phases (c and c ). kinetex v r column (homogenous porous shell) performance was also compared to new ultra ace v r (encapsulated bonded phase) column. sample optimisation revealed that the spe method removes interference from salivary glycoproteins and consequently yields larger peak area ( - %) for all hnps. hnps were extracted by spe with a recovery of - %. the meoh: h o: acetic acid ( . %) provided enhanced (p> . ) hnp - ion intensities. the kinetex v r c ( x . mm, . mm) column facilitated a better separation efficiency of the four hnps as compared to the ultra core super c ace v r ( x . mm, mm) column, the kinetex v r c ( x . mm, . mm) and the kinetex v r c ( x . mm, mm) column. the relative levels of the hnps were determined in healthy volunteers before and after a rigorous exercise regime: it is possible that prolonged strenuous exercise will affect oral innate immunity and therefore also the level of salivary defensins. hnp - are traditionally detected in an enzyme-linked immunosorbent assay (elisa) which does not discriminate between the different hnps due to their structural similarities. there has therefore been a need to develop a mass spectrometry method that will discriminate between the defensins. as part of the method validation, the hnp - level was determined by elisa and the data was compared with the lc-ms data. here we present this cross-validation; the data revealed no significance difference between the two methods (r . ) which confirms that the developed lc-ms method is and equal sensitive method for the detection of these potential antimicrobial markers. this method can easily be adopted for similar molecular weight of peptides as hnps and also for any other biological matrix. moonlighting proteins: relevance for biotechnology and biomedicine luis franco serrano , sergio hern andez , alejandra calvo , gabriela ferragut , isaac amela , juan cedano , enrique querol institut de biotecnologia i biomedicina. universitat aut onoma de barcelona, laboratorio de inmunolog ıa, universidad de la rep ublica regional norte-salto multitasking or moonlighting is the capability of some proteins to execute two or more biochemical functions. the identification of moonlighting proteins could be useful for researchers in the functional annotation of new genomes. moreover, the interpretation of knockout experiments, in which the result of a gene knocking does not produce the expected results, might be enhanced. the action of a drug can also be facilitated because it might have an off-target or side effect with somewhat hidden phenotypic traits. it would be helpful that bioinformatics could predict this multifunctionality. in the present work, we analyse and describe several approaches that use protein sequences, structures, interactomics and current bioinformatics algorithms and programs to try to overcome this problem. among these approaches there are: a) remote homology searches using psi-blast, b) detection of functional motifs and domains, c) analysis of data obtained of protein-protein interaction databases (ppis), d) matches of the sequence of the query protein to d databases (i.e., algorithms like pisite), e) mutation correlation analysis between amino acids using algorithms like mistic. remote homology searches using psi-blast combined with data obtained from interactomics databases (ppis) have the best performance. structural information and mutation correlation analysis can help us to map the functional sites. mutation correlation analysis can only be used in very specific situations because it requires the existence of a multialigned family of protein sequences, but it can suggest how the evolutionary process of second function acquisition took place. we have designed a database of moonlighting proteins, multitaskprotdb (http:// wallace.uab.es/multitask/). from this database we determine the frequencies of canonical and moonlighting coupled functions (being an enzyme and a transcription factor the highest), the percentage of moonlighting proteins involved in human diseases ( % of the human moonlighting proteins in the database) and the percentage of moonlighting proteins acting as a pathogen virulence factor ( % of the moonlighting proteins in the database). correlation between potential human neutrophil antimicrobial peptides (hnp - ) and stress hormones in human saliva nadia ashrafi , frank pullen , birthe nielse , cris lapthorn , fernando naclario university of greenwich (faculty of engineering and sciene), university of greenwich (centre of sports science and human performance) numerous studies have investigated the effect of exercise on mucosal immunity but the focus has mainly been on salivary immunoglobulins lysozymes and hormones (cortisol, testosterone). this is not surprising given that iga and igg are the predominant immunoglobulins in saliva and there is a relationship between mucosal immunity and upper respiratory illness. it is well known that physical and mental stress provoke the release of cortisol from hypothalamic pituitary adrenal axis, by which stress can modulate various immune responses. in general, cortisol and growth hormones helps to induce the activation of neutrophils. to date, this study represents the first study that investigated the correlation between human neutrophil alpha defensins family against cortisol (stress hormone) and testosterone (growth hormone) in human saliva before and after exercise or training. twelve resistance trained athletes volunteered to participate in the study. participants consumed supplements during exercise and the hnp - , cortisol and testosterone response was investigated pre, post and minutes of the workout. the correlation between salivary antimicrobial peptide (hnp - ) and stress hormone (cortisol and testosterone) has been investigated using elisa. cortisol showed no significant (p . ) difference for (pre to min post) between cho and pl (cho: . . ng/ml; pl . . ng/ml) conditions but a strong trend (p . ) was observed for (pre to min post) post (cho: . . ng/ml; pl . . ng/ml) condition. testosterone showed no significant (p . ; p . ) difference for (pre to min post) between cho and pl (cho: . . ng/ml; pl . . ng/ml) and for (pre to min post) post (cho: . . ng/ml; pl . . ng/ml) condition. hnp - showed no significant (p . ) difference for (pre to min post) between cho and pl (cho: . . ; pl . . ) conditions but significant difference (p . ) was observed for (pre to min post) between cho and pl (cho: . . ; pl . . ) condition. the present findings suggested that there is no correlation between salivary hnp - and cortisol for (pl: r . and cho: r . ); hnp - and testosterone (pl: r . and cho: r . ). a worth note from previous study which suggested that using murine skin model (an increase in endogenous glucorticoids (cortisol) by physiological stress reduced mrna levels of antimicrobial peptide (cathelicidin). it is not clear that the correlation between hormones and antimicrobial peptide has been affected by the time interval of the exercise. both cortisol and antimicrobial peptide demonstrated a transient increase after exercise but it is surprising that they are not correlate to each other. one of the hypothesis from the present finding could be cortisol responses slow and it will be interesting to do further research with longer interval. the second hypothesis demands a further investigation to determine the synergism between substances. school of biomolecular and biomedical science, conway institute, ucd., king saud university, sciences, biochemistry department. the crystal structure of a human glucose -phosphate dehydrogenase (g pd) shows that each subunit has two nadp sites; in addition to a catalytic site there is a "structural" site which is distant from the catalytic coenzyme site. mutations causing severe deficiency tend to cluster round and close to the dimer interface and the structural nadp , indicating that the integrity of these areas is important for enzyme stability and therefore for maintenance of activity. in order to understand the molecular basis of g pd deficiency, and to have a clearer indication about the role of some features of the threedimensional structure, a fuller study of the second, "structural" nadp binding site is needed. human g pd controls the first committed step in the pentose phosphate pathway. it catalyses the oxidation of glucose -phosphate to gluconolactone -phosphate, generating nadph which is essential, amongst other things, for protection against oxidative stress. the human enzyme can be active in dimer or tetramer forms. human g pd of "structural" nadp per subunit of enzyme. this tightly-bound nadp can be reduced by g p, probably following migration to the catalytic site. the importance of nadp for stability is explained by the structural nadp site, which is not conserved in prokaryotes. after removing the tightly bound "structural" nadp the enzyme is still active but not stable. the effects of different nadp fragments on the stability of human recombinant g pd have been investigated. nadp is crucial for the long term stability of human g pd, and only one of nadp analogues which is adenosine diphosphate ribose - '-phosphate was able to slightly promote the stability of enzyme. . molecular characterization of specific positively selected sites in mammalian visual pigment evolution miguel a. fern andez-sampedro , eva ramon , brandon m. invergo , jaume bertranpetit , pere garriga grup de biotecnologia molecular i industrial., visual rhodopsin is a member of the g-protein coupled receptors superfamily. this membrane protein consists of a -cis-retinal cromophore bound to a seven transmembrane protein, opsin, by means of a protonated schiff base linkage. it has an important role as a dim light photoreceptor in the retina of the eye. by statistical models, where episodic selection in rhodopsin is tested on one branch of the phylogeny against a background of neutral or purifying selection on the rest of the tree, we have found some significant evidence of specific positively selected sites in early mammalian divergence. we have chosen the three amino acid sites identified with the highest posterior probability of having been targets of positive selection to perform experimental studies, i.e. (positively selected from m to f), (positively selected from r to q) and (positively selected from s to a). we have constructed, expressed, immunopurified and functionally characterized the proposed candidates, f m, q r and a s rhodopsin mutants located at the n-terminus, the transmembrane domain and the c-terminus region of the protein respectively. from the analysis of the molecular features of the f m mutant, we conclude that position is very important for protein folding and also for proper protein glycosylation, since we only could observe cromophore regeneration after its rescue in the double cysteine (n c/ d c) mutant background that stabilizes the n-terminal extracellular domain of the protein. our results also show that mutants q r and a s alter the g-protein activation rate, and hydroxylamine susceptibility in the dark-adapted state. in the case of q r, disrupting critical interactions with the neighbouring y of the conserved d/ery motif, critical in gt activation, could cause the lower gt activation ability. the mutant a s would create a potential additional phosphorylation site in the protein which could affect rhodopsin phosphorylation after photoactivation and, in turn, could affect the binding affinity of arrestin, a regulator of rhodopsin deactivation. this extra phosphorylation site could provide an evolutionary explanation for the enhanced response observed in the case of gt activation. in conclusion, these results highlight the importance of molecular investigations of positive selected sites in rhodopsin evolution and the relevance of structural and functional analysis of these sites in unravelling the molecular basis of visual pigment evolution. natural evolution sheds light on modern drug resistance in protein kinases marc hoemberger , christopher wilson , roman agafonov , dorothee kern the anti-cancer drug imatinib exhibits highly specific binding to the human kinase and oncogene abl with a three thousand fold weaker affinity for the structurally and functionally very similar kinase src. it has been shown recently that the major difference in binding of imatinib to abl and src stems from an induced fit after binding of the drug. to further understand the mechanism of imatinib binding to its target we used ancestral sequence reconstruction (asr) and resurrected enzymes along the node from the common ancestor of abl and src up to the extant kinases. we show that imatinib affinity is gained towards the evolution of extant abl while it is lost towards evolving src. the combination of asr and crystallographic data of the ancestors in addition to kinetics data allowed us to identify a subset of residues involved in imatinib specificity sufficient to switch from an intermediate binder to a tight binder. preliminary data shows that a network of hydrogen bonds and packing interactions stabilize the kinked p-loop conformation for tight binders thus allowing for more interactions between the kinase and the drug. strikingly, many of these residues were identified in human cancer patients as "hot spots" for the development of resistance mutations. further investigation into the identified subset of residues in combination with these commonly found imatinib resistance mutations will allow us to understand emerging drug resistances better. an evolutionary view of the cold adapted catalysis of enzymes vy nguyen , christopher wilson , dorothee kern the diversity in protein function that we see today arose as a result of life adapting to a cooling earth. how did enzymes, the catalysts of many crucial cellular processes, achieve this cold adaptation? this is a challenging question to answer because ancient sequences of proteins that existed billions of years ago are not available. to address this question we used ancestral sequence reconstruction to create adenylate kinase (adk) enzymes from the divergence of anaerobic and aerobic firmicutes towards modern day thermophilic, mesophilic and psychrophilic organisms. adk is a phosphotransferase that catalyzes the conversion of two adp molecules into atp and amp. we make the following observations. first, all ancestral enzymes are active with optimal catalytic rates linearly corresponding to the temperature of the environments where these proteins would have been found. most strikingly, the catalytic rate of our oldest adk ancestor exhibits a higher enthalpy of activation at low temperatures as compared to the modern thermophilic adk. this suggests a large enthalpic penalty had to be paid for reactions to occur at cold temperatures in an ancestor that existed in a hot environment. second, several high resolution crystal structures of extant proteins that we solved ( . Å - . Å), show that the oldest ancestors were more rigid than the modern adks due to an intricate salt-bridge network. this work, thus shows for the first time, the molecular and thermodynamic determinants of cold adaptation in an enzyme over a time period that spans billions of years. induced oxidative modification of plasma and cellular fibrin-stabilizing factor anna bychkova , tatiana danilova , alexander shchegolikhin , vera leonova , marina biryukova , elizaveta kostanova , alexey kononikhin , anna bugrova , evgeny nikolaev , mark rosenfeld n. m. emanuel institute of biochemical physics, russian academy of sciences, institute for energy problems of chemical physics, russian academy of sciences the main function of plasma fibrin-stabilizing factor pfxiii is to catalyze the formation of the intermolecular covalent cross-links between both gand afibrin polypeptide chains. the crosslinking crucially affects mechanical strength of fibrin and its resistance against fibrinolysis. the precise role of cellular fibrin-stabilizing factor cfxiii remains poorly understood. pfxiii is a heterotetramer (fxiii-a b ) consisting of two single-stranded catalytic a subunits (fxiii-a ), and two identical single-stranded inhibitory/ carrier b subunits (fxiii-b ). the subunits are held together by weak non-covalent bonds. contrary to plasma fxiii, cfxiii is a dimer (fxiii-a ) devoid of b subunits. as well as many other proteins circulating in the bloodstream, pfxiii is known to be a target for reactive oxygen species (ros) causing processes of protein oxidative modification. since the conversion of pfxiii to the active form of the enzyme (fxiiia) is a multistage process, ozone-induced oxidation of pfxiii has been investigated at different stages of its enzyme activation. the biochemical results point to an inhibition of enzymatic fxiiia activity depending largely on the stage of the pfxiii conversion into fxiiia at which oxidation was carried out. uv-, ftir-and raman spectroscopy demonstrated that chemical transformation of cyclic, nh, sh and s-s groups mainly determines the oxidation of amino acid residues of pfxiii polypeptide chains. conversion of pfxiii to fxiiia proved to increase protein susceptibility to oxidation in the order: pfxiii < pf-xiii activated by thrombin < pfxiii in the presence of calcium ions < fxiiia. with the aid of massspectrometry it has been demonstrated that oxidation leads to decreasing fxiii-a and fxiii-b coverage both in the forms of zymogen and in the presence of calcium ions. a group of amino acid residues involved in oxidation modification of pfxiii is identified in this study. the oxidation of either cfxiii or cfxiiia has revealed an almost complete loss of enzyme activity caused by dramatic changes in the primary and secondary structure of the proteins detected by the ftir data. taking into account these new findings, it seems reasonable to assume that the inhibitory/carrier fxiii-b subunits can serve as scavengers of ros. hypothetically, this mechanism could help to protect the key amino acid residues of the fxiii-a subunits responsible for the enzymatic function of fxiiia. the study was supported by rfbr, research project no. - - - a. mass spectrometry study was supported by the russian scientific foundation grant no. - - . performance and quality. making microcalorimetry simple with microcal peaq-itc natalia markova , ronan o'brien , mark arsenault microcal, malvern instruments ltd. dynamic interactions involving biomolecules drive and regulate all biological processes. studies of biomolecular interactions are fundamentally important in all areas of life sciences. data provided by isothermal titration calorimetry (itc) enables scientists in academia and industry to directly and quantitatively characterize these interactions in solution. microcal peaq-itc, the latest generation of microcal itc instrumentation, offers a whole range of solutions for addressing current bottlenecks associated with interaction analysis. among the most recognized challenges are the needs to adequately address a broad range of binding affinities and to reliably interpret binding data complicated by the presence of inactive protein fraction or inherent uncertainty in the concentration of a ligand. consistently high performance of microcal peaq-itc enables increased confidence and data resolution when measuring low heats at low or uncertain sample concentrations and complex binding modes. the new microcal peaq-itc analysis software allows for utomated data analysis, minimizing analysis time and user subjectivity in assessing data quality. data quality is determined and advanced fitting performed in a few seconds per experiment allowing for analysis of large data sets of or more experiments in a matter of seconds. glutamine-rich activation domain of transcription factor sp -biochemical activity and structure jun kuwahara , chisana uwatoko , emi hibino , katsumi matsuzaki , masaru hoshino faculty of pharmaceutical sciences, doshisha women's university, graduate school of pharmaceutical sciences, kyoto university transcription factor sp is ubiquitously expressed in a mammalian cell, activates reasonably large subset of mammalian genes, and is involved in the early development of an organism. the protein comprises two glutamine-rich (q-rich) regions (a and b domains) located in its n-terminal half, while three tandem repeats of c h zinc finger motif at its c-terminus binds directly to a gc-rich element (gc box) of dna. in general, q-rich domain is one of the typical motifs found in trans-activation domain of transcription factors together with acidic and proline-rich domains. transcriptional signal of sp are transmitted via interaction between q-rich domains of sp and different classes of nuclear proteins, such as tata-binding protein (tbp) associated factors (tafs) in components of basic transcription factor complexes (tfii). in addition, self-association of sp via q-rich domains is also important for its regulation of transcriptional activity. it has been considered that an sp ! molecule bound to a 'distal' gc-box synergistically interacts with another sp molecule at a 'proximal' binding site. although formation of multimers via q-rich domains seems functionally important for sp , little is known about relevance between biological activity and structural nature of q-rich domains. we analyzed nature of glutaminerich domains of sp by biochemical and physicochemical methods. we found that q-rich domains do not have clear secondary structure whereas they can indicate biochemical activity. detailed analysis of nmr spectra indicated interaction between the domains. the q-rich domains of sp might be one of the intrinsically disordered proteins (idp). chipping away at the yeast proteome: redesigning an e ubiquitin ligase for targeted protein degradation michael hinrichsen , lynne regan one of the central goals of synthetic biology is to exploit biological systems in order to produce compounds of therapeutic or industrial value . often, these efforts are complicated by the many natural biochemical pathways in cells that can compete for the same small molecule precursors. currently, the most common solution is to simply delete the genes coding for the competing enzymes . while such an approach has been successful, it is only applicable to nonessential genes and can produce unintended off-target effects such as decreased cell viability . an alternative strategy is to instead target proteins directly for degradation. using this strategy, scientists would first grow cultures of engineered cells to high densities under permissive conditions (i.e. targeted proteins are stably expressed). then, once sufficient cell density has been reached, enzymes of competing pathways would be rapidly degraded, resulting in the rapid production of high concentrations of the compound of interest. we propose to create such a tool by reengineering the c-terminus of hsp interacting protein (chip), an e ubiquitin ligase. chip recognizes substrate proteins through a short c-terminal peptide tag on target proteins . we have shown that fusing this tag to non-native substrates is sufficient for ubiquitination in vitro (data not published). cellular assays have also been performed in s. cerevisiae, a model organism commonly used in metabolic engineering applications . as a number of native yeast proteins possess c-termini similar to that of chip's native substrates (data not published), it was necessary to develop an orthogonal chip-peptide pair. this was achieved by replacing chip's natural tpr ligand-binding domain with a ligand-binding domain engineered previously in the regan lab . the altered chip construct has been shown to be active both in vitro and in vivo, and produces an altered growth phenotype when targeted against an enzyme involved in uracil biosynthesis. future work will focus on further kinetic characterization of the engineered enzyme, increasing its activity, and introducing the system into a proof of concept synthetic biology application. advances in modern sequencing techniques have resulted in an explosion of genomic data. correctly classifying this new wealth of information can be daunting not only because of the sheer volume of sequence data, but also because the propagation of erroneous and less-than-ideal names and functional characterizations in the current databases gets in the way of functional classification by mere sequence similarity. we are investigating the extent to which protein domain architecture can be utilized to define groups of proteins with similarities in molecular function, and whether we can derive corresponding functional "labels", starting with some of the most common domain architectures found in bacteria. to this end, we have developed an in-house procedure called sparcle ('specific architecture labeling engine') that lets us track and examine specific or sub-family domain architectures, resulting from annotating protein sequences with domain footprints provided by the conserved domain database (cdd), which includes hierarchical classifications for many common domain families. we will discuss how the proteins are grouped into specific architectures, our successes in assigning functional labels, and the major limitations we have encountered to date. while we will be able to assign functional labels to a large fraction of protein models derived from genome sequences, this effort has the added benefit of pointing out insufficient coverage and resolution of the current protein domain model collections that constitute cdd. we will also discuss alternative procedures that utilize pre-computed domain annotation for clustering protein sequences at a level that is well suited for functional labeling. we hope that this preliminary study will help to identify approaches that facilitate rapid and accurate annotation of genomes with a minimum of manual intervention. pegylated amyloid peptide nanocontainer delivery and release system self-assembly of telechelic peg end-capped with hydrophobic dipeptides collagen stimulating effect of peptide amphiphile c -kttks on human fibroblasts self-assembly of palmitoyl lipopeptides used in skin care products bioactive films produced from selfassembling peptide amphiphiles as versatile substrates for tuning cell adhesion and tissue architecture in serum-free conditions influence of elastase on alanine-rich peptide hydrogels interaction between a cationic surfactant-like peptide and lipid vesicles and its relationship to antimicrobial activity self-assembled arginine-coated peptide nanosheets in water toll-like receptor agonist lipopeptides self-assemble into distinct nanostructures approved drugs containing thiols as inhibitors of metallo-ß-lactamases: a strategy to combat multidrug-resistant bacteria references leukotriene a hydrolase -an envolving target. inflammatory diseases -immunopathology, clinical and pharmacological bases the bifunctional enzyme leukotriene-a, hydrolase is an arginine aminopeptidase of high efficiency and specificity lipoxygenase and leukotriene pathways: biochemistry, biology, and roles in disease a critical role for lta h in limiting chronic pulmonary this work was supported by the czech science foundation (project p / / ) and czech academy of sciences the tn antigen-structural simplicity and biological complexity bel b-trefoil: a novel lectin with antineoplastic properties in king bolete (boletus edulis) mushrooms acknowledgements: cynthia leyva-arg€ uelles is supported by a personal grant from conacyt, mexico. this work is supported by conacyt grant ' : and papiit grant in sequence information-based deciphering of biofunctionalities using ism-based techniques has fetched calculation of biological functionalities, designing of biomedical device called computer-aided drug resistance calculator, the understanding of the mechanism of hiv progression to aids [ ], and others. they have compared the efficacies of drugs and vaccines, which formed the basis for the innocentive award (id ) for assessing vaccine potency. conclusions: deciphering biological features without engaging reagents, equipments and animal tissues but biological data such as sequence information is one novel, feasible genotypic hiv-coreceptor tropism prediction with geno pheno [coreceptor]: differences depending on hiv- subtype a reliable phenotype predictor for human immunodeficiency virus type subtype c based on envelope v sequences available: http:// istree.bioprotection.org signal processing-based bioinformatics methods for characterization and identification of bio-functionalities of proteins an empirical framework for binary interactome mapping estimating the size of the human interactome coming to peace with protein complexes? th capri evaluation meeting pj- cabs-dock web server for protein-peptide docking with significant conformational changes and without prior knowledge of the binding site while other docking algorithms require pre-defined localization of the binding site, cabs-dock doesn't require such knowledge. given a protein receptor structure and a peptide sequence (and starting from random conformations and positions of the peptide), cabs-dock performs simulation search for the binding site allowing for full flexibility of the peptide and small fluctuations of the receptor backbone cabs-flex: server for fast simulation of protein structure fluctuations cabs-fold: server for the de novo and consensus-based prediction of protein structure cabs-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site mechanism of folding and binding of an intrinsically disordered protein as revealed by ab initio simulations modeling of protein-peptide interactions using the cabs-dock web server for binding site search and flexible docking cabs-fold: server for the de novo and consensus-based prediction of protein structure cabs-flex: server for fast simulation of protein structure fluctuations aggrescan d (a d): server for prediction of aggregation properties of protein structures cabs-dock web server for the flexible docking of peptides to proteins without prior knowledge of the binding site staphylococcal pathogenicity island dna packaging system involving cos-site packaging and phage-encoded hnh endonucleases the etiology of asdis unknown, but it is believed that it involves genetic and environmental components. the purpose of this work is to assess the possible involvement of food contaminants, such as mycotoxins, in the etiology of asd. the hypothesis is that the mycotoxins ingested with the diet could bind to proteins and expose the entire organism,including cns, to the negative effects of xenobiotics, in genetically predisposed patients. in this study some possible protein targets for the mycotoxinswere identified to evaluate if the bond between any protein target and the mycotoxin in exam could play a role in asd. twelve mycotoxins were selected (ochratoxin a, gliotoxin, aflatoxin b , aflatoxin b , aflatoxin m , aflatoxin m , aflatoxicol, a-zearalanol, b-zeralanol, zearalenone, deoxynivalenol, patulin),which are contaminants of milk and cereals.for each of these molecules,possible protein targets were searched by a reverse docking approach using the idtargetserver[ ].from the results given by idtarget, human protein targets expressed in the brain or involved in brain diseaseswere selected. subsequently, a direct docking was made using auto-dock . [ ], in orderto verify the strength of the interaction between selected proteins and each mycotoxin, and to identify the mycotoxins' binding site on each of the selected protein. finally, the bond of some mycotoxins to selected protein targets has been experimentally tested. for each mycotoxin, idtarget returned thousands of possible protein targets,and only those with the best binding energy were selected and evaluated. among them, human protein targets that are expressed in the brain or that are involved in cerebral diseases,have been selected; moreover the protein targets that were not human but that idtargetselected for five or more mycotoxins, were replaced with their human counterparts. at the end of the procedure, nineteen protein targets have been identified for the following direct docking approach. from the docking results, eight proteins have been selected for experimental tests, having a predicted binding energy lower than kcal/mol. finally, the interactions between acetylcholinesterase (ache), b-secretase (bace ) and neuroligin- , x-linked (nlg x) with aflatoxin b , aflatoxin b , gliotoxin, ochratoxin a and deoxynivalenol, were evaluatedusing fluorescence spectroscopy and microscale thermophoresis. these experiments confirmed the presence of an interaction between bace and aflatoxin b idtarget: a web server for identifying protein targets of small chemical molecules with robust scoring functions and a divide-and-conquer docking approach the calculation of spatial structure and "assembling" of the whole protein from the obtained peptide structures were performed by using molecular dynamics of the protein in the fully hydrated -palmitoyl- -oleoyl-sn-glycero- -phosphatidylcholine (popc) [ ]. the obtained structural model may contribute to identification of ul . active sites and elucidation of its mode of action nmr structural studies of membrane proteins acknowledgments: polish national centre for research and development -grant number pl- functional and mechanistic studies of dysferlin, an essential protein in cell membrane repair references moreover the 'm' parameter, which represents the denaturant effect on the protein stability, is cal•mol- for bgla and cal•mol- for bglb albert einstein college of medicine protein structure modeling, protein-protein interaction computational modeling of ini /smarcb and novel insights into its interaction with hiv- integrase savita bhutoria epsteain bar virus, nuclear antigen) . ini /smarcb has no known structural homologues, and its amino-acid sequence yields little insight into its function. a detailed understanding of structure-function relationships is hampered by the lack of structural information for ini . computational methods that model protein/peptide structures with sufficient accuracy to facilitate functional studies have had notable successes. we carried out combination of sequence analysis ab initio structure modeling and dynamics studies of integrase binding domain of ini and found it to be similar to that of phospholipase a activating protein, plaa. structural similarity with this distant protein suggests divergent evolution of the two proteins. the modeled structure sheds light on various protein-protein interactions of ini . by integrating the experimental studies about the binding, we have shown through docking, how a fragment of ini binds to the hiv- in. molecular docking and experimental studies indicated that two proteins bind tightly through charged/polar residues surrounding a hydrophobic cleft. these studies provide first modeled structure of ini /smarcb or any component of the swi/snf complex, and provide structural basis for in-ini interactions. this molecular interpretation of the intermolecular interactions is expected to facilitate design of inhibitors as novel class of anti-hiv- therapeutic agents ): e . with their catalytic activity towards rna substrates, other biological properties have been reported and evolution studies suggest an ancestral host-defence function in vertebrates. indeed, genetic studies confirmed a rapid molecular evolution within the family, a distinctive trait for host defence proteins exposed to a changing pathogen environment. previous studies from our laboratory characterized the wide spectra antimicrobial activity of two highly cationic human rnases: the eosinophil rnase and the skin derived rnase ribonucleases and have antimicrobial function in the human and murine urinary tract structural determinants of the eosinophil cationic protein antimicrobial activity two human host defense ribonucleases against mycobacteria, the eosinophil cationic protein (rnase ) and rnase the regulatory mechanism that we are reporting will contribute to prevent both nad exhaustion and accumulation of the toxic bal. to the best of our knowledge, this is the first report of a novel reversible covalent modification of an aldh enzyme involving its own substrate anna lewandrowska , aldona jeli nska , agnieszka wi sniewska acknowledgement: this research was supported by the inactivation of the fxr gene reduces aqp expression and impairs urine concentrating ability, which leads to a polyuria or urine dilution phenotype. we have previously found that pon -/-mice exhibit a polyuria phenotype and produce twice as much -h urine as their wild type pon / littermates (borowczyk k et al. metabolism and neurotoxicity of homocysteine thiolactone in mice: evidence for a protective role of paraoxonase development and application of novel non-ewald methods for calculating electrostatic interactions in molecular simulations ikuo fukuda , narutoshi kamiya the most time-consuming part of molecular simulation is the calculation of long-range interactions of the particles. in particular, appropriate treatment of the electrostatic interaction is critical, since the simple truncation cannot be used due to the slow decay of the coulombic function. thus, it is highly demanded to calculate the electrostatic interactions with high accuracy and low computational cost. for this purpose we have developed the zero-multipole (zm) summation method [ ]. in this method the artificial periodic boundary conditions are not necessary and the fourier part evaluations are not needed, in contrast to the conventional ewald-based methods. instead, a pairwise function that is suitably redefined from the coulombic function is used with a cutoff scheme. the underling physical idea is simple: (a) in a biological system, a particle conformation for which the electrostatic interactions are well cancelled is more stable than other conformations [ ]; (b) since such well-cancelled conformations are essentially physical, we should clip a subset of such a conformation out of the conformation within an ad-hoc given cutoff sphere and calculate the interactions only from this subset. this idea is realized by a rigid mathematical consideration that leads to the deformation of the coulombic function. the efficiency of the zm method has been validated in applications to fundamental systems sema a) is a protein originally described as an axonal chemorepellent cue involved in many physiological processes ranging from embryonic development to bone homeostasis or immune responses sema a signal transduction requires the formation of a heteromeric complex with neuropilin- (nrp ) and plexina [ ]. in addition, sema a interaction with nrp is modulated by the furin protease cleavage at its c-terminal basic domain this c-terminal basic domain has also been suggested to mediate the binding to glycosaminoglycans (gags), an association that locates sema a to perineuronal nets and enhances its function in restricting neuronal plasticity and inhibiting axonal regeneration in the central nervous system two peptides corresponding to the highly positively charged regions on the domain were shown to bind to immobilized heparin by surface plasmon resonance (spr) and the affinity dramatically increased when the complete domain was assayed. the binding was confirmed by nuclear magnetic resonance (nmr) and circular dichroism (cd) the conserved cysteine within this motif, necessary for the dimerization of sema a [ ], is also critical for the helix formation. in addition, fluorescence spectroscopy studies showed that the n-terminal region also has a contribution in the binding to gags. we acknowledge the financial support from the european union seventh framework programme (fp / - ) under the project vision semaphorin a: a new player in bone remodeling neuropilins lock secreted semaphorins onto plexins in a ternary signaling complex furin processing of semaphorin f determines its anti-angiogenic activity by regulating direct binding and competition for neuropilin semaphorin a displays a punctate distribution on the surface of neuronal cells and interacts with proteoglycans in the extracellular matrix semaphorin a binds to the perineuronal nets via chondroitin sulfate type e motifs in rodent brains mechanistic basis for the potent anti-angiogenic activity of semaphorin f. biochemistry collapsin- covalently dimerizes, and dimerization is necessary for collapsing activity prior attempts to create functionally relevant groupings of proteins in the crotonase superfamily suggest that this superfamily is difficult to cluster functionally due in part to the functionally diverse nature of the protein superfamily. we have developed two novel procedures to combat this difficulty: tulip (two-level iterative clustering process), a process that utilizes structural information from active sites to cluster protein structures into hypothesized functional groupings, and misst (multi-level iterative sequence searching technique), a process that uses the protein groupings created in tulip as a starting point for iterative genbank searches and further clustering after each search. through these two methods, the total coverage of the crotonase superfamily has increased, and the generated groups contain proteins from subgroups and families that did not have a structural representative. novel hypothesized functional protein groupings have been created, most notably for a large number of proteins that lack annotation data at the subgroup or family level, and for proteins of the enoyl-coa hydratase family fernandes , teresa sorbo , ivan duka , lia christina appold , marianne ilbert , fabian kiessling cnrs, umr , ucibio-requimte, faculdade de ciências e tecnologia e-selectin is a cell-adhesion molecule induced on the surface of endothelial cells in response to cytokines. its upregulation has been reported in many disorders, including inflammatory and cardiovascular diseases, tumor angiogenesis and metastasis [ ]. this profile suggests e-selectin as a promising target to develop molecular imaging probes for the detection of these diseases cyt c with equivalent of fld in media mimicking the cytoplasm. these include % polyacrylamide gel, g/l bovine serum albumin or polyvinylpyrrolidone , and buffer alone for comparison. electrostatic surface representations of the proteins are shown with their in-cell spectrum pl- a search for anti-melioidosis drug candidates targeted to d-glycero-d-manno-heptose- , -bisphosphate phosphatase from burkholderia pseudomallei bpgmhb converts dglycero-d-manno-heptose- b, -bisphosphate to d-glycero-d-manno-heptose- b-phosphate. this is the third step of the biosynthesis pathway of ndp-heptose responsible for a pleiotropic phenotype. therefore, this biosynthesis pathway is the target for inhibitors increasing the membrane permeability of gram-negative pathogens or adjuvants synergistically working with known antibiotics. to find inhibitors of bpgmhb, we performed homology modeling of bpgmhb and in-silico virtual screening with zinc, a free database of commerciallyavailable compounds. tens of thousands of chemical compounds were docked into the active site of bpgmhb. a number of putative bpgmhb binding compounds better than d-glycero-d-manno-heptose- b, -bisphosphate were found using surflex-dock included in the sybyl software package crystal structure of dimeric d-glycero-d-manno-heptose- , -bisphosphate phosphatase from burkholderia thailandensis ewha womans university we have solved the crystal structures of d-glycero-d-manno-heptose- , -bisphosphate phosphatase from burkholderia thailandensis (btgmhb) catalyzing the removal of the phosphate at the position of d-glycero-d-manno-heptose- , -bisphosphate. it belongs to the haloacid dehalogenase (had) superfamily with an a/b rossman fold composed of six parallel b-strands sandwiched between two sets of three a-helices it reveals a conventional rossman-like a-b-a sandwich fold with a novel b-sheet topology. its c-terminus is longer than its closest relatives and forms an additional b-strand whereas the shorter c-terminus is random coils in the relatives. interestingly, its core structure is similar to that of enzyme iib(cellobiose) from e. coli (eciib(cel)) transferring a phosphate moiety. in the active site of the closest eceiib(fruc) homologues, a unique motif cxxgxaht comprising a p-loop like architecture including a histidine residue is found. the conserved cysteine on this loop may be thiolated to act as a nucleophile similar to that of eciib(cel). the conserved histidine residue is presumed to accommodate negatively charged phosphate during enzymatic catalysis leonor morgado , kornelius zeth , , , bj€ orn m. burmann , timm maier bama is a b-barrel membrane protein with five periplasmic n-terminal polypeptide transport associated (potra) domains. the bama structure has been determined recently by x-ray crystallography ( , ), however its functional mechanism is not well understood. this mechanism comprises the insertion of substrates from a dynamic, chaperone-bound state into the bacterial outer membrane, and nmr spectroscopy is thus a method of choice for its elucidation we demonstrated that knocked down autophagy by shrna (shatg , shbecn , and shatg ) and chloroquine (cq) could enhance high dose of uvb induced cell death in odc overexpressing hela and mcf- cells. here, we also observed that knocked down odc in odc overexpressing hela and mcf- cells inhibited autophagy and enhanced high dose of uvb radiation. because of atg can regulate cell apoptosis and utophagy. site directed mutagenesis was used to mutant the amino acid which can regulate cell apoptosis and autophagy on atg , respectively in these two odc overexpressing cells. according to the results fish ß-parvalbumin acquires allergenic properties by amyloid assembly using atlantic cod b-parvalbumin (rgad m ) displaying high ige crossreactivity, we have found that formation of amyloid fibers under simulated gastrointestinal conditions accounts for the resistance to acid and neutral proteases, for the presence of membrane active species at gastrointestinal relevant conditions and for the ige-recognition in allergic patient sera. incorporation of the anti-amyloid compound epigallocathequin gallate prevents rgad m fibrillation, facilitates its protease digestion and impairs its recognition by ige. conclusions: rgad m amyloid formation explains its degradation resistance, its facilitated passage across the intestinal epithelial barrier and the epitope architecture as allergen autophagy could degrade the citrullinated and unfolding protein. herein, padi could enhance autophagy in jurkat t cells and lead to a degradation of p and the accumulation of lc -ii. autophagy and apoptosis are two critical mechanisms which participate against cellular stress, cell activation, survival and homeostasis. pad -overexpressed jurkat t cells caused the activation of th cells to increase mrna expression of cytokines, such as il- , il- , il- and tnfa. cytokines provoked caspase expression and led to caspase-mediated cleavage of beclin- which was an important factor of apoptotic signaling. knockdown of bcen rescued cell survival due to the increase of bcl-xl and the decrease of caspase- . we suggested that padi participated in the activated t cell-induced autonomous death through triggering er stress pathway studies on secondary metabolites production and proteins and enzymes of in vitro cultivated artemisia alba turra and relations with some endogenous phytohormones yuliana raynova , krassimira idakieva , vaclav motyka , petre dobrev , yuliana markovska , milka todorova , antoaneta trendafilova , ljuba evstatieva switzerland aim: artemisia alba turra is an essential oil bearing shrub, characterized with great variability of the essential oil profile of wild grown plants, related to genetic, geographic and environmental factors. it was previously established that inhibition of rooting in vitro caused by cytokinin/auxin treatment affected the essential oil profile of the plant and these changes were also related to bioactive endogenous cytokinin levels in vitro ( , ) cytokinin and auxin effect on the terpenoid profile of the essential oil and morphological characteristics of shoot cultures of artemisia alba terpenoid profile of artemisia alba is related to endogenous cytokinins in vitro salivary hnp - are conventionally measured using an enzyme-linked immunosorbent assay (elisa) which does not discriminate between individual hnps due to their structural similarities. considering the biological importance of salivary human neutrophil a-defensin (hnps), there is therefore, a need to develop an analytical method that will discriminate between the defensins. an lc-ms method has been established for the separation and detection of hnp - . the method has been optimised, validated and applied to examine the relative level of hnp - in participants undertaking a circuit resistance training workout. to date, no studies have systematically investigated the effect of acute (min to hours) and chronic (days to weeks) change in salivary adefensins family before and after exercise by lc-esi-ms systems and models calorimetry showed no difference in dissociation constants at these ph values, while the binding stoichiometry is increased . fold. furthermore, the binding stoichiometry varied fold among the two alginates corresponding to their difference in average molecular weight and in addition fold higher binding affinity was found with the high as compared to the low molecular weight alginate. in conclusion, the binding stoichiometry of b-lactoglobulin with alginate increases by a factor that correlates to the average molecular weight of the alginate and also a much higher affinity was found for the high molecular weight alginate. acknowledgements: this work is supported by the danish council for the presence of mucins and other high molecular weight glycoproteins in saliva makes the direct analysis of defensins difficult. the lc-ms method was linear for concentrations of hnp- between . and ng/ml (r . ) with a lod of . ng/ml. inter and intra assay precision was . - %, respectively. saliva sample were clean up by solid phase extraction (spe) and without-solid phase extraction (wspe) . mm internal diameter column in relation to the method transfer . during lc-ms optimisation genome-wide docking database (gwidd) provides the most extensive data repository of structures and models of ppi on a genomic scale. currently, we are expanding the gwidd dataset to , ppi in , organisms, up from , ppi in organisms in the previous release. the ppi data were imported from intact and biogrid databases and were subjected to in-house modeling pipeline. gwidd current implementation contains , experimentally determined complexes, and , sequence homology and , structure homology models of complexes. the user-friendly interface offers flexible organism-specific search with advanced functions for a refined search for one or both proteins. the new gwidd version includes also a new interactive visualization screen that allows to view search results in different residue representations with the emphasis on the ppi interface refolding and activation of recombinant trypsin i from sardine fish (sardinops sagax caerulea) amyloid is detectable in human dental plaque and is produced by both clinical and laboratory strains of s. mutans, further supporting a functional role. s. mutans lacking p demonstrates residual amyloid forming properties, however, a mutant lacking sortase, the transpeptidase which covalently links p and several other proteins to the peptidoglycan cell wall, is defective in cell-associated amyloid-like properties. the objectives of this study were to identify additional amyloid forming proteins of s. mutans and to evaluate the effects of buffering conditions and ph on the ability of the identified proteins to form amyloids. a p -deficient mutant strain was grown to stationary-phase in defined minimal media, and secreted proteins from spent culture supernatants were fractionated by ion exchange chromatography. partially purified protein fractions were tested for binding of the amyloidophilic dyes congo red (cr) and thioflavin t (tht), and for characteristic birefringent properties following staining with cr and visualization under crossed polarizing filters. proteins from fractions that tested positive for amyloid-like material were separated by sds page, and identified by lc/ms. these included wapa, gbpa, gbpb, smu_ c and smu_ c. recombinant proteins were expressed in escherichia coli, and purified for confirmation and characterization of individual amyloidogenic properties in vitro. recombinant wapa and smu_ c displayed all the biophysical characteristics of amyloid, including visualization of fibrillar aggregates when viewed by transmission electron microscopy. in contrast, gbpa and smu_ c produced amorphous aggregates. wapa and smu_ c form amyloid at different ph, smu_ c under acidic conditions and wapa under neutral to basic conditions. this suggests that the prevailing environmental ph may represent different in vivo triggers for amyloid fibrillization of different s like other small gtpases, the activity of rheb is dictated by its guanine nucleotide binding states: it is active in its guanosine -triphosphate (gtp) bound form and inactive in the guanosine diphosphate (gdp)-bound form. rheb proteins play critical roles in regulating growth and cell cycle, and this effect is due to its role in regulating the insulin/tor/s k signaling pathway rheb interacts directly with fkbp and prevents its association with mtor in a gtp-dependent manner. moreover, fkbp bound to gtp-g-s, a nonhydrolyzable gtp analogon, has a much higher binding affinity for rheb than the gdp-bound form the second study contradicted both studies, since they could not detect any interaction between rheb and fkbp [ ]. to clarify whether there is an interaction and if it is nucleotide dependent, nmr monitored interaction studies were performed employing a c-terminal truncated construct of human rheb ( - rhebdct) that cannot be farnesylated and the biochemically defined binding region on fkpb (fkbp -like fkbp -bd). based on our data rhebdct -gdp does not significantly interact with fkbp -bp. n-fkbp -bd titrated , we observed a weak interaction between rhebdct bound to a gtpanalogon (gppnhp) and fkbp -bd. mapping of the observed spectral changes on the structure of rheb-gtp suggests that fkbp targets the switch region, loop - and the neighboring b-sheet region. we further analyzed the backbone dynamics of rhebdct -gdp and -gppnhp using n relaxtion data (t , t and heteronuclear noe) .based on these data the phosphorylation loop, the switch regions and the loop around residues - show increased backbone dynamics that modulated by the nucleotide binding international centre for genetic engineering and biotechnology tdp- is an rna processing protein that can form inclusions of debatable nature implicated in neurodegenerative diseases. within the putative aggregation domain, repeats of residues - can recruit endogenous tdp- into aggregates inside cells . recently, we showed that a coil to b-hairpin transition in a short peptide corresponding to tdp- residues - enables oligomerization . we have used a broad battery of biophysical experiments, including chromophore and antibody binding, electron microscopy (em), circular dichroism (cd), solution and solid-state nmr, and x-ray to shed light on the nature of these aggregates. based on these findings, structural models for tdp- ( - ) oligomers have been constructed, refined, verified, and analyzed using computational methods, ranging from docking and molecular dynamics simulations to semiempirical quantum mechanics calculations. interestingly, tdp- ( - ) b-hairpins assemble into a novel parallel b-turn configuration showing crossb spine, cooperative h-bonding and tight side chain packing cellular model of tar dna-binding protein (tdp- ) aggregation based on its c-terminal gln/asn-rich region structural characterization of the minimal segment of tdp- competent for aggregation structural evidence of amyloid fibril formation in the putative aggregation domain of tdp- kyungpook national university scolopendin , aglqfpvgrigrllrk, is a -mer peptide derived from the centipede scolopendra subspinipes mutilans. to investigate its property against fungal and bacterial pathogens, antimicrobial tests were performed. we observed that this peptide exhibited antimicrobial activity in a salt-dependent manner and showed no hemolysis. the circular dichroism (cd) analysis observed that a-helical structure properties. we determined the mechanism(s) of action using flow cytometry and investigated the release of potassium. the results showed that the microbial membrane in escherichia coli o and candida albicans was permeabilized with loss of potassium ions. additionally, the bis-( , -dibutylbarbituric acid) trimethine oxonol [dibac ( )] and , '-dipropylthiacarbocyanine iodide [disc ( )] assay showed membrane depolarization. using calcein-encapsulating giant unilamellar vesicles (guvs) and fitc-dextran containing large unilamellar vesicles (luvs), scolopendin disrupted the cell membrane and the damage size is between . to . nm against composition of microbial plasma membrane of e. coli and c. albicans. thus, we demonstrated that a cationic antimicrobial peptide, scolopendin , possesses broad-spectrum antimicrobial effects that formed pore on the cell membrane. structural and functional investigation of the far c-terminal domain (ctd) of the bifunctional enzyme trai using nmr spectroscopy protein structural biology structural and functional investigation of the far c-terminal domain (ctd) of the bifunctional enzyme trai using nmr spectroscopy b.krishna chaitanya, evelyne schrank and klaus zangger institute of chemistry/organic and bioorganic chemistry university of graz, austria corresponding author email id: krishna.bhattiprolu@uni-graz.at bacterial conjugation is a complex process for the horizontal transfer of single stranded dna from one cell to another. this mechanism also leads, for example, to the spread of antibiotic resistance genes and virulence factors among bacterial species. multi-protein complexes formed at the origin of transfer (orit) region of dna and at the cytoplasmic membrane of the bacterial cell, initiate this process. inside the membrane, the relaxosome identifies the single strand for transfer in a plasmid dna, relaxes and unwinds it, whereas the transferosome is involved in pilus formation (type iv secretion system) and transferring the gene through the cytoplasmic membrane. these events take place in the donor bacterial cell along with several other auxiliary proteins [ ] the bifunctional enzyme trai of plasmid r plays a crucial role in the relaxosome activity, as it contains both a relaxase and helicase domain. to exert its functions on dna, trai works in close co-ordination with other relaxosome proteins like tray, tram and the integration host factor. trai is a residual protein and contains major domains: n-terminal relaxase domain, a central helicase domain and a c terminal domain (ctd). the structure of the c-terminal domain until residue has been solved by crystallography, while the structure and function of the remaining residues remained undetermined [ ]. there are saxs models and crystallographic structures for different parts of trai and also for the full length protein. prediction of cleavage specificity in hcv ns / a serine protease and adv cysteine protease systems by biased sequence search threading gonca ozdemir isik , a.nevra ozer department of bioengineering,faculty of engineering,marmara university proteases are enzymes which recognize specific substrate sequences and catalyze the hydrolysis of designated peptide bonds to activate or degrade them. due to the biological importance of proteases, it is particularly important to identify the recognition and binding mechanisms of protease-substrate complex structures in drug development studies. the assessment of substrate specificity in protease systems is crucial, where interpreting the adaptability of substrate residue positions can be useful in understanding how inhibitors might best fit within the substrate binding sites and aid in the design of potent selective inhibitors. substrate specificity is generally determined by the amino acid profile, structural features and distinct molecular interactions. besides experimental methods, computational tools for prediction of natural substrate cleavage sites, such as threading, have emerged as useful alternative approaches to provide valuable insights into complex enzyme-substrate interactions. in this work, the substrate variability and substrate specificity of the hepatitis c virus (hcv) ns / a serine protease and the adenovirus (adv ) cysteine protease was investigated by the biased sequence search threading (bsst) methodology. using available crystal structures of the proteases, the template structures for the substrate-bound proteases were created in silico by performing various peptide building and docking procedures followed by energy minimization and molecular dynamics (md) simulations. bsst was performed starting with known binding, nonbinding and some random peptide sequences that were threaded onto the template complex structures, and low energy sequences were searched using lowresolution knowledge-based potentials. then, target sequences of yet unidentified potential substrates were predicted by statistical probability approaches applied on the low energy sequences generated. the results show that the majority of the predicted substrate positions correspond to the natural substrate sequences with conserved amino acid preferences, while some positions exhibit variability. for ns / a serine protease cleavage, the significant selection for pro at p and cys at p positions is zearalenone is a mycotoxin produced by fusarium graminearum and related fusarium species. f. graminearum is a powerful plant pathogen and infects major crop plants around the world. acute toxicity of zearalenone is low, but due to its structural similarity to b-estradiol it has binding affinity to the estrogen receptor, which results in interference with hormonal balance. typical effects seen in animals include symptoms like hyperestrogenism and reproductive disorders (reduced fertility, reduced litter size or swelling of uterus and vulva). to reduce the risk for human and animal health posed by the ingestion of contaminated food or feed different decontamination strategies have been studied, including biotransformation. today many microorganisms are known to degrade zearalenone, but for most of them the degradation pathway and formed metabolites remained unknown, hence it is unknown if this degradation also means detoxification. only for the fungal strains trichosporon mycotoxinivorans and gliocladium roseum zen degradation has been studied in detail and loss of estrogenicity of reaction products has been confirmed. we screened for, and isolated zearalenone degrading bacteria from soil samples. the most promising new bacterial isolate was taxonomically assigned to the species rhodococcus erythropolis and designated pfa d - . the zearalenone catabolism pathway of pfa d - was found to be identical as known from g. roseum. the primary reaction product, hydrolysed zearalenone, has so far only been postulated in g. roseum. we prepared hydrolysed zearalenone by preparative hplc and showed loss of estrogenicity in assays with the breast cancer cell line mcf and the estrogen reporter yeast strain yzhb . a genomic library was prepared and screened in zearalenone degradation deficient r. erythropolis pr . the gene encoding zearalenone hydrolase was found and named zena. the hydrolase was identified as member of the a/b-hydrolase family and named zena. it was cloned, recombinantly expressed in e. coli and purified by x his-tag mediated immobilised metal affinity chromatography. activity of his-tagged and untagged enzyme zena was compared in cleared lysate and zena was purified for enzyme characterisation. the influence of ph and temperature on enzyme activity and stability was evaluated and kinetic parameters were determined. a new biding site for snake venom c-type lectins?maria cristina nonato costa , ricardo augusto pereira de p adua , marco aurelio sartim , suely vilela sampaio university of são paulo, fcfrp c-type lectins are proteins that bind different glycan molecules by interactions with a calcium atom present in a carbohydrate recognition domain (crd). many organisms (plants, bacteria, virus and animals) use these proteins in various biological events like lymphocyte adhesion, erythrocyte agglutination and extracellular matrix organization. the c-type lectin fold is plastic and possible for about different sequences, what promoted its adaptation to diverse functions, similarly to the observed for the immunoglobulin fold ( - sequences). it is comprised of about - amino acid residues that folds in two fourstranded b sheets sandwiched by two alpha helices. interestingly, c-type lectins present in snake venoms are possible anti-cancer agents since they are toxic to cancer cells and inhibit the adhesion and proliferation of various cancer cell lines. therefore, we have purified a lactose binding c-type lectin from the venom of bothrops jararacussu (bjcul) to study its structure and binding properties to different sugars. bjcul crystals were obtained by vapor diffusion and the structure solved by x-ray crystallography to . Å resolution. bjcul structure is a decamer formed by a pseudo fivefold axis rotation of a dimer hold by a disulfide bond. each monomer binds a calcium atom and possibly another metal at a second and opposed binding site.